Fundamentals of veterinary clinical pathology 2nd Edition

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s, ....... L S'ockham, DVM. MS. Diplo"",,,,. American Cou.g. ofV."'rinary Pathologist. (Oinical P:othol· ogy): Prokssor in tho o.partmtion. Colt.g. of V."'rin:uy M. dici"". Micbig>n Sto.. Univrnity. Ea., Unsing. Michigan. 0 2008 Bl:ocn",U Pubwhing All ri&Jt'" r""""d Bl:ocn",U Publi'hing Prof""iorW 2121 S.... Avrnu •. An=. low> 50014. USA 1.800.862..6657 Officr: 1·515·292·0140 F",,: 1·515·292·3348 Wrb .i..: W'VW.bbcXw.Uprof"..iorul.oom

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Bl:ocn",U Publi'hing ltd 9600 G .... ington Ro.d. Orloro OX4 2DQ. UK TrI.: +44 (0)1865 n686s Bl:ocrnU Publi'hing AU. 5'50 Sw.n.ton Street. Carlton. Victoria 3053. Austr:olia TrI.: +61 (0)3 8359 lOll Autboriz. tion to pbotocopy itrrm for int ..mJ or penorW ..... or the in..mal or personal .... of 1peCific cli
c...loging.in.Pub~""tion 0...

Stoddt:un. St... n L Fund.mentals of ""..rirury c~nical p.thology I S~ L Stoddt:un. Micb..t A. Soott~2nd rd. p.:cm. Includes bib~ogr>phical refr .. J>CeS :and in""'. ISBN.]3: 978-0-S138·0076-9 (:d.k. p"per) ISBN·IO: O-S138·0076-5 (:d.k. p"per) I. V... rirury clinical p"thology. I. Soott. Mid.. el A. (Michoel Abn). 1957- II. Title. [DNu.t: l. Animal Dis....... p"thology. 2. Cliniul LIDo"'OOY Tr<:bniqueo-vrterinary. 3. Pathology. V... rinary----nmbods. SF 772.6 S86if 20081 SF772.6.S762008 636.089·607-
CONTENTS Prcfocc

vu

ix

Admowlodgmcnts

1

Introductory Concept>

2

l
3

Erythrocyt<>

4

PJ"d
5

Hcmosusis

6

Bone Mmow and Lymph Node

7

Proteins

8

Urin' rySys,cm

9

Monov.!m. FJcctro~~cs and Osmo[,Jity

3

H

W7

m m 323

369 415 495

10

Blood Gas"" Blood pH, .nd Strong Ion Diff
11

Calcium. PhO!phorus. Magn",;um, .nd Their R
12

Enzymes

13

Liv
14

Glucose, K<1oarnincs, and Rd .. od Rogu!'tory HonnOD'"

15

&ocrine PancJ<'2S and [n,,,,,ine

16

Lipid.

17

Thyroid Function

18

Adrenocortical Function

19

Cavi..'}' Effusion.

•

Color

559 593

639 615

701

739

763

pl.,,,,

783

S05

831

Iv"""" pag~1494 >In. 495

Ind"" 869

,

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PREFACE Writing thi, =nd ~di{ion v..,., motiv"ffl by our de';", to synthesiu .nd incorporat~ n<we. information rdarn! to fundament:'!, of _"inary clinical p.thology, lind it WlI< drinn by the ",me goals a, the lim al ition: to uptain the physiologic, pathologic, :md an. lytical ronditiollS or diwrders responsible for abnornul J.bor.Hory datil, lind to do so using con';swlt terms .nd II uniform forJrult. Wh.n~r possible, di!U.le< :md conditions.", groupM by common m«ha. lIi,ms or processes to promou "COII""pruaJ understanding oflaboratory d.to thai can be, gener_ aUy appli.,j across many s~es. Th. content of the lim .clition w>s largdy p .... rval. but the =nd ,"",irion ronrains :addi. tional disorder>, diagnostic tests, illustrations, "&",,,=, :md pathophy.iologic explanations. There .'" four major cru.nges: {I} th•• ruolytical a'peen in Chop,,, 2 of th. first «ii{;on "e now

dinributal imo th.i, '''']l«,ivr ch.pters on I. ukocyt .., erythrocytes, .nd plald.ls {Chaplnd volume ",..ould b. r«J.uirrd to do juni", to it. Lastly. matrri:d i. ag.in restrictrd mo,tly 10 dogs, cat., horsts, and cattle b.caus< th ... spni., provide the basi, fot • fundamental und.manding of nterinary clinical pathology. Rq:re'llably, we willlikdy uncover mistakes in th. =ond .clition as ~ did in Ihe first. Corrrctiom for Ihe.. mistakes will b. .vail.ble .t this textbook', WdJ ,ite {s... reb FYCP Slockh.m or FVCP Scott}, which i. linkrd to th. publish..-, Web .ite and tho authors' Web .it.,. Also available in th. textbook', Web ,ile ... lim of suggmrd .~ific objrctives that may b. used as ,rudy guides. and drctronic copi.. of the book's figures .vail.ble aclusivdy for .clucation:d purp<>5<s. On", again, we found th. writing of th. =nd .clition to b. a gr.... t I... ming exp"iena. and ho~ that our efforts facilitot. th. I... ming of others.

'"

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ACKNOWLEDGMENTS FIRST EDITION Many ~pJ~ dir~ctly .nd indi=:dy assi,t.d in the writing of this book. W. thank our faman;c f. milies for providing th. lIHI., ""planation., and their anS\nrs provid.d insight ;1110 what thtffi th~ chall~n~ of writing thi. text without the ~rsisunt ~ncouragem~nt of a longtime &i~nd, oollragu~, and a""llent t~ach~r-Donald A. Schmidt, DVM, PhD, Diplom.t~. ACVP, ProftJ.50r Emenior author} w~re atremdy fonu""te to h.", Dr. Schmidt a. a texh~r. The specialty in veterinary clinical p.rhology gr~ out of the increased use oflaboratory test. in vturinary mffii"in~, WlI.I promotffi in th~ early 1960. by. society now known as the Am~ri_ can Society for Vet
W. ar~ nilJ greatly .ppr«iativ~ of th~ many pwpl~ who directly or indirectly ..sisted in the writing of th~ first edition, .nd we extend ,in"".. thanks to coU~agu .., ,rudenu, and family who mpponffi our .fforts to produ"" th~ oerond ffiition. W•• ppr~ciat~ th~ faculty in our depart_ menU and collq:.. who value th~ ",holarly activiti .. of t.,..ching and the writing of a textbook, and we are .. peciaUy thankful for the unw.vtring support of our spouses and for th~ enrouraging f"""back off~r.-d by v~~rinary nud~nts, ... id~nts, and fdlow clinical pathologist,.

"

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Chapter 1

INTRODUCTORY CONCEPTS Clinical Pathology, ......................... . • . • . • . • . • . •...................... 4 Samples .... ..... . . . .................. . ....•.• .•.•. • . • ...................... 5 I. Blood Samples and Specimens ... ..... • . • .•.•.•.•...................... 5

II. Urine Samples ......................•.•.•.•.•.•...................... 7 III. Other Body Fluid Samples ....... . .... • . •.•.•.•.•...................... 7

Major Types of laboratory Assays .........•.•.•.•.•.•.•.•...................... 7 Signif icant Figures ......................•.•.•.•.•.•.•.•...................... 9 Units............... ................. •.•.•.•.•.•.•.•.•..................... 10 Reference Intervals ........................•.•.•.•.•.•.•..................... 16 Quality of Laboratory Results ............ . . .•.•.•. • . • . • . • ..................... 20 I. Major Determinants ............ ...............•..................... 20 II. Analytical Properties of Assays ..................•..................... 21 III. Quality assurance .............. . ....... .......•..................... 25 Differences in l abo ratory Methods and Thei r Results .......•..................... 29 Comparison of Assays ....................................................... 31 Which Laboratory Do You Use? .......... . . . . . . . . ............................. 35 Evaluating and Validating Laboratory Met hods .................................. 36 Diagnost ic Properties and Predictive Value of Laborat ory Assays ................... 38 Receiver Operating Characteristic (ROC) Curves ................................. 45 Herd-based Testing f or Cattle ................................................. 48

3

4

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

Table 1.1. Abbreviation. and ,?',nools in lhi. chapler

[IT.J.. [xl BHB

FIN thyroxin~ con~lItration by '"'Iuilibrium dialysis x cona.ntmion {x = ana/yt. }

Ca' + CLSI CV

Calcium Clinical Laboratory Stand",1s [n!limt. Co.fficicnt of variation Echylcllffli.minmtr:>ac<:{ic acid F= ioniud calcium False nq;at;"" False pmitivc

EDTA ,y FN FP fT,

~.Hydroxybutyratc

F= thyroxine [ntwlational Falcr:nion ofClin;co] Chemistry [mcrn>tional Union of Pure and AprHa! Chcmi.lly Dipotassium salt of ethylcntdiaminclctraacrtic :ocid T ripou .. ium salt of cthyicnffii:mlinetci"",c;.,{ic acid OJ,odium .all of cchylcnaliaminmtr:>ac<:{ic acid N>lional Commi{{~ for Clinical Laboratory Stmdanb Nonesurifial fmy acids (fany acid.) National Institute ofStondanh and Technology Pralictive v:llu. of. negatin usc PVH Pralictive v:llu. of. pmitin test PV(+} ROC R."",inr o~rating characr..istic Standard d",iation ,d Sl Syst' m. Inumational d·Unite. T, T riiooothyronin. ,C." Total colcium Total .. ror allow.bl. TE. T ru. n.gativ. TN TP T ru. pmitin TRH Thyrotropin_rd=ing hormon. Total thyroxin. V Im.. national unit Umal stondard d",iation VSD WRl Within .. ft ..n~ im"v:ll Noto: s... T . bIe 1.4 fOr . bb",vi. tioru of units of ",.,.,we"",nt .nd the figure kg.., ... for . bb""" .. lFCC JUPAC K,EDTA K,EDTA Na,EDTA NeelS NEFA NIST

,T,

oons unique to 611"=.

CLI N ICAL PATH OLOGY I.

What is clinical pathology? A. Ckfinitions' l. p"",,,u,gy i, th. "branch of malicin. that d",ls with the b",j, of di ...... ''P'''ialJy tho .. strucrural and functional ch. ng.. in organ •• nd tis.!ues cousing or ",usa! by. di ...... • In gtne",l t.. m,. it is th. study of di ......

1/ INTRODUCTORY CONCEPTS

5

2. Clinkal patm,/ogy is • "mrup«iahy of pathology that drals with the u", of labora_ tory metho,j, (clinical chemistry. microbiology. hematology •... ) for the diagnosi. and treatment of di",=. " In general temu. it is the study of di",= in the clinical environment by UM of l.boratory '=)'1 B. A:J cumntly ""rtifi.d by the Am"ic;m CoUege ofY~"inary Pathologists. wmiNlry c/iniral patlwlogim ar< .p«ialim in the discipline. of b;;nic p.thology. hematology (nudy of blood). clinical chemistry (study of physiologic and biochemical reactioru). cytology (.tudy of ""ns). and surgical p:uhology (nudy of di=K vi<> microscopic analysi. of tiOSlle samples obuin.d during surgery). C. Veurinary clinicol pathologists .nd oth" laboratory profe1Sionals (m.dical trchnolo_ gin.. mrdicallabomory trchnicians. and '~trrinary trchnicians) often work in • clinical laboratory th.t limits its as>ay "menu" (offeral tests) to hematologic assays. clinical chemicol .... ys. urinalysi •• and clinical cytologic or histologic examinations. Othu assays or diagnostic laboratory p~ur .. a" off".d by .p«ific labomori.. «.g .• microbiology. histopathology •• nd toxicology) th at ." mprrvis.d by microbiologim. hinopathologists. and toxicologists. r<>p«tivdy. II.

Laboratory tests should k used with oth" diagnostic proc.dur<S. Before laboratory tem a" u...d to purme a possible diagn<>..ssify pathologic sum that denlop in domestic ma mmals. Some body systems (e.g .• integument. nermus. skeletal. and cordiovaocular) .'" rdativdy ..... ily evaluat.d via visual or imaging methods (physical examination. radiography. and ulumonography). whe"as oth" lxxIy .ynem. (e.g.• hemic. immune. urinary. and endocrine) are kller evalu.t.d by laboratory tem.

Ill.

Whot." the m. jor " • ..,ns for analyzing patient sample. vi<> laboratory proc.dures? A. To d~ect.n unidentifi.d pathologic st.u B. To define. classity. or confirm a pathophysiologic disord" or di..,= nate C. To diminate (rule out) • possible couse of the anima!". illness D. To assess rn..nge. in • pathologic .tate eith" due to natural prOf:ression of the disease or kcau .. of mrdical or mrgical therapy

SAMPLES I.

Blood sampl.. and sp«imens A. Mon clinical laboratory assays are design.d to d~ect or quantity substan= or ""lis in blood sampl..; the sUNun"" or ""JJ of int"en is caU.d the aNllytr. Obuining useful results for the analyte ""luir.. appropriate umples. Whenev" th"e is doubt about the appropriate .. mple for • particular test at" panicular laboratory. the laboratory mould k conuct.d prior to sample collection. (Stt Quality of uboratory Results. section 1A.) B. Blood l. Blood and iu m.jor components are frequently used as sampl.. for laboratory """)'1 Blood mun bt collect.d and processed properly so that assay results reflect the true composition of blood rathu than .nifactual chang ...

6

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY 2 Blood is romp<=!']",., alld five Jruljor la>kocyt. Iyr") and pl=. Blood withdr:lwn from ~ blood vessd must imm«iiatdy k mind with an anlioo,,!:ulmt to prevent in;ti.otion of clot forJrult;on ~nd to maimain "",Us and omu oompon.nu in m5Jl<'fl!ion. 3. Analysis or proct:S!ing of whole blood must k rdativdy rapid b«:au.. the ctlls di. within • f~ houn, and thus a sample wiU krom. un'ccqJubJ. for .nalysis, What constitute, . d
e. Pwm.

I. Pl..,m. is the fluid component of blood th . t is ru.rv<Slffl .fw ctntrifug.tion of an amicwguJ.t.d blood sampl... PjasJrul will contain the .ntj~Jant th.t can illt.rf.t. with some :uu)"l. 2. Anticoagulants ust called antithrombin Ill), which then inhibiu the activiry of several coagulation factors {including thrombin}. It :oIso forms .n ionic bond with C~". but its major action is through .ntithrombin. {I} Usrd for several .peciallaboratory =ys (such as blood g:as an:olysi.) and can bt used for many clinical cheminty assay> {2} M.jor disadvantages (a) Alters morphologic fe.tures :md staining of lrnkocyt.. {b} Allows dotting:as efh:t. are slowly o""rridd~n by the coagulation system {c} Allows platd" dump' to form

1/ INTRODUCTORY CONCEPTS

7

3. PI.. ma h.. two IIUjor compon~nts. a. Water: .bout 92- 95 % of plasma volume; 100 ml of pl ..1IU contains 92- 95 ml of H,O. b. Solid" about 5-8 % of plasma volume. Most ",lid. ar~ prot~in' on a _ight per volume (w~ightlvolume) basis. Other ",lids are glucose, ure., electrolyte., and other chemicals. 4. G..nerally, the chemicol compo.ition of plasma is very ,imilar to interstitial fluid in moll tissues. Plasma and interstitial fluid are the a tracdlular fluid. , one intraVll5Cll_ lar and one atravascu.lar. D. Serum I. s"rum is the fluid component of blood that i. harvrned after centrifug>tion of. coagulued (clotted) blood sample. As deKribed in Cru.pter 5, the clotting involves platelets .nd coagulation protein,. To 1:"1: the ffillimal .mount of ""rum from the clotted sample, centrifugation should not be ,uned prior to the retraction of the clot (which typically uke. at I.... st 30 min if. clot actiVOltor is not pr=nt in the tube). If samples are centrifuged prior to clot retraction, some ""rum will be tr~pped in a ",ft fibrin clot. 2. s"rum ha. essentiaUy the 1mle romposition .. pl"'ma except ""rum does not contain most of the coagulation proteim. Th~ Imjor prot~in {on. weight/volume b",is} that is .bsent in ""rum but present in plasma is fibrinogen. 3. During the clotting process, substance. rel=ed from "d is .her the analyte roncen_ trations in ""rum. For aample. platdets rel......, K+, and thu. ",rum [K+] is greater than pl .. m. [K+] (..,. Chapter 9). [I.

Urine Sampl~s A. Other than blood, urine is the most common sample .nalyzed by laboratory """'ys. AI with blood, urine must be collected and processed properly", that the =y reruh, reflect the true composition of the product of the urinary system. B. To prevent .nifactual changes in urine, it ,hould be processed soon .fter collection. Gen~ral guidelines for the collection and processing of urine for routine .nal)"'e5 are d=ribed in Cru.pter 8.

Ill.

Other Body Fluid £amples A. Pleural fluid. peritoneal fluid, synovia, and cerdJrospinai fluid samples are collected to characterize body cavity effusiom, joint di"".,e., and central nervou, system dilOrder.'l, respectivdy. B. The processing and ru.ndling of covitary fluid. are deKrib.d in Cru.pter 19.

MAJOR lYPES OF lABORATORY ASSAYS I.

M.ny laboratory tests or assay. involvt the an.lyli. of body fluid. (blood, ""rum, plasma, urine, peritonral fluid, pl~ural fluid, cerebrospinal fluid, and .ynovia), tissue 1mlples, or feces. Most clinicall. boratory procedure, fall into one of thrtt large groups (aamples follow .ubdivi,iom); IIUny procedures rould be cl.... ified into more than one group. A. Clinical hematology assays: Most assaY' are completed on whole blood sample •. I. Quantitation of cell concentrations in blood: total leukocyte concentration {count}, erythrocyte concentration, and platelet concentration

B

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY 2 s"miqlLOmitation of cdl con~mrations: ",lculata! abwJuu J.ukocyu coII<:entn tion and pl.lde! mimat. from blood film ex.:lmination 3. Ddining or classifying ""lis by microscopic fratu ... : toxic neutrophil" ,eaa;n lymphocytes, polychromatophilic erythrocytes, poikilocyw, microcytes, hypochro_ mic erythrocytes, .nd J.uhmic ""lls

4. Ass=ing th. co"l:"l. tion p,ol'mies of blood: cloning times and platdet function

="

B. Clinical chemislI}' assay.: Most assap ... completed on .. rum or pla,ma .,ruple<. l. Det«ting or quantilYing the coII<:emration of. chemical subst>n"" a. Quantitatin :maly'is: Iksuh, at. clost to the true coII<:emralion (e.g., .. rum ron""nir>tions of glurost, iOdium, proUill, creatinine, .nd u=) and typically are .. poned with. ~fic numerical val"" {Ott the Significam F~r •• =cion}. b. s"miqu antitati", analr'is: !«:suit, are · within the ballpark" (~.g.. urin~ gluco ... prot~in. and bilirubin concrnmuiom by rrag<'nt pad chemistry a~) and Jruly b. .. poned as approxin",e. num"ical .... lues or in a categorical scale (e.g .• 1+. 2 +. or 3 +) th .c rq.restnu ranges of numerical valu••. c. Qualitati... analY'is: R~sults indicae. that a .ubstancr is or i, not pr=nt (e.g .• f.c det"'t~d microocopically in pleural fluid with a Sudan suin) and ..~ reported as "present" or "ab..,nt" or as "politive" or "neg>ti"'." 2. Det"'ting or quantifYing the activity of a chemical subnan"", a. Quantitati... analysis: Iksuhs a.. close to true activity (~.g .• meas ur~d activities of serum enzym.. mch as alanine aminot"'ns~",,,,. alkaline phosphat=. lactate dehydrog~n .... and creatine kin...,). b. Qualitati... analY'is: R~sults indicat. that activity i. or is not p.... nt (~.g.. hem, ', ptroxid... activity or leukocyte est"... in urine). C. Clinical microscopy I. Clinical cytology: Th. study of cdl populations and their micro=>pic ~atu ... in an attempt to define or classifY abnormal ti ...... or fluid {~.g .• lymph node aspirates to diagnose lymphoma or histopl asmmis. aspirae. of a skin tumor to determine wheth" it is an inflamJrultory or nalplastic lesion. and analysi. of pun or fixed tissue in an attempt to define or classifY abnormal ti!SUe {i.~ .. inflammatory. nalplastic. and toxic dilOrde ..} and ptrhal" est.blish an etiologic diagnosis 3. Urin~ sediment aruolysio: Microscopic ex:mlination of urine to det"'t or .. miqu.ntifY the presc:ncr ofl~ukocytes. erythrocytes. casts. Nct"ia. cryuals. or oth" Uructures 4. Clinical parasitology: Micro!COpic analysis of fecal. urine. blood. or oth~r sample to det"'t ova. larvae. or other microsropic form. of po.rasites [I.

Actual descriptions of the numerou. laboratory m"hods are beyond the scopt of thi, textbook. How .... r. an understanding of basic principl.. and method. i, frequently nttded to interpret ... ults of a laboratory a.. "Y. Such principles are l"""ted in "",eral parts of th~ textbook. A. Chapters 2- 5 contain the basic principles .nd roncrpu of the rommon hematologic

=".

B. Ch apter 6 contains the guidi~ principles and overview of the analysi, of bone marrow and lymph nodes. the two major ti .. ues inmlved in hematopoiesis.

1/ INTRODUCTORY CONCEPTS

9

Chapt~rs 7- 18 havr shon =:tions that d~5Ctibt analytical ptincipl~s that apply to oth" individ,",l malytes. D. Chapt~r 19 romains Wsic principles ""naining to th~ .nalysis of avitary dfluions.

C.

S[G NIFICANT FIGURES I.

Results of many loboratory assaY" COlI!ist of • numbtr. ofr.n calcu1.tffi •• ccompanial by • unit of m.am", and ",f,,~n"" im.rvals. Th~ numb" should bt r~portal to th~ appropriat~ numbtr of digits by foUowing thIN basic rules for "'porting .ignifiam figu",". A. Reuin only as many significam figures in • result as will givr only on~ un""rtain figu",. uro i. not a .<>ning "'mlu from multiplicotioll! or divisioll! that u.~ two or mor~ numbtrs with appropriat~ signifiam figu",". th~ final calculatal ... ult should havr no mo", significant figures than th~ numbtr{.} with th~ f.~t .ignifiam figures. For 1.23 X 2.4 = 2.952. th~ product should bt rq><>rtal as 3.0. C. Wh~n tq>Oning "'mlu from additions or mbtractioll! that use two or more numbt" with appropriate signifialm figu",". th~ final calmlatal r~.ult should havr no more significant figures than th~ numbtr{s} with th~ f.west signifialm figures. For 1.23 + 2.4 = 3.63. the sum should bt ",]>Onal as 3.6.

II.

[t is f"' 1.2 X 10' 2. 1250 --> 1.2 X 10' 3. 1145 --> 1.1 x 10'

Ill.

How many significam figures.", in a numbtr? s." T.bl. 1.2.

[v.

U.
10

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

Table 1.2. Enn'plc. of significant figure.

Nurnkr

Signi/ic;ont figures

'"

3 4

'"

2 or 3

120.0

4

124.0

0.12

,

0.02 0.020

,

All digits imply 'ignificanct, Th~ uro was not nmM for the whole num!>.r. [15 addition indicol.. that it is a significant figure. If the uro is JUS{ coIIstrving 'p"""'. it is not a .ignifiant digit. If values arc usually rq>On.d to Ih. on•• pl."" in a particular .. ning (e.g., I 21). then the uro is a significant figure. The uro in Ih. tenth indicat.. it i. a '~nific;ont figure. Thus, the uro in the ones pi""", is also a significant figu,,", The uro is only pr... rving a sp''''' and i, not a .
'1"'''''

'pa""

are un""nain of th~ t~ns valu~ .nd mould r~pott th~ m",surffl valu~ to th~ n~.rest t~ns unit (~.g .• 130). D. Und~tstanding significmt figur~s b«:omes imponant when J.boratory data ate intet_ pretai If one under.tonds th.t we are un""ttrun .bout th~ J..t teponed .ignificont figute, then it is less likely that a 120 in today'_ .<;;Imple and. 130 in tomorrow' • .<;;Imple will k considered a true biologic chan~. Unfottunatdy, th~t. at~ srvtr:ll t ...sons that all r~sults "" not reported this way: l. It llliIy not k the policy for some loboratories. 2. Analyzers and data IlliInagc:ment programs may not foUow rules of significant figures and may not be: modifuobl~. 3. Th~ appropriate numbe:r of pia"". after the d«imal point may vary for a particular analrte, de~nding on the IlliIgnitude of the value, but analyzers and softwar~ may not allow such variability. An aampl. i. shown in Fig. I .1. UNITS I.

51 units nrsu. non-SI {conventional} units A. For several d«ades. th~re has oon an attempt to switch to a metric syst~m of units throughout the world. Other than th. United Stotes, the ronvu.ion i. mostly complet~ in a modified practical form. During the 1970s and 1980s, ......,ral organizations in the United States .ttempted to convert m~mkrs of th. m~dical communiti.. to 51 units but had limited su=>. Because there i•• lock of consistent us<: of the 51 unit system, veterinary medicol professionals nttd to k familiar with both 51 and non_51 units. B. In the contat of unin used for laboratory data, th~ basic units of measur~ment are lined in T able I .3. Many clinical laboratory and prof~ssional organizations have agr~ed to us<: Ii," as the preferred unit for volum~ insc ...d of the 51 unit of cubic ~ b«au .. •uch a volume as th~ latt~r (I m' = l<XXll) is rardy clinicolly relevant. Even the u.. of liter for a volume unit has limited rel<"VlUlcr in the clinical l.boratory wh~n the sampl~ volume for many ......Y' is less than 0.1 ml.

11

1/ 1NTRODUCTORY CONCEPTS

Measured values WBC con~mration _ 21.5 X 109/L WBC diff. coum: 73 % neutrophil., 12 % lymphocyt ... 9 % monocyt ... 4 % wsinophil •. 2 % basophil,

Calculation. and re.ultam significant figure. Ncutrophils: (215 X 1091l) X 0.73 _ 15.695 X 1091L _ 16 X 1O~IL Lymphocyte.: (21.5 X 1091L) X 0.12 _ 2.58 X 1091L _ 2.6 X 109/L Fo, tlm~ 1'rnI1tt: 3 lignificanr figurrf x 2 lignifiamr figurrf - 2 signifICant figum Monocyu.: (21.5 X 109/L) X 0.09 _ 1.935 X 1091L _ 2 X 1O~IL Eosinophil.: (215 X 1091L) X 0.04 _ 0.86 X 1O~IL _ 0.9 X 1O~IL B..ophils: {21.5 X 1091L} X 0.02 _ 0043 X 1091L _ 004 X 109/L Fo, tlmn,,,,Itt: 3 ,ignificanrfigurrf x 1 ,ignifiamrfigurr - 1 lignifican. figurr How~nr.

mon comput~r_basN rqx>rIing 'yn~m' will r
Total WBC N.utrophil. Lymphocytes Monocyt.. Eosinphil, Basophil,

Results u,ing • ignifiamt figul't rules 215 X 10jiL 16 X 10~IL 2.6x 10~IL 2 X 10~IL 0.9x 10~/L OAx

10~IL

Results u.ing consinent daoimal poim • 21.5 X 109/L 16.0x 109/L 2.6 X 109/L 2.0 X 109/L 0.9 X 1091L 004 X 109/L

Fig. 1.1. Significon, figures. Th •• ignificant figu= of ,t.. mea:5ured mel c:tlrul. ted Y11u .. of. leuIrogr:un .'" provided. Ho~.,.. oompu,.,._b"",d reporting ~.ms may not .n.b!. th. a ..ibility to rtpon only.ignifi_ con' figures. diff.. diffir.ntial: mel WBC • .,rut< blood ",U (lcu\rocyt<).

Table 1.3. fuamples of measure.n,ent in SI uniu and conventional uniu

Amoum of subnana.

ungth

M,.

Time Volume

SI units

Conventional units

mol~

gram (g) y:ud. foot, inch pound {lb}, grain minute {min}. hour (h) lit~r (L)

{mol} met.r {m} lcilogr:nn {kg} second ('1 cubic meter (m')

B=u"" th. reporta! units for .mount or cono::mration of mbstances ""'Y consid... bly, a vet~rina'Y ma!ical prof... ional ,hould know th~ common .bbreviations for th. major unit, (Table 104). C. The Nationallnstitut. of Standard, .nd Ta:hnology (NISI) provid.. rul~, .nd nyl. conventions for th. use of the SI units to r~du~ .mbiguity in oci.mific communi",_ tions. Exampl.. of th. NIST unit connmiono u..d in thi, book are in Tabl. 1.5. D. T able 1.6 contains th. formula. for th. conv."ion of analyt. values from non_SI units to SI unit,; only analytes p..".nt~d in this tatbook art induda!. Th. tabl. cont aim two types of formulas: (I) formul .. thai ,how th •• impl~ conv.rsion factor that is used

12

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

T able 1.4. Common unit
mol.

mol

millimole, 10-' mol micromol., I mol nanomole, 10-' mol picomoJ., 10- 11 mol femtomole, 10-" mol anomol., 10-" mol

mmol

cr

,mol nruol

pmoJ fmol amol

L

liter

dL

decilitor, lO- 1 L millait"', 10-' L microlit .., [crL nanol;,«, I ()' L piroliw, 10-" L femtoliter, 10-" L anolit", 10- 10 L

mL ,L oL pL fl.

.L

,"

kilogram, 10' g

m,

milligram,lo-'g

"0,

micrognm, 10-' g nanogr:ml,lo-' g pirogram, 10-" g femtogn m, 10- " g

pg

""

w· m

aooll·m, lo-IOG

omer £lYle" NIST style

Non_NIST

Symbol, un:dtc=i in the plural

20 PC

20 pg.

Symbols not follow
""

Table 1.5. Example. of NIST" Rul.,

Th". is

llyiC

for writing units con'pared

sp''''' betwttn the numeri",] v:due and unit symbol

10

6~

"0 n 6 yr.

W"

W"

37"C

37"C,37"C 15 gldL

15g1dL

• N .. ion:ol In .. itut. of S,mdords ..,d T .cltnol<>&y • Tb. NIST .y"om Ius : for enmpl.. 5 % to 10'10 or (5 to 10) '10. To ..... 'I"",, :and to confOnn to roJIUDOn "'" in ve .. rinuy ~"r1nuo. tho numb.n..iU b. displ.~ .. folio"" in this textbook: 5-10 %.

to colcuuto the num"ical value of the S[ unit,' :md (2) formul ... that mow the conversion of tho numorical value .nd units. When av.ilable. the r«:<>mmend.d ,m.ll.., ",port>ble inc.. ment of the SI unit i, provid.d.' Similar information is oonuin.d in the .nalytical con<:ql' •.crions of eam mapt ... [I.

Amount vers", cona""tntion: One imponant basic oonelly will not ~ d",,,,as.d. and thus the dog i, not initially .nemic. Mt.. fluid mifu resto .. plasma volume. the dog will have ftwu erythrocyt.. in iu body and a low.. erythrocyte rone
. - ••

T ..... '.6. C-........ ol .....sI-n. .. S1 .....

A.odyl" Aa . . .

,cr"

,>t.

..".'""'-, .-'.'-

.-' .,,-, -, • --,-, ..-. -, "'-, -, .-' -, " -.' .'" ........

~-

AIOO . ......

l\-HydoOI)l>'",...' oci
('~

"'''

."" .",

~o~ ~

meldb ,., JUDOlI"'C' to dliL ~ .. L~ " ~I pelpmol . looo .. llL = --tiL ,ldL • to dliL = tiL I'CidL ~ .J'O.\ ""~ .... . lOOO ...... rp-,t • 10 dUL

'"

17.7"

"

",,"

.""

000.01\86

"

~

","

c:.. ........

~.

Fnritio

"0.

0.""'17 lJJ.Jll 17."

.-

h·

meI"L . '.7 JUDOlI"'C' to dliL = ~moUL melL ~ J92.6 ~ . lOOOl'Cimc = JUDOlIL meI .. L ~ J!ll"; mel""'" • 1000 mUL = mmoUL meldL ~ \"U mel ...... . 1000 ~_"""'" • 10 dUL = JUDOlIL meI"L ~ l"'-7 mel ...... . 10 dllL = .....vI. mE,tL • I .....umE~ = """",II. meI"L~l~\ mel....,. . 10dliL = _ L mmH, • Ill.l1' f"'<'I~tnmHc = p"'" I'CidL ~ .l
.-

",,"

".<

meI"L ~ 11M = ,-L

.""

o.7l7" 0."'"

0,

","

O.INI

~mL ~ IJj~ pelpmol. 1000 mllL = pmoUL meI"L+ ...... meI ...... . 10dllL = .....vI. mE,tL • M .....u.. Eq = mmoUL I'CidL • omNI ~_'" • 10 ollL = ..-.11. ocImL, , ""1000." 1000 mUL = ""L meI"L, V'OOO "'C ' to dliL = ,II.

... · Iom;"

~.

W

"ono

~.,

~

c".

'?-fl«' "",=Do. fuo

2.\47 2.147 11.10

(,~

.-,

M"'t:M~t

~,

"'~ ",,"

om

mel ...... .

1000 ~_"""'" .. 0 dLIL

_me

.... " ,.' '

to .-.IlL

0.2..-.11. 0.2mmoUL l~_L

0_<15 """",II.

,-" ,-"

to_L

'"-

to .-.IlL 0_01 """",II. 0.<11 """",II. I~_L

to I'CiL 0.1 VL Coo,;" ....

•

. - ••

T ..... 1.6. COO ........ 01 000..sI _

M", Fib.u..p ~,

IT, (ljot..i . . ~-,

"oo.~

"-'" ,~

""'" MCHC

L-Ioct",

",' ",'

,,"' ,,", "" "

PI"d«

=

",,"

"'~

"'''

.'" ",," .'" ",.,

~.,

~UlmL

."

~.,

",," ",,"

.'"

~.,

",,"

~"

"" od"

",,"

*.ooo".L

•• lo'l~L

to $I

.oUt> (c-.; . ..... ,

-.-' "-, -, ".." .-'-,-, -, "-, -, -, --, . -,

M..!tpyti om" ,~

IU7

"

Omjjl

"

7.17\ 17>'" o~~

mcloL. "'''.1 mel""'" • loollL = .....tIL

mE,tL • , .....umEF madlL

,ldL • to dliL = tiL ~ ..L. 1000 pel", • HlXImUL = ""L ~UlmL + llM ~Ulpmol • 1000 mllL = pmoIIL I'dL + \.001 ~,~ • ,,>XI ~ = r""""'L mE,.,L • , .....umE~ = ...-.Ill "",01 + l~ mclmmoI • 10 dllL = ......... L meidl , Lll _

",

0,))43

*. ,o'lL

= pmoIlL ,ldL • to
,ldL • to dUL = tiL

M 0 71

•• IO"IL

mc/_L , o~"""'Imc ' IOdLlL ""mL . +ILl ""ODd • ,,>XI mLlL = ' - L "".L. Tl7.tIa->I • IOOO~.....! . to dliL

0.1 "

"'W

O..l21~

",

.-,

Compkt, " " , = D o . _

.."

_.,

....'

,~,

'

" ,-, ,"""L ,

o.I..-.IIL

to

'' .... '' .... o.•

..-.IIL 0,[ """""L

lodUL = _ L

mE,.,L • I .....umE~ = madlL "","1 . >l melmmol • 10
,-, ,-,

mE,tL . madlmEq = _ L I'CidL . I7.4 ""~ .... . to dIlL = ..-L "","L . )'o97 mel ...... . 10 ol1L = ......tIL

• .000".1. lo'~UL = # • ,0'11 #. lO'I ~L . H"~UL = "

10"1L

j~_L j~_L

OAll mmoIlL 1O.1001L

..J..J

,,'"

"""

..J..J

00

00_00_0

_ _ 00

11 ' '11 ~~ 11llt aa.aa~~1 ,1 1~~ , .,

5

'il

...

;'~ 1 1" tIl,' "" "ll

......" !I~

m

'" il"ii"l ill ::! s:::: ::: 11

~ l

s:~ '" ~ ''' ' g~

.. HllLl; -

U 00:

tl=tl~tfl !l ~- .... " j

~ &l~~~?~~~hl " 'ii'i-o;j,,;"" " " ' -' " ' 'iio;jo;j " " ta}~,}}} "

'
•

~ /3: :; ~ ~ ."'''' .".;.;",,-,,-

$- ......

...

~-..,.,<>~Q;:

"'0:;lIUlIl1 -'"' "" ..

'1

"

11 11111,ll1 111~ it

..J...J

..J...J

." ~ o;j ."

:
..J

.. ;j 'il

latht}}

.

,t·

.:.=

~~~f~~~~,='

"

16

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY ar~ told that a horst', .. rum ~lIzrm~ J~d was d~",asffl. Does this m""" th. horst has Ie .. of thai mzym.? It might. However, it could ~ chat th. amount of ~nzyme {the protein} was not d~r<"~. but th. enzyme'. activity w as inhibit"'" or moyb< th. nructur< of the enzyme was defectin. D. YOU.r< told th at a COlt'. reticulocyte ptrctmage is iller.aM more myeloid cd[., ft~r erythroid <:tlls,

C. You

or both? Or is th. ratio incr • ..,.d ba:;,u.., th~ numkr of mydoid cd], i. increasM mor~ than th~ incrns. in erythroid cdl,? A colrul atM ratio i. alw:lY' a rdati"" numkr and mu,t k int~rpr~M acrordilll:ly. F. You ar~ told that a dog·, urin~ h" an incr~aKd prouin con""'ntr>tion. Ikcou.~ th~ oon""'ntr:llions of all .ubnan= in urine d'1"'nd on Ih~ conservation of w:l1~r by th~ kidn")'•• the incr.ased prouin con""'ntration could ha"" =ultM from incr•• sed w:lt~r conservalion and not incr• ....:! prouin loss via th. urinary .ystem. REFERENCE INTERVALS I.

R.f.ren"", interval. and Ih.ir purpo'" A. Results of laboralory tests {laboratory dato} on p:lli~nt ... mple, would k very difficult to interpr~ without "firm« inrrrwUs. which are the results we ex~t to find in healthy animals. Th~.. intervals are used to help d~'""t pathologic ,to",. Other term. thaI are used as synonyms includ~ normals. normal valu". normal'dnt'. and "in""u:r rangr. B. In.n atlempt to ~st.bli.h uniform uuge of term •• the following "rms and definitions have been recomm.ndM by an Expre .. nt the refer~n"" population 4. Rifrrmu !!dlur. • value {result} obtainM by oburvalion or m~asurem~nt of. panicular ,ubstan"", in a ref~rencr individual 5. Rifrrmu dhtriburion: Ih. distribution of r~f~ren"'" value>, which i. not n=sarily Gaussian {a kll.,haprd curve} 6. Rifrrmu limitr: the lowes{ value {Iowa ,ifnmu limit} and the highest value {upp" "fi,au:r limit} of the rrftren"'" interval, a. derivM from a ref.rencr dinribution 7. Rifrrmu inurVdl: an int.rval be-iw..:n and including th~ two ref~ren". limi" 8. Qb"""d !!du.r. a value ob,.inM by observation or m.asurement thaI i. to k comp.red to the r~ftren"'" interval C. Use of th~ term "fi,ma mngr i, discouragnl for two r"'Kln,. l. Sutistically, a 'lint' i. th~ differen"" be-iWttn high'" and lowest observations: for example, the r:mge i. 40 if th~ high'" observation wa.< 50 and Ihe lowest wa.< 10.

1/ INTRODUCTORY CONCEPTS

17

2. Some comid" a ranu to indude all m.-amr the values betw..,n two "f."ncr limit .. D. Using the t"m. nCrmIll and "bnDrmIll to de",rib. laboratory test result, am b. mi,le"". ifll: and i, discouragffi. I. A l.boratory ..suit con b. WRI but niH rdlttl a pathologic proct:M. For aample, • strum sodium concrntration that i. WRJ in a ddtydrat
""It"

II.

Esublishment of referen"" interval,; A complete description of the process of enabli,hing ..f... nc< intervals i. b.yond the scope of this book, 10 r..d." a.. "fe.. ' in the process~" as follows; A. Select crit,,", for ",feren"" individual •. Crit"ia could includ•• pecies, age, and method of det"mining health .um •. I. Initially, thi, may seem like a .imple uok b.amse the criteria define clinically healthy adult animal •. Generally, we w>nt to umple ~ b"",d group of .nimals 10 that the referencr interval is IJ.stful for • br",d group of patients. How"","r, it can become mo" complex wh.n pot.ntially clinicaUy rel...... nt diff".nc<. occur ~Ust of .... riation. in; {I} br=i (e.g., some Akitas have .mall" .rythrocytes), (2) physiologic state {e.g., milking venus nonmilking catd. or high altitude v"su, .... levdj, or {3} nutritional sUte. 2. E""n if a laboratory is mc=sfuJ in esubli.hing ref.",n"" int.rval. for .dult ~nimals, how about appropriate ..feren"" interval. for neon.t .., nursing animal., or w.. n· ling.? Critical ....... m.nt of pati.nt values ..quires th at crit"ion.match
FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

18

nri.tiolls nNdrd for repr=ntatin ",f~r~n"" int~rv:lh. For ex.mpJ~, r~f~ .. n"" int~rv:ols

h:dic ""in nr,m indwdliJll: cathff
of s;ompl. rubdoontain.r or antjco~.nt and volume of the sample 3. StOf:og. tim•• nd ump
2.

Ty~

=".

2. Thi. ""p"n .. i, magnifi+ oon<: ISO, . nd 2.5 % of values ~'" < 50, so th. r~f... n<:< limits a.. 50.nd I 50. Th~ ref~ren<:< interval would b. 50-ISO. 2. Wh~n data h.", bttn shown to fit a Gau"ian di,tribution. pa.. m~ric or nonpara_ metric method, con b. usn! to establi,h ..f..~n~ limits. r.ram..rric m..rhods ~n~rat~ valu .. that r~pr ..em th~ mean ± 2.d. Nonpa"'m~ric m~thods may b. mor~ .ppropri.t~ than parametric method. wh~n th. numb.. of ref~ren<:< individual. i. low. 3. Wh~n th~ di.tribution i, not G.u ..i. n, th. dau should ~ith .. b. tr. n,form
1/ INTRODUCTORY CONCEPTS Gaussian distribution

"

-

Positively skewed distribution

"

, , , , , , ,

--

n 'll.

•

19

I,, :'1

2.5 'II.

j

,

,

~

cantor 95 % mean:l 2 sd Analyla concentration

f---

----t

I--

-

,, '

~

centor 95 % mean:l 2 sd Analyla concentration

----t

Fig. 1.2. Rd'e=>c< dinribution .

• la tho left graph. tho rm"''''''' ....1ues oonform
III.

U.. of "& .. a",, interv:ll, A. It i. imporunt to uaduSland that m~it ",fura"" interval. rep .... m ..",lu exptaed in 95 % of the h.althy .nimals (i .•. , ia 19 of 20 healthy animal'l. I. Therefo", I of20 healthy aaimals is a~ed to have a measured value ouuide of th. "'~"'n"" int.rval. The value dut is outside the r.f..en"" imerv:ll but still rq>r=ats a v:du. from a he:dthy .nimal i, 'I~ted to be clo.. to the ",f..en"" limits. A marked diffe",n", from the ..f... ner imerval probably "'pre",nts a pathologic state. 2. When aamining multiple "n rerults for oae .nimal, it is imponam to ",member that thi, 95 % cruner of ..... lue from a heolthy animal falling WRI applies to ram rn;ult "'pa"tdy. a. Thus, ia • panr! of 20 assay>, each with • 5 % chan", of being outside of the ..f... ner imerval ""en without pathologic crung. , the" i, a 64 % crun", for at least one value to be outside thr reference inte",:d (Ott Reference [nterv:lb, ""'t. 1ll.A2.b). With • pand of five mulu, the", is. 23 % ch.ner. How""er, that v:dur mould be clo"" to the lower or uppt con also be explained using multi .... ri." comp.ri",n colculations.' B«ause the", is a 95 % (0.95) probability in he.lthy animals th., .""h re",lt is

20

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY WRI, th~ probability th at :011 "n" v:du~. ar~ WRI is 0.95", and th~ probability that not :011 v:du .. are WRI is I - 0.95" (wh,,~ n is th~ num~r of ob5trvation.). Thus, for .. pand of 20 test results, 100 X {I - O.9S lO } = 64%, so there i. a 64% chane;., that at lean one value is ouuide the ..f,ren"", interval. B. Not all en"" illt.rv:.!, accurately ,eO,",,1 aptctal findings in .. poP"J.tion of

,.f..

int.rm. This may k kall,., of such problem, .. an in"'kquau pJ'OC«!ure of establish. ing the va[ues, a diff... nct btlWttfl r.f... nct and test poP"J.tions, or .. chang. in mHhod, or instrumentation by the labor>tory without an approp'"'te chong. in

r.f... m,. inte",:.! .. C. Laboratorie; should provide information .bout th.i, .. values when ~l1e'tal. Sum requ.u ••", p"nicularly appropriau when Ii",! cotmmpl.ting Ust of .. loboratory. This enables Mll.{;On of how likdy it i. that th~ values .. fl~ct h""lth in th~ populo. tion of int~ ..n. R..quested information might indud~ the following: l. N umb.:r of ref~ .. n"" individual. 2. Age •• br..,ds, .nd genders of r.r.r~n"" individuals 3. Criteria for inclusion as ...f..~n"" individual 4. Methods for ~nablishing "'~rencc: inurv.ls

r"."ct

IV.

!Ufo",u-r limitl nrms tkrision thmholds (limits) A. As ddined above (Refe.. no:: Inte"",ls, ~t. 1.B.6), "fomur limitlar~ th~ high", valu~ or lowen valu~ of th~ ..fe",n"" int~rval as derived from a re~r~no:: distribution; a r~fe .. no:: interval includes value. expected in 95 % of the h~althy animals. These valu .. a.. usa! diagnostically to hdp drtect p>lhologic states in ill .nimals. A pati~nt value that i. above or b.:low the r~fereno:: limiu may indicot. the preseno:: of a pathologic naU. B. A tkcirion .hmhold (somrtimes coUed tkcirion limit or n""jJpoint or cuwjJ wlur) is • value that is used to d ..... ify ...suit as po.iti"" or neg>liv. for. di ...... or to decid~ wh~her to """t or not to treat.'" AJ deocrib.:d in the oection on Diagnostic Propenies and Predicti"" Value of Lobor.uory AssaY', a decioion threshold may not b.: a refereno:: limit. o.-cision th=holds con .ho b.: used to estimat~ • cost to b.:ndit ratio for diagnostic methods. C. The t~rm drrision limis is also used in the rontext of a rriti,"l tkci,ion limit, whielt is • value that is used for m.king critical therapnllic decisions. For example, a critical decision limit for .. rum calcium rono::ntration may b.: 15.0 mVdL, m""ning that agg .."in th~rapy is initiated whenever ...rum calcium ron""ntration i. gr~at~r th an 15.0 mgldL. In rontrast, th~ uPI'" rifrrmct limit might b.: 12.0 mgldL, and a tkrisitm .h",hold for a h~rcalo::mic disord .. might b.: 12.5 mgldL.

QUALITY OF U.BORATORY RESULTS I.

Major drterminanu: Loooratory test results will b.: of the mon b.:ndit if they a.. oomis· undy oorrrct from patient to patient, day to day, and month to month. Thr.., major foctors determine whether result, are valid: (I) quality of .ampl~ . {2} quality of .nal)"is, (3) quality of laboratory .nd patient records. "When result. are not what a vnerinarian expecu, h~ or.m~ might "'y, "I do not believe tho .. results; th ..e mun han bec:n a lab error." "When melt oonclusion, .'" formed, it i, important to r.m~mb.:r th.t there are many pot.ntial r""""n, for an ~rroneous laboratory test remit. Errors may b.: preanalytical, analytical. or ponanalytical.

21

1/ INTRODUCTORY CONCEPTS

A. A laboratory tell ... ult con only ~ .. good .. th~ .,mpl~. Pmlnalytiwl (rrrm, which u~ rd.tively common, ui", from probl~ms with umpl~ collection .nd proassing. Th..., .. ron con ~ minimiud with ~tt~mion to th~ following: l. S.mpl., collection a. Pro~rly p.. parM pati~nt {~.g., the animal should ~ faSlM for .r l=t 8 h prior to collection of most blood .,mpl~.} b. Pro~r coll=ion techniqu~ (~.g., >traumatic ""nipunctu.. to minimize: h~molysis ~nd activation of clotting pror.ins ~nd platd.,.,.) c. PfOJ"'r coll=ion contain~r (.,.g.. st~ril~ v~r.",s nonn~ril~ or clot rub.: vc:rsus an EDTA tu~) d. Pro~r .ntico>gulants wh~n n~M {•. g., EDTA vusu. h~parin, or wdium heparin vorsus lithium h~parin} •. AdaJuat~ volum., for ous;oys (•. g., to obuin I mL of .. rum, • volum., of at l=t 3 mL of blood is usuaUy n~MM) 2. S;,mpl~ handling a. Pro~r lobding of all specimens (".g., .nimal id~ntificotion by nam~ or number, dat~, and tim~ of collection) b. Samples kq>t at appropriat., t~m~mur., prior to and .ft~r proc=ing, during shipm~nt. or during stong. {•. g., 25 'C, 4 'C, or - 25°C} c. Prompt proctlsing {•. g., for a labil~ analyt~, proct.. immMiatdy so th., .nalyt., does not d~t~riorat.,} B. Analytical n-roys should be minimi=:! by att~ntion to th., following: l. M~thod appropriat~ for species {•. g., an instrument designed to m...."'.. th., rdativdy brg.. human ~rythrocytes may not provid~ """unt~ m~..... r~m~nt of small~r ~rythrocyt.,. from the domestic mammal.} 2. Quality of innruments and aJuipment (i.~., g~n ..ally 'you g~t what you pay for," but • qu. lity ifi!trum~nt remain. a quality instrum.,nt only if it is properly maintainM) 3. Quality of rrag<:nu (~.g., fresh .nd within the apiration d.>I<, ~ing u..d a.ccording to instructions) 4. Quality of loboratory ta:hniqu< (e.g., ~rson_to_person variation, training of ~rson, and inherent pro=:lural difficulty) 5. Quality control program {quality ... u"once program} {e.g., the laboratory ~nonnd SlIb5Cri~ to and adh~ .. to internal • ...,ssm.,nt proctdut .. to assur., that all pam of the quality an. lysi, of the .ample ar. m. intainM daily} C. Posillnalytiwi (rrrm con be minimiud with mention to the following: l. Tranocriptional.,rron minimi=:! {".g., .. ron am ~ mad~ duting manual transcrip_ tion of ..",lts to • loboratory report or during kryboard .ntering of dau into comput~n}

2. Dau p.... ntM in a report chat is e-asy to rrad .nd thm rMuC<::! incorrect int~rpr~ution II.

th~

likdihood of

Analytical pro~rties of a=ys: From the .nalytical ~npectivc:, th~ ~st clinicall.boratory assay i. one that consistently m~~.ures the true concentration of the .ubnana: and at concentmiom that are clinically rd~.nt. Wh~n evaluating and romparing laboratory assays, fj"" pro~rti .. con ~ assessed: A. Analytical pruililm:' th., ability of . n assay to get th., .,m~ r<::'lult if a .,mpl~ is analyzed sev~ral times; .lso callM rtprgt/udbiuty or rllndom Ilnalytiwl trror {Fig. l.3a}

22

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

~ ,,0,,,, a. Analytical precision (systematic error)

'/e\

,'b. Analytical imprecision (random error)

'/ ::::

@ ,'-

~

c. Analytical precision and accuracy

'/'

~\

':Cf '\.

/'

/@' ,0

~ .

'/

d. Analytical

e. Detection

specifidty

IimH

Ill",.mio", of :orWytical properties of ....Y'. ('1'_';' <m>r): Jl..cauo< tb. "'n hoJ.s in th. tngot >t< tightly & ..",,«1, tho .boote, w>s pr.a.. ~n though oons""'ntly iwocum •. b. A",,}ytiml ;"'1""';"" (NIt"'- """): &c. .... tho hoLe. in tb. OJ<.....,nly distributed. th..venge of ten shot> is onctly in tho middl< of tho t>
Fig.

l.j.

"'ge'

a. A...Jyti,m pr«jM ~

'ug<'

,

,

lmp~.

<. A...Jyti,m pr«jM ~ """ """""'Y' Bee."", :oil 'm hoJ.. in th. th. t:o"il"~ tho .hoot.. _ ace"",'" :ood pr.o...

""D.,.

W'il'" or. tightly dU""'M in d.. middk of

d. A""Jytiml tp«ijinty. Of .... ten holes in th. urg.~ .. round, tions, d.. .hoo1<' probobly or.."'" =< .m..lLest hoI. th .. you can ",li.blr detKt is th. em «liilily
0"'"

differ..,t .i"", of aU holes. th.n tb • ....,.;tivity ~mit b.. not ~ ",ach.d. If your
l. The n=:l for .nalytical preei,ion dep"nds on the degr,"" of variation that can bt a=ptffl .. random v.riation (uror). When =n~ment of a""",,, ",
1/ INTRODUCTORY CONCEPTS

cv (expr~ssaI as a ~re;.,nt"l:~) =

23 standard drn..tion

X

100

{LL}

m~~n

a. A

m~thod·,

CV i, deurminal from rq>li"'t. analytis within an .....y run and run, of th. sam. assay. A wimin-tion. A brtwun-aJ.kl} CV r~presents th. random error within one run of the assay plw th. error from additional run, of th. assay by wing th. sam. sampl •. The within_ass», CV wiU be mtaller th an th. bet~n_assay CV. dini",l rdevane;., of an assay'_ CV i, deurminal by many factors. If critical dini",l da:isions are made when changes in an analyt" cone;.,ntra_ tion ar~ minimal, th.n the .....y must have a very low CV {high pra:i,ionj. Otherwise, th. diagnostician would not know if th. chIDge in analyte cone;.,ntration is due to a tru~ change that occurm:! in the animal or if the ch ange rq>resenu aruolytiml ~rror. An """y' _ CV wiU typically vary with the analyte·, con"'ntration. High~r CV valu.. may be found at the lower and up~r limiu of the .....y', IDalytiml range. Within th. analytical range, CV values are typimlly higher at th. lower analyte ron",mration, kause CV values are exp=W .., ~rcentag ... (a) If ID assay has a CV of to % ~t all rone;.,ntrations, then the .... y', nandard d~iation would be 0.1 mgldL at an analyt~ cone;.,ntration of I mgldL, I mgldL at an analyte concentration of 10 mgldL, 10 mgldL at an analyte cone;.,ntration of 100 mgldL, etc. Depending on the analyt. and th~ amount of biologic variation, a CV of 10 % am be completdy unaccq>tabl~ aruolyticaJ variation or be very a.c<:eptable. {b} If an ..,say', standard d~iation is 10 mgldL at all concentrations, the assay', CV values would be 50 % at a m.... n ron""ntration of 20 mgldL, 10 % at a mean of 100 mgldL, and I % at a m~an of 1000 mgldL. Knowing the amount of analytical vari ation for an .....y i, helpful wh~n trying to determine wh.ther a change in a pati.nt', data represents analytical variation or a true biologic change. On. way to approach thi, decision is using an assay', USD. USD is an average of standard d~iation valu .. from 3-6 consn:utive month, of quality assurane;., values. If a change in a patient·, data is I... than twie;., the USD, th~ change may be only analytical variation. If a change in pati~nt data samples is gr .... t~r tru.n three times an """y'_ USD, th.n th. change it probably due to biologic variation. The .hru titNl USD is an .nimate of'ignifi"'nt change limit;'· a change in data of at least thr« times th~ USD is probably a signifiamt biologic change and not simply analytical v~ri.tion. (a) If the USD for sodium roncentration at 150 mmoUL is 1.5 mmoUL and a patient', sodium cone;.,ntration is 150 mmoUL on day I and 148 mmoUL on day 2, th.n the change of2 mmoliL may be due to

~..,n djff~rent

{I}

{2}

b. The {I}

{2}

{3}

24

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY analytical v>riat;on since it is I... than twice the USD. How.nt, a value of 145 mmoUL on day 2 (a chang<' err.le, than Ih,,,,, times th. USD) probably r.p ....,nts a true biologic clung... (b) If th. USD for blood neutrophil concelllration is lOOO/j.lL at a lIn1tro· phil concentration of IO,OOO/J.IL th.n nrutrophil ronctmration in a =nd sample n~s to bt < 7000/j.IL or:> 13,txXl/j.IL to bt rdi.bly

oonsid,,«1 a biologic chang<' jf the first s;;ompl. had a neutrophil oonctntmion of IO,OOO/J.IL (4) The CV ~rctn~ and USD valu .. may vary considerably ktWttIl :assay methods, .nd the ~rctntagrs or values .'" frr=nt the mean roncentration d.termined by numerous oo..rvatiofiJ using .n =y that i. accepted .. the best avail able. 2. Typically, accuracy of a clinical assay i. assessed by compuison of iu results to the results of.n a<=pted ",ference mtion group •• providing a true v:due. OptimaUy, the assay that i. a<=pted .. the refe .. nce m
1/ INTRODUCTORY CONCEPTS

25

Th~ pres~",:" of gluco .. may ~ d~{a:ud when a chemical r~3oCtion producc,. hydro!:"n ~roxide {H,OJ. A subsuno: may int.&r~ with the a=y by having the sam. oxidizing prop"nies as H 2 0,. {2} In ¥ctrophotommic a,,~, the p.... n"" oflipids, bilirubin, or hmlOglo_ bin =y imerr.re with light transmission through a sample, and thus the remits of the :way a.. chang.d by artif. ctual .p«tral changes and not by. ch.mie>l r....ction. 4. D'p"nding on how. suMan"" int..f.... with.n a=y, it may lead ~ith .. to falsdy incr ......,J or to f:d ..ly d= ......,J cono:nt.. tions--positin im..f..~n"" or n
{I}

{Fig. 1.3~} l. An .... y's d.ra:1ion limit involves the ability of the assay to diff... nti.r. b. ckground

"noise:" from. true change: beca<= of the pr=n"" of an .nalyte. The der.ction limit ddin .. th.l"",·est value of.n assay'. analytical rang~: andlyrim1 rangr is the range: of values ovc:r which the :way can provide rdi. ble resulu. 2. If.n analyte i. rdatively .bundant (e.g., .. rum N.'), then. d.ra:1ion limit of 100 mmollL =y ~ adequat~. For subnances that ... rdativc:ly ra .. (e.g., aldon._ rone), a d.ra:1ion limit of 100 pmoliL =y ~ n=i.d to ~ dinically useful. E. Andlyriml lmlitivity (I UPAC d.finition):' slop" of the calibration rurw and th~ . bility of an .n:dytical proctdu.. to produce a chang<: in the lign. l for a d.fin.d chang<: of the quantity l. In other terms, an ......y. analytical .. nsitivity i. how much chang. of th•• nalyt~ {cono:nt.. tion, prop"ny, l rang<:, wh ....... dnutum limit appli •• to the lowest limit of the .nalytical range:. lll.

Qualityassuran"" A. Quality '!SUtan"" con",,!,ts I. Ev.ry laboratory:way has th. por.ntial to produ"" erroneom results becau.. of a vari.ty of analytical f.ctors {e.g., r~nt det..ioration, pip.rting errors, inrub.ition .. rors, .nd de<:tronic int.rfer~n""}. To deta:1 .. rors, .wry laboratory or dinical practice that .nalyzes .amples should have a qu:dity .ssm.n"" program that i. d.. ign.d to d.ra:1 unaccepubl...rors in its .,,~ .nd assure that only quality ... ults are ge:nerated. 2. Each l. boratory assay h.. 5Om~ random an:dyticalerror (= Quality of laboratory Results, sect. HA). Typically, m. nual:ways h. w more random .. ron than do autom.t.d m.rhods because: people gen.rally cannot .. produ"" their work a. well a. a machin. can. Variation. in results that are cau..,J by random .rror should ha"" a Gaussian or normal distribution (•.., Fig. 1.2). With th>t distribution, 95 % of the results should f:dl within the int.rval that i. equ:d to m.... n ± 2.d. If the same sample i. analyzed 20 times, 19 of20 ... ulu a..

26

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY ""~{M

to '" within that int~rvaJ. But it js .ho imponant to ~lIiz< that I of20 remits is exp«:tffl (0 bt ouuid~ of int~rv:ol----N
cru.t

wdl! 3. Anothrr k." roncqJI in qu<>lity .mIraflC. progr:u m i. that a""publ. random .rror for a dinic;ol .~ should bt bo.,.d on wh.t is ronsid.ral to bt a biologicaUy significant chang. in Ih. resuit. If within_individual and bttwttn_individual biologic v:ui. {ions of a particular .nalyt••", small, and clinical d«isions .r. mad. wh.n th". is only a minor chong. in" l.boratory trn ,emit, Ih." Ih. random .rror of tho =y liNd, to bt r.htivdy ,rnaU. How""",. jf clinical d«ision.... mad. only who" th". is • markal changt in a J.bor>lory (est .. mh, a Jarg<'f ,""dom orror may bt

a=ptabl•. Two ""ampJ.. illu!!,,!. thu. roncq>u: a. ~rum [Na'] in most h....lthy ma mmals is n= 150 mmoUL If a patient has a .~rum [N . +] of 150 mmollL 011 day I .nd 160 mmoUL on day 2, most clinicians will ronclud. that "'m~thillg has happ, Th~r~for~, for th~ strum Na+ assay to ~ clinically v:duabl~, its au. da::isions may ~ m ade kau"" of random an:dytical ~nor,

b, ~rum [gluco ..] in most h~:dthy mammal, is lleor 100 mgldL If a pati~m hal a ,~rum [gluco ...] of 100 mgldL on day 1 :md 110 mgldL 011 day 2, most clini_ cialls w ill ronclud. that such changt. are within npectffl biologic variatioll; that is, there has not bttn a chang<' for biologic, pathologic, or therap, The ..fore, for th~ strum gluco ... a .... y to ~ clinically valuable, its '=pcabl. random onor can ~ largtr thall tho ralldom .nor of the .. rum Na+ :way, B, Westgard rules. James 0, Wmgard, Ph,D" is • leader in tho fidd of quality :wurance progr.ms for clinical laboratories, H e has d.vdop«l a system that i. desigll.d to da::ide whether an =y'. run <:dl patiem sampl.. =yn! at th~ ... me timo as the comrol ... mpl~) i, "ill control" or "out of romrol" [Hcai], of thi, .ynem are beyond the KOp< of thi. book, but an excellent Wd> .ite npt.im th~ k." concepts {http://www,wostgard, rom}, L A quality as.mrance program should ~ d •• ignffl '" that it detects analytical erron that llliIy ~ of clinical significallce, All measuremellts colltain erron, and tho two IlliIjor types are random error and S)"tematic «ror, Random error i, cu m by facton that randomly affect tho meamrements, such as vari ations ill disptnsffl ""lum. of rea!:"m or umpl~, SysullliItic ~rror is a rq>roduciblo inaccuracy that will ronsistently muh in value, that are too high or too low, Th~ Westgard system is d .. igllffl to detect both types of erron, 2, W'..gard ",k, a", a stries of rules that are appliffl systematically, oith~r sillgly or ill combinatiom," This >ystem i, appliffl to tho results ob[ained for control ... mples that a", analyzed roncurremly with pati~m samples, In his abbreviation >yn~m, "s" , cands for ,candard devi ation, a, I,. rule: A rull is rrjecud when a .ingle control m~asurem~m nettds the m~at1 plu, ls or the m ....11 minus ls of pr.vious control sample values, or it .. rves :as a warning system [0 illitiat~ additiollal inspection of control data. b, I" rule: A rull is .. jectffl when a .ingle control m~asurem~m nettds the m~at1 plus 3s or the m ....11 minus 3, of pr.vious rontrol ,ample values, This rul~ is primarily ... nsitivt to random ~rror,

1/ INTRODUCTORY CONCEPTS

3.

4.

5.

6.

27

e. 2,. rul~ A rull is ..j=a! wh~1I two colI!«mive comrol m~asur~ments nettr.ble random .nalytica.l error. The less impr«ise the ''''''y is, the mo .. likdy it is that. high or low control ... mple v:due will truly indicate a problem with [he assay and the I.... string<'m the Westgard rul.. neal to be to k.." the ......y in rontrol. The more impreci .. th~ ......y is, the mo .. difficuh it is to kllow wheth .. a high or low control umple value is caused by :m:dytical variation. 10 more millg<'m Westg ..d rules will be requirM. Whell.n """ys rull is rej"'tM, the result. for the colllrol ... mpies w..e unacceptable and thus th~ ..",I" for the p:uiem umpl .. may be erroneous beau.... of all an:dyti_ cal error. Whell the comrol sample ..",hs a.. unacceptable, laboratory persollnd should isolate the factor thaI i. causing the error. The error could be rela[M [0 ( l) the operator, (2) the protocol, (3) the reag~nu and materi:d., or (4) the :m:dyur. Whell th~ problem i. foulld, appropriate corr.cri"" actions .re t.kell (e .g., cllang<' reagtnts, clean pipe"e, or r«alibrate [he m:ochille), alld then the rolllrol .ampl .. and patiem "'mpl....e re.n:dyz.ed. A common m.,hod of monitoring the ..",lu of the rontrol .. mples is to plot them in a LNey-Jenllings control eru.n (Fig. IA I. However, such plots do nor help us d",id~ wh at is .u:q>table or unacceptable variatioll in a clinical laboratory ......Y'. Dr. Westg ..d has .lso .ddressed [hi. i,,,,e ill his work using concepts of tot:d qu:dity m:magement .nd Six Sigma Qu:dity M anagemem {http://www.weseg.rd.coml sixsigmabook.html)." Th... concepts ha"" been au:q>[M by lOme internatiollal groUp' as th~ p.,&rrM quality .,,,,ran,,,, method. The und~"'tanding and applica_ tioll of the Six Sign» Qu.lity Managtmem synem requires collsiderable effort: a few key COllcepts introdu"" the [opie: a. Sigma re~", to the Gr""k letter o. which stands for standard devi.tioll--a ,tatistica.l illdn uta! [0 n p""'" random drna[ion from the mean (""ntr:d tendeIlCY). b. A sigmil cla.. ifica[ion for. test ..fe.. to its precision ",lati"" to the WlOUnt of .cceptable error (total random .nd analytica.l v..iation that is .u:q>table). If, b.saI on knowl~ of biologic variations, wr imerpret two v:du .. that ..e within to mgldL of ...ch other a[ a d",ision threshold to be the same. then we will colI!id.. such error as acceptable or .Jlov,able. Thu., the allowable total error (fEJ for .n assay is 10 mgldL a[ a d«i.ion [hreshold. Erron of [hat magnitud~ or less will not .ff"'t interpretation of the results. TE,. must be d~termillM at dinica.l d",isioll thresholds, the values a[ which d",isiolls about the p....llce or ab",n"" of diseaR are made.

2B

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

'" '00 ,,-

<'>t

~

00_ ",-

•

V~1ue5

"';lhjll M:09plablo limits (90 • 110 mgldL)

. . V~1ue5 ba)'DJld accopI1IbIo limit.

"'unIl'"

Fig. 1.4. L."'Y.J~nning. control mort for • gluc<>oo controllOlution. Qo.Wity do.. for 12 d of ''''ring orr di'PJ:oy«I: locb <X>lO<] in tho control..,lution i, pLonNl on tt.. elm•. P, .. iow ..wy... .. ublished ,bot d.. . 000pubL. limi" for th. rontrol ..,luoon are 100 ± 10 mgldL (mean ± 2 >eci!"nt ..,t, md dun piptr:otion (2) ~ within ":""pubLe limit> .., v.ru .. "",..unt< uulyzM with .,m .., of p.tient umpLes, .00 ,esults of .:och control . oluno ... ar< pLotted in .. par ... u..y-Jrnnings oontrol cbar"'. For other ....Y'. rontrol solutions might b. rnalyud with eadt shift (•. g., .10", • p.,iont', lomlt i.s u=I lOr di.gnostic or thonp
c. For ,heoretical "-=y A with a TE. of to mgldl, aruolysis of control ",lution with .n analyte oonctmmion near the decision thushold re~. a st:mdard drnation of 5 mgldL. As.m ming the", is no symm. tic error (bunl , this assay wou.ld be call«i a twfI-lif."'a assay beau,", the TE. divid«i by its stondard deviation is 2. When designing a quality assuranct program for this assay, there would be miet criteria estoblish«i with SN

tion of the instrument for patient ... mpl... a. Th. ~rson who will analyze the p. tient "'mple mould an.lyze the control mat",ial b«:.u.. one of the most common sources of ~nalytical ",ror is the o~rator.

b. Th. rontrol mat",ial should test the .ntire o~ration of the analyzer .nd not just be, what is called an rlut"nir crmtrol, • control th. t may .imply .nm.. that the innrumem's circuitry is functioning without actually running a .. mple through it.

DIFFERENCES [N lABORATORY METHODS AND THEIR RESULTS I.

Not only do vet",inarians fa"" challeIll:" of species ... riations, they .ho must deal with the diff",em results gtn.rated by diff",em laboratori.. and diff",ent laboratory methods. The.. a.. Jrulny potential "~wns for the diff",ences; wme of the diff",ences :ore due to random errors that are not prevent.bl., oth.rs are caused by . nalytical or syst.Jrultic ",rors (bia.), and others are b.rnu.. of poor . nalytical methods that produce in. ccurate ... ult •.

[I.

Ex>rnples of diff",en""s A. Figu", 1.5 illustrates r.mlu of a survey completed by the Vet",inary Laboratory As5Oci . tion {VIA} '" part of. quality ...mran"" program. For the survey, the VIA dinributed aliquots of. pooled .. rum to laboratories th.t subscribe,d to its quality assuran"" program. Mter anal",i. of the distributed sample, ....ch subscribing laboratory ",m its r..... lts to the association for compilation and analysi •. Th. '!JOCiation then dinributed the ... ulu of the :maly..s. The type of program is wm<:times referred to as a p"ficitnQ 'ifting progr.m and can be, u..d to det",mine the validity of a laboratory's a=y. Ideally, the mult. of an individual laboratoty could be, compared to the result. established by • laboratory that analyzed the ",me "'mpl. by using . =ogniud ..fe .. n"" method. Without such . value, the p"firitnQ tifting provides a method of periodically monitoring.n assay's perforJruln"" compared to oth", laboratories.

30

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

....

,~.

ALT (U1.) Allumin (gldl) ALP (U1.) AMS (un.)

2(1 ... 8.8 . . . . . . . .

107 385

a. (mmollL)

00

!!,"8

x"""""" '"

q.

SOl

k82XtMnnnXxxgxxi Rhh;g.ggyyyyiS\lO n .. n •• 8M . . . . . i'lxg22X2XXXX !

,J! .........

ChoIest!ll'Ol (mgldl)

185

CK (un.) Croabni"" (mgIdL) GUcos& (mgldL) LipaS& (Un.) Phosphaus (mgldL) ~ssium (mmeln..) Sodium (mmoln..) tCa"+ (mgldL) teO, (rnmoln..)

1.7 WSlQYVWl , .., ~. 69 138"*12 x2) 53 Ph· •• S'jXXXXM 4.6 3.6 L?I?SNSNSNSlS?W 129 I'Y'\(WW?Y??i"Y"'iY"'iY' i ~ 7.6 V&S&'2Z&9QSM66N-L2'Z70J

Total protein (gldL) Urea (n-.WdL)

!

7 P;XxxXXXXXaneguogE8x 8 X3

QI2''''!o''''' M2I&I' n

34 h66MIY?i6d

M

"

280 1258 111

uxxxxxx"xxxxxXXXXXS I 239

............ .....................

5.1

..

Highest

>t:)Q\N'QI

.

3.7

978 6.8 5.4

153 lOA 17

6.8 51

c:::::::J R&.uIIs of ... methods (II • 7l--126) ".t>iV R&.uIIs of sa"", method 00 diffemol instruments (n • 19-111) _ R&.utts of sarno method 00 oa"", instrumool(n. 12- 15) Fig. 1.5. Resul", from umilar:and differ.nt .. soy m
B. Careful ~.w of ch~ data in Fig. 1.5 am b., disturbing ba::.u.., th~r. are mark~d diff~ren"". in th~ resuhs g.n~ra,ffl by labon'ori ... It it beyond ,h~ JCOp" of this tatbook to d=rik th~ po .. ibJ~ reasons for the diff~ren= in detaiL In g.n~ral. th~ r~sul" may va'y ba::.u .. of on. or mor~ of th~ following: I. CommuciaJ standard solutions vary and may b., poorly calibr:n.,j. Al'hough th ..~ ar~ strict requirem~nu for d~dopm~nt of human "laYS, ch~« are not ,h~ sam. requirem~nu for vet~rin.ry '!laYS.

1/ INTRODUCTORY CONCEPTS

31

2. For assa". that m.......... mlym~ activity or d~nd on ~nzymatic activity within th~ ""'y, th~ .. COlI "" marhd diff~ .. nct. in ~nzym~ ~ctivity kcau .. of diff~ .. nt ""bur~tes {..agtnts}, r....aion um~ratures, or pH of =y .".{<ms. 3. E""n though two labonrories mighr h. "" th. sam. ch~mistty instrum~nt and .......y method, thelabomori .. might purchas. r~agtnt. nom diff~r~nt compani... Th~ ..agtnts might ha"" th~ sam~ constitu.nt. but at diff~ .. nt conctntration •. C. Ikcaust of the pot. ntial marhd diff~r~ncts in ... ulu d
b. Ref..~nct int~rval, providM by the manufaaur.. of.n instrument should "" corefully KrUtiniud. The n",nufaaurer should provide detail. about the ..f..enct int. rvals, including th. ait~ria u...:! to sdect .. f~ .. nct individual., the num""r of .nimal. in th~ .. f~renct .ample group, and the m~thod of det~rmining th~ ref..~nct limits. If th~ manuf. cturer'... fc:r~nct int~rvals.re going to "" u...!, th~n .plit sampl .. mould"" an.lyzed by th. m. nufa.ctu .. r and your laboratoty to aocc:n.in whHh .. th ... i. actll~nt agr.. m~nt in the =".' ..suit •. Alt. rna. tivdy, samples nom 20 healthy . nimal, could"" ",saYM: only I of 20 values should fall out.ide th~ refe .. nct interval if it was based on th~ central 9S % of th~ .. fc:renct popul.tion. If mor. than one value is outsid~ th. manufactur .... ..f..~nct int~rval, the refe .. nct int~rval may not"" appropriate. 4. A good diagnostician dHect. and confirm. abnormalities. If. l.boratory result is not coII!i,tent with your imp ..... ion of th~ co.., you should .,k th~ l. boratory to repe . t th. =y or you mould .ubmit anoth ....mple to confirm •• ignificant .bnormality. 5. You .hould ~.tablirn a strong professional ..btionship with your laboratory'. pe"",nn.1. The quality laboratory will sui"" to provide you quality and timely results. You .hould .tri"" to provid~ th~ laboratory with quality samples. COMPARISON OF ASSAYS I.

A. descrikd in th~ prrviou. section (Quality ofLaboratoty lksult., .ect.II.B), it i. K1mHimes n""ssaty to comp"" the results of .....,... to d ... in agrttm~nt when thq product ess.ntially th~ same .. mit from the ",me ..mple. A. Importantly, results may"" mongly corrdatM but not in agr.,.m~nt. Line~r rq: ..... ion con "" used to establi.h • relationship ""tw..,n independent .nd d~ndent v.riables. If two ....."..re m~mpting to me~JUre th~ sam. an.lyt., thry should ha"" a relationship, but neith.. measur~d value i. a d~ndent v",iable. B. The m
32

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

,

NeCLS Bias Plot

Comp,~"""

•

~

~

II,

~

! • 0

0

-,~

0

, ., •,

Altman-Bland Bias Plot

ComparIson of R~sul'" 01 Two HCO,- AMays

0

0

·

,

of RIo ...... 01 T'MI Hco,- Assays

o ,

•

o o~

0

•• " 11; ~8' o

1 . '<>•••• o ~o 0

.,

Q

' • ____ 'i10 _____

~

Method 1 (1flIfd/L)

~

0

.", :'1 n !

., .,

~.O{j :t~~ ...

•••

•

•

-,~

0

~

• • •,

o

0

•

Av... """ d1118N""9

0'

, 0 __ • ..3>. . _____

~

•

"

Av""'1/8 dl'le.9"""

~

0

0

•

"

MoQn of "",\hods

~

~

I ..-.1 2 (!I"fIlOlIL)

Fig. 1.6. NeelS rnd A1tnun_Bhnd bi .. plo",. Tho mults ""'. "bul.ted rnd "'''l'' (~hod 2 - mC< fOf all umples is c:olcub..d: in this comparison. tt.. '''''''go diffeteJ>C< is 2 mmollL (bia., - 2 JIUDOUL). li.... "'pr<,serond "",thod is oong comparNl to . ",",.one< method or an es",bJim.d IImbod . • n,. A1tnun_Bhnd bi.. plot is th. urn< os tho NeeLS bi.. plot ""oopt th.e .""ngo of th. to.<> me. mted V:O/.,., (o...... g< of omhod I and mrthod 2 concentr:ltions) an plotted on tb. I4IU. Th ••dvmug. of tho Altman_Bland bi .. plot i.s tbat ""it"'r omhod i.s oomide"'" to be tb. mor<:terur.It< metbod. HCOJ-. biurbon ....

II.

Th". an four major m.. hods of assesIing 'gr,""mem of assays. It is b.yond the =!'" of this book to .ddress th. applicotion of th ..e methods, bur v.teriruorian, should be aw;on of what th ... m.. hod, provid •. A. Bi.. plots (Fig. 1.6) I. NCCLS or CLSI bias plot {NCCLS is an abbreviation for Nation:d Committ,"" for Clinicol Loboratory SUnd",!. that has oon nnamrd the Clinical Loboratory SUnda,,!. Institute} a. Th. NCCLS plot display, the degr,"" of agINmem becw,""n a new method and th. old mrthod (or ..f.ren"" method). Th. value! d..ermined by the old! .. f... nce m.. hod are plon.d on th. x_axis, and th. diffe .. nce becw,""n values d""mined by the new and old method is plotted on th. y_aIis. B.. ides plotting th. paired data point" the mtan and nandan! deviation! of the differenCe! .n .lso plotted. b. Th. graph displ.ys a positi"" bi.., (m.... n differ.n",,)o O) or a nq:ati"" bw (mean diffe .. nce < 0), and may indicate .ith" a ronslanr bu., (a diff.nnce betWttn the ..suit, th., is ronsUnt o""r all values) or • proportio""t bu., (. diff".nce be~n th. results th., chang.. proportionately with th. magnitude of th. re!uh). 2. Th. Altman_Bland bi.., plot is ,imilar bur differem: the awrag~ of th. two v:du .. it plonw on the I _axis .nd the diff.rence betWttn th. two value! is plOltw on the y-axis. This method d~, not a!S\1me that eithu th. new or th. old method is mon accuute.

1/ INTRODUCTORY CONCEPTS Deming Melhod Comparison Comparison 01 ResUl5 01 Two HCO, - As ... ys

13 Passing & Bablok Method Comparison ~risoo

oIRmult. 01 Two HCO,- A.... ys

• •

~

•

,l'-_ _ _ _ _ 16 _

25

1 (ImIOflJ

~

,l'--_-_-~

:J\

Fig. 1.7. D.ming .00 Passing 6< Bablok method comp.mons. Th. remJ", ""'.. tobubtprn. Th. analy>i, of th. two gnp!.. , " " , I r a mikl proportional bi.., tbat ill. th. . !>solu« differrnc< be""""" th. 1lison ill very .. miJ ... to th. D.ming nmhod, but tho. impncision no-.d not ho. normally distributVer th. ump~ng nngr. 1be 9'5 % confidm"" interval of .... b.st_lit ~". is :Wo disph~. HCO,-, bic:uboll1tr.

B. Deming method comp.riwn (Fig. 1.71 I. In the Deming method comparison, the v:du .. obyinffi for a given ... mple ... plonffi with the new method on th. y_nis and the old (or comparison) method on the I_axi,. An eq.u>tion for th. ~,,_fit line (y = mx + b) and the 95 % confiden", int.rv:ds for th. dope and y_int"",p" are rnkulatffi. a. A proportiOntlt bias i, det«tffi if the 95 % confid.n"" interval for the slope d<= nOt indud. 1 (i.e., ,]ope of the identity lin.). b. A ('fI",tIlnt biA, is det«trd if the 95 % confiden", inter ... l for th. y_inte=pt d<= nOt indud. 0 (i.e., y_imer""pt of the identity lin.). 2. The regression equation (y = mx + b) has bttn uK
34

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

c.

Th~

corrdat;on codlicj~m d~p"nds on th~ nng~ of dna in th~ .. mpJ~." A b,1:" rang. of data will tend to Ita"" a higher correlnion than will • ,null ",~, ~.n

when most v..!ues ... dUSI.fa! in a !l<>rrow rang<' and a single value st,etch •• ch.

rang. more bro:odly. d. Figu... 1.6 .nd 1.7 show excdlent oorrd.tion (f = 0.98) ~tw""n d.na ~5, but th. bi", plots illustrate th ... js a bias ~tw,""n the ''''''Y'. 3. [n Ih. Deming method, imp=:ision is allowed in both method, .nd the variance" must ~ normally dinributed {i .•.• it is. p>':mle'lric aruolrsi. method}. Th. point, fallon one side of the identity line. then th~ relationship ~ttn the two methods may not be linear. D. Kapp. >g1"ttment {Fig. l.8} as l. Thi, method can be used wh.n laboratory result, are in a categorical scale 1+. 2+. 3+. and 4+ or trace. mild. mod~rate. and marked. A 2+ value may not be twi", that of a 1+. and a 4+ may not be twic. the value of a 2+. 2. The calculated kappa value provid.. a guide to the degree of agrttment. The size or degree of di~""'ment is not considered with the standard kappa m..rhod. but it is with the weighted kappa method.

rum

Comparison of Urine Heme Reactions: Reneclance Photometric versus Visual Grading

H > " 0

-,,,

""G"~v"

I ""!Jat.....

R.m"'ctanC8

, ",

,~

"" 0 0 0

, , ",

, ,, ,

ic Grltdinll

0

0 0

0 0

0

Wetghted kappa value. 0.78 Interpretation guidelines

0.20 ~ 0.39

F";r

0.OO ~ 0.19

Poor

, I , • 0 0 0

,

Fig. 1.8. K.pp •..,.groement d ... for urin< he"", ,u"tion. Aft .. dipping the ruction pad in tOO u,in< ''''''ple.. the color cltrn~ in a horne-,e:o
1/ INTRODUCTORY CONCEPTS 111.

35

Aftu the quantitative "'p«t, of the =rs are comparM, a decision i. made", to whether the dq; ... of :ogrttment i, acceptable or un:occepuble, A, Visual inspection of graphM dou =y reveal th.t the remlu diff.r sufficiently so that the two =rs cannot be con,iderM the ",me, Thus, there would need to be different refe",nee intervals .nd decision thresholds for the two ousays. B, Because both assays will inherently not be 100 % precise, some diffe .. nee. are exp«tM e",n if the =rs have exeellent :ogrttment, The amount of inherent difference can be a.sses..d by calculating. combined inhe.. nt CV," C. Th. a=ptability of a n~ assay can also be a"....,J u.ing • mMical decision chart,"

WHICH lABORATORY DO YOU USE? I.

Many factors must be considerM before you decide how you wiU obuin laboratory dota for your patients, Ensically, there are thr.., major options: in_hoUie laboratory, veterinary ref.renee laboratory, and laboratory in local human hospital, Four m.jor question> must be considerM: A, How will the r.. ults affect the au. of the patient? B, Can the laboratory consistently provide quality .nalysi, of the "'mple? C. How much will it cost to .nalyze the "'mpl.? D, How important is a short turnaround time?

[I.

In_house laboratory (in. veterinary dinic or hospital ) A, AdVlUltag<"

], May allow 24 h

.= to laboratory dota

2, Shorter turn. round times, which are sometimes required for emergc:ncy ca ... 3, Fresh "'mpl.. and thus f.wer concerns about "'mple deterioration

B, Disadvanuges ], Capital expenditure of several thousand dollars is requirM .nd equipment depreci_ ates rapidly, 2, Need to =intain inventory of ~nts and supplies 3, Need personnel trained to operate and mainuin equipment 4, Need to follow a qu ality ........ nee program (You may be legally respomible for documenting quality of laboratory data,) 5, Typically need high .ampl. volume to generate enough incom. to co""r expense. 6, Refc:renee intervals typically are provided by the manufacturer of innruments or ousay and =y or may not be quality ",fe",nee intervals, 7, May need to ship. malfunctioning instrument to the manufacturer for re!",irs and thm n.."j • rq>l acement instrum.nt or backup methods C. Excellent in _hou .. testing can be: providM, but the quality and value of the remits .'" often inf.rior to those providM by ""telinaty reference laboratori.., Sources of error include the following: L Inappropriate "'mple is testM rather than rejected (e,g" became of wrong tube, wrong volum., or interfer.nt pr... nt such as lipemi.), 2, Sampl. proc..sing is inappropriate (e,g" sample not mixed .dequately), 3, Analyzer is not mainuinM adequatdy, 4, OutdatM "'agents a", u...i, 5, r."onnd do not follow a nandard operating procedure, 6, Control samples are nor tenM or are not tested frequently enough to trust results,

36

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY 7. For h~matology. microscopic .valuation is not don~, or it is done by .omwn. with I... exptri.nct th,n ..ferr:'! laboratory p
III.

Veterinary .. f...""", labontory A. Advanragr-s I. Loboralory ptrsonnd are typicaUy trainffl to provide quality .n:'!ysi, of _"inary sampl.. and r~ni", sampl.. that =1 lnd (0 inaccur>te 2 Di:ognostic mppon =1 b. .vail.bl. &om nteri",,'Y clinical pamoJogim in the laboratory. 3. Ref.ren"" illt.rv:.!, should b. .ppropriat. for the .~j ... 4. Mon laboratori., have a qu:dity m~m.nt .yn.m in pi""", to hdp enSUf. qu:dity results. Th •• yn.m oli.n includ.. proficiency testing by an at.rnal group such that p<,formalla can be, com"" •..! to that of J>ftrs. 5. Many more di.gnonic assays ... typically available than "r .vailabl. in .n in_hou .. laboratory. 6. Th. eost for sample analysi. i, more clnrly determined and thll'l can b. chargffi to th. e1i.m on ...mpl~by_sampl. basi,. B. Di..dvantago' l. Not .Jl.n:olyt..." st.bl•• nd thus ",m. dff.riorat. during shipm.nt. Som•• n:olytes requi .. sp«ial shipping. 2. Turnaround time, V:lf}' with location: wme ... av.ilable the .. me day, .nd most a.. a..... il.bl. th. next day ucept for .peci:ol um. Somelaboratorie. off.. couri....",ice and n:JUlu "ported via fax ma.chin.. or e_mail.

,,,,,,,It•.

[v.

Laboratory in loc:ol human hospit:ol A. An adv.mage i, that turnaround times am be within hours. B. Di..dvantago' l. Quality ..f..ence interval, fur veterinary .ampl.. may not be establiohed by the laboratory. 2. )"say mffhod, may not be appropriate for veterinary sampl ... 3. Technician, .nd technologist, may not be trained to rorreccly idemity .peci.. variation, or di,..,..." that are unique to veterinary sampl... 4. Pathologists who specialize: in human pati.nts frequ.ntly a" very int.... t.d in hdping, but lack the training in th. di"'.... of domncic animal, .nd variation! lttn in veterinary sampl... 5. Th. f= charged by laboratories may be "latively high. C. Testing in local laboratori.. that .peci:oliu in human m.dicin. should gen.rally be avoided.

EVAlUATING AND VALIDATING U.BORATORY METHODS" I.

Re"",ns for <"Valuating laboratory method, A. New or rev:i..d l.boratory methods may be eval uated for e1inic:ol u.. when th. fuJJow_ ing ... true: (I) r"",nt r"",arch findings suggest a better .pproa.ch, (2) n.....er ..... Y' might be mo .. accurat., mo" precise,less upensive, or less difficult, or (3) an inuru_ ment was purchased to .. place an outdated or m:olfunctioning innrument. B. A dinic:ol perspective need. to be ron'idered for new or rev:i..d laboratory method •. [, the method pra.ctic:ol? Wh.t are the equipm.m .nd .pace need.? A.. personnel tr.ined

1/ INTRODUCTORY CONCEPTS

37

for th~ method? Will it improv~ pati~nt all< at a r.asonabl~ cost? Has th~ .. bttn ad~quate nudy of the method to prove its clinical value or i, the method stiU in th~ devdopm~nt stage? Are the fetJ.uil<m.nu for .,mpl~ collection. processing. and handling pnctical? II.

Wh.>t .re th~ sources of . nalytical ~rror? A. Each =y system has it own inh~ .. nt rllnN""

aror {= Quality of Labomory Results.

=to IlA}. Typi"'Uy. th~ more prea .. the assay is. the ~ner it is. but sometimes the

moll precise methods ar. too upensive or tim~ conmming. B. Besides r.mdom ~rror. a method may not provide aa:unt~ r..... lts becau", of I)itmtlltir aror {bias}. For exanlple. the m~.n concentr.otion of a n~ method might ~ comis_ tently 5 mmoUL too high compared to th~ mean value deurmined by ...fer.nce m~thod.

C. MeumI)' is • rdative term. and . n assay's . nalytical accuracy i, sometimes difficult to ....... in a clinical laboratory. An ....y's result may ~ compared again!! one of two "tru~" values: l. Th. me.., concentntion determined by the r~ference m~thod (. gold standard) 2. Th. me.., concentntion det~rmined by numerous analy... by comparative methods (all laboratories using th~ .,m~ instrum~nt and reagenu) D. Acceptable analytical performance l. Th. most accurau. precise. specific. or ",mitive =y typically is not nttded in. clinical environm~nt. Howevc:r. the a.. ay doe:, nttd to m~et requi"'m~nts for a clinically u..ful =y. 2. eriter;" for accepuble performance of clinical =y' have bttn proposed. and they ""ry oomide.. bly :mlong analytes. A hematocrit method might ~ oonsid~..d a.ca:ptabl~ if it provides results within 6 % of th~ targtt value (•. g.• 37.6-42.4 % for a hematocrit of 40 %). Or a cortisol method might ~ consid~red accepuble if conilOl concentratiom ... within 25 % of target valu ....·" III.

Validation method" Mter .vail.bl~ information has led to • conclu,ion that. new or r~ised assay should ~ evaluated. thr.., lIage' of validation have: bttn recommended. A. F.mil;"ri7.;;ltion: Thi, 11"1:' includ~s establishment of. v.orking pJ"Oce
[V.

Implement>tion pha.. A. [f results of the validation pJ"Oce
3B

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

DIAGNOSTIC PROPERTIES AND PRED1CTNE VALUE OF lA.BORATORY ASSAYS I.

A. m~ntjonffl ~~rli~r, a frequent purpost of ~n:dyzjng a patient', sample is to detect or confirm Ih. p,... n"", of. dis..,. .. sUI<. But if a laboratory test ,e,ult is ouuid. the .. f".n~ im.rral, how likdy is it th.t the patient h ... ""ruin disorder? SimiluJy. if a [.bomory te,t result js WRI, how ".nain

"e we

that the anim:.! d""",', have a ~rtain di..-a,. or p atho_

logic nat.? The following inforJrultion is.n introduction to th. diagnostic value of 1.00,.,. tory """'Y' .nd the predictin value [hallY. which involn OOIl""PU or proadures du, ... u..d to answer [I.

th= questions.

Th". are four dassifjc;;otions of test , .. ulu ,.loti... to the presell"" or .b..""" of. di,.,.~ A. TP (true p",;t;",,): • result thai correctly identifial a pali,IIt •• having a sptcified di ..... B. TN {true nq;ativel: a r~lt that corr«dy id~ntifial • pati~nt as not having a sp«ifial di..,~",

C. FP (faJ",

p",itiv~):

•

r~lt

that incorr
id~ntifial

a p~ti~nt as h.ving a .p«ifial

di,~.",

D. FN (faJ", negative:): • result th at incorr«dy

id~ntifial

•

pati~nt

as not having a .p«ififfi

di",~",

III.

To classify test result. into one of the four categories. two factors mu!! b.: known: A. What criterion is used to ",,,,rate . positive: from a negative: ... ult? Is it • po.itive result when the result is .bove: the .ppropriate ",fc:r~n"'" intaml? Or i. it • positi"" result when the val"" «ems. certain d«ision th",shold th.t could b.: within or ourside of the r~f~"'na: interval? B. Wh.t determines whether an animal has the di""", of interest? That is. what is [he goJd stand.rd that allo,"", u. to Jay thaI the .nimal definitdy d"". or does not have: the di"" ..? For many .pontaneou. di""ses. there may not b.:. PU'" gold standard. and thus the diagnostic value data must b.: consid..ffi to b.: rdative: to an imp
IY.

After ten results are appropriardy classififfi as TP, TN, Fr, or FN, .. ve",J calculations are mad. in an ."empt [0 cha",ct~rize the diagnostic prop
. . . .. ( ) D lagnosllc ",nJIIlVlty .. % =

numb.:r of true positive numb.:r with .p«ified di",...,

· . sp«l·fi· "~"omo"'C'C"ofc'oruO'C"C"'f"c'i"~,::-", D lagnosllc clly ( .. % ) = -::=, _ numb.:r without sp
(1.2a.)

(1.2h.)

"

l / INTRODUCTORY CONCEPTS

Di"l:nostic accuracy( .. %)

numl= corrrcdy classifird numb.. of .nimals in study

TPif + TNif

~C;,"~;C",c:C-"C; TPif + FP if + TN if + FN if "" ( !'2eo)

. . . numkr of true po.itive Pr«i,cuve value of posllvt test ( •• %) = all positivt resulu

(1.2<1.)

. . value of n~llvt . t.st (as %) = -"o,o,m,"' C"'Co,rC"ru",-,o"1""';"" Pr«i,ctlVe all n~tivt """,lu

( !'2eo)

B. DiagnDific Iprofirit) I. Definition: the frequency with which . Ust is n~tivt in p>tients th .r do not have the di",... of interest (Eq. 1.2b) 2. A test tmot has high diagnostic .prcificity COlI k a good one for confimling tmot an .nimal has • dis..,... kcau .. the ten has vtry few FP results. If the result is positivt , ther<: is • high probability that the animal will havt the disease. 3. Diagnostic . prcificity must k diff... ntiat«i &om :malyticl specificity.

C. DiagnDltic aen",,,), I. Definition: the frequency with which. test rorrrcdy classifies . n animal .. having or not ru.vi"i: the di...,. .. {Eq. l.2c} 2. A test has high diagnostic accuracy when it has reL.tivdy few FP and FN results comp.red to TP .nd TN results. D. PV(+ ), the pr«iictive value of. positivt t.st, pDlitiw p"dktiw v"lu~ l. Definition: the probability that a p",itive test result indictes that the animal has the disease {Eq. l.2d} 2. A test tmot ru., a high PV{+} is one rru.t has very fc:w FP compared to TP results. Thus, • positivt Ust result strongly suggests the pr..ence of the di ....... E. PV(- ), the pr«iictive value of. nq:.ti"" test, n,ca.it't p,dittiw ""/;;, l. Definition: the probability that a n~tivt test result indict.. that the animal does not ru.vt the di ..~ .. (Eq. 1.2e) 2. A test tmot ru.. a high PV{- } is on. that has vtry fc:w FN comp"r«i to TN r.. ulu. Thus, • 1Iq:.ti"" test r.. ult nrongly mggests the . b.. nce of the disease. V.

Basic concepts of pr«iictivt valu... Thr.. mojor quenion. are colI!idered when the diagnostic properties of an assay are rvalu.r«i: A. Wh>t is the prevalence of the dis..,... in the studi«i population? The effect of prev._ lence i. illustrated in F~. 1.10. Basically, when the di ..... prevalence is very low, it i. more likely that there will k FP resulu. Conversely, wh.n the di ..~ .. pr<:""lence i, very high, it i. more likdy that the .. will k FN r.mlu. B. What method is used as the gold nandard for mablishing the p.... nce or .b",nce of di .....? Having .n excdlent gold st.ndard is sometime< very difficult for spontmwus di ..ases and thm comp.rison ag. ill!t ~ poor gold sund"d moy l.~d to questionable results. Also, colI!id.. this quenion: What would k the remlu of a romparison study if the new =y is .ctually ktter than the existing gold sundard?

40

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

,A !-

-I",", 0

0

•

-._.

B

-

, , , TN :FP

"

,!-

-

~

j"

~-

.'" • 0

"

'/

,C

'"

, , N',

.-. -

j-

j 1.

~ ..

-.-

.;; 1~ 0

TN

Ih....

r"-::: TP

_)'I. OOO ..... Moo

. : decision threshold

"

Fig. 1.9. Effects of dilfer.." <Jecis;on thresholds on d:usifying 'os, rem] .. , [n th... ",,:ampJe...... diltribution of obs • distribution may OJ m.y not b. tru< in ",01 .ru<\ies. n •• distribution of in the di ..a>.. are not G . ussian but rruy not b. ,Uw.d as shawn in this "". mpJ.. A. Tb. d..<:ision ,bromoJd is ... at the mun an.Jyt< conomtr .. ion found in tho rnimili without th. di ...... With ouch 1 d.ciUon ,b=boJd, • Tbe di.gnostic oe",itiv:ity would b. 100 % bocau .. th.r. are no FN remlt., • Tn. di.gnostic spt< no FN ""ulu. a n•• ""'ision tbmbold is at tho high.1t v:;d"" fOund in rio. .rumrn without tbo ru.e.... • Tbe di.gnostic ..",itivity would I>< poor (.bout 60 '10) b.caw. tb.r. ace ",I.. ivoly mrny FN , ....1".

wt:o

• • • •

Tn. di.gnostic sp< 100 % b.au.. ilion :or< no FP r.",la. Tn. di.gnostic :oco.u:ocy "fOUld I>< poo' b.a.,.. of tho. mmy FN ,.,..,1". Tn. PV(+).....,uId be 100 % b.a .... tho."':or< no FP ""ula. Tn. PV(- ).....,uId be poo' bee..,.. tho. .. "'. mrny FN , .. ultl.

C. TI>< ""'ision tb,.,hold is 1t. ronc<", tb. I.... t """,bp bmw.n tb. groups OCCWll. • Thit decision du.. hold "'p,... na . compromit< to obtoin tl>< 00' rombin .. ion of di:.gnostic ..",itiv:ity:and di.gnostic spC
C. W)uc i, going to

~

the da:ision th",mold th .. q>:mu •• a positive reru.lt from a neg .. ive result? Extentivo evaluation of.n atS1)' i. som<"limes nttded to find the best da:ision th=h.old. Th •• ffa:ts of ffiIDging th. da:i.ion th ... hold .'" illustmed in Fig. 1.9.

VI.

AppJiC1tion .nd interpre ... ion of predictivo ""lue concep" A. The dinical value of the calculated diagnostic propenies is influenced by the p ...... l.nce of a diseas.. For aampl•• A .nd B in Fig. 1.10 .... um. th. diagnostic ..,nsitivity of. t... i. 90 % and its diagno"ic .pecificity is 80 %. I. In ex.mpl. A of Fig. 1.10. with. disa .. pr.... l.nce of30 %: a. Th. PV(+ } is 66 %: for all positive test remit •• 66 % wiU ~ TP results. b. The PV(- } is 95 % : for all nog .. i", telt remits. 95 % will ~ TN results. 2. In example B of Fig. 1.10. with a diseas. prevalence of I %: a. The PV(+ } is 4.3 %: for .ll positive test ... ult., 4.3 % will ~ TP results. b. The PV(- } is 99.9 % : for .ll nog .. i", test ... ulu, 99.9 % wiU ~ TN =ults.

PredK::tivo value 01 positivo lest (ao %)

Pm
'"

TN # + FN#xlOO

"" '"

-

xlOO _ 95%

,.", PredK::tivo value 01 positive test (ao %)

TP # 9 TP# + FP#xlOO - 20y xlOO - 4.3 %

TN # 792 Pmdctivo value 01 oeQalivelesl (30 %) _ TN# + FN#xlOO - 793xlOO - 99.9 % Fig. 1.1 0. EumpLe. of diognostic propem.. of """y". For •• m =mpLe. tho v:llues for diagnostic ..... itivity ..,d spocifirity.,. 90 '10 . 00 80 '10. "'E-'ivdy. • l~ exa",,u A • ....., discov<...d (vi •• gold ".., b."" tb. ru.e.s... 300 dogs b.Y< tb. ru.e.s.. ..,d 700 dog. do not. k the diagnostic .. nsitivity is 90 %. then 90 % (270) of tho 300 di .....,.[ dogs willlu"" • po .. ill b""" neg'';'" ,<suIts (TN) .00 140..iU b.Y< • potitiv< ,,,,uJ, (Fl'). • Sup 2: Add tho m.... fo, tho numb.r of po<. &..d on the p=,u.""e . ...b.. is tho ....., pooiti"" prNlictiY< v.ru.i WIt .. is the t .. t·, neg:otiv< p,Nlicrivo v:ol...? • Sup I: Construct . ubi. from tbe :ov:oiJ:obLe information. Bec.""" 1 % of the dogs b.Y< tho dio..... "'n dog. luvo the dis. ... 'OO 990 dogs do not. s.c..... the di:.gnostic .. ntitivity is 90 '10. 90 '10 (nine) of tho "'n di..uod dog, wiU b.Y< • positiv< tes, ,....It (IP) ..,d lO'IO (on<) .. ill b...., . neg .. i"" .... resul' (FN ). Bee ..... the di. gnostic opeciJicity is 80 '10. 80 % {792} of 990 dogs willluvo neg:otiv< ""uJts (TN} .oo 198..iU h . ... . potitiv< r.. uJ, (Fl'). • Sup 2: Add tho v:;d.... fo, tho numb.r of po
test·,

41

42

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY 3. By comparing th. results of the two aampJes, thest conclusions are formal: a. k the prrval.n~ of the dis"" .. droppffi from 30 % to. I %, the PV{+ ) d.a.~=' from 66 % to 4.3 %. Thus, the PV(+} is less when th. pr~.l.n'" of the di ..= is low.r. b. As the prrvale"", of the dis"" .. drop~ from 30 % to. I %, the PV{-) incr...aI from 95 % to 99.9 %. Thus, the PV{~) is grrater when the p=:den", of th. dis..ase is low". B. Th. predictive v:olue oonctpu are usa! to. romp'''' the di"l:nostic v:due of two labora_ tory tests. To. illustrate this application, d ar. w... ""tmctal from an :micl. th at oompar.d the di:agnonic v:due of ..,rum [IT.J and [IT.J .. for di:agnosing ftline hyp 48 nmolfL IT, results we .. dassififfi as "",iti"" if [IT.].. W 5 I pmoUL Decision threshold. "",ruentffi the up~r ..f.",nee limits d~~rminffi from 172 healthy cots. b. Rc:mlts we", clas..ififfi .. negati"" if they did not m..,t positi"" criteria. 3. B=d on th= criteria for the IU8 cots, a. 917 cot. had hy~rthyroidi,m. Of th=, 837 hood an incr...ffi [tT.] .nd 903 had increased [IT.]... b. 221 cots did not have hyperthyroidism. Of these, none had incr~ ..d [tT.] and 14 had increased [IT.] ... 4. From the data providffi, t. bl.. we.. conmuctffi to show the dassificotion of test rn;ults. From the tabulatffi data, the diagnostic pro~rti .. and predicti"" valu .. of [t T..] and [IT..] .. were calrulatffi (Fig. l. II). S. B=d on the "",lruuion of rq><>rtffi data and .ppliCOltion of the afo .. m~ntionffi gold standard and decision th ..shold., th= conclusions COIn be drawn: a. Serum [ITJ .. had better di
[);agnostic ,..nsitivi!y (as %) _

Diagnostic spIICificity (as %) _

[);agnostic IOCCU"'<¥ (ItS %) _

'" '"

TP I + FNl

TNI + FP l

'''' '" '"

x100

_

xlOO _ 91 %

x100

221xlOO _ lOO%

.ow _ _ xlOO _ 93 %

.c.-~'"'C'"·,'," ;;'c-,,,. WI + FPI + TNI + FNI , '00

Pmdictiv .. valu .. of posti'ffl 1&51 (as %) _

""

TPI + FPl

x100

"'"

837 xlOO _ lOO %

'" ""

..'~ "~'",XlOO _ 221 x 100 _ 73 % TNI + FNI

P",dictiv .. valu .. of ""ll"~v" t9.t (ItS %) _ "

[);agnostic ,..nsitivi!y (as %) _

[);agnostic sp9Cificity (as %) _

[);agnostic accuracy (a. %) _

'" '"

TP I + FNl

TN I + FPl

x100

'" on

-

xlOO _ 98%

x100

,TP ,'00 ","~"'~''' ;;'c-,". I + FP I '+,'," TNI + FNI

Pmdictiv .. vlllu .. of posliV8 I&s\ (as %) _

'"

TP I + FP l

x100

"" '"

_

1110 x lOO _ 98 %

"'" xlOO _ 98 %

P",dictiv .. val"" of ""ll"tiv.. 1&51 (II. %) _

Fig. 1.11. Arulysis oftb. d.iognostic propc. p..,...,ted in tb. _to o [tT.] d... • From tho
.v.".

43

44

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY c. ~rum [IT;.] .. had kiter di>gnonic accuracy (98 %) chan did ",rum [IT.] (93 %). Thus, if only [IT.] or [IT.] .. can be, dewmin«i, .. rum [IT.] .. has a beuer crun"" of corrw:ly cl:usifYing the at '" h~ing or not having hyperthy_ roidism. How~er. measuring [IT.J.. j, more expensin than mnmring [ITJ. d. The PV{+} for incr .... sa1 [IT.] was 100%, .ndit w:as 98% for [IT.] ... •. Th. PV{_ }, for [IT.J and for [IT.] .. w". 73 % .nd 94 %, respectivdy. Th= ..suits indicate thu . [IT. J.. thaI is WRl would strongly suggest th.l. cat dot. not han hyperthyroidism kcau5t th... wu. v.ry few FN result.. f. Not. thai Ih . ..suits of surn nudies may vary wilh diff.rent diff... m populations, diff...nt gold ,undo,,!., and diff... nt drci,ion ch ....hoJd •. {I} [IT.] was usa! to d~.rmin. the pr=nct or ab ..""" of hyptnhyroidisffi. Th. gold nandard u...,j {or m",hod of esublishiJ\i: the p ...en"" of hyperthy_ roidi,m} in the study is colI!iderM an excellent method of mablishing the pn=n"" offdine hyperthyroidism, but it v,ould ha"" bttn interesting to lram wheth" the diagnostic value dota for [tT.] would changr if [tT.] hod not bttn u..d to hdp determine the presen"" or absen"" of hyperthyroidism, {2} Mon cats in the study had bttn referred to 'p«iali<{, for t=tment of hyperthyroidism, Thus, th"e was a high prevalen"" of hyperthyroidism in the study', populotion, A high prevalen"" inere"",s the PVl+} of diagnostic tem and lo~n the PV{~) , Accordingly, the predicti"" values of [tT.] .nd [IT.] .. would probably be diffe"'nt in • nonreferral veterinary practi"",

=ys.

VII. Applic;otion of the aforementioned method. to evaluate and rompa", diagnostic methods requi .... c;oreful planning, . ppropriate choices of the .nimal popwatioll! (disea.ed """''' nondisea.ed), and .vailability of:m ex""Uem gold nandard, A, A deficiency in the diagl105tic properties and predicti"" value theori.. is that a positive result is gi""n the same weight or importan"" if the value i, only slightly iner.....d or if it is extremely increased. Such a ~ighting proceu {i,e" lack of ~ighting} &equently is not . ppropriate in the clinical deci,ion process, B, After. diagnostic test {...... y} hOI! bttn thoroughly .tudiM, six criteria .hould be fulfillM before the diagnostic properties (prMicti"" value, diagnostic acrur:ocy, etc,) are transferred to another clinical setting:" L The definition of the disrase is connam, .nd the diagnostic criteria . '" applied in the same w:oy ouch that two dogs with the .. me .bnormaliti.. in two different settings would be diagno..d with the same disorder, 2 The .. me test is USM, As d ..crikd in ""rlier .section., the .. may not be romplete agreement betWttn two analytical assaY' even if the .. me instrument and rrag<:nu are USM, 3, The decision thres.hold. between c;otegories of test results are constant: that is, two different obse"",,,, ,hould agr.. on remlu that are positiVi and on results that a.. mgatiw, 4, Th, distribution of ten ","ules in the disea .. group is ronnant; that is, the data should ha"" the .. me . ""rage and .. me distribution curve, It may be difficult to fulfill this criterion- perh . p" .nimal, are presented e.rly in an illne.. in one location, but, in another location, animals . '" not p"""'nted until the :mimals are """erdy ilL Data for animal. with • mild di",... will probably be different from those of a group with seve.. di"""",

1/ INTRODUCTORY CONCEPTS

45

Th~ distribution of tm =ults in th~ nondi..,= group i. constant; that i., the dau should ha"" th~ ""m~ '''''rat<' and ""m~ distribution curve. It may b. difficult to fulfill this criurion---<:
5.

RECEIVER OPERATING CHARACTERISTIC {ROC} CURVES'· ... I.

ROC curv.. wer~ originally denloptd to assess the ability of radar imag~s to detect ~nemy airc",ft in World War II, and therefore to a = the ability to d~tect tru •• ignal. from background noi ... In th~ context of laboratory um, th. ROC curves display the rdation_ mip b.twttn • TP raU and a FP me. A. TP "''' is equal to diagnonic ",mitivity expressed as a decimal. For aampl., when the diagnostic ",nsitivity is 90 %, th~ TP me is 0.9; result. are positin in nin~ of t~n di..,a.=! animals. B. FP "'tr is equal to I minus th~ diagnostic specificity expressed '" a decimal. For aampl., when the diagnostic .pecificity is 70 %, the FP m. is I - 0.7 = 0.3, and thre~ of Un nondi ....ed animals would have an FP re",h. Wh~n the diagnostic specificity is 100 %, ther~ v.ould b. no FP result. and thus th~ FP rate would b. 0.0. C. Figure I .12 show. theoretical r~sults from th~ comparison of two all'YS: .....y A and assayB.

II.

The clinical valu~ of th~ comparison of diagnostic procedures by ROC cur=; d~pc:nds on many factors. Major issues to b. add""""'" includ. th~ following: A. Th~ '!lays should b. analytically ... lid and .pplicoble to clinical investigations. B. Selection of th~ comparison group'should b. clinically rdevant. Both group' mould han similar dinicol f~.tures {~.g., both h.n polyuria, vomiting, or anemi.}.o th at th~ ability of the assays to differ~ntiate disorde" i. evaluated. Conversely, comparison of a sick group versus. healthy group i. probably not appropriate or neffied {the animal w .. defined as he;;olthy without laboratory uml. c. The .c:<:uracy of the rompariwn i. vc:ty d~pc:nd~nt on the ac:<:uracy of th~ gold nandard procedure that is used to differentiate di"''''''_present from di",.", .. bsent groups. As th.re ..~ ""ry few "pur~ gold" procedures. results of the comparison neffi to b. int~rpmed .ccordingly. D. ROC curves can b. used to rompar~ th~ diagnonic accuracy of assay•. I. A visu.l impection of Fig. 1.12 .."..,als that the ROC cu"'" for .....y A i. doser to the top l.ft rorn~r than assay B, .nd thu. assay A ha. b.ller diagnostic accu"'<J than assay B. 2. A more objecti"" comparison can b. :K:COmplished by calculating th~ ."'. under the curn for earn ...... y.,..., How.""r, th... valu .. should b. int~rpr~ted carefully if th. shapes of the ROC curves differ. E. ROC curves can b. used to hdp establish a decision threshold to rule in or rul. out. diagnosis {Fig. 1.13).'" Other than b.ing used to establish decision threshold., Fig. I. 13 also provide> information that should b. considered wh.n interpreting th. un results of assayc.

Assay A Distribution CurvQS

Iv:.say B Distribution Curvos Do-..", ab ... nt

Di .... a.~ nt

r~-.!:pr~

...

12345 AMiyte cor.cenlralioo

2345 AMiyte cor.cenlralioo

, , , , ,

A..',say B Diagnostic PropArtiAs

Assay A Diagnostic PropArtiAS Decision th",shoId Dx. '9nsitivity Dx. 'p"cifocity 1 • Ox. specifidly

1

2

3

1.00 0.98 0.00 0.50 0.60 0.00 0.50 0.40 0. 10

4

5

Decision threshold 0.80 0.60 Ox. sooo.ilivily 0.'" 0.70 0.'" 0.97 1.00 Ox. spocifidly 0.52 0.70 0.'" 0.03 0.00 1 • Dx. spocificity 0.48 0." 0.10

Assay A's ROC Curve

0.35 0." 0.97 LOO

0.'" 0.00

Assay B's ROC Curve

LO

.,

.it- _ 0.8

,,

, ,,

,

,,

,

,,

, ,,

,,

,,

li Ji Ii .. 0.6 .>

,,

0.2 0.4 0.6 0.8 1 • o;agoos~c spIIcificity (fal ..... positiWl ralo)

,,

•

a.S.

.il§. 0-

1.0

0.4

,,

, ,,

,

, ,,

,

,,

0.' 0.2 0.4 0.6 0.6 1 • Diagno.tic s.pocif,city (lal ..... posi~V9 ral&)

1.0

Fig. 1.12. Comp:uUon of di.gnostic nl ... of two m.or .. ic:d ....Y' by ROC ru ....... • n,. initioJ ... "I' of th. ....m. tion is tho an.Jrsis of umpt.. from two groupo of arUrrW.s (dioe ... p, ... nt ...d di ..... . bo.nt) by the two ossoys. Th. p.... nC< or .bs<m" ofdis. ... isesublish.d by .goLd.n:u xbrd proc (top ruIV .. in figur.). To g1ther d... fOf th. ROC rurv<, multipt. d.cision tlrn-sholds.,...lec",d tlut wiU provide dilf<,ont di'llnostic .. ruitivity ... d specificity voJ ..... T h. d.ci,ion tlrn-shold, ol' then ....d '0 clusify . ctlUl ..... ,urod ronamtr1tions .. being TP. FP. TN. or FN r..ul... Fo, tbe iUus".tion in tho top gnphs. fiw d.cision tb,.molds ""',., .. leered, :and the d..hod ~ .... rop, ... nt tb. sq»r>tion of positive rnd no... ...ul.. at •• cb decision th=boLd. • From the clusifiod d .... th. di1gno'tic ...."itivity (IP .... ) rnd specificity v:ol.,., ol' c.Jrut.",d fOr . ..It decision tb,odtold. (Fo, rbi, ij[wttition, tbe number of rnimili in botb groups wer. osti .... red from tho gnplu .. itb ." ""umption tlut the totol numb.r in ••cIt group was O<)UoJ.) n.. FP "'''' is ",lrut.red by SIlbtr:oCting di'gno,tic specificity from I. • n,. docim:d f..ctiont for TP ",,,, :and FP .... ..., plott
46

,. •••

., ..

Assay C'S ROC Curve

, ---,------------------

•••

~ '1 ! 0.8

,,

.

B

,g.!

, ______ J.llJll~ _ ,, 2 mgldl , , ,,

~l 0.5

.., :50"'y1,.- // ., .., ,~ r~~ I

,,

,,

, ,,

,

,,

o.A

,'

, '

0.1

.' i i

0.2

0.3

0.4

0.5

0.6

0.7

O.B

0.9

1.0

1 _ Oiagno5lic spe<:ificily (fa"'~ nil.)

Fig_ I."'. SelOC'!ing decioion threshold. with ROC cu ..... In ,his th.eo< .. ical assay C. the onol)'le concentn_ Ii"", ""' plotted on tbe ROC c:u ..... for -.y C . • Th. top-Wtmoot point provide..1or b... bmn"" ~n diogn_ic ....uilivity ond diagnostic .pecificity and is oft.n d.,.." .. a dKision thre.hold to oond uock that . p"iorn, tu... purirul ... oondition. V.Ju .. of " .j(I "'g/dl woold be """';dercd 0Yidoion threshold, rho - r ......,.,\d h.:o~ a diagnonic .....,;.il)' o£73 .. and • diagnooric opecifici 20 mw'dL wuuId 1U1lJi:'" tl>. p~ of tM w..-. With this d.cision thr.. hold. ru oouywould hzw. high diagnootic ...".j,m.,. {90 'MIl, but . diagnootic oprcificity of only 37 ". • F«. con6, .... toly ,eo" .... dec.,;.", ,hmhold 10. ~ m.y be .., •• ~ mg/dL 10 accept OJ>ly 10 '" Fr. (i..,., 90" d"~ic ~dficity. <>< an FP .... '" 1.0 - 0.9 = 0. 1). Voba of> ~ ""ldL .... gg ... 11... the onimaI hOI tbl! dismK. With thi.. docioion tbr..bold.. the diognooOc .poci6dty....,..jd be higb (90 'III). but tbr <Wgnootic I SO mgfdl would be cono.idekd likdy to have the di_. • B.-i on this theomial aampl. . ...tu.t ""u]d be the doci.ion thr.sholdo if you drcick to ooo.pt ~ 'III FN. ood 5 'III Fr.?

oct...,,"' .....

41

48

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY ColIsid~r a patient with clinical signs suggtnive of ,he di~ of inurest and an =y C muh of 40 mgldL a. At 40 mgldL, assay C has ~ FP me ne~r 0.22, or a diagnonic sptcificity of 78 % . In oth" of 100 dog. that do not h. "" ,h. di ••""" of into=t, about 22 will han .n analrto cona.ntmion of:> 40 mgldL (22 % of rhos. without the di",..e classifi«i as di=snll . Thud".. , patient mayor may not hove d~as • • but disn .. is morelikdy ,han nOi. b. AI 40 mgldL, a=y C has. TP rat. I><'lU 0. 73, or a diaglx»Iic omsitivity near 73 % . So, of 100 dog> chat do h."" th. dilmSt of interest, 73 will Ita"" an analyt. conctntration of:> 40 mgldL (73 % of thOst with th. dilelSt classifi«i as di..-asal). Therefore, d.. pite. value chat is nol:;> 40 mgldL, the !"Iliont may S{iU h. "" the dise>st btc.ust 27 % of ""limn with th. di ..= h;,,,,, valu .. of 40 mgldL or I. ... c. This example emphasizes that for many diagnostic tests •• nimah with and without the di"'''''' of inte..st may have the .. me .....y result. 2. Anoth" key concept rdated to this illustration is th . t e.ch ROC curve .. prestnts the resulu obtained from one sampling of the defined popubtions by using on. assay:u it compares to one gold nandard. Anoth" sampling ming the same sdection criteria, the same assay, and the same gold nandard probably will not produ"", the ... me ROC curv• . F. F.cton oth" th.n the probability of correctly classifYing .n aninul .. having or not having a di"'''''' can influ. n"", where decision th ... holds "'" ost . blished: I. If the monality rate is very high for a dis ...... that is not treated promptly, the decision thr..mold may be changed 50 that th"e ... few.:r fal .. negative>. 2. If the dist ... is known to cause I"'in or unreasonable discomfon, the decision th ... hold may be ch . nged so that there are few" f. lst negati=. 3. If the tr .... tm. nt i. known to ha"" "",ere .ide effects, the deci.ion thr..mold may be ch anged so that the", are few" f.l", positives. 4. If the fin:mci
I.

woro"

,h.

,h.

HERD_BASED TESTING FOR CAlTLE I.

Oth" than usifll: r..... lts oflaboratory assay. to hdp detect or mablish the p.... n"'" of pathologic st . tes in individual :mimals, clinicall. boratory :usay" .ho are u.sc:d to evaluate the nutritional and met . bolic ,tatus of group.! of cattk Th... mu . tions should compl._ ment other management tools such .. ration evaluation, milk composition analy.is, and body condition scoring. The goal is to detect .ubclinical bbor.uory .bnormalities that reOect rorrectible nutritional m. nagtment practi""" so that changes am be innituted prior to .dverse dinical and economi" consequences. Metabolic profiling may be mo,t applicable to lorge herds where ( I) adequate .. mple sius are .""ilable, (2) costs a.. diluted across more animals, :md {3} correcting a mboptim.l nutritional stat. could tramlote into consid"able economic savings.

II.

Th. approach to group testing differs in ..""",I w:I}" from individual aninul testing with diagnostic profiles.'......, A. Emphasis is ploced on evaluatillJ: and monitoring subclinical mh" than clinical di ... ",. Clinically ill cattle a.. excluded from herd_based tming bemuse their result, may be indicative of.n individual dist... problem mher than a herd problem.

I / INTRODUCTORY CONCEPTS

49

B. Individuals to bt t~stffi must btloJli: to ddinffi group.< of similar "i:~ .nd lactation st"i:~ under similar environm~ntal conditions .nd nutrition<>l man"i:~m~nt (~.g.. prefre;h~n _ ing cows and po"calving cows). S~fic group trend. or .bnormaliti.. may not bt app .."nt if inclusion criteria are too broad. thu. aUowing a gr~at .. dq;"'" of physiologic variuion. C. U..ful information ~uires .d"'luat~ group represenution .nd th~refore .n ad«Juate sample .ize fur the group of int.... t. £ompling multiple animals from a definffi group hdps rffiu"", variation unrdat~d to nutrition<>l state. I. S.mpl~ sizes of7- 12 havt been .dvoallffi as reasonable pools to provide u",ful inform.rion fur mon purpo .... regardl.... of herd size. Howev... great.. numbt .. of t.. t individuals v..ould provide re;ults with tighter confiden"'" limits and may bt nea .... ry to ad«Juatdy "pre"'nt target groups in lar!:" h..d.. 2. Herd size may limit the numbtr of .nimal, in a rdevant group th at am bt t.. tffi at one tim~ (~.g.. too few p..fresh cow.). How",,",r. u ..ful information may bt obuinffi in these group.< by t.. ting over time as animals enter th~ group. Interpretation mun then includ. ronsider.otiom of tempo",l variabl... 3. Economic pressurr:s ~noouragr smaller test groups and pooling of samples. but pooling may bt und..irable. a. In one nudy. 5 of21 .nalytes {including tCa" and BHB roncentratiom) had poolffi mean v.lu .. that diff..ed from mean valu.. of the individual .nimals in some pools.'"' b. Unapectffi markffi abnormaliti.. in one or more sampl.. may bia. the pooled res ults ... pecially if un"'lual vol urn.. of sample from earn individual ..~ used. c. Individual animal d.ta cannot bt generatffi. '" only mean valu .. rather th:m rang .. of values or proponions of .bnormal remlu abovt or btlow a decision threshold can bt a=d (S« Herd_based Testing for Cattle. sect. lll). D. Analyt .. measured by h..d_ba..d m.tabolic profiling t<:Sts should refleet merabolid nutritional status. Typically. they ar~ not tightly regulated by hormonal or other physiologic f.ctors. For a:mlple. metabolic p>thways allempt to maintain glurose oon""'ntration within a cenain rangr ",,",n if th..e is a marked diff..~nce in the intaJ.:~ of carbohydmes; thu ..... rum [glucose] i. not a good an<>lyte fur ....... iJli: carbohydrate intake or digestibility. In cont""t. plasma [BHB] is not tightly regulated. and incr.....d con""'ntrations refleet a deficient en..gy natu •. Even if an<>lyt.. meet this criterion. it i. al", important that :malytical variation is small compo.m:I to rn:mges cau..d byalt..ed nutritional stat... Exampl.. of t<:Sts and ",.u!ts that may bt useful include the following; I. Low blood herrultocrit values in the first 8 wk oflactation suggest the nm to evaluate energy .nd protein nutrition. 2. s"rum [ure.] at any lactation st"i:~ is :m .....ssm.nt of ruminal balan"", of ~nergy :md available protein. 3. High ... rum P_hydroxybutytate (BHB) oon""ntration at about 5-50 d into lactation is associated with suboptimal energy :md glucose balance .nd. clinically. reducal milk pnxluction. incr.....d clinical hto.is. di'placal abomasunu. :md reducal fenility. £ompl...howd bt collected at • consistent time rdative to fming; for nample. 4-5 h after the first meal of the day. when rumen butyric .cid production i. high. 4. High .. rum or plasma non .. t..ified fatty acid (NEFA) con""'ntrations in prefresh. dry cows in th~ last month of gestation (~.pecially 2- 14 d prior to calving) ar~ evidence of a negativt ~nergy balan"". £omples should bt collecred shonly btfore feeding time. One can draw .nd fr=.e multiple .amples and ... nd chose that fulfiU the 2_ to 14-d precalving criterion on"" this is known.

50

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY 5. low .. rum [tCo'+] 12- 24 h . fm calving by mu[tiparou, cows illdic:ot.., clinical or subclinical p:orturi.. m hypocal""mia.

III.

Imrrpretation A. Abnormalities in herd.basM 1m results must be, jlu.rp.. ud with mention 10 . 11 th .. variabJ .. that COli aff«t thrm, !lot juS! nutritional f. eton. Other f.Clon indud. pr..~ruoIytical ... mpJ~ variotion, a,",lytical variation, biological vari.tioll, circadi.nl pr>ndi al variation, ..asonal variation, variation in phpiologic states, occurr..I1"" of po.thological , Ut .., and olh .. , environm.nrn .nd managtment f. eton. Mon of [hest vari.tiol" a", controllal by careful group "[«:lion criteria ~nd appropriat~ procalures for .. mpJ .. coU«;tioll. processing. and

.nat)";"

B. Man h~rd_baKd test resuJu will f~Jl within di.gnonic ref.."'lIu interval. ~u .. the .amp]aI individual. Jack clinical sigll! of di, .....,. Th"ero.., a diffemlt ~ ofint.. pr<,ti"" guiddin .. i. n~=ry to d~t= subclinical abnorm:.!itie;. Cur",ntly. interprrti"" guiddine; ar~ ,ugg~stal =mm~ndation.s by ex~rts in the fidd ba.>N on exp"ri~n~ and r...euch. Giv~n th~ int~rloboratory ",rittn ~valuata! in ~~ral ways. I. M.,n h~rd v:.!ue; truly b. comp.ral to mean ",lue; of ",f~ren"" h~rds. When metabolic profiling was first describal. th~ ..f..~n~ int~rval for an a""lyre was defina! as th~ m.... n of its me' n! for r~f.."n~ h~rds. ± 2 sd. How~""r. th~ .. h~rd ",f..en~ inte",:.!, .re not re.dily .",il abl~ beau", of the ex~n .. of te;ting and diffirulti~, id~ntifying ,uitabl~ ref~ren"" h~rd,.

2.

M~.n h~rd

v:.!ue; truly b. comp..ed to an e;tabli,hed expecra! m....n result for the and group of int.... t. For exampl~. it has !>ttn 'ug~ned th .r m.... n urin~ pH ,hould b. 6.0-7.0 in prefre,h row, fa! anions to hdp prevent milk f~~r. A m~.n pH of 6.0-7.0 mpports appropriat~ acidification of th~ group. When the herd·, m.... n ± an un~rtainty int~rval doe. not includ~ 6.0-7.0. a h~rd·. urine pH i, cons.ideral inappropriate. When th~ herd·, me.n valu~ is not 6.0-7.0. but the mean ± un~nainty int~rval o""rlaps thi, tatg"t. the results are cons.id",al bord..line. A 75 % confiden"" int~rval ha, !>ttn mggesta! as a u",ful guid~ for metabolic profiling and a reasonabl~ compromi'" becw",n 95 % confid~n"" and practicality. 3. The !",/"Centage of individual results abo"" or b.low an establi,hed d«i,ion threshold may b. calculata! and compara! to what i. con.sid~ral an a=ptable !",/"Centage of high or low ",lue;. A p"rctntag~ (± un""rtainty int~rval) gr~at .. than th~ a.cctpted p"rctntage 'ignal, a problem. A p"rctntagt and confid~n"" int~rval overlapping with the accq>ted !",/"Centagt is a bord~rline r~.uh. D«ision thresholds may "" "'m~h at artificial in that the riN m~abolic profiling.re also imp"rf«t. How~.r. this doe, not prevent th~ acquisition of u",ful information. a""lyr~

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Chapter 2

LEUKOCYTES Physiologic Processes Involving Loukocytes..................................... 54 Analytical Principles and Methods ............................................. 60 I. Complete Blood Count (eBC) ......................................... 60 II. Leukogram ......................................................... 62 III. Principles of Determining Leukocyte Concentrations ...................... 62 IV. Relative versus Absolute Changes in Leukogram Results .................. 69 Abnormal Leukocyte Concentrations in Blood ................................... 70 I. Abnormal Neutrophil Concentrations ................................... 70 II. Abnormal Lymphocyte Concentrations ................................. 81 III. Abnormal Monocyte Concentrations ................................... 85 IV. Abnormal Eosinophil Concentrations ................................... 86 V. Abnormal Basophil Concentrations .................................... 88 VI. Abnormal Mast Cell Concentrations .................................... 88 Leukogram Patterns ......................................................... 90 Abnormal Morphologic Features of Leukocytes .................................. 92 I. Changes Associated with Inflammatory Diseases......................... 92 II. Leukocytes That Contain Miscellaneous Inclusions ....................... 94 III. Organisms in Leukocytes ............................................. 95 IV. Leukocyte Agglutination/Aggregation .................................. 98 V. Hereditary Disorders That Have Leukocyte Inclusions ..................... 99 Other Nonneoplastic Leukocyte Disorders ..................................... 100

53

54

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

Table 2. 1. Abbreviation. and [xl ASVCP BFU·E BLV

cs. CBC

CFU_Baso CFU· E

CFU_Eo CFU_G CFU_GM CFU·M CFU_Mm CFU_M~

CLP CNP

EDTA FcLV f.MLP G_CSF GM·CSF

19E 1L-, INFy LTB. MalNP MLP MNP MPS nRBC

PAF ProNP SNP TNFa TNF~

TNP URL WBC WRI

syn,ools in lhi. chapler x conctmmion (x = analytc) Am
PHYSIOLOGIC PROCESSES INVOLVING LEUKOCYrES I.

L,"/wn: all leukocytes in an animo!, including l.... kocyt. precunon, leukocyt.. in blood and lymph vessd., and ti1SU< lnikocytes A, Bone marrow oonc.im pr«urson for n.... trophil" eosinophil" ba50phih, monocyt.. , Iymphocyt.. , and man ceU, (ron'idered tissuelrukocytesj,

"

2/ LEUKOCYTES

B-Iymphocyto T_lymphocyto

.-

Platelet

CFU-Mog

. ~ .~ BFlJ..E

CFU_E

o

Erythrocyte

Neutrophil

•e

,

Eo.ioophi

,

Basophil

CFlJ..Eo

CFU-Baso

e '

CFlJ..Mast

Fig. 2.1. Difhr.nti1tion of plwipotenti,j stem ""u. to tho oommitted ""J] lines of tho b.rrutopoic. p>rt oftlM: J.ukon =q>t tbos< of tho
l. leukopoi~sis is pan of h~matopoi .. is. which is a rompla 'y,um involving "~m cdls that >c. c:opable of sdf_ren~ or diff~rentiation tow ..d a oomminffl cdlline {Fig. 2.1). 2. St~m ""lls look lik. "",:011 lymphocyt .. via light and transmi«ion dectron micros_ ropy and at< prestnt in blood and omer tissues. including bone marrow. spl,""n, and li""r. 3. Spa;ific S{imuJi and regulotors govern l.ukocyte diff~t
lymphocytes, and null_lymphocyt ... C. Leukocytes in blood at< in transit from ,it.. of production to ,it.. of function or destruction.

"

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY D. Tissue l.ukocyt.. l. Granulocytes (neutrophil., a)sinophil., and Wsophils) ptrform their role. in host

d.£.Jl5t and di•. 2. lymphocytes may undergo bl:astogt'nesis, mum to blood vi. lymphatic ~" or die. 3. Monocyte. tr:msform into hisliocytes or macrophage; that :if. cop.bJ. of mito,;s, p",form their host d.f."", functions, and di •. 4. Mast cdl pr~nof1 diff.rent",u into tissue mast cell., ptrform th. ir rol .. in hon d.f.nsr, and di•. Sumet< """ known. [I.

Neutrophil pools and kinetics (monllY[I{ of neutrophil.) (Fig. 2.2l

©

:@

:( J~-

-

~

@ @J

---

CNP: !roo IIowinll MNP, adoorad

....".

Fig. 2.2. N..,trophiJ kinetic" in health. Murow bas tlu ... mojo, J> (Mme), band n bind to adhesion pro,eins on endothe~:d ""lis. they mign" into ,i .. "", to fOfm the TNP. In ,be TNP. ,be .....'ropbils perform tbei, prot«:t:iv. functions:md die.

2/ LEUKOCYTES

57

A. Murow neutrophil. l. IL.I, IL·3, IL..6, GM.CSF, and G·CSF .rimulat~ CFU·G to diff~ .. ntiate into the neutrophilic cdl line .nd to enUr th~ ProNP {mitotic pool}. 2. Mydoblasts, progranulocyt.. {promydocyt..}, and mydocyt.. divide and matu", in the ProNP. Programm":! cdl death ocru ... in the mydocyt~ stag< to limit neutro· poi..i. in h~ahh. During enhanc.d neutropoi ..is, f.,.·.. cdls die in thi, n'4:~, .nd thY! more ~nt .. th~ MatNP (postmitotic pool). In hralthy mammals, neutrophil preeuuors a.. in thi, pool for .bout 3 d. 3. In the Mat NP, metamydocyt., matute to b:md neutrophils .nd then to segment":! neutrophils. Sq;mented neutrophils that a", ready for relra .. to murow sinulOid. ue in a subpool coli..:! SNP (storage neutrophil pool). Neutrophilic cdls are in th~ M.tNP for 2- 3 d in dogs .nd for 4-..6 d in people. The M.tNP:ProNP ratio is about 4-6, meaning that the number of ceHs in the M.tNP is 4-6 tim~. the number of cells in the ProNP. 4. In h~alth, ... gment":! neutrophils a.. released &om the MatNP to murow.inu ..., :md th~n to Jl"ripheral blood. Neutrophil.rd~.oing factor.; indude chemoattractanu (CSa, IL-8, f.MLP, LTB. , .nd PAF) and cytokine leukocytosi, factor.; (IL-I, IL-6, TNF«, TNF~, G.CSF, and GM.CSF). B. Blood neutrophil, l. In h~alth, neutrophil, have a blood half.life of .bout 5- 10 h b.fore they ~nt .. tis,m ... 2. Blood neutrophil. are dinributed into two pool, as det .. mined by their locotion in

-".

a. Neutrophil, that are f..., Howifll: in blood {and thus wHeeted in blood .ampl..} ..~ in • CNP (circulating neutrophil pool). b. Neutrophil, that temporarily .dhe.. to endothdial cell, are in th~ MNP (margin. at":! neutrophil pool), which is locot":! primarily in small copilJari.. and nim in which neutrophil, havt the moJi opportunity to cont.ct endothdial cell!. Aft~r adhesion. neutrophils may break 100.. :md retnt.. the CNP or migr.te into th~ TNP. {I} InHammatory cytokin.. (induding IL-l .nd TNF &om m.cropru.g.. and IFNy from lymphocyt..) nimulate endothelial cdls to produce and express oodh.,ion proteins (sdectim) that m..:!i.u migration and "rolling." {2} End~nous chemical m..:!iator.;, induding LTB, and PAF. activat~ neutro· phi]" lrading to exp ..... ion of h~h.aflinity membrane imegrin', which bind to endothdial cell receptors th at m..:!i.t~ the proc= of migr.tion into tissu ... c. In most m.mmals, the MNP:CNP ratio i, near l. In em, th~ ratio is 3.' d. Major proc""", th. t inHuence mrasured blood neutrophil con""ntrations {I} Production (a) Stem cdl proliferation :md differentiation {b} Effectivtm:ss of m. turation in the mydocyte nage {2} Rde.... from marrow: The olden or most matu", neutrophil, preferentially Iran th~ marrow. {3} Distribution of neutrophils betwetn the CNP and MNP {4} Migration from blood to tilSUe:s: Compared to immatu.. neutrophils, matu .. segmented neutrophils have mor~ po"mi. l to emigrat~ to tissue.

58

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

Blood vessel

Fig. 2.). Lymphocy« kin
'0

C. Tissue neutrophil, l. Ch~mot;;octic substance; such.., C5.,Il.-8, LTB.. and PAF promote neutrophil migration to spaifi, ,ires. On"", in Ii",,,., nrutrophil, ~n~raJlr do not mum to

blood. 2. [n the .bstact of di..,=, most of an .nimal'. neutrophil, die in r.spiratory and alimentary tissu ... Ill.

Lymphocyte pooh and kinetic> (Fig. 2.3)

A. Lymph node, and oth" lymphoid tissu .. I. Mediator> stimulnr diff... nt;",ion .nd prolif".{ion of B.lymphocyt.. and T .lymphocyt... 2 lymphocyt~, Jeav. lymph nod.. vi<> dr.rent lymphatic v....d, and .nt
2/ LEUKOCYTES

"

C. Lymphocytes ill lymph lIod.. I. lymphocyt.. migr>t~ to lymph nod~ corti= via .p dT~ .. nt lymph atic """ds. and ",rurn to blood. 2. About 25 % of blood lymphocyt.. ~nt~r lymph nod .. ~ach d"Y through postcapil. lary v~nul... which han uniqu~ tall ~ndothdial edl. and r~tor>. D. Lymphocytes ill oth~r tissu .. I. lymphocyt.. ~migr:lt~ to ti ....... to ""rform functions. Th~r~ thry moy und~rgo blmogtn..i., ~nt~r lymph atic v....d. to mum to blood. or di~ . 2. Lik~ n~utrophih. migr:ltion to tissu .. invol"". lymphocyt~ ch~motaxis .nd bindillg to ~ndothdial cdl ~tors. E. M.jor processel that influ~n'" m~asurw blood lymphocyt~ con"'ntratiollJ I. Production a. Sum cdl prolif~ .. tion and diff~ .. ntiatioll b. BI.. tog~lI ..is 2. Distribution of lymphocyt.. k!w""n th~ CLP and th~ MLP a. Migmioll from blood to lymph nod...nd oth~r tissu~. b. Migmioll from lymph nod .. via ~ff..~nt lymphatic v....d, to blood F. Lymphocyt~ lif. .pall varies from hours to )""'Irs. Monocyt~

IV.

pooh and kinetia A. Monocyt~. and naltrophil. sha.. a common bipot~ntial st~m cdl {CFU.GM} that i• • timul>lw to diff~r~miat~ by inflommatory cytokill". B. Monocyte. dn-dop from mOllobwts and promollocyt... Wh~1I rd~ased from morrow. monocytes distribut~ ~n m..ginatw .nd circulating poot.. C. Lik~ oth~r leukocyt... monocyt .. ~migrat~ to ti ..!U~. aft~r binding to .ndothdial edt.. 011'" in tissu ... monocytes may diff~r~miat. into cdl. of th~ mOllollucl~.r phagocyt~ .ynem: macrophages {including Kupffer edl ••• lvrol.. mocrophagcs. and type A .ynoviocyte.l. microglial edls, or d~lIdritic ",H•. I. Th~ rming ma.croph~ is sometimes callw a histiocyt~ or fin-d macroph~ 2. Delldritic cdls ar~ .ntigen.pr=nting ",lis. Examples includ. the ung~rhans ceUs of the .kin and im..digiming ",lis in lymph nodes.

V.

Eminophil pools .nd kinetics A. Eosinopoi..is is stimulatw by 'J"'Cific medi ator>. illcluding IL-5 {msinophil differ~nti •• tioll f.ctor} and GM·CSF from ma.
VI.

Barophil pools and kill.tics A. BalOphih originate in bolle marrow. wher~ th~ir production and diff~re miatioll is controHed by IL-] and other cytokine,. B. Th~ marrow trallsit tim~ of w.ophils is .t 1<"llSt 2.5 d, th~ir circulating half.lif~ is .bout 6 h, .nd thry may survi"" for as 10Ilg" 2 wk ill tissu ... C. BalOphil .migration to tissu .. i, promoted by IL-I. TNFo.. and ~ndotoxill and i, .imilar to th~ proc" , used by naltrophih. Basophil! at< activated by IL.] alld IgE

60

FUNDAMENTA LS OF VETERINARY CLINICAL PATHOLOGY

binding. B..ophil granules rontain suNtan"". that promou hyp<,..,nsitivity i"H>IIlffi" tory tractions and mrac{ eminophil •.

VII. Mm "",U kin"';cs A. M..,I cdl p=rsors :He found in m ..row and "'. d.riVffl from a oommillffl n.m cdl that jt diff.",m from Ih. basophil's oommiu..d n.m edl. B. Undiff"rt, d but committal /lUSt cdls [ran marrow and circulu . in blood but ar. r«:ognized only by sp«ial m<'lhod. that ... not usa! dinically. Finding diffu.miat..d mast cdh in Ih. blood of dommic mammal, is oons.id.ral a pathologic !IaU. e. On~ in tissu.. , m ..! cdl pr«ursors diff"r<miat< into m:ast cdh, und.rgo mito,;,. or di •. M:m "",Us play roJes in hyptr..",itivity "actio!,., fibrolis, and oth., inflammatory "'pons", in tilSUes. Mast ""lis are oons.id.r.d long_liv..! ctlls.

ANALYTICAL PRINCIPLES AND METHODS I.

Complete blood count {esC} A. Numerical and micro,ropic data of blood leukocytes a", compon.nts of mammalian CBC ... uh •. Th. primary purpo"" of the CBC a.. ro ..:r..,n the hemic s)"lt<m for abnormalities or its respon'" to a di",= and to confirm or define the pr... n", of a h.matologic dilOrder. B. Major compon.nts of the CBC I. L(ukngram {meaning "leukocyte pictu"'"}: results of l.bomory .,.lay< and calcul.tffi data that char>cteriu the leukoc)'t<s in blood 2. Eryrbrngram (meaning ".rythrocyte pictu .."): results ofloboratory =ys and calculatffi da1> that characterize the .rythrocytes in blood (Ott Chapter 3) 3. Throm/wgram {m.aning "platdet picture"}: ... ulu of laboratory =ys and calculatffi data th>t char>cteriu the plateltion is increased. the di",.,e is causing at l.ast on. of the following: a. Inc",.,ffi production of that ",ll type b. A shift of that ",II t)'~ from a no~ or noncirculating pool to circulating blood c. An incr.....d circulating li£. span of the cdl typ<: b.cau .. the m e of ",II loss to tissues or rat< of cdl de.th is deer•• "'! 3. If. cdl ron"'ntmion is decr..... d. the disra,. is causing at least on. of th. following: a. Deer • .,ffi production of that ",ll type b. A shift of that cdl t)'~ from the circul ation to a noncirculating pool c. Deer • .,ffi circulating life .pan of the ",II ry~ 4. If morphologic f...tures of a givc:n cdl ry~ a", abnormal •• ither (I) a d.frct in hematopoiesi. has cau..d th. production of abnormal ",lls, or {2} morphological abnormalities have b..,n acquirffi as the cdls circulat< in the bod)'. D. Blood .lample for leukocyte evaluation l. Blood is collectffi into a tube containing .ither K,EDTA or K,EDTA and immffii_ atd), miud b)' slowly inverting the tube at least t.n times. To obtain complete and acrurat...sults, the sample must be f"", of clots and pl.tdet dumps.

2/ LEUKOCYTES

61

2. Stability of ctll.: [WBC] is usually coruid~rffl to be, stable for ~ral hours .t 25 ·C and for up to 24 h at 4 .c. E. Microscopic aamination of nain.d blood films I. Microscopic enmination of a nain.d blood film should alw"}'"' be, • pan of a CBC. rvtn if an instrum~m provid.. an .utomatM la>kocyt~ diff",ential count. A d.tailal description of th. prrpantion. staining. and enmination of th. blood film is be,yond th. SCO!", of this chapter. Th. basic componenu of the p r = arr as follow.: a. A blood film is mad. to obtain an ",en distribution of cdls and an ""equate "counting wiltlbw" (i. •.• that part of the sme.r whe", there is a monolayer of erythrocytes that occasionally touch each other and where nucl.,,, and cyto_ pl,umic feamrr, ofleukocytes.rr distinct). Cd], will be, .. pamal morr in the counting window of blood film. from anemic individuals. b. Th •• ir-dri.d blood film is .rainal with. Romanowsky_ty!", stain (e.g .• Wright. W riglu_Gi.ms;;o. Diff_Quik. or Quik_Dip) that provides diffe"'ntial staining of c.lls. c. A blood film examination includes scanning with 4x or lOx objectives .nd mor~ criti",l evalu ation with high-dry (40x) or oil objectives (40x. SOx. or lOOx). (I) Au the cells ,"""nly dimibut.d .nd pro!",rly st.in.d? (2) Au there .bnormallargt' structutes in th. blood (f=iu~ntly concentratffl in th. feath~r.d .dge) ,uch as microfil.ria. pl atd.t clumps. macroph~ • • pithdial cdt.. endothdial cdls. or mq;aka,yocytes? If so. record their pr=nce. (3) Do the .rythrocyte .nd la>kocyte densiti .. correspond with the known cdl concentrations in the sampl.? If not. check the accuracy of the cdl roncentrations. (4) Enimat. Ihe p[aldet d~nlity in several 1000 X oil fidds and compa", with the expectal ... lues. Record th. presence of giant or iliift plaldets (pl atd.ts larger than erythrocytes in most species) or pl atd.t inclusions. (5) Evaluat~ .rythrocytes for abnormal ius. colors. inclmioru. or associat.d organisms. (6) Complete. leukocyte differential count (..., Analytical Principles .nd Methods. sect. Ill.E) and record it. Evaluate th. ob..erv.d la>kocytes for morphologic abnormalities and record the findings. 2. Microscopic enmination of a nain.d blood film is especially imponant in sick patients .nd Ihose with .bnormal concenlr.tions ofleukocytes. erythrocytes. or plaldeu. o.,fecu found in th. blood cells.", described in Chapters 2-4. F. Staining of blood ceUs I. iWmanoWiky 'taim. which .re the best for staining blood cells. were described by Romanow..ky (1891) as. rombination of eo.in and methylene blue to produce. spectrum of colon from blue to raldi.h orangt'o depending on Ih. pH of the cell·s rontent. a. Wright IMin i. a combination of "".in and oxidized methylene blu •. The oxidized methylene blue nains are call.d /Wi" dytl. b. Other Romanow..ky stains include Giemsa. W,igh,_Gi.msa. and Wright_ Leishman. which are ... rious combinatioru of a7.llrr dyes and "".in. c. Generally when staled or w,itten. "Wright" nain ..f.rs to • Romanow.ky_type stain and not the otiginal W,ighl stain.

mapes.•

62

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY 2. Wh at."" th~ oont~lIIs of <,<,Us th . t ."" nainffi? a. Acidic Slructure. (e.g., DNA and RNA) aUnCl th. basic nu •• dyes. which .uin ,tructure. various colon from purple to blue. b. Alkaline structure. k.g., hemoglobin, .nd U>5inophil granules) allnet th. acidic a>sin dye and ,c.in Slructures from ral or pink 10 orang•. 3. T.rms usa! to doscrik colon or staining proptrties a. Nrumphilje: n(urro (n.ilhor alkaline nor acidic) pJu.s phiuc {"loving"j b. E~linqphilk: loves «>sin {.cidic} dre; will'" rw to orang. c. &',ophilic: Joves b.. ic (:dkalin. :md nur.) dy.. ; will k blue to purple d. Azurophilic: Ions ..ure dye; will h.ve a blue to purple to reddish purple to pink oolor dq>
n.

lnIkogram A. Morphologic (microscopic) .valuation l. A microscopic .valuation of .uinN leukocytes is.n importmt part of th. leuko_ gram, "p<eially wh~n th~r~ i, ~ l~ukocytosis or l""ko~n",. It hdps confirm or ",fut~ automatffl quantitati"" results •• nd provid .. additional diagnostic and prognonic inform.rion that connot be obtainffl without microKOpy. 2. Lmr stetiom of thi. crupt.. d~""ribe th~ clinical signilicon"" of l""kocyt.. with abnormal nuclei. cytoplasm,. lizes. inclu,ions. or oth.. f... ture>. Bts.id.. tho .. ch.nges associatffl with pathologic nau" in vitro changes amsffl by poor s;omple handling con mah accur:ll< identificotion of leukocyte, n... rly imJ""Sible. B. [WBC] l. [WBC} i, the number of leukocytes ~r unit volume of blood {in clinical jargon, commonly r.ferted to as WBC rount}. The [WBC] by it..,lf is of limitffl value without asses.!ing the con""ntrations of e. ch ty~ ofleukocyte. 2. By wme method" the [WBC] actll<>lly i, ~ total nucle.tal cdl con""mr:ltion. If it i. , the [WBC] must be correctffl when nRBCs.", pr...,nt (0« correctffl [WBC] in Eq.2.l). 3. Unit conversion: "'J.IL X 10' J.IUL _ " X IO"L (SI unit) C. Differential leukocyte count l. A diffirmtial ltuktlcyu count is ~ method of determining the rda ti"" numbers (or the ~r""nt:lge.) of the l""kocyt .. in blood. 2 Raw data :He reportal ., ~r""ntage. (e.g., 80 % neutrophil,). These ~r""ntage. plus the [WBC] :He USffl to calCllJ.te th~ con""ntrations of each ty~ of leukocyte in a blood ..mple.

Ill.

Principl.. of determining l""kocyte concentr:ltion. A. Hemocytometer method l. A hemocytometer (Fig. 2.4) is u..,d to determine [WBC] by diluting the blood {mually with the Unopette system} .nd then dispensing the dilutffl blood into the hemocytometer chambers formffl bene . th • cover glass. The leukocyte diluent contain, a lysing agent that rupture. the erythrocyte. 50 they do not interf~re with the nucleatffl cell enume.. tion. 2. The chamber is examinffl with a microscope:, .nd leukocyte, at< countffl within the 0.9 J.Il volume of Huid dem:Hcotffl by the nine large ""Il<>res. To obtain. leukocyte concentntion, the number of leukocyte. countffl is lim muhipliffl by l.l to

2/ LEUKOCYTES

63 Hemocytometer

~ Elf

1- ------ - --- -- ---

r' , ~

~

l mm <:

, (0.1

"'-'"

Fig. 2.4. H.mocytomet~r. Th~ I..mocyrom
cakulat~

a con<'<'ntration in th~ dilut«i blood This product is th~n muhipli«i by the dilution f. ctor {• .g .• lOO} to cakulat~ th~ [WBC] in th~ blood 3. Counu should be don. in both chambers of th~ h~mocytomec~r. and ch~ mean of the two ... lues .mould be u...:!. Mark«ily diff~"'nt valu .. on the two sid.. mggest un~~n sample dinribution and potential ~rror. B. lmpalan<,<, cdl count~" (e.g., Coulter count.". Bahr Syn~m, Cobas Minol ST_V<:{. M:oscot Muhi'pecies H~matology System. CELL_DYN. and Heska CBC_DiffVeceri_ n>ry Hematology System) l. Basic principles (Fig. 2.S) a. Wh~n. nonconduai"" partid. {• .g., <,<,U} p~s through an a~rtu"'. it uutes an d«trical int.rf~ren'" in a cur",nt th.t is flowing through. conducti"" liquid. b. The number of panides det«ted within. ddined volume of dilut«i blood represents. ",ll {partid.} con"'ntntion. Th~ dq:ree of interferen", de~nds on the appar.nt volume (and possibly sha~) of the partide, and .pparent ""lum.. a", u...:! to diff..~ntiau partid... The ""lum. of .""h partid. i, record«i 10 that .n a"""'1:e volume can be calculat«i. c. Most impalan'" cdl counters are designed to """luau human blood cdls and n.«i to be modifi«i to Nat uat. domestic mammal blood Erythrocytes of some species (• .g., gnats, sh"'p, .nd lOme horses) are too small to be rdi.bly detect.d. AI"", latg<' platdets (as commonly .... n in em) may be count«i as erythrocytes inn~ad of platd<:{s. LysiIll: ""lution. and proc«iu ... may al"" n.«i modification because of 'peci.. diffe",n",s in SIlocq>tibility to cdllysis. 2. Typically, blood is processN along two paths. a. Erythrocyte path: m~.n ",II volume, [RBC], and platd<:{ con"'ntmions a", determin«i in dilut«i blood. b. uukocyte path

64

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

Fig. 2.5. sro.m.tic "pR'Sg< volwn< ncmtr:otion. Afu" addition of Jy.ing _gmt to r=>< J.ukocyt. nocki :and fluid i=' through:m ap<,nu.:and tbr innrumrnt oonsiders tho>< particLes to I>< J.ukocyt .. , Righ" How cytomrtry principLr: Blood ""Us.."prndrd in:m isotonic dil.,."t:ur injrctod into. >peCioJ Ilowi"ll fluid, The Iluid dynamics in thr 'J"t<m ",eat< a ,I><.tb uound tl>< dil""'t to form a umplr ",.. m, eeUs in tl>< .....pLe st,. .m pass througb a w., br:un, monly 0 . . . . . . timr, Each erJl "'....... tho light in dilfuod through tl>< I.... brant,

"",ud

'p""

{I}

Th~

[WBC] .nd th~ blood [h~moglobinl are d~rrminffl in dilutffl blood

aftrr cdl, (l~ukocyu" ~rythrocytes, :md platd~u) .'" Iy=' but nucl~i r~main as !""tides, {2} In da .. ic impffiancr m~ho{h, th~ [WBC] i, r~ally. total nucJ..at~d alJ ron<Jrmr>tion b«au.. the nucl~i from leukocytes .nd nud~~tal ~rythrocyt~, a,e not diffe",miatffl, C. Optical or la,., How cdl cytometer> {e,g" CELL-DYN innrument', ADVIA, .nd IDFXX LaserCyte Hematology Analyzer} L Basic principl. {Fig, 2,5}

2/ LEUKOCYTES

65

a. Lastr light i, satt~rM when it hits. cdl. Th~ tyP' of scatt~r d~pends on cdl .iu. inr.mal structu... gr:mularity. and mm."" structu... and th= f.... tures ar~ USM to diffe .. ntiar. cdls. b. Instruments may ~ abl~ to diff... ntiar. the major l.ukocytes. but comput'<'ific for ....ch spl~ r...ctions that produ"" • black pr"'ipitau in tho .. ""II.. Th~ leukocytes (,uinM .nd unsuined) then I"'ss through a beam of tungsten light. and .. nson del"'t increase-d light .bsorban"" (due to stained cdl.) and light scatt~r (due to the ,iu of cdlt). The peroxidase ch:mnd provid.. a [WBC] .nd per""n~ of eosino· phils {3+ peroxida.. J. neutrophil. {2+ p'roxid...}. monocyt .. (1 + peroxidase). lymphocytes (,mall. negative: peroxidas.). and la'l:" unsuined cdb (large. negative: peroxidasr). Because cat rosinophils lack p'roxid....ctivity. they must ~ d ... ifiM by using a special ,uin (ox.:lz.in~ 750) that i, also used for d,,"'ting reticulocyte .. Eosinophilt of lOm~ greyhounds. Gr~at Dan... and other dogs have: low peroxidase .ctivity .nd ar. mi.d ..sifiM as monocyt ••. S."'phih a.. not accuratdy classified in domestic species. b. Basophil channd: By using a diffe .. nt Iy.ing agtnt. "rythnx:yt•• and leukocytes oth~r than basophils {in human umples} .'" IpM. and then the fluid pa!SeS through a lasrr bram. Light scatter is used to diff~ .. ntiate particle sizes (inuct human basophil, ve:nus other ""U nucl.i) and nucl~.r d"nsity or lobularity. Th. b''''phil ch annel provid~s. [WBC] and per""nuges of minimally lobulatM {lymphocyt... monocyt ... immature granulocyt ... and bl>stic ""lls}. basophils (in human sampl..). and polymorphonucl.... r cdls {neutrophils .nd eosinophils}. Conine and fdine b"",phib a.. not delectM by th" basophil ch:mnel. c. If th~ .. are erythrocytes that are wi",:mt to lrsi •• the [WBC] of the peroxidase channel will ~ falsely incr~• ..d. but th" basophil channel [WBC] should ~ .ccurat~. The pr=n"" of nucl~atM ~rythrocytes is suggested if the "polymorpho. nucl~.r ""II" ron""ntration from the basophil channd i. greater th.n the mm of th~ neutrophils .nd eo.inophil con""ntratiom from th" peroxidase rnannd (u""pt in cau. because th"ir eosinophilt lack peroxidase activity). d In cont .... t to th" microlOOpic differ~ntialleukocyt" count. this el~ctronic diff"rential count differentiates thous;ond, of I.... kocytes. and thus sampling "rror is typically not a problem. How"ve:r. th~ el"'tronic methods mun ~ species specific and may not ~ a.ccu .. te when atypical ""lis {•. g.• toxic neutrophils. and r...ctive lymphocytes} a.. pr=nt. Automated differential counts may ~ con· sidered reliable wh"n ""II populatiom are d ... rly >qlaraud as indicated by ""ruin d"finM crit~ri .. D. Quantitative buffy CO>l {QBC} an.lpi,'· {QBC V.tAutor ...d}

""U,

66

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

Butry coat

EryIhrocyt...

Fig. 2.6. Scbem. tic "'p,ostion of ...hoI. blood. G.ntrifug..l forces "'I',..t< II.. compo"""'" of blood into fiv< 1.J"'J'lI (pWIIU, pl.",,,, .. , agr=ulocytes, grrnulocym,:md rryth.rocyt"') b....d on 'Mil r.uti ... densin... Tn. buJJy coat (coml"""'d of pb"Le<. :md Leukocyt"') is upand«! by. Hoat that b... d..nsity ,imilar.o buffy coat ""It.. L'f"'" orr =ogniud by US< of 8UOI
l. A .pa;ial capillary tu~ that contains a plaSlic cylinder or Hoot is ustd. Ikcau .. of

Ih. cylindds density, it will float in the .. gion of th. bullY coot cdls with ""mrifu_ galion. 8«>...., of the cy\indd. di:nneur. the length of the bullY { is exp"ndffl around the f1O<1i (Fig. 2.6). Fll1Ores""n"" is ustd to diff... nt""r cdl lay.", on the b",is of lipoprotein, RNA, and DNA content,. 2 If the quantity of blood th.t is put into th. capillary !Uk i, oonstant and Ih. mr m ",,]um.. of •• ch cdl typ" .re r~lotivdy connam, th~n the conctmr>tion of a given ""U typ< will corrdat~ with the thickness of th~ loy~r for tb." cdl typt. cdl conctmration. 3. Results g~n~r:lt<"d by th~ IDEXX QBC yrtAuto...,.d h~matology .yst~m:md the basic principles of.ach .va]Ut<"d by adding th., granulocyt<:md rombin<"d lymphocyte' monocyt~ conctmration •. b. Granulocyt., conctmration is d.t~rmin<"d by the thickn~"" of th~ cdl lay~r with charact~rinic DNA and RNAllipoprottion may ~ rq><>rtN for dog. and cattl. bn::.u,", th~ir msinophib .how det=ably mor~ RNAllipoprot~in fluornan"" than do their nrutrophils and basophils.

2/ LEUKOCYTES

67

c. Lymphocytefmollocyu collcelllntioll is d"'"millffi by th~ thicklles.! of th~ cdl l. y" .bo", th~ gr.lllulocyt~ byer th.t h .. the DNA fluor""""",, char:lct~riS{ic of lymphocyt.. and monocyte>. 4. Th~ . ccuracy of th~ p"nial leukocyt~ diff,,~mial coulll d.".,nd. on the .milling "':lCIiOIll of the leukocyt.. and their d~",iti ... Atypicol stainillg or d~",itie> will /"<SuIt ill ~rronlypical lymphocyt.. ",rsus monocyt ... 3. Accuracy and p=ision of th~ microJCOpic leukocyte diff~",mial coulll a. Assumillg prop<:tors.' b. Evalu.tioll of th~ data in Tabl~ 2.2 "",ai, ,,,"oral ~ collcepn. {I} Th. collfid~II"" im~rval b.rom.. unallor as th~ lIumb.r of ""US ill the diff~"lIlial coum illcr.-ases. However, it i, 1I0t pr:lcticol to do a ]ooo-cdl microscopic diff~",mi.l count in most blood film,. (2) Th~ collfiden"" interval, . " large ~tIOugh (.. pecially for th. ] oo-cdJ coum) to yidd collsid=ble ulI""rrainty in th~ r..wts (T .bl~ 2.3). Thu" th. blood of an animal m.y ha", aactly the """~ l.... kocyt. oon""m1>tion, on 2 diffrr~m days, but the diff~r~mial leukocyu COUlltJ may diff.r mmid .... bly .imply b.cau", of .lImum.ring diffr",m ""II, during th~ d.iff...~mial cdl mullls. c. Th~ abwlurt or calculotffi concelllr:ltiom of the leukocyu, at<: much b.{{~r valu.. to illl~rpr... oomp"rffi to the diff~"lIlial coulll pl" in most CBC remh., alld thus diff~t<:nces (mmp. r<:d to "f~r~II"" im~rvals or comp"rffi to .. rial ... ulnl .hould b. illl"p"ud accordingly.

6B

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

Table 2.2. Th e 95 % confidence limiu' f.,.. pen;cntages obtained in differential leukocyte count."

" , 0

0 0 0 0

Num~r diff~"'ntia{~d'

."

"0 4 6

0 0 0

,

, 4 6 7

0

."""

"

, " " " 5 " ,

0

, , , 5 ,, "" , "" 45 , " " " " "" " " 3

9

4

4

6 7

3

6 7

3 3

>3

4 5

3

9

4

>7

>3

4 5 6 7

30 35 40

9

."

Num~r diff~ .. nt",{al

"" ,"'" , " " "" " """ "" "" """ " " "" ""36 " 4

6

>3

>7

30 35 40 46

30 35

39 70

37 43

"" " '" " " ""

33 39 44

33

53

6>

73

4>

46 77

49 54

"

• Confid.nc< i"",,,,:tIs for thl . roW pt:ig< of • given J.ukocyte if th. 'p«ified numoo of ",Us ""'" dilfe""ti. "", md II.. obs.rved per=>ug< _
T able 2.3 . Polential differences in absolute leu kocyte concen.trlnion. due tn random error of microscopic differential leukocyte counts if Ib e fOt.l1eukocytc concen.tr.ltion was 20.0 X lo-'/ j.l L

Low oonfid~n"" limit ""lu.. Neutrophil, Neutrophil, Em.inophil, Eo1ino~hil,

""

X

10'Ij.lL

70

14.0'

30

6.0

4 0

0.' O.~

Actual v..!u..

,,'

X

1001j.lL

"40

16.0'

"

'.if

0

'.0

O.~

High oonfid~n"" limit valu ..

"" "" " 4

X

1001j.lL

17.6<

10.2

3H 0.'

Cmin • .. f,,~n"" int.rv"! X

10'1j.lL

3.0--11.5 3.0--11.5 0.1-0.8 0.1-0.8

• P.. cnfid..nc< limits obuinr
F. Corr«tion of [WBC] for th~ pr~ .. ncr of nUcln!M.rythrocyt.. (nROC,) if th. [WBC] i. r~..!ly. total nud'>!M cdl oon""ntntion 1. Microsropic and som~ d«tronic mrtho,j, drt~rmin. [WBC] by counting nud.atM ""US or nucl~i {.ith~r ofleukocyt.. or nRBCs}. By thosr m",hod. , a mnsurM [WBC] rrprosrncs th. sum of nud"'!M crlJ concrntration, (leukocytrs + nRBCs),

2/ LEUKOCYTES

69

ob",,,,ffi during th~ n:lmination of th~ blood film. th
>r~

oorrected [WBC) = m=ured [WBC) X Exampl~:

"',.,-:;:c"""':;;;;""'" "" 100 + ffnRBCIIOOWBC

(2·1.1

m<:a.rured [WBC] = 20.000IIlL. nRBC = 251100 WBC

0,,'0'0, 0

oorrected [WBC) = 20.000IIlL x-;100 + 25

'00

20.000IIlLx - = 16.000fIlL

'"

100 l.... kocytes w~ .. rounted. 25 nRBCs were also rountffi. Thus. th~ .. w~r. actually 100 leukocy", out of. total of 125 nucl ... ted a:lls. Th~refo ... 100 of 125 nucl~.tffi cd], {4 of 5} must ~ leukocytes. b. If th~ m• ..,ural [WBC) w;o. 20.0 X 10'11lL. then 4 of5 of the nucl~.ted ""U, w~re l.... kocytes. and thus the correctffi [WBC) is 16.0 X 10' l. ukocyt~"J.lL. 5. Th~ cona:ntrations of th~ individual types of leukocytes ar~ calcul.tffi .fter calcula. tion of. corrrcted [WBC). a.

[v.

Whil~

Rdative ve:rsus .bsolute rnangc:. in leukognm results A. ~pt for the "",ntrifugation m~hod. the leukocyte con""'ntrations a.. calculated by multiplying the total leukocyt~ oona:ntration by th~ pera:ntages obuined from th~ diff. .. ntial count. These Glkulated ronuntrationo are used to d~ect ah",/uu changes in the leukogram. but it is importont to understond that th= calculated cona:ntrationo con only ~ .., accurate •• th~ values used to colcul.te them. B. Rdative: changes in leukocyt~ populations may not rdlect tru~ changes. I. As shown in Tabl~ 2.4. d.r"'tion of true changes in • leukocyte popul. tion should ~ b...d on ""aluation of th~ calcul.tffi CDlUrmra';Dm of th. individual l.ukocyte

-.

2. [f a l.... kocyt. pera:ntage is incr..... d or d",re...d romparal to r~fe .. nce int~",als. the finding GIn ~ d~"'ribed as • rdative: ch. ng.... that is. rdative: n.... trophilia or .d.tive: lymphopeni •. How""er. !Urn de"'riptiollS ar~ not rerommendffi bn::;ou", th
70

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

Table 2.4. Relative venus absolute danS"" in leukocyte values in CBC rc.u1Ci

Dol:' Units

Neutrophil. Lymphocytes

% %

WBC. Neutrophil.

X lo'/Ill X lo'lllL X lo"l~L

LymJ::hocytes

'" 30 70

"

3"

4'

Rcf,,~n~ int~rwl

30

70

70

6"-'0

70

30

30

20--35

...

'.0 0.6

50.0 15.0

'.0

L4

35.0

0.6

50.0

35.0 15.0

6.0--1 7.0 3.0--11.5 1.0--4.0

• B...d on p«=>tages, ",l"in ntil'< Iympbocytwi, "'" p.... nt. n.., .. is • low n
"r."'J>C< internls. Th< dog b.. :on """olu« ... utropbj~. >ltd

ABNOR.,\1AL LEUKOCYTE CONCENTRATIONS IN BLOOD

I.

Abnormal neutrophil con~mrations

A. ufl shift l. A "fi shift {shili: to Ih. HI} is .n inc""....,j cono:mmion of non"'gm~med n..... tro_ phil. (usually baud,) in blood. When th~ .. i, """at d~maud for n..... trophil. in ti"u~,. younger stages {m"':mlydocytes. mydocyu •• and rarely progranulocytes} Im}' b. pr=m iu th~ ldi ,hift. a. u,ft mift, occur when rei..,. .. of u..... trophil, from marrow diminishes the SNP . • nd younger cdl, a.. then rei..,....! from th~ MatNP. The capability of ueutro_ phil, to respond to stimuli .nd migrate iner....' .. they m.,ur~, thu •• ..gmented n..... trophil, respoud fi"t .ud immature forms respond J.t~r. b. Becau.. it usually occur> in ... ponse to rd.,ivdy intense, often acute inft.mma_ tory stimuli, • left shift i. fr"'lu~ndy con,idered the hallmark of ""ute inflamm._ tion. Such infl:mlmation i, rypiGlUy cau...! by inf'n:tiou, "Cenu (~.g., pyo~nic b.icwia, or fungi) but can b. caused by noninf..aioUl ditord~" (e.g., u,""rosis, immuue_medi.oted di ..=, or ueopla'ia). Glucoconicoid hormon .. and eudo_ toxins alto ,timulat~ rd..,. .. of n..... trophil, .ud thu, m.y GlU,. a mild l~ft shift. 2. uft_shift classificatiou. a. Severity, Two diff~""nt f~.tur~s might b. consid~red wh~u d=ribing the !lNerity of a left shift. Siuce th~ t~rm' can describ. differ~nt fiudings, one must b. car~ful iu imerp..,.,ing or using the term •. {I} Immaturity of noutrophils in th~ l~ft .hi&' bands {I+ or slight}; baud and metamydocyt~, (2+ or mod~r.ue), and b.nd, metamydocyt .., and mydo_ cytes (3+ or marked)' {2} Magnitud~ of nonsq:memed neutrophil oono:mmion" mild « 1.0 X lo-'lJ.ll), mod~rat~ (1.0-1O.0 X Io-'Ij.ll), and marked (> 10.0 X Io-'Ij.ll). R,mg.. are provided as examples .ud mch guiddines vary with species.

2/ LEUKOCYTES

71

venu.s degt'lluati"" l~ft_shift d""ilications lim d~=ikd by Dr. O.W. Scru.lm (fath~r of vet~rinary h~matology). spmlic criteria for dassiJYillg ld't .hifts w~ .. not providnl.' (a) A rrg~lI~mive l~ft .hift is "cru... ct~riznl by a leukocytmis du~ to neutrophilia alld with th~ apptann"" of immatu'" lI~utrophils in ~riph ...l blood" lp. 272). (b) In. d~!:"nemive ld't shift. "toulleukocyu count ",mins within th~ normal rallC" or is ollly slightly d~ud. whil~ th~ occumn"" of young gunulocytes in th~ circul>lioll is promin~nt ' (p. 272). {2} In th~ 1986 rdition of St:lu.lm·, Vnninmy Hr""''''~, th= nates .'" de=ikd as follows:' (a) ' ~n~rative l~ft shift i. char.ct~riud by a leukocytosis due to naltro_ phili •• nd with th~ .p~aran"" of immatu .. neutrophil. in p from multiple days will ben.. characwiu th~ llrutrophil response:. If th~ ..!:""e.. ti"" l~ft .hift p (endogenous or exog~nom) deer",,, emigration of nrutrophils to tissue by down_ ..gulatillg adhesioll molecules and thu.s can cou,. • right .hift. b.

R..g~lI~ntive

(I)

Wh~1I

72

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY Table 2.5. Di..,a..,. and conditions thai ca u.c neutrophilia Inft.mmatory neutrophilia '[nf=ions: bacterial, fungal, vir'!, protozoal 'Immune hemolytic :onemi. 'N«rosil: hemolysis, hcmorrh3J:c, inf:lJ'Cl', bums, neoplasia, ,{incphrinr Chronic ncutrophili" [cuhmi. rarallNpiani, neutrophilia Oth.", or unknown m~nism' Neutrophilia of leukocyte adh ...ion ddicicncy G_CSF administration Emoge" {oxicmis (carly) • R.btivoly common dis. ... or condition

2. Ocher causn ofa right ,hift: PoodJ~ marrow dyscr:asia,' F~LV_associated mydodyscr:asia; '"'Iuin~ idiopathic hYP"r"'gm~ntation,'''" vitamin B" d~ficiency in giant sdlllauu", with an inherited nulabwrption .yndrome," folate deficiency in a cot," occasionally chronic inflammatory di..,a~, and in vitro :oging due to ddayed analysi., C. Neutrophilia (incr~a..d mrasurrd blood naltrophil ron""'ntmion) (fable 2,51 L ACllte inHamm.rory naltrophilia a, This naltrophilia resulu from changes in neutrophil kinetics cru.t ar~ cousrd by 'CIIt~ inflammatory mrdiators. Th~ net re",1t i. an increa.M CNP that may contain a ld't mift {Fig, 2,7B}, {I} Rdea .. from SNP OCC\Irs within hou", after on.. t of inflammation and muse. initial neutrophilia if reins. U""'MS neutrophil emigration to inflamed tissue, {2} Rdease from MatNP cau... a left shift and OCC\I'" aft .. rrduction or depletion of SNP, {3} Increased production from th~ myelocyte u,,*, It takes 2--4 d before effect. are ... n in periph~ral blood, {4} Increased production via stem cdls: Thi. will lead to granulocytic hyper_ plasia. It takes about 5 d before effect. ar~ =n in peripheral blood, b, Acutr refer.; to th~ rype: of inflamnutory reaction and not the duration of the di ......, 1be acute inflammatory pattern con be = in an anim.! with a prolongrd infum_ matory state if th~", rmuins an acti"" need for IleUtrophils in th~ inflamal tiSSlle, c, Infl.mmatory mediators must enter systemic blood and uimulate marrow a:lls for a naltrophilia to devc:lop,

A Normi:ll 118ulrophil

• B. Arute innammatory neutrophilia

•

"""'

MalNP

iii

•

"""'

MalNP

--

D. Steroid neutrophilia

•

"""'

, - , MII1NP

,

,:@~:, ,

E. Physiologic (shift) neutrophilia

•

"""'

MII1NP

F. Chronic myeloid leukemia

,

"""'

MII1NP

~I

I, :

Fig. 2.7. N ... trophilia kinetic>. A. Neutropbil kin
S. hu.. in8aaurl1tory neutropbi~a: N ... trophi~a oeru" t-:. .... th. ,.Le... of neutrophils from tb. marlOW < migntion of n< migntion of neutrophih to th. inflamN tw,,,,,. A Left shift may not I.. p"",nt bee ..... g,anulocytic byp<rpwia maintaim tb. SNP. D. Steroid neutrophilia: Neutrophilia 0CIP. dec,.as«l migntion of neutropbils to ,;.,"". and "'I.... of ... utropbih from th. SNP and ",meti""" th. MatNP. E. Phpiologic (.bift) J>Ntrophi~a: N ... trophilia occurs ~ oftb • •bift of neutropbils from tl>< MNP to th. CNP. F. Chronic m)""Loid leukemia: Neutrophili. 0Cgni...d .. being of neutrophil ~"" • .

73

74

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

T able 2.6. Species differences in ,he magnitude of lnflaDlmatory neutrofhilia Ikf~ren~

Common

Occasional

interv'!,

m~nitud.

JruI!2!itud.

Dog,

3.Q.-11.0"

"'" Ho= 01.01.

2.0--12.5 2.2- 8.6 0.6--4.0

12.0--30.0 13.0--25.0 9.0--20.0 4.0--10.0

30.0--60.0 25.G--40.0 20.0--30.0 10.0--20.0

Uncommon ffi:!i,;lIitude

>60.0 :>40.0

>30.0 :>20.0

• N ... trophil oon=>tr>ooru >co ""press
{I} A neutrophil;" is np<'CIffl if th". is .ub,t;>ntial acute inflammation of SUOCucaDroUS {i!SUes or imernal tissues (respir.tory InCl. paon.... p<,i{o. neal or pleural covity, and occasionally ul
urine or skin} or do Dot leave the prot,""ta!environment (brain or spin.! and). d. Th~ m"l:nitud~ of infLommatory n~utrophilias vari~. among sp«i ... and thu, int~rpmation of sptcific ron""ntrations differs among ,~i~. (T .ble 2.6}. e. Th~ inflamm>lory bovin~ neutrophilia mult. primarily from incr~asffl production (granulocytic hJ'P"rpwia) .nd not from rei..,." of storrd neutrophil" b=.use c;ml~ Ita~ a .mall SN P. f. Th~ Urm kuknn~;d mpon~ has bttn used to describe .ny extrem~ inflammatory lalkocytosi, that i, leukemi._like but prov~n not to be leukemic; Th~ Urm am be applird only retrospectivdy. {I} Classically, di",rd~rs '!JOCiatrd with. markrd inflammatory neutrophilia includrd focal .uppurati"" l.,jon. (e.g., Glnine pyometra, pleuriti, or pyothorax, peritonitis, prostatitis, pnalmonia , and .1>=..) and hemolytic an~mia (e,~ially immun~ mrd iatrd). {2} Oth~r dilOrd~", recognized as Glming .n atr~m~ inflammatory neutrophilia include amin~ rectal nwplasm,," cmin~ pulmonary neoplasm,," Glnine baksiosi,,'· and Glnin~ hepatOl.OOflO,is GlU..ffl by H~potoZIJ~n II",mCtlnum. g. An animal that has an acute infl:unmatory neutrophilia usually Ita. a lympho~ nia, oft~n an eosinopenia, .nd occasionaUy a monocytosi, or toxic neutrophil .. In dog" • mastocytemi. may be found. 2. Chronic inflammatory n....trophilia a. This naltrophilia result. from chant'" in neutrophil kin",i", wh~n an inflamma_ tory proct:SJ oontinu.. for at l~.... t • WM duting which inflammatory mrdiators stimuJ.t~ d ...dopm~nt of granulocytic hYl"'rpluia {Fig. 2.7C}. If th~ SNP i, .. pl~nishrd, .. gm~ntrd neutrophil, a.. rd..,.oed inn~.d of band naltrophih, and thll'l the left shift dimini,hes. In addition, oth~r d~f.n .. m~.nism' b.com~ acri~, and thll'l the neffi for nrutrophil, in tissu .. may dimini,h. Ho_""r, if the inflammatory stimulu, i, mo .. int~nse, a l~ft ,hift may persist, or a chronic inflammatory pattern may never be reocha!. In these disorders, th~ .. it incr..... rd production of neutrophil, bec..,.. of granulocytic hyperpla'ia and increased n.... trophil rei..,... from SNP in respon .. to inflamm>lory mrdi>lors. When th~

75

2/ LEUKOCYTES

rat~ of namophil rd~~", from marrow is grrat~r than th~ rat< of neutrophil margin.rion and ~migr>!ion to tissu .., • neutrophilia develops. b, Chronic indirectly rd'",. to the duration of the di"", .. ; the infLommatory process has ~r:sistM long enough to r.sult in granulocytic hYP"rplasia, c, Wh~n.n animal has a chronic inflammatory neutrophilia, oth" lrukocyt~ abnormaliti.. may include lymphocyto.i. (with or without reactive lymphocyt..), monocytosi., ..,sinophil"" I>=>philia, a I.ft shift, ~ right shift, and toxic nrutrophils, 3, Steroid {mess} neutrophili a a, This n.... trophilia r.. uhs from changes crratM by th~ effeet. of endogenou. or nogenom gluroconicoids on n.... trophil kin..riu {Fig, 2,7D}, Although this nrutrophil", is frequently callM a itrN n(urrophi/ill, it mould not be confusM with th~ """iologic (lhifi) nrotroph;{id COUsM by the me,,_indu=l rd.~ .. of catecholamin.., {I} Neutrophil. shift from th~ MNP to the CNP b.cou"" the production of oodh..ion mol<cul.. is down_regulatM, {a} This proc= con potentially doubl~ th~ mrasurM neutrophil concentra_ tion! in canine, equin~, and bovine blood G ..... t~r incr..... in m~ .. urM neutrophil concentration! may occur in fdin~ blood b.coUst of th. larger fdin~ MNP, {b} 8«;ou"" of thi, mift, few~r n.... trophil! ~migra" to tissues, .nd thm neutrophils have an incrrastd circulating Ii£. .pan, Th. old~r neutro_ phih may becom. hyp'nia, monocytosis, .nd eo.ino~_ ni • .' The magnitud~ of n.... trophilia vari .. with diff",.nt gluroconiroids and dosages, The ill in gluroconiroids, c, Blood may have. mild Ide .hift (typically band neutrophils < 1<XXlIJ,lL), a right shift, or no shift, d. Typical gluroconicoid ~ffect. diff" .mong 'prei.. of .nimal, (f.ble 2,7),

Table 2.7. Ezpccted leukocyte concentration. in animal. with steroid leukograms Ltukoc~e

concentrations

Total WBC. (x 1001J,lL) &grIt.ntM n.... trophil R.nd n.... trophil Lymphocyte Monocyte Fmino/:hil

""'.

Cat.

15,0---35,0

20,0---30,0

Ho= 15,0---20,0

;

;

; WRI -L-WRl WRI WRI'

WRI- ,Iight i

WRI

1

-L-WRl

1"

WRl- i WRl '

WRl-;

Cattl. 8,0- 18,0 WRl - ; WRl

1 -L-WRI -L-WRI

• [hcr ....d
76

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

4. Physiologic (,hift) lIa1trophilia a. This neutrophilia result. from .ff,""cs of cat«:holamines (typi",JJyalJOC"'tM with fear, neir.ment, and aerei .. ) that cau.. a .hjft from the MNP to the CNP (Fig. 2.7E). Th. shift may be, c>uW by the chang. in fluid dynamics that mult. from increastd blood How rat., espa;ially in lungs." Nrutrophil :odh..en"" to

endothdi:d cdh =1:'!so be, rffiucffi. " b. Th. m"l:nimd. of neutrophilia =y be, up to cwi", th. URi for canine, «J.uine, and bovine blood, .nd potentially up to 3--4 X URi for a cal kcau .. of. eat'. I"'get MNP. c. It is ,,,,,n most &«J.uently in h.:dthy animal. and mostly in cats. uukocyt< conctntration, mum to ""f".nct illt..""l, relativdy f:m (within .n hour) if the stimulus dj,..p~.rs. d. B«aust th. incrrasnl blood flow "Ie .JUts kinetiC! of olh" l. ukocyte., th.", .150 may be, inn.=<:! oonctntrationl of olh" J.... kocytes, espn;ially lymphocyt ... Th~ ~lIIir~ respon'" results in a physiologic leukocytosis. 5. Chronic mydoid leuhmi
77

2/ LEUKOCYTES Table 2.8. Disca.... and conditions that ca u.e neutropenia Inft.mmation 'Ov~rwhdming Mcr ..;.,1 inf«tions: '"'Iuine salmondlosi, 'Some viral inf.crions: canin~ and fdine parmviru., aJuin~ influ~n:u 'V>r;~ty of inflamm.tory Sl ales in caltk mastitis, pnalmon;" P~riph~ra1 dmruction Immun~.ma!iata! n~utro~nia

H~mophagocytic .yndrom.. Granulocytic hypoplasia ·Inf.crious: parvovirm (dogs .nd calS), F~LY, T"""pfil=, EhrfkhiA Nwpl astic: primary or metastatic Toxic ·P.. di=bl~: emog~n, ch~moth..apeutic drug., chloramph~nirol (cats) Idiosyncntic: ph~nylbuurone, brack~n fern, grioa>fuIvin Murow n=ruis Mydofibrosi. In~ff.crin production Immun.... ma!iated n~utro~nia Diphenylhydantoin and phcnylbut37.0n. toxiro.is (su.pected in .nimal,) Chronic idiopathic naltropen;" {G.CSF deficiency} Cyclic h~matopoi .. is Cyclic hellliltopoiesi. of grey oolli.. Cyclic hematopoiesis :wociala! with F.LY • A ,.tll;v
{2} N.utrophil.rdea,ing factors .timulat•• sudd~n rd", .. of nrutrophil. from the SNP. Aft.. th. SNP i, deplet.d, <:<:Ih in th~ M.tNP are rd~ased, and thu. a Idi shift i. typically present. How~~r, th ... may not ~ • left shift """n with the rd~= of edl, from the MatNP, kcau .. th. total measured nrutrophil con""'ntration llliIy ~ I.... than th. URL for the Mnd neutrophil ronccnlration. If bon~ marrow i, examined during the iIIn~". <:<:11 popula. tions .. fl«t the cru.ng~' in the Ialkon. (a) ~Iy: depletion of SNP .nd po .. ibly decmsed M.tNP {b} 2- 3 d: increased ProNP without inc",ased MatNP {c} 5-7 d: granulocytic hyperplasia from ill<:.. as .. in both ProNP and MatNP c. Inflammatory neutropen;" i, common in .dult catll~ kcau .. they have: a rdativdy small SNP. On<:<: th ... i, • delllilnd for neutrophil., the .. is not. sudd~n rei ...... of neutrophil, from bon~ marrow to cr",t•• neutrophilia. Also, experimental data indicau th at it takeslong~r for catd. to gtncrat~ neutrophil. on<:<: th~ r... rv. pool i, depleled!' {I} Th. following Kquencc i, the expected bovin. respo"" with ""ut. inflam. mation induccd by introduction of Enm-obafUr Iltyggmf1 inlo lacrating quart~".'

A. Normal

~ I --f;. B. Inflammatory neutropenia

•

'roN'

-- -

C. Endotoxin neutropenia

,~~,

•

, V_,

'roN'

,~

!:,' I~i I

-

---;',,

•

,

,~~

;

- -

E. Granulocytic-hypo lasia neutro

•

'roN'

F. Ineffective-production neutropenia

•

'roN'

<

••

-0

~:3 -

@

•

-

, -- ,

, =' , ;@~

; - -

Fig. 2.8. N..,tropeni. kinetics. A. Neutrophilltin.ti", in h..lth (a,..wc& v.,..ion of Fig. 2.2). S. In8:munatooy nKat>< the margination """"ed tho ,01.... of noutrophih from lIUlro..... and migntion of nouttophit. into tho infl1lIk"<\ C. Endotoxin noutropeni:o: N..,tropeni. 0CCIl" bee..,.. . ndoto:tiru ulmuu", tho margination of noutro_ pbils (.eq....'ntion of nouttophih in tbe MNP). With the concu,,,,nt inJlamm. tory '''po''''''. ' v:uioty of ch. nges in nouttophil kinetics .,. possibJ.. Endotoxin. auy aho alJect m. rro.... ""lis to crus< inc, ...od 'et.... of nKau.. n'. perhaps boa .... of 1l1tin.utrophil1l1tibodies. If peni...",. gnnulocytic hyperplasia ....iU

tis,,,,,,

-"".

Eo G,.nulocytic hypoplasia ,...,tropeni:o: N..,tropeni. occun bee ..... """trophil prodoction is deer • ....!. F. IndiKti", prodoction noutropeni:o: Neutropenia 0C
7B

calif ••

2/ LEUKOCYTES

79

{al Initial n..... tro~nia (within 6 hl {b} Signifi<:ant Ht shift (by 24 h) {cl N.dir of neutroptn;" {by 31 h} {2} The ...... rity of the n..... troptni. typically wiU be greater in b.cteremic than nonbacteremic <:atde with m. niti,." 2. Neutroptni . <:aused byendotoxemia (Fig. 2.8C) a. When~er an infection with Gr;om_neg. tin org.nism. cou..,. an endotonmi •• endotoxins initi.te chang.. in the leukon. b. This n..... troptn;.. occurs because endotoxin! cou.., the rapid shift of neutrophils from the CNP to the MNP. Thi, effect lam for 1- 3 h after a single eXpo.'lure. Endotoxins al50 indu", the rd ... .., of inHammatory medi.tors (e.g.. TNF and IL_I) that promote the adhaion of neutrophil, to endothdial ",lis. Concurrent .ctivation of the neutrophils may cou.., oxidati ... dam~ to endotheli. l cdb." c. B«ause of ...... ral !actors•• n..... tro~nia may no longtr be pre..,nt when a v<:terill
{2} Endotoxim stimul. te incr ...,ed production of n..... trophib. which affect. blood neutrophil con"'ntrations in .bout 3- 5 d. {3} Other inflammatory mediator> may concurrendy alter neutrophil kinetics. d. Most ,tudi.. of endotoxin_indu",d change. in n..... trophil kinetics inmlved injection of endotoxin .. In .pont:meou. infection,. other medi. tors .re prob. bly .lso .ltering neutrophil monment. 3. Peripheral destruction neutroptni. (Fig. 2.8D) a. Immune_mediated n..... troptn;.."'17 {I} Antineutrophil . ntibodies bind to neutrophil, th. t . re then dmroyed by the mononud ... r phagocytic system. {2} F.ctors that indu", the pathologic pr<JC<:S.1 are not esublished in dommic mammal.. {3} Animals m.y be re.pomin to glucoconiooid therapy or other immunosup_ pr=ive >gent,."" {4} Examin.tion of marrow may r......1 gr;onulocytic hyperplasia. b. N ..... tro~nia of hemophagocytic syndrome""'" {I} Neutroptni . may be one of multiple cytoptnias .l.IOCi. ted with phagocyte hyperplasia. {2} In people. the .cqui...d ,yndromes are associ. ted with a variety of infectious and neopla.nic st.tes." 4. Granulocytic hypoplali. neutropenia (decreased_production neutro~nia) (Fig. 2.8E) a. Granulocytic hypoplasia occur> when either stem cdl, or of the marrow microenvironment are dam,,!:ed. The hypoplas;" cam.. decreased neutrophil production. b. This form of n..... troptni. is usually differenti. ted from other neutro~nia, by ptrsisten", of neutropenia (usually without. left mift. though secondary infections m.y cau.. left shift.) and bone marrow exarnill
",n,

BO

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY ~ d,""r~ Th~ .. =1 not k a lymphopenia, !>«au.. lymphopoi .. is in oth~r lymphoid {is.su~, may not be, impairffl. c. Ch. mOlhuapeutic "l:en[, can b. cytotoxic to many rapidly dividing cd]" including nrutrophil p,,",,,rsors. Thu., neutropenia em =ult from the admini •• tration of ch.moth.rapnnic ~nt, such as vincrislin., doxorubicin, cyclophos_ phamid., cispl.tin, and carbopl.t;n. Th. onset of th. neutropenia d.".,nd, on ,N...d factors but typicoJJy occurs 7- 10 daft" the la't do ... 5. Indfectin production neutropenia (neutropenia of indfectin lI.utropoi .. is) {Fig. 2.8F} a. In.f&ctin production occurs when neutrophil precursor.; .'" def=in or

damagffi and die kfo .. th<7 are rd.a.=I from Jrulrrow. b. Lock of an ord.rly matur.uion seque""" in marrow sampl.. ,ugges{, ther. is • maturation ar=t k.g., hyperplasia up to on. n~ .nd th.n hypo,,]"';' th. t stage}. Th,,~ :.!SO m.y appnr to bo • f.ilur~ to rd~ ... (i.~ .• th~ anim:.! may h. ve • persist~nt neutropenia .nd a concurrent gr:mulocytic hyperpl",ia in th~ marrow). Th • ..., p.tt~rns occur in 5Om~ dogs in which immun._mffiiatw nrutroptnia is mspectw'"' and paraUds findings in som~ dogs with marrow_ di=tffi immune_mwi>tw an.mi • . c. In~f&ctive nrutropoi ..is i. m.ract"ized by a persin~nt neutropenia {with little or no l~ft shift} concurr~nt with marrow hyperpl",ia prior to th~ d~f'""tive ,ue<'. A monocytosi, m.y bo pr • ...,nt boom.. stimulation ofCFU_GM =
.ft.,

2/ LEUKOCYTES

B1

Table 2,9, Disca.... and conditions thaI ca u.e Iymphocytool. Chronic inflamm.tion 'Bacre",,1 inf<'CIions, espeei:dly.napw.m.t (e,g" Eh,!imid r .. nu) Fungal infections, pri/IUrily symmic Viral inf,""tions: FcLV, BL V, equine inf,""tious 'Mmia virus Protozoal inf'""tions, espcc;"lly babe.i:d .nd theilerial Physiologic (shift) lymphocytosis 'F;y.t-or.llight response: ncitement, fright, pain, exercise, anxiety Catecholamine inj'""tionl: epinephrine or norepinephrine Lymphoprolifer,ui"" disorder> 'Lymphoma (BLV, FeLV, .nd idiopathic), leukemic phase: Lymphoid lrukemia Per>isrentlymphocytosi. of cotde (BLV) Hypoadrenororticism • A r.t1t;vely oommon disc ... or condition Not<: Lins of .ptr>tio ... than do "",n"" animals of the =p«:tiY< >peCies,

cytokin.. or r«:eptors), resulting in d,""rrascd hrm>lopoiesis, When nrutro. peni. drvriops, there is less dasu se.inducffl inhibition of hr/IUtopoiesi., thu. allowing a new v..ave of progenitor> to devdop until the cycle repealS, b, Cyclic hematopoi •• is associalw with FeLV infection"" [I.

Abnormal lymphocyte concentrations A, Lymphocyto. 30,0 X 100IJlL in dogs), d Concurrent lrukogram abnormalities commonly include a neutrophilia (mature, perruops with right shift or left mifr) and/or a monocytosi. and <>=LSionally.n eosinophilia andlor basophil;", 2, Physiologic (shift) lymphocytosis {Fig, 2,9Cj a, This lymphocytosis is co.....! by shifring of lymphocytes from the M LP to the CLP (especially from the .plrc:n) that i. promoted by nogtnous or endogenous cotecholamines, Thr lymphocyte shift i. probably mediated through both increased blood flow rate .nd d,""rea.=i lymphocyte adherence to endothriium," b, The m"l:nitudr of lymphocyto.is may be up to 2 X URL, and the lymphocytosi. usually lasts minutes to hours, c, Morphologic chan~ in lymphocytes :lie not expeered, bUlthe.. arc data to indicotr that the cotecholamine effect is mosdy on natural killer ceUs, which may appc. r as granul ar lymphocytes (also called large: gr:mular lymphocytes),"

B2

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

,I ,'

, I

,C!'

Fig. 2.9. Lymphocytosis ki""Ocs. A. lympho
B. Chronic in8.mrrl1'agmt. n •• lymphocytosis it • port of lymphoid hyp<rpwia. C. Physiologic (.bift) lymphocytooi~ Lymphocytooi. oeru" b.a.... of the .hili of lympho:x:yks from tho MLP to the Cll'. D. LymphoproJifontiw <>«>pmi>: A lymphoid J.ukomi., .... lu from:on unoontrolled pro~f
d. As with shift n. utrophilia, shift lymphocytosis is ~n most f=iu.ndy in cats, young ho=., and h..,.lchy '!Iim.!,. 3. lymphoprolif.ratin di,ordm (Tabl. 6.5 and Fig. 2.9D) a. This lymphocytosis usually i, ausa! by. neoplastic prolif.ration of lymphoid cdl. in lymph nod•• , bon. marrow, or oth" tjuues. b. BLV .od F.LV ..o known to indue;., ntoplastic transformation oflymphocytes. but not aU form. of lymphoid nwpl,,,i. in cats and aml~ ar< C>Usffl by th ..~ virme;. c~<= of lymphoid ntopl",", in oth~r domestic mammal, ar~ not known. c. BLV inf=ioD! moy al", indue;., a p
2/ LEUKOCYTES

83

d. In domestic mammal., lymphoid lru~mi", usually .. prestnt th~ leukemic manifestations of lymphoma; that is, th~ nalpl,.. ia stmro in th~ lymph nod .. (or other lymphoid tissues) and 'pread to th~ blood. e. uuk~mia is d""ically chuacterized by a markro or ""t",me lymphocytosis with many lymphocytes having abnotmal or "immatu .." morphologic f.-atures. Th~ diagnosis is ~a,iest if both erit..ia ue pr."'nt .nd difficult when they a.. not. 4. lymphocytosi' of hypoadrenoronicism (Addison·, dise;o",) a. This lymphocytosis may"" em=' by an absence of glucocorticoid hormones, which normally inhibit lymphocyt~ pnxluction or alter lymphocyt~ distribution in the body. b. Dogs with hypoad .. noronicism Jruly haw. lymphocytosis. A/wIIJS think of hJ'PO'ldrenororticism if an obvioudy stressed dog h .. a neutropenia .nd lymphocytosis {stress should em", th~ opposite-neutrophilia and lymphopenia}, especially in arotemic dogs. c. The da .. ic leukogr>m of hypoad .. nororticism consists of a low_normal to decr..... d neutrophil concentr>tion, high_normal to increased lymphocyte concentration, • normal monocyte concentntion, .nd a high_norJrulI to inc",ased alsinophil concentration. 5. "lymphocytosis" of young animals a. Puppies, kin~ns, calves, .nd foals have great.. blood lymphocyte concentrations than do adult animals. lymphocyte concentrations in canle increase until about I yr of~, and then graduaUy dec",... owr the yt"3-rs in adult • .' b. Compared to adult refe .. nce inter""],, the app ... nt lymphocytosis may "" up to 2x URL B. lymphopenia (dec",ased mearured blood lymphocyte concentration) (Table 2.10) l. Acute inflammatory lymphopenia {Fig. 2. lOA} a. This lymphopenia is caused by changes in lymphocyte kin~ics nimul>led by .cut~ inflamJrultory medi.tors that reduce the ClP.'"

Table 2. 10. Di""UC5 and condition. that cau"" lymphopenia Acute inflammation 'Acute bacterial infection> 'Acute viral infectiom Endotoxemia Steroids 's.-. the list for neroid nrutrophilia in Table 2.5 Depletion lymphoid e/fusion; chylothorax, .nd fdine cardiomyo!",thy loss of lymph; alim~ntary lymphoJrul, enteric neopl",m., granulomatous em..itis, puatu""rculosis, protein_losiIli: ~mero!"'thy, lymphangi«tasia, ulceratiw enteritis lymphoid hypopl .. ia or aplasia lmmunosuppr=iw drug, or whole body irradi.tion Destruction of lymphoid tissues; multicentric lymphoJrul. g<'neralized lymphadeniti' Combin~d immunodeficiency ofho=. (Arabian and Appaloosa) and dogs (b>Sse"l hound, Cudig.n Wdsh corgi, and Jack Russell terri..) Thymic aplasia of black_piro D. ni,h cattle • A rel. ti",ly oommon dioe .... 01 a",dition Not<: lilt! of sp
B4

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

,,I ,,'

Fig. 2.10. Lympbopeni. kineti",. A. hut< infbmmatory lympbopmia: Lymphop to lymphoid tiss""., and (3) d.ecre>sed movement of lymphocytes from lymph nod.. b.cit to blood. B. St .. oid lympbopeni:o: Soon .n.r . dmininr.tion of g1ocooo"icoids. lympbopeni. 0CCIl" bee ...... of th. movemmt of lymphocytes to lymphoid tis""" and pl.,i", lymphopmi. occ",", b.cau.. of
{I} [ncr~asffl ffiugination and ~migration of lymphocytes to infbma! {iSSll~ {2} Homing of lymphocytes to lymph nodes by inc ...... ing the raU of migntion

through postcapill.ry high endothdial

~ll,

{3} Reducing the m . oflymphocym lnving lymph nod .. via ~ffe""m lym_ phatic nssth b. Most .cut~ inHamJrultory leukogr;oms with neulrophil", or n~ulro~nia also hav~ lympho~nia. Dis;;op~mm"" of lymph0l"'nia is gener;olly ronsid~ral a good prognostic .ign. c. Historically. lympho~nia w;o, oonsid~=' to ~ causal by th~ .1....' associalal with acut~ inflammation nth" th:m causal by th~ inH.mJrullory process itsdf.

2/ LEUKOCYTES

2,

3.

4.

5.

III.

85

Th~ stress of an illn .... m.y indu,,~ th. Iympho~nia, but documentation of ruch • pathog..n ...is w;o. not fOund, Stuoid {mess} lymphopeni. {Fig, 2,IOB} a, This lymphopenia is ",msn! by th~ "hang<" in lymphocyte kin~iCl c;msffl by ~ndog..nous or aog..nous glucocorticoid" {I} Imm.di.tr: shili oflymphocyt.. &om the CLP to oth.r pools, On~ th~ df<ecs of th. glucocortiroids diminish, the lymphocytes r~urn to blood, Som. rq>Om indicat~ the Iymphopeni. is caus.d by d=....s.d .fflux of lymphocytes from lymph nod ..," wh.,.... oth., d. u indicat. a ralimibu_ tion to bon. marrow,"'" {2} Ln.r: Lymphotoxic .ff.cts caus<: lymphoid hypoplasi. and thus d=e.s.d lymphopoiesis. Sensitivity of lymphocyu. to glucocorticoid. vari.. with the sp«:i~. and also with th. st~ of Iymphocyt. d.vdopment." b. Typical mu ... are th~ sam~ '" m~ntion.d fOr st.roid n.... trophilia, and it is usually consider.d th~ most common lymphopeni. in all speci... Th • ..-verity and duration oflympho~nia are g..n.rally proportional to dose: and/or duration of incrras.d glucocorticoids. D.pl.cion Iymphopeni. (Fig. 2.IOC) a. This lymphopeni. is produc.d by a 10... oflymphocytes &om the body via a 10... of lymphocyte_rich lymph or because of an incompl~~ lymphocyt. circulation pathw;oy (•. g., rq><"3.t.d removal of chylothoracic fluid from. cat). b. Disorde", that caus<: thi. Iympho~nia are not common. lymphopenia of lymphoid hypoplasi. or .plasi. (Fig. 2.IOD) a. This lymphopeni. is causn! by .ither cong~nital or acquired lymphoid hypo_ pl. ,;. or apl""ia that d=..,es lymphocyt. production. b. Bemuse most blood Iymphocyt..... T_Iymphocyt.., .dective apl""ia ofT_ Iymphocyt.. will musc: • more ..".... lympho~nia th . n will a ..,Iective aplasia of B_lymphocyt... lymphopeni. oflymphoma a. Lymphopeni. is common in animals with Iymphonu. b. Th~ lymphopenia may be caus.d by d""."".d production (damage: to lymph nod ..) or alt.,ed lymphocyte kin.cics {disrupt.d circuution patterns}.

Abnormal monocyte roncentrations A. Monocyto.is (in"'~. sffl measured blood monocyt. con~ntration) (Tabl~ 2.11) l. Inflammatory monocytosis a. Acute and chronic inflammatory di ....s'" may cause monocytosis by "ytokin~ stimulation of monocyt. production . nd "'I•• ..,. b. Monocytosi, ge:n~raUy refl<ecs • n..d fOr macrophag.. in di",ased ti,m. or blood. 2. Suroid {mess} monocytosis a. Glucocorticoid hormon.. or drug. are a common cause of monocytosi. in dog• • nd cats, but thq muse minimal to no changes in ho",~s .nd catd~. b. Monocytosi, is probably caus.d by. shift in monocyt .. from a marginat.d pool to a circulating pool. 3. Nwplastic monocytosi. (monocytic I.... hmi.) a. This is typically characterized by a mark.d monocytosi •. Monocyt.. may . ppear . bnormal or rdatively normal, but wh.n atypical . nd immature, they would prob. bly not be calegoriud "" monocyt ...

B6

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

T able 2. 11. Di""a..es and condition. {hal cause monocytool. inft.mmalion '[nf=ions: b.ct~rial {including anapJ..,mal.nd rickensial}, fungal, protozoal ' N«rosi.: hemolysis, hemorrh"l:c, nNpl,,,i., infuction, lrauJrul Suroids · Slre.. (physical or nrurogenic) Hyperad""nororrici,m 'Glucoconiooid thc,""py Ad""nOCO{lirolropic hormone . dmininr>lion Neopla,j.: monocytic [cuhmi. ~condary 10 immune_ffiMia!ffl neulrop
b. This is • rdativdy uncommon form of [cuhmi. when compared 10 gn nulocytic .nd lymphoid leukemias. 4. Mcondary 10 immun~mnliat«i ncutropy occur duting neutropenic cyel.. and her;old incr ......,. in blood neutrophil concentrations. 6. G_CSF: This may be administered to promote nrutropoiesis, but it wiU also promou monocytopoiesis. B. Monocytopenia: This is difficult to document beau.., healthy domestic mamm>ls may havt rdativtly few blood monocyt ... Monocytopenia i. not con,idered a diagnostic problem.

[v.

Abnormal eosinophil concentmionl A. Eo!inophil", {incr.....ed me• • ured blood eosinophil concentmion} l. Mon eosinophilw .ppe:ir to be reL.ted to eosinophil .nti_inllamm>tory function> or to the .ttraction of eosinophils to ti ... u.. after mast celJ or basophil degranulation. 2 An eosinophil", sugge;ts the po .. ibility of m>ny di..,,,,,, nates (Table 2.12). a. In the hyper",nsitiviry disorders, typically there are clinical .igns associ.red with the involved tissues: for nample, pruritus with II .... bite or naphyloroccal dermatiti •. b. Both internal and extern.l parasit.. are frequently blamed for .n eosinophil"" but many .nim>l. with similar p.rasitic infections do not have an eosinophilia. Per..inent mild eosinophil", is oe<:a
2/ LEUKOCYTES

87

Table 2.12. Discascs and condition. Ihat cause eo.inophUia Hyp<=n>ilivily (.JI~rgic) disord~", 'Fl~_biu dmnalilis Hyp<=nsilivily 10 S1aphylococc:.J or m~ploroccal prol~ins Milk aU~rgy in ruminants Anhma and eosinophilic r~spimory di""rd~", P">silism Eclo!",rasitcs 'Hrartworms "Tissu~ n~malodes, It"m.'od~., and proiOZO' Dogs: Di"'fi"'';'', Arllmmu hrilonmtll (DiJmalonmw), Spirrxrrcfl, Strcnt:JloidLs, Trichuris, .nd PIl""tfm;mul inf~ction.; larv:ol migr.,ion of hookworms and roundworms; HIlb",nmw uts: PIlrllt,mimuI, klru,.",mmgylul Hot=;: Strcngyluiul unl~: SIlrr«J1tis 'Mast ctll d~granul,'ion alUsN by inHamllUlion: cutaneous, ... piratory, im" ,iruol,

gc:nil:ol, utin.ry Hypo.dr~nocortici,m

Idiop>thic =inophilic condilion> 'Dog: eo!inophili, mynsili. , =inophili, gmr""m~rili., eosinophilic !",non~ilis, eo>inophilic pnrumonili., eosinophilic granuloma complu in Sibtrian huski .. 'UI: eo.inophilic granuloma romplu, =inophilic ~nurili" hyp<mninophilic syndrom. Paranwplastic =inophilia (including mast ctll neoplasm.) Eosinophilic leuk~mia • A lel1t;vely common di..... 01 condition Note: Lins of sp
d. Eosinophilia is found in "'m~ dogs with hypoadr.nocotlicism.

Th~

pathogc:nesis

probably involns the lad: of conisol. at<" ,,,",,,:01 idio!",lhic =sinophilic condilions (T abl~ 2.1 2). T ypicolly, Ih~r~ ar~ clinic:ol sign. associatM wilh Ih. involvMlissu.,.. f. Idiopalhic hyp, and ho"'......... (I ) Th~ syndrom~ is characurizrd by pr, but animals cbuifiM .. having hyp 20.0 X 1001IJL 10 bt hypnw,;nophilk. 3. P.ranroplastic eosinophilia a. Mast <:dl neoplasms and .djaa:m lissues &«iu~ntly comain eosinophil! btc;ou", of ch~moallraclants relrasnl from mast <:dIs. Th~ .. may bt • concur ... m eosinophilia. •.

Th~r~

BB

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

Table 2. 13. Di""a..es and condition. thai cause eo.inopenia 'S{~roid, (Stt Table 2.5) 'Acute inHammalion Di= causing a hypoplastic to 'pl.'lic marrow • A r.t1tivcyt< diff.",n,ial coun .. :and rometrn... 1 Jow., «kr= limit of OIJ..tL

b. Eosinophili. may also bt linhd 10 other n, inducing faclors (e.g., im. rlcukin 5). r.ranry lymphoma: and hor.,,- imcstinal lymphoma.

4. Eosinophilic [.uk.m"- (Tabl. 6.5 ): This form of chronic mydoproliferatin dis.,... i. ""ry uncommon. In cats. it has bttn linhd to a .. troviral inf«tion." B. Eo.ino~nia (d«r~asffl m=ural blood ro.inophil cone
v.

Abnormal basophil con""ntmiom A. IWophilia ( incr~asffl m...su=i blood w.ophil con""ntration) l. Only subsuntial or ~rsin~nt mild incr..,.... in luwphil con""ntrations abo"" 200300/IlL should b. con.id~..d d.finiti"" ba"'philia., b.cau.. of th. impr« ision of l.ukocyt. diff~"'ntial mum. (.. p«ially wh~n d~aling with a minority ""II population). 2. A causr ofM.IOphilia may not b. 'ppa"m but can b. .uoci..tw with all~rgic, para.itic, :md nroplastic stat.. (Tabl~ 2.14). B. B>!Op
VI.

Abnormal mast ""J] con""mration. A. Mastocyt.mi.. {mast ""lh det«tN in ~riph ..al blood} l. Finding on~ man ""U in a blood film or buffy coat pr~p'lfation of dommic mammal. i. comid~..d to indicau th. pr~ .. m'., of mastocyt~mia. Th~ t~rm mMf«Jrnniil i. p"f.. =i ov~r ma""<"JfO'js b.cau .. manocytosi. Jruly "f~r to incr~• ..d mast ""U numb.rs in tissu .. oth.. th:m blood. Eith~r t~rm may rd'.r to nroplastic or nonnropwtic mm cdl populations. 2. Disordu. that Jruly cau.. a mastocyt~mia (Tabl. 2.15) a. Mastocytmis in caU occurs in two form., 'Y't~mic .nd spl~nic, and mastocyt~mia may b....,n in eithu ........

2/ LEUKOCYTES

"

Table 2. 14. Discases and condition. Ihat cause basophilia AU~rgic r~a.clions (immMiat~

or dd.)'bI) Drug" food" inhalonu, and iO=I stings or bile, P.rasiti,m F1~ .. Ganrointestinal p"""ites ouch a, nem. ,odes 'Vascular pamil" such a.! Dirofildro. i",,,,;til and AammtxluiumrmJl (Dipttilumnnaj rrconditu", Neoplasia Basophilic lruhmia M:ast cdl neoplasia Fdin~ m}'doprolif~ .. tive di",ase; Lymphonl
Table 2.15. Disorder. reported 10 be associated with maslocytentia Neoplaslic disord~ .. Cmanams maS! cell neoplasms Vis"", ..1 m",' cdl neoplasia M:ast cdlleuhmia Nonn~oplastic di50rd~ .. in dogs • Inflammatory Ent~rilis, .. p«ially parvovirus Fibrinous pilu,"" associaud wilh a.cme infl:munalion Skin di ...=: fl ... _bit~ hypc=milivily, alopy, sarroplic mange, :md food allugy; some wilh =ndary pyod~rma H~morrru.1:" =ndary to hemophilia in dogs Gaslric Ion ion in dog, • A ,.I.ti""ly oommon dise ... 0' condition

b.

Cut;m~ou,

maslocytonl
90

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

and not quamitotiv. kaUst Ih • • mount of blood aaminrd is not constant or is unknown. For ""amp!.. finding 30 mast ~ll, per sJid. on day I and 15 mast cd], r
Ih. quality of blood film on day 2 P"""'filM • comp]"" a.mination. B. 8«;oUst no m:lSt cdl, our n~ffl in th. blood ofh.althy domestic mammals, d,""reasM oon",mntions do not occur.

LEUKOGRAM PAlTERNS I.

Although rach typ< of l.ukocyt. is uniqu., alloratiom in blood leukocyt. OOIl",mrat;ons frequ.mly occur in prffiict.bJ. ""norm that an summ>riud in T .bl. 2 16. Th. crurac{
[I.

Hemic neoplasia A. L<:ukogram pamrns in :mimals with hemic neoplasia at<: nmmdy v.nabk l. Ext",me leukocytosis due to numerous poorly differentiated cdls i, indicative of.n arut~ l.... h:mia. Occasioruolly, the initial microw:>pic findings su~ the poorly different ;"ted cdls are oflrukocytr lineagt, but .dditional testing mablish .. the neoplastic cdls to bt of erythroid or mq;.karyocytic Jin.-ag~. 2. Ext",me leukocytosis due to numerous wrJJ-diffe",m;"tw "",lis is suggc:nive of chronic leukemia, but. marked infLommatory leukogram mun b.: rxduded 3. low numb.: .. of atypical cdls in !",riph~ral blood, I"'rticularly if accoml"'niw by cytop
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92

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY Light_.c:m~r pan~rm produced by som~ h.m>tology analJ=" {e.g., the CELL-DYN 3500} may provide clue. as to the t}'J>C' of hemic neap!"", prestnt. This is !>«:au.. neopJ:.stic ~lls often han similar light_samet prop at. typically not ddinit;ve. C. Multiple m...:hod. are usffl to character;", neoplastic hemic ~ll, in .n anempt to <"ubli,h th. ir lin~•. Olh.. th . n th. microscopic a.mination of routindy '!:linffl blood, marrow, or lymph nod. sampl... =r>J ocher method, .te ustd to rvaluate cdls (= Chapter 6). I. Enzyme cytochemimy or hinochemi
B.

Ill.

Recognizing .leu\rogram pmern:lids in the undemanding of an animal'. illness. However. not all disord~rs will produ~ classic p,lt{~rn', concurr~nt proa::s.= em complialt~ p~tt~rn', and som~ p"tt~rn' ov
ABNORMAL MORPHOLOGIC FEATURES OF LEUKOCYTES I.

Chong~' associ ata! with inlUmmatory di ..~",s A. Toxic n.... trophil (pl.t~ IA- C [for all plates, ..., th~ color =tion of this book]) I. Toxk n(utrophiu a.. neutrophil, with any or all of the following cho",ct
2/ LEUKOCYTES

93

h..,.hhy cm may ha... nrutrophil, with Dohl~·. inclu,ion bodies. Th~",fo ... fdin~ DohJ~·. induoion bodi .. without othor toxic ch.nges g~n~tC'ntine_inducM inflammation." .nd in dy=ia. inducM by ""fonicid or cdaudon . ... How~... r. it ,hould be noted tru.t ju,t th~ p..",nu of. l~ft mift i, not a toxic chang<'. 5. Toxic chang., am be graded b=d on types and d~, of toxic change.. but classification .)"t~m' h . ... not oon nandardized. Proru.bly th~ more RV~" the toxic ch.nges .... the mo", ,..,or. the inflammatory ,Ute i. in the animal. s.v~ .. toxic ch.ng<' h"" bttn associated with roo"" prognosi,.·' 6. Toxic neutrophil, {.. pecially band .nd mffamydocyt~ stag..} am be mnfwed with som. monocytes. a. If. uJ] i, a toxic b.nd. oimilar toxic ch . nges .hould be present in segm~nted nrutrophil,. b. If . u J] i, a monocyt~. it ,hould be dininguishabl. nom • segm~nted n~utrophil tru.t has th~ clrar to !"lIe pink cytoplasm. c. In KIm. immatu.. toxic n~utrophil, cannot be reliably dininguished from monocyte> on a Wright_stainffi blood film. In th. ", c..... 'CCUt
Appar~ntly

='.

disea, ... B. Giant neutrophil. (Plat. IC) I. GidlY nrutrophils a.. sq:m.nted or band nrutrophil, with a diam.ur of 14-20 ).lm. Th")" . .. larger than typical blood neutrophil.. with diamet~ .. of 11- 13 ).lm. Th")" probably form because of a ,kipped cdl divi,ion in nrutropoiesi,. 2. Giant n~utrophils may be "-""'Ciatffi with inc",ased n~utropoi~si, caused by inflam_ mation. in which = th")" represent a toxic chang<'. Th")" a", ... n most commonly in cats. 3. Th")":doo a", ... n in the poodl~ marrow dy=ia. FeLV inf.aions. and aftor adminiq:;_ m~nted n~utrophil, have oon observed in dogs aftor cyclophosphamid. t",.tm.nt, {authors' ob",,,,.tion} .nd eJtp
94

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

th. blood of a!lim:.!, with a variety of ""ute .nd chronic inHamm. IOIY di ....... ,. Ik.ctive lymphocytes, which "e rno .. common in chronic than .cute inflammatory stat.., cm k found in mon blood ",-ruple,;, but an jnCJ~ numbtr of them con indicate an inlhnurtatory di",asr . Thq.", common in young a!lim"',. 2. Ik.crj"" lymphocyte:! may ha"" a variety of structural f•• rure, that.", oonsid,,«1 to bt ""Clive changes: an incr.asal amount of cytoplmn: .n enh.ncnl cytop[",mic bawphili.; a perinuclear halo; a prominent focal Golgi zon.; .nd =ntric, .nl.rgffl, cl •• vM, oonvolutal, lobllJ.tM, or baok<aran~ a", usually associatffi with syst~mic inf=ion. such .. hinoplas_ mo.is. ehrlichiosis. or leimmaniasi,.

II.

t.n.kocyte. that conuin miscellaneous indusiom A. Sideroleukocyte (Plate IJ) l. A iidmJ/i"lwcyu is • neutrophil or monocyt~ containing hemo,id .. in. On Wright_ stainffi blood films. h~mo.id~rin is a blu~_grttn or ydlow_brown pigment. Its p..,..n~ can bt confirmed with .n iron nain {Prussian blue}. 2 Sideroleukocyte. a.. rarely found but c;on bt =n with hemolytic an~mias (e.g.. equin~ inf=ioU1 an~mia or immune_medi.tffi hemolytic .nemia) and aft~r transfu_ sion. Th~ h~m",id..in in blood l~ukocytes may r~pr=nt hemosid~rin ~ngulfed by tho,. cells while they we .. in a splttn or other tissues." B. Erythrophage (Plm IK) l. An rrytltrophdgr is typically. monocyu or n~utrophil that has ~ngulfffi an erythrocyt~.

2. It is occasionally =n in immun~ h~molytic anemias such as idiopathic immune hemolytic anemia in dogs. equine infectious anemia. and neon.tal is""'rythrolpi'. C. LUpU1 eryth~matosus (LE) ~JJ I. ALE ull is • neutrophil that engulfffi nucl~ar amigtn-amibody complexes. With. Wright st.in. thi, mat~rial 'pf><ars ., pink or pale blue homog~nroU1 inclusiom of variable sizes. N~ m~hylen~ blu~ suining produces mo .. prominem LE indusions with homog~neou. staining.

2/ LEUKOCYTES

95

2. Rardy s,""n in dommic mamm:ds even in th~ LE cdl t~st d~sign«i to produ"", th~ cdl in vitro. D. N~utrophils rontaining rill.. t cdl granules: Rardy. blood and maJIOW neutrophih ronuin JIUS{ "",J] gram,] ... In on~ describnl a1S<:. the a1t had splenic mastocyto.is and ""e intact mast cdls in the ~riph,,:d blood." III.

Organisms in leukocytes A. Roct~ria oth" than those: in the family A""pfmmaIaUll( l. Rardy, b.ct~ria may be: found within n~utrophils ofbacte.. mic pati~nts (pt." IL). 2. When th= bact"i.... found in blood, one must attempt to d<{"min~ wheth" the bact"i... pre.. nt a bact"~mi. or a ... mple conuminat«i with bact"ia. B. Roct"ia in th~ family Andp/as""'flUrar (ord" Rick~mial ..): Morulae of Ehr/khi 20) are a1U«i mo,ufilr, which typically have d",meten of 1.S-4.0 J.Im and suin p:de to dark blue-grey with a Wright stain. In g",nulocytic forms, th~ .. may be: mo .. thm one morul a per leukocyte. Morul a. should not be: confustd with ruhle', indmion bodi.. , platdets lying on leukocytes, or blebs of nud~ar memb",ne. C. Canine dist~m~r indusions (Plat~ 2D) l. Indusion! appear as monomorphic or pleomorphic, red to purplish rro or pale blue, cytoplasmic indusions found in neutrophits, monocyt .. , lymphocyt~s, .nd erythro_ cytes (..., Chapt" 3). Quick stains (~.g., Diff_Quik) stain the vi",l partid.. be:n" than do traditional Romanow.
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2/ LEUKOCYTES

97

inf~«i dogs include fevtr. p:nn. l. meness • • nd ocular discharg... The illn= is SNere. ~nd the progno,i, is guard«i. 2. Finding gametocytes of H. amrrinmum in blood leukocyt.. i. very uncommon in clinical ='; when present in periphe ... l blood. th ... is typically a very low perant_ age of infected leukocytes. Gametocyte> are ov:d to dlipticol. p:de blue. and ... about 9 Jlm X 4 ).lm. They may fiJ] a cdI'. cytoplasm and cou .. peripheral di'pla",,_ ment of the cdl's nucl...... The organism Im}' escap" from the ""J] and l..,ve a nonstaining are~ surrounded by a copml•. 3. Enc)"ted forms of the protozoa ... common in skdet:d mu",le •• nd a skdet:d muscle biopsy i, one method of confirming. diagno'is. 4. Common J.bo ... tory data in clinical =s include neutrophilic leukocytmis (sometimes atreme or leukemoid). a nOffilOcytic normochromic nonregene ... tive anemia. thrombocytmi •• incre ..«iserum alkaline phosphat ... activity. and hypogly""mia (which /lUy be coused by gluco .. cofi!umption by leukocytes in vitro). hypoalbu_ minemia •• nd corr.. ponding hypocal""mia. There i, poor cross-re~ctivity between H. "mrriCllnum antibodies and H. ,,,nis antibodies. E. HrpafllZMn rlll';1' l. This i, • protowon who .. g.Jmtocytes infect canine n.... trophil< and monocytes. It is found in dogs of Europ". Asia. Mrica, .nd South America and possibly in B... z.ilian cots. 2. Inf~ed dog, frequently la.ck obvious clinical signs but con be febrile ~nd lethargic. In contrast to H. amrrit'lmum. gametocyte. m.y be commonly found in the blood leukocytes; sometim.. a high p"rcentage of the neutrophil, .nd monocyt.. a.. inf~«i. Vi~ light micrmcopy. the gametocytes a.. very similar to tho .. of H. amnicanum a""pt that H. can;, structures~... little lon!:"r {.. ll!Jm}. Encysted forms of the protozoa ....... in skdet:d mUICle. 3. In Brazil•• very low percentage of inf~«i leukocytes con be found in dog, inf~«i with H. ranis {or a very similar organism}.~' A nearly identical organism h .. been found in domenic cou."'" F. H;'fllpkuma ,ap,u"'",m (Plate 2F) l. The yust pha.. of H. c"fMu"'rum can be found singly or multiply in the cytopl.,m of monocyte. {or ma.crophages}. neutrophils. and occasionally rosinophils of p"riph..:d blood. Cdl, containing Hi'fllp""ma con mu:dly be more ....,ily found at the feather«i «i~ of blood films (beca""" l. rger ""It. are con""ntrated in the feathered ~) or on buffY coat prep"'. tions. 2. The org.nism. are round to oval. about 2-4).lm long. with a prominent ""ll waU. Their internal nructures~ .. commonly a:ctntric ~nd st:nn pale blue and/or dark pink to purple. 3. When found in blood. the inf~ion has advanced to disseminat«i hi,topl .. mosis. and organisms can typically be found in macrophages of bone /lUfJOW. spleen. liv... lymph node, •• nd oth.. tissues. G. Lriihmania 'pp. (Plates 91 and IOH) l. Lri,hm.mia is a kinetoplastid protoroon th. t is found primarily in M«iiterran .... n. C.n!r:ll American, .nd South American countries. However. it hal been found in restarrn coloni.. in Ohio and Oklahoma."'" in Tu.. and Maryland dogs."'"' and in dogs from other Southe~"ern ,cates." 2. Organi,nu (ama,tigotes). which app"" '" oval to t .... rdrop strucru... in m. cro_ phages •• re 2- 3!Jm long with a:ctntric r«idish nuclei .nd ./lUll cytoplasmic r«i

9B

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY bar. (kinffopl:.su). L~islm"mu. ammigo{~' must ~ djff,,~miatffl from amastigot.. of T. cruz; and the yeast form of Histuplllmw. which js .bout the urn. ,iu and has

~

chich, ",II wall but doel not hove a kinetoplast. 3. M'Cfoph>ge. laden with amastigote •• '" umally numerous in bon. marrow, lymph nod .., 'pJ,""II, and lin•.

H. Trypano,uma cruzi l. Two ¥ci .. of TryfJ.rations of. puppy with trypanosomiasis; th~ blood also comainal mmy trypom .. tigot..... I. Myccbactr,;um 'Pp. (Plau 2G and HJ l. Rardy,' myrobxurial inf=ion may ba:om~ syst~mic .nd mycob.ct~rial organisms will bt found in p<>rtal ob5.rvation during n.tural infn:tions. K. T"""pfil= t!'ndii (P1. Ir 21) l. T .chyroilr. of TIJXOpfil""" t!'ndii are rardy found in blood neutrophil, and mono_ cyt .. of dogs and cots with .cur. toxoplasmmis. 2. Thq ..~ mor~ common in mxrophag .. of inf'""tffl ti"" .. (lung .nd intestine).

IY.

Lrukocyte agglutination/aggreg. tion A. CX:casionally, small aggregat .. of 3- 15 l.ukocyt~. are found with th~ body of a wdl_prqmal blood film. Typicolly thq ... mixtures of neutrophil. and small lympho_ cytes with f~·~r .osinophil, and monocyt ... Th.,. should ~ ,uSp
B. The publishal studi.. ofleukocyte aggregation are for human .. mple .. The :aggregation of neutrophiJs might dep
2/ LEUKOCYTES

99

tioll mayor rruoy 1I0t occur in citratffl or hq>uiniud 'amples ...... Wh~n aggrrg.tioll w:as Sttll ill room t~mp'ratu", ,amples but 1I0t ill ""mpl~, ~pt at 37 ·C. th~ lI.utrophil aggrrgatioll was due to an [gM ." C. A major rta'lOn for r
v.

H.ralitary di""rd~rs that han leukocyt~ illdusiolls l rar~} A. Ch<'di.k_Hig",hi SYlldrom~" l. This is. hrrfflitory dirord~r ofblu .... mok. Pe,,;"n ats. H~rrford cattle. othrr nondomestic rruommal •• alld peopk 2. Diagnostic f...tures indude large specific gram,] .. in cytoplasm. of n~utrophils. eosinophils. alld b;,,,,phib. Abnorrruol gflmul.. r~Hect fusion of gr.nules (ly",,,,m..). B. GM, gallgliosidosis"''' l. This h~ralitary disord .. ofFri";all cattl • • Siam~", alld Karat cau. Ellglish sprillger spallid •• mixffl_brrffl be,agles. Portuguese water dogs. and Shiba [nus is ""USffl by. d.fici.ncy of P1:alactO!idase. 2. Diagnostic features indud~ small. distillct. de.. cytoplasmic vacuol.. ill lymphocytes in Wright_stainffl blood films. C. GM, gangliosidosis .... '.. l. This is a hrrfflitary disord.r of Yorkshire pigs. Germall ,horthairffl point .... golden retrieve". alld caU th at is au...i by a ddiciellCY of p_hexosaminidase. 2. Diagnostic f...IUres indud. dark blue granules in neutrophih alld prominent azurophilic grallulation or v>cuoliution ill lymphocytes. D. Hrrfflitary anomaly of n~utrophil granulation in Birrruon ats'· ' l. This .nomaly is .n auto",mal recessi"" trait in pur. brffl Birman aU. 2. Diagnostic f... tures indud. promin~nt fine eo'inophilic granul.tion ill the cyto_ pl"'ms of neutrophils that mull be, diff.rrntiatffl from toxic granul...nd inclusions of MPS type VI. Uhr:astructural morphologic f...tures of granules ... normal. E. MPS type P""'·J l. This is • hrrfflitory dirord.. in domestic cats. dog•• and peopl~ that i, caused by. d.ficiency of u_L-iduronidase. 2. [n som~ studi... leukocytic cytoplasmic inclusions were...,n via tran.mission da:troll microscopy but not in routin~ light microscopy. Oth." hove found that fdill. n~utrophils have .bllormal cytoplasmic granulation {.rruoll pink granules}. F. MPS type IllB'" l. This is a hrrfflitary di",rd.. in schipperk.. that is au...i by a defici.ncy oflyso_ ""mal glycosidase N_a""tyl-a- D_g! ucosaminidase {u_N """""trlgl urosamillidase}.

100

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY 2 Blood lymphocyt.. :md marrow m3oCrophag~', lymphocyte., and plasm. cd], we", d.""ikd as having abnormal dork_,taining gr:mulation. G. MPS typt VI'" l. Thi, is • hedmid•. H. MPS type VII''''-'· I. Thi, is • h,,«iiYry disorder cau,al by • drfici. ncy of p-Wucuronidas.. 2 Neutrophil, of dogs and cm with MPS typt VII have inclu,;ons lih thost found in MPS~V1.

I.

J.

Fucosidosis'" I. Thi, is caustd by. ddici.ncy of CI._L-fucosida.. , an enzyme in glycoprotein metabolism. 2. Lymphocytes of dogs with furosid05i, havr cytoplasmic vacuoles. Oth~r n0r3i:~ di,,,,,..,. hav~ bttn =gniud in domestic m.mmal,. but l.ukocyt~ abnormalities havr not bttn =n in thest. Th"", includ. MPS type lilA in wir.haired dachiliunds"o and • N~ ilalond hunuwooy dog.'"

OTHER NONNEOPLASTIC LEUKOCYTE DISORDERS I.

Lrukocyu adhesion defici~ncy (UD}'" A. u\D has bttn =gniud in Iri,h rtd and whit. ,m. .. and in Hol,t~in cmk th. diwrd... "'~ .. f~rred to as cOllin. u\D (CUD) .nd bovin~ lAD (BUD). Both dilOrd... "'~ a1~ by d~f«t, in th. int.grin CDIS mol«ul. that r.,ult nom ,ingl. nucl""tid. subnitution. that at< diffe .. nt in the two .p«i... Th. d.frct pt
II.

Bon. marrow dyscra,i. of poodl..'" A. Thi, i, • h..tditary disord~r =n in miniature .nd toy poodl ... The drvdopm.ntal def«t in ",J] proouction i. not known, and th..e i. not a clinical problem .uoci<>ttd with th. syndrom•. B. Di>gllonic f""tu ... of the .yndrome l. Hypersegmemtd or gi<>nt n~utrophils 2. Macrocytic normochromic erythrocyu. without an.mia or rrticulocyto.is. Their m•• n cdl volumes usually at< 80-110 fl (in most unaffrcttd dog" 60- 77 IL), their m.. n cdl h~moglobin ron",mration,.t< usually WRI, and th.ir m""n cdl hemo_ globin valu .. a.. iner"",..! (compartd to tho .. of most unaffrct"! dog,).

2/ LEUKOCYTES

101

Ill.

[diop. thic hyptrsq:ment«i neutrophil, of hor...'"'' A. The di"l:nostic feam", of this syndrome is hypmegmented neutrophils and has bttn d=ribed in two hor... of the qu .. ter ho= brttd. In one rq><>rt. neutrophil nuclei h.d 3-11 ddinitin lob., "p,... trd by filoments, and .bout 70 % of nuclei had 7- 10 lobes. Oth.. cas.. han b.en .imil... B. Hypt=gmemation i. not :woci. trd with the common COUIe; of hyptnq;menution; that i., hy~rad .. noconici,m (Cmhing', di ..... ) and aogenous gluroconicoids. The co.,.., of the .yndrome has not bttn det .. min.d. The po"ibility that hy~rs
[v.

Pdi:"r-HuI't .nom.ly"....,.., A. Thi, i, • h..editary disorder that occu". in .....e",1 br=:l, of dogs, domestic .honhair cot" .nd in Arabian hor .... Most clinical cas.. involvt heterozygotes for the .nomal"., a homozygotic kitten wa, ,tillborn.'" B. The disord .. is cha",ct..iz.d by h}'ll'OS'glllemation of n"'trophil, ""inophil, and basophil nuclei. The nuclei .'" round, oval, .p«:tade ,haprd, or occa!ionally havt parti. 1 ..gmemation. The nudrar chromatin may ap~.r hyptrchromatic or normo_ chromatic but lacks the pal.. ap!"",,,,nct of typical oond neutrophil, (Plate 2J- l). The v.riou. nuclrar sha~ can b. g",d«i (round or oval to two or mo .. lobes) to charnct..iu the drgr.., of hypo..gmemation. "UJO C. Drc..a..d lobulation of monocyte nuclei h .. bttn d=ribed, but recognizing the change "'Iuir... grading .y,{<m th.t i, not commonly usffl. ". D. Pdgtr_Hut't neutrophil, .hould not b. classifi«i a, band neutrophil. {or youngtr form.}, ba::au,," such classificotiollS will ,"",ult in a left .hift .nd thus indicote.n infl. mmatory di ......

V.

Pseudo-Pdi:"r_Huet neutrophils .nd ""'inophil, A. P.. udo- Pdgtr_Hut't neutrophil. a{<...,n in row,,'" occasionally in dog, with =rr inflammation, and in cot, with FelV_inducrd mydoid I",nmi .. J>]' The tran,ient defrct "pr..em, •• ynchronou. neutrophil maturation; some n",trophi], look like Pdger_Hut't cells, but others do not. Usually other toxic changes ... pr..ent with ........ inflammation. B. Pseudo- Pdgtr_Hut't ~inophil, havt bttn described in cattle and a ho=."·'''' In cotd., they we .. found concur.. nt with pstudo- Pdi:"r_Hut't neutrophil,. [n the ho=, they we", found concur",nt with. markrd at .. me inflammatory eo.
VI.

NOIISuining eosinophil granules (grey eosinophib) of do!}""'''' A. Canine =inophil, typically rontain numerom round ~inophilic granules of varying diamete"., .long with a ftw cytoplasmic "vacuol... " [n some bre«i, (es~ally grey_ hounds, but also golden .. triNe". .nd Shetland ,h..,pdog'), the eosinophils contain eith.. poorly nainrd granules, or the nonsuining granules ap~ .. a, cytoplasmic vacuol.. th. t ,ometimes han central foci of eo.inophilic staining. The cytoplasms of these ""l:",nulor" conine "",inophil. K1metime, .p~" grq and thus the name grey eosinophils. A[terrd suining r"Present' a mooifi«i chentical compo,ition; the alt ..«i composition is associat«i with ultrastructural changro (authors' unpubli,h«i data). B. Th... i, no known dinical or p. thologic ,ignificonct to the presenct or ab .. nce of these eo'inophil, oth.. than the pot.mial for incorrrcdy identifYing the cell,.

102

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY l. Microsropically, th~."" rometirnes incorr«dy identifi«l ... toxic neutrophils or

monocyte,. 2 Affrct.d a.'inophij, also han low ~roxjd..., activity and at. usuaUy misclassififfi . ,

monocyte. when .n:dyud in th. ptroxid ... clunnd of the ADVIA (,.., the Analyti_ cal Principles :md Methods &ct. 1lI.C.3) (.uthors' unpublirn«i
Refe re nces I. 1'._ KW, "" .... "" ML. ~ FK. 197J. BIood ..... trophlIk ~nn"1ocyt< JciD• ...d "",.... J Am Vrt M«Ihw< 18~670-67l . S. Riim"", a.. 19S9. V.,ial.ility of ........ in difi<..",ti.J "H mun .. "" blood .... u". T riand' 4, 1S4-I SB. 6. Sod..J", OW. 19<>1 . V............, H,_""'v- I . odhloD. 1'hit..<J">Oi. in ",;"iatu".npl.ilo;". k....,. I Am Y" Med Ju.oc 17B,J(l3- lO S. II . It.maiah SK. H......,. JW. Gi&ul>, S. F.."klio RP. Ctawfo..J PC. 200}. 1"..... KuJ .. homoIyoio -.-.dated ..ith Iiv... cytk.....,.;. n b, attaroopo,,01 citooL.ti.on of blood tJ.roudt. tilioooiUn GM. G. ", FI!, Di... """, RP. 1m. Di1l"c.mliatio" of and "" ... bort.tcmk """'" ..it!. >0"'" ",Iifoot", m .. citia. I", ~ of th, 17t!. ACVlM Fo.um, Cbiap>. 693. 2S. W"P""' IG. Roth RA. 1999. NnotropNJ "';vatio" dwi,,& ,..do..,,,,,,,, ... I lnokoc BioI 66.10-24. 26. a.ic1","" WR. 1'0..., KW. 1981 . Immo"" mediated _pcnia in ",,,,,....J .nim.~, A",; .... Y.. Clio P.tbol 10.6-16.

..n•.,

ri.,,'

"""".n,"""'wtin,

<»"""

au,,,,,

..u.,,,,,,

b",rn,,,,,,,,

y"

2/ LEUKOCYTES

103

Jain NC. Y
1990. Mrtbodo fo. drnoctioo of~m«liated ..... p
Yrt)

~h52-57.

29. Hinoh YM. Mitdtam SA. 0..",,)K. I ~3. M..hlpk <J"pntia ••• _i·."! ...ith. m<>n<>1ili ....... in • doc&Yrt CliD P.""'I l},I6-l(). }O. Srodtb.o. C. Sb.ppnodd R). 1m. H"mopl",~.>o" <J'~ fondjo,," in .. dc,:. &nd • = ...ith. ........pha~ pplain b"""", ..... tropIill ,lott." m....,;.,.",. T ""do Mol Med JlH6.J.-170. J.6. S.......,., CL. K<.ciba G). O·Ked< DA. c,;'p MS.)aoobt RM. R.ojIID IL 1937. Cydk htmatol"'''' • ...dated..,.jth kli", bluuoia .ino. i.,f",tion in ...., ca". I Am Y" Med A_ 191,9.J.-96. l7. ImbofBA. Duooo D. 1995. L...koqt< migation and ~o.Adv I""".""" SS,J .. j......jI6. }8. BJom."", Ii. W,in.dclo S. Sdo,~ ¥TA. 1990. T'" infI"""" of pnx\nitoIoo, on do, .«iKulation of pnipb,"[ blood Iympl.oqtko A. 2001. Di.....,;o •• od ~pbiIk ditt." "o=b/i." idiopothi< loypt""tinopbili< ~m' io. dc,:. Y" R...o 1"~JS6-~9. "J. c.rn" SA. Randolplo )F. &b HN. Rrt", S. 1?90. Eooioophilia in th, ca" A <,.....;'of.od blood "ll. io. ca •. Y" P.""'I 2O.6.J.8.-6.(O.

50. W",l.. RE. 1978. S,.."mk.....rocyt<>oi. and ......<>oit ... ca~ Rrmit.;"'n aft .. opkn«Oi. in 16 dop.) y" In"", Mod h7S-SO. 55. Srodtb.m SL. &..1 DL. Sdomidt DA. 1986. M .. ""ytnoi. iD dop..,.job KO" infI""",.",.". diocw
104

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

S8. ,\1"M.",.. PM. 1m. F"'I"'D<J' and ..... nty of .... ""'Y' ...... in doc • ..itlo. and ..itl.ooo, mt" cdl """"'G120 """. (19'91- 1997). J Am Vot Mod hOD< 2IS,JSl-J57. 59. F.. .....d .. Pj, Modiano JF. Wojci.."1" /' Tbom .. JS. B.noon FA. Sonith R HI, A"""f AC. B.""" Re, Boo..., U, Jok ...... Me. !'i,,,,, KR. 2002. Uo< oftb, CrJl.Dp. 3500 ... pmdkt kuk....k «H ~ in p<';pl.,,,l bIood<>f doo", and cat>. Y" Clin P.oboIjbl67_ 182. 60. Go ..... KA. C .. ako_ Me. 198• . Elf"" of EDTA on mo

oIoa of n...-"h.iI. oH ..ltby doc. aod doc • ..ith i<>llanuaatio ... Y.. ain r.d.oI. l},2Z--ZS. 61 . Arod. I, KJ.m.nt E. s.z...G. lOOS. Oininl, bi.od.....ico.l, and ....... toIockal cb"act..iotia, di..... P'~ and """""'" of doc. I"",.. oti"ll: ..ith D""roph.il p«O f""", EJn./NhU ...."pi and ~ ",...u. I aiD Mkrobiol 33,2746-27t9. 70. M......" RF. O"""IH . Roa D. R...J KD. p"",,= M. Bjo<.odop«W7iD, EJ..iNhU "!.; and hu,.."" uanuJocytic d..lichiooi • • , "" iooI.,eo from No.""'" Calihnia. I aiD MiaobioJ )8,1J.64-1l69. 72. yinc.nt·I""""'" NA. M.cin';'" DK. UDwn B1.. 1m. A n"" H _ """". from doe~ Deocriptioa of th, co .... ti'" "C of =in< H,, ___ .pccieo from &nil. P•...itoI R... 97m - 9}. 75. P.Judo GR. o.ll·Porto A. "" Cutro , T .iDdad, AR. M"M"" ... C. Fdcd,.."" H. 200}. H".,...... . pp.' Rq-, of '00" "'"'" in d.np in B... ru •. B.ci[. Yo< P.",i"] IIS,M}-2ts. 76. F......... MD. T.ttri .. KR. Sce6,[dA. Eli"i C. YotoL. KS . F,.,.......,. KR. Unl..", GF. E...in, SA. "t.-ud C1.. 2007. Mol","" do"",,,,.u.tion of H" ...""", '1'. from B.uili ... doe. and it. phylo~nrtk "Jatio",hip .,j ... <>tho. H,_" .pp. Yo< P....itoIltS,21_JI. 77. Rub;"; AS. do. Son"" Paduan K. P"" RR. RiboUa PEM. 0·0..,., LH. 2006. MolocuJa, doanoct<>i .. tion of fdino H,_" op. SJ . G ...... fooD TL. R.od CM. lo"li: TM. !>kG..,..,. PB. P'pp" MG. 19S4. Cutancooo. lei ... ,.....;.... OO<J...i
,,,,all,

c'

2/ LEUKOCYTES

105

Bj. Ban SC. v ... &ok 0 . CUUdo-N........ MS, Lop..,JW, IG.d.I.offLV. AUi"", N, l.a;.c A. at om ... f....... p.ppy. V" Clin P.d.oI JI,9-12. B7. Fa,.. R. 1m. M.ltipIiwion of s..>'«<]"u ••""""'is in ob. ""';n. blood."...... ) P.... iool6j,980-982. 88. Fa,.. R, Lc.k RG. 1979. s..""""is t ..n....itted b, bt.-J " • ...ru.;"", ) P.....itol 6j ,890-89J. 89. O'Conn,,, SM, Sh.rui KA. Lo",. GL 19'91 . Spwi.ow nnnropol.oO< and bioc.I.....iad anal"; •. V" P.d.oI 26,114-120. 97. K<>....kr SD. Pi=< KR. Ba, WW. 1978 . Clinia.1....t biod..miaJ .boonnaliti" in p<> In"", 16o }9--4'. 100. y......., 0, M ... ~S, Tab" K. U"oub. K. SatoI.. H. Sitoda T . Baba y, Y............. A. Kat<> K. T.bb .. ki K. Y.........ki M. Nab,..... H. Doi K. M.edt Y. Or.... H. 200 • . G",rvncli.ooid.ooio ...i... , 0 (SutdItolf·1dr dj ......) in • family of Jap""'" dom"tic ca ... V" IlL.o I jj,7J9-744. IO! . Hi..,!.. VM, Cw.oin ~ .... TA. 1984. H .. edit.., ....,malyof ..... tt<>pI.il "",oJ.tioo in BitnuncatL AmJ V" It.. 4j,217{1-217". 102. Sk.H RM, H,1man RG. Spdlal!7--49S. 10J, H ......... ME.~, GD, p,k PF. D....it.k RJ, P.... ...,.. DF. 198J. n, patl.olo", ofdtt klint modd of m"""polpa«h • .ido.i. I. Am) P.tl.oI. IIh27- J06. 10 • . Ellin......,.j NM, W."I!: P. Slotn T. Sb..p NJH, Ct.taM, D.ch. S, Ed ....... NJ, Bublo, I. TltompoonJN, B...b W. Hacd.m E. H.oki ... ME. G iu< U. 2OO}. A modd of m""""""".cd",ido, i. lllB (Sao/iJipp<>.,..Jrom, '1P" lllB), N~I-«-D -duooo aminid .. , ddKi..ncy in Sdoipp"kt dop. J I...... it Mrtab l)j, 26,.(89-SOol. lOS. H ......... ME. Jnyk PF, D. ..... Jy.....d.."idooio typ< lilA in N", z".Jand Hunta",,), dor; •. G"",m;'" 79, 15O- IH. 112. G~ Ye. Ba.ttTRJ,. A.chnaano MR. Smith CWo Kd..~ ME J., S - MF, Hi.tbtdn DD. 2004. T!.. "ntcic immwooddici
T., . . _ . . . ;

K.oba,...,.

'00

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

1[J. D ............ 51. MilIinp><> A. Kij .. j, And..""" 1. Bi",,, M . 2002. c..,j", J,~ ~ d.&iW0< ... in oir/rt affected ...im.~. V .. Q 160Zl- J}. 118. c...&1d PI, W"""" AD). 1989. Invacl",tion. ,,(boo, man<><' dy."",ia in • poodk ..ru. moao';'. J Comp P • ....,] 10 [,269--278. 119. Fddman BF. Ram .... AU. 1976. 10, Pdc .. -Hui't ....,maIy of p .. ulocy
""p

},ll-- }O.

120. L.tim<. KS, R..wl.od GN, MahaIf.y MB. 1988. Ho"""'n""" P.!&<._Hu" ..."m.1y and d.",ndrod,..pl .. ia in • •ciUbom ki"6I_n. 125. latimt. KS. Duoca., IR. Ki.d.t. 1M. 1987. Mo

c and qroc.l., .. i.try ol P~ •. H"" blood ctlb. Y" Clio P.dooI. 16,9 (abo,,_). 126. Od..m BI. Gltnn BL 196I!. Acq";'od PdC .. ·Hu1t ....,mat,. in atd.. I Am Yrt Mod A.ooc 15H 1_ 16. 127. T oob SR. Ooioo. DE. I."," O. 1986. H;"opatboJocia.l and ~",,,.toIo&iaJ Iiodinp ;0 mydooid !tuk....;. iodocod by ....... ftlint !tuk....;. vh. ioolatt. y" P.dooI. 2},t62-470. 128. 10"" RF. Pui. R. 196J. ptybowtd oooioophiL I Small Anim Pna .. (s...PI'~,l9-JJ. 129. luhik MC. Co.to CG. 2005. M""pI.oloVc doauctcri .. tioo of optdlK pan.lrt in prybowtd ",, ';oophi~. Y" Oin

c...n..

n,

P.""'] )4140-1t}. I.J.O. G=n6,[d CL Mo.. id.IB, Soh .. PF. Sdud[" OJ. 1999. latkoptoi.;o'" b"W.y Bc]~ T ....... ,o. I Arn Y" Med A.ooc 21M 121_ 1122. 131. T..,. GC, Y~rt Ie. T "",ovakl GH. 19S0. c...;"" ,~didtio.i " A """tpt e. B...... v. lutt H. 1997. Ubo"to'l' findio ~ . in ="'. an.. ap«immtal ;ofectioo ..;t!t ~;"1''''''1' '~1... Clio Di.~ Ub 1......""14,&B_6t7.

Chapter 3

ERYTHROCYTES Physiologic Processes ... ... .. ... .... ... .. .. .... . ..••• •• ... . .• •••• • ••..... .. 110 Ana lytical Principles and Methods . ... .... .. .. .... . •• • .... ... • •••...... ....... 120 I. Complete Blood Count.. ... .... . ... .... .... . ... ... .... . ... ... .... .. 120 II. Erythrogram ....... ... ... .... . ... ... ..... . ... ... .... . ... ... .... .. 120

III.

Methods ... ... ... .. ... ... .... .. .. .... ... .. ... ... .... . ... ... .... .. 124

Morphologic Features 01 Erythrocytes: Clinical Significance and Pathogeneses .... .. I. Assessment. ....... ... ... .... .. .. ... .... .. ... ... ... .. ... ... .... .. II. General Features.. .. ... ... .... . ... ... ..... . ... ... .... . ... ... .... .. III. Erythrocyte Color ... ..... ...... ..... ....... .... ....... ..... ....... IV. Erythrocyte Org~nlSrTl$ •........•••........ •••• ...... ••••••• ....... V. InclusloM O1her Than Organism•........... •••• ...... ••••••• ....... VI. Abnormal Erythrocyte Vol ume ... ..... ..... • •••• ..... •• •••• ••....... VII. Abn ormal Erythrocyte Shape ... .. .. .... ... . •• •• ... .. ..••• • ••.. ... .. Anemia ... ..... ....... .... ....... ..... .... ........ .... ... • •• • •••• • ....... I. General Information .................. ... ......... ... • ••••• •...... . II. Classif ication. of Anemias .......•••.........•••...... ••••••• ....... A. Classification by Marrow Responsiveness ............ •••••• • ....... B. Classification by Erythrocyte Indices........ ... .... . •••• ••• •. ..... . C. Pathophysiologic Classification ... .... ... .. ... .....•••• ... ... ... .. Nonra-geoneratiye Anemias ..... ..... ...... ..... ....... .... .... •• ••••• •.. ... .. I. Gen eral Concepts ...•••........•••.........•••...... ••••• • ........ II. Disorders That Cause NonregeneratiYe Anem ias •........ ••••• • ... ... .. Blood loss Anemias .. ....... ..... ...... ..... ....... .... .... •• ••••• •.. ... .. I. Causes of Bl ood loss ... ... .... .. .. .... ... .. ... ... ..•• • _ .. .... ... .. II. Classifications Based on Du rati on and location ....... .... ....... .... .. Hemolytic Anem ias ..... .... ....... .. .......... .. ......... .... ....... ..... . I. Concepti and Classifk:lItion•.....•••.........•••........•••......... II. Hem olytic Disorders and Disease•...... ...... ..... ...... ...... ..... . A. Immune HemolytK: ArnHTlias INot Associllted with Infectionsl ... ..... . B. Infectious Hemolytic Anemias . ... .... ... .. ... .... .. .. .... ... ... .. C. Erythrocyti c MetabolK: Defect. (Acqui red or Inherited) ... ..... ...... . D. Erylhrocyte Frllgmenlati on in Blood Cre~ting s<:hilOCytet, Keratocytes, or Acanthocytet ..••...........•........••..... . .... E. Hemolytic DiSOrders of Other or Unknown Pathogeneses ........ ... .. Erylhrocytosis and Polycythemia .. ... .... .. ... ... ... .. ... ... .... . ... .... ... .. I. Tarms and Concapts ..... ...... ..... ....... .... ....... ..... ....... II. Erythrocytotic Disordert and Conditions .............................. Other Erythrocyte Di.orders ...•••........•••.........•••........•••......... I. Methemoglobinemia ...... ..... ...... ...... ..... ...... ...... ..... . It. Cytochrome-b, Reductase ICb,R) DDficiency ... ....... .... ....... ..... . III. Familial Mathamogl obi nemia Associa ted with GR Deficiancy in a HoflUl ... ...... ...... ..... ...... ...... ..... ...... ..... ...... . IV. Hereditary Stomatocytosi • ...... ..... ....... ... ... .... . ... ... .... .. V. Hareditary Band 3 DDficiency in Japanase Black Canle ...... ... ... .... .. VI. Harooitary Elliptocytosis of Dogs Due to Protain Band 4.1 Deficiancy .. ....... ... ... .... .. .. .... ... .. ... ... .... . ... ... .... .. VII. Elliptocytosis in a Mixed-brood Dog Dua to Mutant Spectri n . ... ... .... ..

135

135 136 131 138 138 142 143 151 151

lSI 151 153 158

159 159 160 167 161 161

110 110 116 116 181 186 191 191 193 193 193

198 198 199 199 199 200 200 200

101

108

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

VIII. Speclrin Def iciency in Dutch Golden Retrievers ........................ IX. Megaloblastic Anemia ............................................. X. Siderobl aslic Anemia in Dogs ....................................... XI. Distemper Inclusions in Dogs ....................................... Laboratory Methods for Assessing Iron (Fe) Status .............................. I. Serum [Fe ]. ...................................................... II. TIBC and UIBC ................................................... III. Percent Transferrin Satu ration (% Satu ration) ......................... IV. Stainable Fe in Macrophages of Marrow, Spleen, or Liver ............... V. Serum Ferritin Concentrations ...................................... VI. Reticulocyte Hgb content (CHr) and volume (MCVr) .................... VII. Comparative Fe Profile Results ...................................... Blood Typing and Crossmatching ............................................ Methods for Detecting Erythrocyte Surface Antibody or Complement ..............

201 201 202 202 202 202 205 206 207 207 208 208 208 211

Table 3. 1. Abbreviation. and .",nbols in thi. chapter [xl x conctntration (x _ analyt~) 2,3_DPG 2, 3_ Diphosphogl~rate AID Anemia of inft.mmatory di..,~", ATP Adenosine triphosphau Be Conjugatffl bilirubin Bu Unronjug. tM bilirubin C3 Complement protein 3 Cb,R Cytochrome_b, rffluct= CBC Complet~ blood count CH Cell (corpu.cut.r) hemoglobin content mc-an CHCM Cell (corpuscut.r) hemoglobin concentration mean CHCMr Cell (corpuscular) hemoglobin concentration m... n of rC"ticulocyt.. CHDWr Cell (corpuscular) hemoglobin distribution width of rcIiculocyt .. CHr Cell (corpuKllt.r) hemoglobin content mc-an of rcIiculocytes CRP Correctffl rcIiculocyte percentage DEA Dog erythrocyte anti!:"n DNA Dfflxyribonucleic acid ECF Extracellular fluid EthylenffliaminC"tetraacttic :ocid EDTA ElAV Equine infectious anemia virus Erythropoietin E"" Erythrocyte ,urf.ce4!.!OCi. tM immunoglobulin ESAlg FAD Flavin .d~nin~ dinudrotide F, Iron, eith.. Fe'"' or Fr' Fo" F~rrous iron F~rric iron FeLV Fdine leukemia viru. G6PD GluooH_phmph. te dehydrogenase GR Glutathione rffluctase GSH Glutathione

F,-

H~moglobin con~lIIration

HC HCO,·

Bicubon. te

Hcr

H~matocrit

HDW HDWr

H~mog!obin con~lIIration

distribution width H~mog!obin con~lIIration distribution width of rHiculocyt .. H~mog!obin {iron in F~l<- st>t<}

H,b Hgb_F'" Hgb.....

19A IgG IgM IL-, IMHA MCH MCHC MCV MCVr

NADH NADPH

NI NMB nRBC p.co, p.o.

PCR PCV PFK pH

PK RBC RC ROW ROW. RMT

RNA RP 51

nBC T NF UIBC WBC WRJ Not<:

lin individu:d ~rythrocyt ..)

~

M<'th~mog!obin H~moglobin ddt. (meas,m of fr.., plasma [Hgb] by ADVJA) Immunoglobulin A Immunoglobulin G Immunoglobulin M 11II~r!ftIkin I, 6, 8 Immune_ndi.ted h~molytic .n~mi . M~.n cdl h~mog!obin Mean cdl h~mog!obin con~mration Mean cdl volum~ M~.n cdl volum~ of rHiculocyt .. R.ducM nicotinamide .denine dinucleotide R.ducM nicotinamid~ .denine dinucleotide phmpb.te Neonatal i""'rythrolytis New m<'thylene blue Nucle. tro erythrocyte Partial pressure of carbon dioxide in arteri:d blood Partial pfessur~ of oxygen in .nerial blood Polyme""", chain reaction P""kal cdl volume Phmphofructokina.. - log [H+] Pyruv. te kin ... R.d blood ~(( l~rythrocyte) Reticulocyte roncentration R.d ~(( distribution width R.d ~(( distribution width of feticulocyt .. Reticulocyte maturation time Ribonucleic acid Reticulocyte per~mag~ Syst~me [nt~m.tional d'Unite. Total iron_binding a1pacity Tumor necrosis factor Unbound iron_binding a1pacity White blood ~(( (leukocyt~) Within the r~ference illlerv:ll

.... ligwe kg.., ... fo, . bbrrullions d..t

.."

unique to tt.. ligw ..,

109

110

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

PHYSIOLOGIC PROCESSES I.

Erythron: all

~rythroid

cdls in an .nimal, including pr«ursors .nd

~'Yd"ocyt~.

in blood

vessds and sinu.= of spl«n, Ii","" and m:urow (Fig. 3.t) A. Erythrocyte p=:unon I. Erythropoiesis is p:m of hematopoi ...is, which is a romp]'" s)"um inmlving stem ""US and cytokin.. {Itt Fig. 3.1}. ~eral cytokin.. work .ynergisticolly with Epo to nimulote th. rq>]icl.tion and diff.rentiation of blast.forming unit--.,rythroid (BFUE) to rommiuffl stem ""US (•. g., mlony_forming unit--"'Yduoid or CFU.El, which respond to Epo eith.r by dividing or by diff... ntiating toward rubribJasts. Epo inhibits apoptmis. 2 Epo is produ=:l mosdy by th. f=] Ii"", and th. adult kid"q.''' IkruoJ ptritubul>r interstitial cells product Epo in =pon .. to r.nal hypoxia. Renal hypoxia moy ~

Fig. j.l. &ythrocyte lWmia in h..Jth: Tho erythron con";", .!uN m. jo, pools: erythrocyte prerurso" (mosdy in marrow), blood erythrocytes, md .p!.nic .J}'throcyt.. , Afrer .timubtion by Epo, colony_fonning uni.--"rythroid ""Us (CFU_E) differ. ntia.. into rub,ibb.", (Rb) md th. ptKUl5Ol'S prolife .... (vi:. mit"'is) and .... tu'" until erythrocytes (E) ... fo,mod, An ord.rly maturation P'DCe.U produces a pynmidd distribution of .rythroid ",U pol"'lations (only th.e top half is .mown), rele ... to th. blood, erythrocytes circuh .. in tbe v=u~ system.o ttmsport 0, to tiss ...., A resen'< pool of erytbrocytes is ""'I"",..nd in th.e ,£,leen of mOlt mom"",h, So .... ",nt erythrocytes"", d.stroyed by macropboge', M
Aft.,

111

3/ ERYTHROCYTES

cau,t .. into a rubribwt, two major cdl proce.... oo:ur, a, Cell. undergo mito ... to produce mor~ and .maller cell., Evidem,e suggests th at at [Hgb] increases in th. devdoping cell, D NA .ynthesi. deerea, .. and thus fewer mitoses occur,' b, Cell. produce m.... nger ribonucleic acid (mRNA) for .ynth..i, of Hgb and cyto,kdeul prot~ins, 4, [n th~ J.t~r .tages of mammalian ~rythropoi .. is, a metarubricyu nucleus is extrud d.teriorate 50 that glycolysis becomes the ATP_ producing p:llhw:oy in moJi aniJrulI, (ncq>t pig., which UK .n alternat~ ~n ...gy pathw:oy involving ino.in~), B, Blood .rythrocytes L Major feam ... of ~rythrocyt .. in differ~nt .peci....~ rompar..! in T.bl~ 3,2, 2, Erythrocyt~ destruction in health (.. nescence) a, About 250 million .rythrocytes die ptr hour ptr kg body weight, b, Old erythrocyte> han nry linle m.tabolic m achin.ry (~nzym.. ) to k... p them_ .dv.. functioruol and deforrruoble, N~ar death, they may become more rigid, ,pheroid, and I.... e>pable of passing through sinu",", c, Age-rdat that are bound by naturally oo:urring antibodies that may mn be within its spl..,n, Damaged or less deformable erythrocyt.. are ",moved by m acrophages th at ar~ adjacent to .inusoid. or pr... nt in th. red pulp, Wh~n .plenic contraction occurs, ~JYIhrocyt...'" forced into the 'ynemic blood,

Table 3.2. Conlpadson of typical blood erythrocytes in ma ture bealthy animal. Do~

"

Reticulocyte concentration (x lO'/lll) RBC concentration (x lO'/lll) 7 RBC lif~ .pan (d) '00 RBCs in blood' (x 10") 5.6 RBC diam.ter (j,tm) 7 RBC volume (IL) 70 RBC central ~allor Promin~nt • Counting only oggregat< mirulocyt .. • E..,im ..... ore bosed on 40 ml bloodllb body _igb, ond Not<: All numb. .. ,ef...""" int
repffSffi{

em

.,.

Ho=

,

Cattle

7- "

9

7

,.,

,SO

,SO

70

6

."

4S

4S

Mild

5-"

Non. to mild

,

'"'>-6

"

Mild to moderat.

ovnag<"iz.ed rnimili, .pproxim. t. ovnag<' to illwtr::lt< similari,;'" ond diffe",nceo, Tbey ... fK)t

112

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

2 Cal 'pi,""", are thought to ha"" cloud circulation (blood d.,., lIot How through rM pulp), which is l~ .. efficient at removing damagw erythrocytes. Th1 for caul.

n.

Erythrocyu kin<'lia A. A blood [erythrocyte] js estabJish«i by the ,dati",

rat .. of ''Ydu''''ytr production, ,hjfting of erythrocyte. to and from splenic ,inl1Se'l, .nd erythrocyte destruction. B. Erythrocyte production d'p"nd. on Ih. degr.., and duration of Epo nimulus and the copability of pr«ursor cd], to respond to Epo.

III.

Hgb: strucru.. , function, .ynthesis. and dq;radu ion A. Hgb structur. I. Hgb is a ["'ramer with globin link'"'" to • """rate heme thaI bind, 0,: its ,d.t;", mol~>r DU.<S is n""r 64,000. Globin. are polYp'plides and, in maCU"" h~~hhy m:mun~h, rach Hgb mola::ul~ contains two Cl..dl~ins ~nd two p-chains. Approximatdy 95 % of.n erythrocyte is Hgb on a dry weight basis. a. Rat .. of synth.. is of heme .nd globin a" bal:m=:! and r produ"", .bnormal amino .cid "quen"",. in the globin" an .nimal has a hemoglobinopathy. 2 F~ in heme is in the fe"ou. naU (Fe'"') and is kept in the ralucal S{ate by eITZY" m atic "'~ction> cotalyud by C~R (NADH .. methemoglobin ralucta .., commonly coiled juS{ methemoglobin ralucta ..) :md NADPH diaphor>" (NADPH .. methemoglobin rMuctase). 3. Amino acid. in the globin ch.ins :ore m.imainM in • rrou=:! S{ate by rMuai"" "'action> involving GR and catalase. B. Hgb function (Fig. 10.3) l. Hgb (with F~") tr>nsports 0, from lungs to tissues. In health, Hgb i. ]00 % satur>tro with a, in an ..ial blood. Hgb .. F~ does not tran>pon 0,. 2 Hgb plays two major rol .. in the tr>nsport of CO, from ti<su.. to lungs. a. When CO, diffu ... into erythrocytes, corbonic anhyd .... cotalyzes in reoction with H,O to form H+ and HCO,- . Hgb xts as a buffer (H + + Hgb- --> HHgb) to r~mo"" the H+, and HCO,- diffuses from the "",Il to the pl:uma. The buffer .. ing of H+ by Hgb f.cilitat .. the additional conversion of CO, to HCO,-. When erythrocytes return to lungs, ,,""tions .re "",ned .nd CO, i. rdeased for expiration. About 70 % of the CO, formed in tissu .. i. tr>nsportro to lungs vi;, this system. b. When CO, diffu ... into erythrocytes, ",me binds with Hgb to form carbamino .. hemoglobin. About 20 % of ch~ CO, formro in ti ....... is tr>nsportal to lungs vi;, this system. C. Hgb synth..is l. This occurs in erythrocyte pr<cursors (rubriblasts through reticulocyt .. ) in a .. ri.. of "'action> (Fig. 3.2).

.am

3/ ERYTHROCYTES

113 Hemoglobin Synthesis in Erythrocyte Precursors

lhci>o • succinyl GoA

5.ALA synIhilse & Be

protopo
5 porphyrin maclion. protopcxphyrin IX • Nt"+

..

• '-"" porphobiioogoo n

--

&-ALA • f>.ALA

• • • •

~

4 Iorrihemo!I • 4 globin.

metoomogiobin

(... ';home

methemoglobin hemoglobin

Hemoglobin Degradation in Macrophages

,,,.

bil.......an

__

bilirubin (ooconjuga!9d)

00 Fig. .}.2. Hemoglobin 'ynth ..i, and degr.od.tion . • Hgb 'Y"thesis in etytbrocyt< pu. 'Y"tbe.;' of Hgb hOI thrN major ' '''ge." (t) a ..m.. of porpbyrin r",ctions. (2) incorporation of F<'+ into protoporphyrin IX to form 1>."",. md (3) binding of fow ferrihomo and four globin mok.moglobin . • Hemoglobin d.gr. d.tion in mocroplug .. : In l>..Jth ... n<>e and 1>."", i, 'plit from globin cboins. H."", i, dq;roded to bilirubin. F.'"'. rnd arbon monoxide (CO). The globin cboins "'" dq;r>
:ocid .ynth ... i, th~ mojor rat._limiting ~nzym•. It ..-quires viumin B. (pyridoxin~) :as a cofactor. and i. inhibit"'" by ~ater h~m~ cona.ntration.. 3. 5_Aminol~vl.tl~nic :ocid .ynth .... porphobilino~n .ynth..... fwochd . u ... and roproporphyrino~n oxida .... ~ inhibit"'" by l~.d. a. In l~ad toxicity. inhibition of th= ~nzym .. lead. to gr~ter cona.ntrations of hem~ p.. cunon in erythrocyte •. CoII«tivdy. porphobilino!:"n throllf:h protopor_ phyrin IX m coli"'" porphyrim. b. POrphyrill. which i• • condition in which rona.ntr;Uions of porphyrins in ~rythrocytes, plasmo. or mine are inc ......""'. em be acquir"'" (a. in lead toxicity) or con!:"nit"'. Anim:ol. with porphyri<> a.. prone to photosensitivity. 4. The mammion rat~ of erythrocyte pra::ur.;or.; i•• ffect"'" by [Hgb] in th~ir cyto_ pl"'m .. If Hgb .ynthesi. i. incompl~u. addition. 1 mit""'. during a a.1I·. d""dop_ ment will produa. .maller ~rythrocytes.' D. Th~ IfP" of Hgb synth~.
5_Aminol~vl.tl~nic

114

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY E. Hgb d~""atjon and bilirubin m~aboljsm: Aft~r erythrocyt. dtlth in macrophage< of 'pl""n, liver, or marrow, Hgb i, dq:,..d.d to Bu, amino .cids, .nd F. (Figs. 3.2 and 3.3). Bu and Be a.. ""creta! or dq:;r:odal. wh...... Ih. amino .cids .nd F•• '"

r«yd.d.

i

i

HIIJ"'tocylo ~

'"

.1

• •

~"

~

•

~

• •

I

'''"

'.

, ,

Fig. .l.3. Bj~rubjn "",..boIism. • In h..lth, rJY1hrocytr d .. truction in moaoplug .. of .pWn, ~,..r. 01 IJUlrow <=.II" in Bu fonn1tion. SrrWl and wwJly clinically insignificont >moun'" of Bu "'" formgr. it fonns 1 nonrov:olm, :ISSOCiation ...itb 1lbwnin:and i.s tnosported to hopatocytes. Bu is «btivoly ........ insoJubJ. prior to binding to albumin (Alb). • Wb.n Bu .,",," tho Ii"", ond it> protein-p'""""bJ. ,;nuses, it probobly bin,h to hopat<>toJ'll of th. orvnic .mon tnnspon po/yptocyt'" .. i,bout .tbumin. and binds to y.protein (livndin, .!so c.JLed g1utothion< transr. .....) or z..protein (f.ltly ocid-binding protein), Bu probably ~t.". t..potocytes by 0 p ... ivo but fxili....d procou: binding proteins ~hIDC< tt.. pr0<J<S5 by '«lucing the dIIux of Bu bock to tho unwoid.J plasm .. • Witbin t..p.t<>, Bu dif'fus.. to the .ndoplaunic , .. irulum, it is conjugoted .. itb glOCllronid. (> 60 %..ith glueos< in bo,..,,) to fonn bi~rubin monogluooronide or bi~rubin diglururonide, o>lt..:· tively c.JLed Be, It tt..n diffuseo to the con.Jiru.b, membnno, • Be is mmpolt
whe,.

M"

3/ ERYTHROCYTES

[v.

11 5

F~

A. Total body F~ i. distributM in thr,"" major .ites in h..,.hh: (I ) about S()....70 % in ..yd"ocyt~ Hgb. (2) about 25-40 % in 'to~. and (3l the r~maind .. in oth~r mol",ul .. (~.g .• myoglobin. cytochrom... and ~nzymes) .' B. Physiologic proc= or con"",pts (Fig. 3.4) C. Th~ inl<stinal ab"'rption and cdlular rd~as<: of F~ are regulatM by h'lxidin, a ,mall pq>tid~ hormon~ that is synth~.ized primarily by h~patocy"'. Canin. hq>cidin hal a mol"""' .. muctu .. ,imilar to that of human h~pcidin. · Wh~n hqx:idin bind, to m~mbran~ ftrroponin (a prot~in involvnj in F. ""pon fiom v~rtdJrat. cdl,) on the basolat~ral m~mbranes of smaU intestinal villu. ~nt~rocyt .., th~ f.. roponin is internal_ ized and the "",U los .. its ability to ""pon F. &om the cdl to th~ circubtion. Aocumu_ latrd intraa:lIul.. Fe inhibits th~ ""p.... ion of divalent meul transport~r 1 and f.rric rrduct"",, (duod~nal cytochrom.. b) on the brush bord.., '" F. absorption into the a:ll i. alro d"'.......:!. Th~ .ynth~,i, of h.pcidin i, influ~na:d by hypoxia, F~ availability, and 11..-6.' I. Wh~n h.patocytes d~rct hypoxia, th~ d",re.. rd formation of h~pcidin causes a low.. [hq>cidin] and thu, inc.....,. the a""ilability of Fe for erythropoiesi'; that i., the incr..,....:! mov.m~nt of Fe from .nt~rocyt .. to plasma and increased rde... of F. from macrophages to plasma through a simil ar m",hanism. 2. F. loading by di~ or tr.",fi..ion inc...... the formation of hqx:idin. wh~r..,.s Fe d.ficiency d"'r..... ' its formation. 3. During inflammation, cytokin.. a.. rd~...d, including 11..-6. 11..-6 promot.. the 'ynth~si, ofh.pcidin; it bind. to ftrroponin v..hich is th~n internalized. Thu, the amount offerroportin . ""ilabl. for the ""pon of F~ from a:lh {including macro_ phages} is reduced. Thi, process tends to 5e<Juest.. F. in macrophagc:., and thus it is I.... available for ~rythropoi ..i. and for inf"'tious agtnts.

v.

erythrocyte m~abolism {Fig. 3.S} A. Glucose i. the major .nergy sou,""" for matu .. erythrocyt.. in most .peci... B. Pig ~rythrocyu, lad: a functional glucose transpon~r and usc: ino,ine inst..,.d of gluco ...

VI.

Reticulocyt .. A. Rni",l~ryln are nonnucleatM, immatu.. ~rythrocyt .. with stainable cytopl"'mic RNA l. When ~rythrocyt .. a.. incubatrd with NMB prior to th~ making of a blood film, the NMB precipitat...nd stai", the RNA .nd mitochondria to yidd punct.te or r~iculatrd {hena: the term rttinou,cyu} Wsophilic muctu ..,. 2. On a Wright_suinrd blood film, the RNA will givc: the polychromatophilic erythro_ cyte ito blue or basophilic tinctorial properties. Not all reticulocytes have: enough RNA to be d~rctrd on a Wright_otainrd blood film. B. Th~ lif~ 'P.n, of circul ating reticulocyte! of non:memic dogs and ptopl~ ... 1- 2 d, aft .. which the a:lIs a", matu", erythrocytes. Th. life 'pa", of cat reticulocyt .. are mo .. ""riabl•. [n health, cattle and hoIUS do not havc: circul ating polychromatophilic erythrocyt.., because maturation is mo .. compl~te in the bon. marrow. How~vc:r, some hematology analyu", d,,"'t low cona:ntratio", of .. ticuloc),! .. in hor..... ,. C. T YP'" of reticulocyte! l. [n most speci.., :my nonnucl..,.trd ~rythrocyte th at conuins RNA {stainable with NMB, oth.. vital stai"" or Wright stain,} i, callrd a rmruloryu.

M a tur~

Blood

Iorritin

8

F ig. J.4. "" kinetics in healthy rnim .... • Absorption: Dim of domestic mamau/. may con";n F." 01 """. lngest«l ~ it convort«! to Fe" by f<' d.y i.s • wry ",wI p<'''''DUg< of tot1l body Fe sto= . • T n mport: Ne. rly all "" in pl .. ma i. bound to apotr:onsf degr>OUnts of stored Fe . • Frrritin mrui.ts of :opoferritin oompleud....ith J;,l':md i. • "'htivrly ",lubl<:. mobil<: "'ure< of ~. Tbrre :or. =ter:d fornu of :opoferritin b.c..,.. of .....ious combin. tions of H or L SIlbunits. Ph""" ferritin i. • glJ'<'O'Yhtrd polym<:r th .. i. ",l.. ivrly ~ poor. Ti.n.. ferritin. whicb i. nonw",ybted rnd rrt.tiv.ly F.: rich. is prodoced by mrny cells. prirrurily lJl1croph>g"'. b.". ""'Y""'. in,,,.. in:d muoooal rpithe~:d «lis. and erythroid pr=,.. Synthesit of .pof i. incr"""! by in81fturl1tion {. pofrrritin i. . positivr . cuto-ph ... pro",inl :and when F. sto .. ge i. incr......! . • Hemosiderin it .r.bti""ly insolubl<:. poorly mobil<: .ou= of F.:><- rnd rep .... nts tbe m.jor stonge form of F.:. Hemosiderin i. . compJa of protrin :and F.: o:tides tlut i. found prirrurily in l)"OSOmet of .... cropb~ in the spt.:..n. ~""r. rnd tn1frow of moot tn1ffim .... A b.. ltby cot'. tn1ffOW d .... no. b.... enougb hemosiderin to h< detKtrd by routine .uiniog methods. • Tist ... form" A .. btiv.ly .mull qU1fltity of F.: i. prrsent in myoglobin. "'t:oWe. pe=id ..... rnd cytochro ......

,i...,..

"6

3/ ERYTHROCYTES

117

2. [n em, miculocytes stainal by NMB may k d:wifial by two .yn~ms. a. Th~ mo .. commonly u~ .yn~m diflrrentiates .. ticulocytes into punctau .nd aggrq;at~ forms." The deg= of polychromasia =n on • W right_suinw smeor tend. to corrd. t. with th~ aggrrg.te RP." {I} Punnatr: ~lls with two to six small granules of miculum {2} Atu'gatr: cdls with large :.ggregates of reticulum b. Ty"" [, II, and 1lI reticulocyus lJH {I} Typ' I (lightly reticulatw, oldest form): Cd], a.. uniform in size .nd nain light gr,""n with faint blu~ nippling. Th~ir circulating life span is 3 d, and the maximum con~ntration ocam about 1()""12 d .ft~r the on,.t of anem .... {2} Typ' II (mod~r.otdy reticulmd): Cd], vary som.wrult in siu and stain light gr,""n with large dark gr.nules. Th.ir circuloting life span is .bout 12 h, .nd th~ maxim.l con~ntration occurs about 4 d .fter the on~ of anem .... {3} Typ' III (heavily rrticulat.d, youngest form): Cd], ~n ..ally are larger than nonmiculat.d ~lls and have blue-grttn cytoploum .nd a h~a"Y dark blue granular network. Th~ir circulating life span is 12 h, and the maximal cono;,ntration occurs about 4 d aft .. th~ on..,t of anem .... D. Reticulocytosis: inc......d RC, CRP, or polychromui. I. A rrticulocytosi. i. th~ best ..,miquantiuti"" .viden~ of increas.d erythropoi..i. (in .prcirs oth.. than horses). a. C,ttlr: Aft~r .cute blood lOll, a reticulocytosis is rx""cted within 3-4 d, and ptak production is rxprcta! in 7- 14 d. A f.w l .~ or shift .. ticulocytes may k s,""n in the blood kfore • miculocytosis is p ....,nt." b. Dogs: The ""pon.., .ft~r .cute blood 10.. i. not cl~.rly establishal but is g.nerally rq")fiw to mimic th~ bovin~ pattern. Som. report th.t the """k r.. pons< am k =n prior to 7 d. c. c,u: The arut~ removal of .rythrocytes by """,. ta! phl.botomi.. to cr .... te • Hct of 50 % ofb...,lin. in five em resulted in the following:" {I} An aggrq;ate miculocytosi, occur=! by day 2, ptaka! at day 4, and murn.d to bastline by day 9, "",n though the alU we .. nilJ .nemic {Hct in the low 20s}. {2} A slight punctate rrticulocytosis wa. pre.. nt by day I . • ptak miculocytosis occurral from days 7 through 14, and punctat. RP did not .. turn to IuKlin. until aft .. Hct .. turna! to p.. phlebotomy values (aft.. 3 wk). d. Horses: They very rardy ha"" circulating polychromatophilic erythrocytes. The clinical valu~ of .utomata! miculocyt~ cono;,niratiollJ in a..... ing equine anemias is in""nig.tional. 2. Th. drgrtt ofincr......d polychromasia .hould correspond to the deg= of rrticulo_ cytosis in dogs and catd. and to aggregat~ miculocytosi. in cats. VII. Blood groups or typ.. A. G.n.ral cona-pH I. Erythrocytes can k groupal into diff~rent blood groups or typr". ba..d on the pr=no;, or ab,.na- of .rythrocyte .urfa.cr antigens, which are som.time callw

G""'~

ATP~HK

ADP --1

GM

HMP Shunt G6PD

PGD

-------';!';:C- G-PG

l ATP~:K · I i G-3-P"'---~-3-P eo' t== .",. u :g •~• ::t"G .-

NADP+ NADPH

/ ProI.s·S-ProI

j

NADPH

"'10. Prot_SH

.

~ ,~'C>, XHgb-F.J+ - - LMB " ,.,. NADP' : FAD

Cb <> ""F..,"Cb"

-----

AlP

NADP'

~

NADH

ADP 1,3-DPG

7'.

GSH~G:~

ADP - 1 F.l,6-OP

~

~ R·!).P -

7--;:

DPG Shunt

• 2,3-DPG_

' : ', NADPHD :

Hgb

"'' '''" 1.18

.~.

NADPH

•

--- ."""" h. Hgb

i

---

0,

2_PG

J "'''--1""

ATP

--1 PK Pyruvat" NADH

):: "',.

c.o."

Fig. J.S. M.jor bi<><.h.mic:d ,..ctio ... in . rytbrocytes. • Aru.robic glycol}" .. (also known .. th. Embden_M"J"'bof p. tlt.....y) provid<> th. bioch.mic:d ,k.Leton for orythrocyte mouboJi,m and ATP and NADH. s.c..... of th. 'p~t of F_I.6-DP into tw<> molecules of G-3-P for to.<> par.JLeJ pathW>Jl". gIyroIym <X>JUum.. two molK:uLes of ATP rnd produ= fow mokcuLes of ATP. PFK j, tb. me-limiting mzym<,:and in .ctivity is ~b:on=l by :dbIemi. :md ,< [1St re:oclion, resulting in . nr FAD). GSH .,.d NADPH .,., tb. aujOf «ducing :ogis. • CIro,R wes FAD rnd NADH to cot:dyu tb. convemon of two mo&.cule.. of F<'+Ch, to two molecule.. of F;+Ch,. n,. ",'+cb, reduces Hgb-F." to Hgb in 0 no<>t:dyres convemon of Hgb.F .... to Hgb 000 thus it IDOtbe, meth ....oglobin 'MUCUS<. Ho~.,. this is 1 .... ry minor r.. ction in pby>iologic ... "'" rnd "'qui'e! rn .lectron """"pto,. Tbe ..peuti. c:dly. MB Cln b. used .. tho. .lectron 1cceptor.,.d i.s convened to L\1B: LMB thon ,.""'" nonenzyrruti. cnly to «due< Hgb-"''' to Hgb.

Il""'"''''

'"

3/ ERYTHROCYTES

119

bUllui-group fofflJn. N=lyaU blood.group factors >r~ detmnina! by gtnetia. and ...ch sprcies has diff~r~nt .ntig~ns. In ptopl~. blood.group f:octors Im}' function •• m~mbr:m~ transpon~rs. r"""ptors. lig.nds. or structural prot~ins. 2. A v..i.-.:y of nom~ndatu'" .ystem. han bttn UIM for som~ blood types. Th .. ~ .'" th~ mor~ common syst~m' u,a! B. In dogs. at 1~:lS{ a d"",n blood groups h.ve b«n d~scrikd. and nin~ are currently nIDla! using th~ in the United Sm.. h an DEA 1.I.nd20%haveDEA 1.2. e. Cats h ave on~ blood.group .yst~m. th~ AB SJ 90 % in n=ly aU bretru}.:md tho.., cats han a natural .ntj.(B h~magglutinin) antibody. Type: B i. ra .. in m.ny br«ds but rdativdy common (25-50 %) in a fc:w (e.g.. D~von ""'. Cornish rex. and exotic and Briti,h monhair). and those: cats h.n • natural antj.{A h~maggluti. nin} .ntibody. Typt AB caU are rare (< I % of cats) and lock natural antibodies againu blood.typt .ntigtns. D. The blood·group .yn~ms of hones a", r~lotivdy complex. with over 30 identifia! blood factors bdonging to at lease th~ seven blood "Y't~ms that h.ve b«n recognized by th~ lnternatioruol Society for Animol Gen.-.:ics: A, C. D. K. p. Q, and U. Blood t)'pt. are descrikd by. capical letter to denote the system •• nd a lowerca.., lett~r to denou the specific f:octo[". for example. Q •• Qb. and Qc. The incidence: of some blood t)'pt. varies markally .mong bretds. Factors Aa. A£. Ca, and Q. ha"" particul .. clinical rd~vance:. Horse. that lack Aa. A£. or Ca moy have ruotural .nti.Aa. anti.Ac. or anti·C, .ntibodies. so plasma donors .hould be positive: for the.., factors so that the n.tural antibodies >r~ nol pre:s<:nt. Typt:'l A:. and Qa are commonly ."""iala! with n
Fig. J. S. '"'"'ti~ud

• Thr dipbosphoglyc.b.ring cyclr) provitr>tioru . .. bigh in dogs rnd ho""" but very low in c.. dr rnd co ... '" 1.3·DPG. 1.3..dipbosphoglyc<....: 2.3·DPG. 2.3..dipbosphog/)=ntr: 2--PG. 2--pbOlpboglyc<"'tr: 3·PG. 3· pho.pboglycrr>t<: 6.PG. 6-phospboglurona..: ADP . • denosinr dipbwpb...: ATP. adenoone tripbwplu..: Cb,R. cytocbromr.b, reduct=: DPG. dipbospboglyc.rr.otr: F.l.6-DP. fructos<.l.6-dipbwplu..: F..6.P. fructo ....6-pbo'!'lu..: Fr"Cb,. frrrocytocbromr h~ F.>hospb. ", debydrogrrw><: GS H. gluutbio .... uducrd: GS.SG. glutothionr dimlfidr: Hgb. drozyhemoglobtin: Hgb.F?". mrtbrmoglobin: Hgb.Q,. oxyhrmoglobin: H K, b""oltin=: I·IMP. brxosr monopbospb. t<: LMB. lrulwmrtbylr ... blur: MB..... thy. lrnr bl",,: NAD· . nicotinunide adeninr dinuclrotidr: NADH. uducrd nicotin:omidr :odrni... dinuclrotide: NADP"'. nirotirumidr . drni ... din"clroridr ph"'pb. te: NADPH D. NADPH drhydrogr: PGD. pbwphogluoo ... t< debydrogenasr: PK, pyn>""'" kin ...: PO.. phospb. t<: Prot..s H. protrin ..itb uducrd.mlfbydryl groups; Prot.S..s..pro~ protein .. itb di",lfidr bridg ..: and R.'5-P. ribulooe-5·phospb ....

""

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

E.

Th~ blood_group 'Y't~m' of c:o{d~ .'" V~'Y rompla, with over 700 known blood_group f.ctors in .t l~ast II blood syst~ms. With this dq;r.., of g~n",jc diversity. it is esstnti[{l~ with th~ um~ blood typ",

ANALYTICAL PRINCIPLES AND METHODS

I.

Complete blood count {CBq: Major collap!> J>",toining to a eBC are in Chapter 2 (Am/ytiCl.I Principles and Methods, Ket. I).

[I.

Erythrogram A. Morphologic ~"'uatjon

l. A microscopic rvaI",,{ion of suinal erythrocytes is .n imporunt part of the nythrogram, espa;i:dly when .nemia is p,,,,,,m. 2. [n the Morphologic h.tures of Erythrocyt.. stcrion, the clinical .ignificollcr of erythrocytes with abnormal rolol'S, .jus, ,ha!>"" inclusions, or alh" f....tu ... i.

d.""ikd. B. Hcr (synonym, PCV): hnnaro_ ("blood ") -nit {denoting ".qm.tion"} l. Hct is th. pe'''''ntage of blood ""lurne filJal by uythrocytes and th.r.fo .. a m= .. of th. O,-carrying cop3oCity of th~ blood. If th~ .. a", 100 ml of blood with a Hcr of 45 %. then ~rythrocyus occupy 45 mL 2. A Het will accuratdy r~A'""t th~ [RBC] in a blood sampl~ if th~ Mev is WRl. 3. A Het will accuratdy r~A'""t th~ blood [Hgb] in a .ample if th~ MCHC or CHCM is WRI. 4. Unit: vol % (oommonly jun %): and 40 vol % = 0040 {the SI apression is. unitles. decimal fmetion} C. Blood [Hgb] l. Th~ blood [HgbJ is th~ grams of Hgb ~r 100 mL of blood ~ntially all Hgb in blood i. in ~rythrocyu. ex<:qlt in a f~ pathologic nat.. (e.g., intrav>scul.. h~moly_ si. cousi"l: h~moglobinemia) or aft.. t... tllYnt with Hgb_~ 0, corriers (e.g. , Oxyglobin). 2. Th~ blood [Hgb] will ac<:uratdy reA'""t the [RBC] :md Het if th~ MCHC (or CHCM) and MCV .r~ WRl and hemoglobin~mia is not p"'.. nt. Th~ blood [Hgb] is a mor~ direct m~",ure ofblood·s o,..corrying <:ap>eity than is Het or th~ [RBC]. 3. Unit oonversion: gldL X to dUL = gil (SI unit, nearest 1 giL)" D. [RBC] l. Th~ [RBC} it the number of ~rythrocytes ~r unit volume of blood (in clinicol jargon, oommonly .. f~rred to a. REe ...."nt). 2. Th~ [RBC] will acruratdy ",A'""t Hct and the blood [Hgb] if the Mev and MCHC (or CHCM) a", WRI. The [RBC] is used to caleulau MCH and am be u=i to calculau the Mev if ch~ MCV is not directly me,",ured. 3. Unit oonversion: {If X 1001J.ll} X 10' j.lUL = If X 10"IL (S f unit) E. Wintrobe's ~rythrocyte indices: Mev, MCHC, :md MCH l. Th~ W"mrroh(·, (')"1»"«)"( ;ndkrt a", thrtt values that a", used to cha",cr~riu: erythrocytes in ~riph ..:d blood The MCHC (or CHCM) .nd Mev ar~ used to classifY anemi", (.... Anemia, sect:. [I. B). a. MCY is the volum~ ~r an"'i:e erythrocyte apr.....:! in femtoliurs (IL) or cubic micromeurs (j.lm').

121

3/ ERYTHROCYTES

b. MCHC is Ih. edlul" Hgb con"",mration ~r ,v,,>gt "ythrocyt~ np~ '" grams of Hgb ~r 100 mL of ~rythrocytes (gldL); gldL X 10 dUL = gil (SI unit) Co MCH is th. qu. mity of Hgb ~r .""~ .rythrocyt~ np=snl in pirogranu {pg}. 2. In mosl blood specim.ns. not all .rythrocyt~ ,,~ th. sam~ (i. •.• Ih •• rythrocyt~ h.n diff~",m volumes. Hgb ronumration ••• nd Hgb rom~nu). It it important to ",m.mb" Ihat th~ Mev. MCHC. and MCH "'I'",,,,m th~ .""r:ocr> for all ~rythro. cytes in th .... mpl~. 3. Rd.lionship of indiu. a. Brau", th. MCH "'Pr=ms how much Hgb is in .n av,,"C<' .rythrocyt~. :md th. MCV rep ..",nts th~ volurm of.n .v.~ .rythrocyt•• th~ MCHC of.n avtr"C<' .rythrocyt. can b. ca1eulolal by dividing th. MCH by th. Mev (Eq. 3.la).

MCH MC HC = Mev

(3. 1a.)

Enmpt.: MCH = 20 pg. Mev = 60 fl MCHC =

~

6011..

=

MCV = HctXIO [RBC]

20xlO- U g 60xlO"L

=

2O, OOOXIO-"g 60x 10 " L

MC HC = [Hgb] X 100

Het = "M"CV "-C'C[ReBOC"J

H"

333 gIL = 33.3 g/dL

MCH = ]HgbJx 10 [RBC]

(3. lh.)

(J.l c.)

" originally deocribrd by Wimrob.. th. indices .. pr~,.mal th~ ca1culotal intmdationship" of Ih..., m.am=! valUe!: Het (.. ponal as a %). blood [Hgb] {rqxmal", gldl} •• nd [RBC] {r~ponal .. lO' Ij.tL} (Eq. J.lb). 4. Th~ Wintrob. formulas ..~ u...:! to ca1culat~ 5Om. of Ih. results g~n~mal by imp
Wh~n

122

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY {2} Th~rdo ... you have ]0 g of Hgb pt. 33 ml of erythrocytes. or 30 g pt. ]00 mL of uythrocytes. and thus the MCHC = 30 gldL c. Rdationship of Hgb and Hct {with oonnntional units} {I} When th. MCHC is 33.3 gldl, the blood [Hgb] numerical v:due will b.

one_third of th. Hct numerical v:due {•. g., Hgb = 15 gldL and Hcr = 45 %, or Hgb = 8 gldL and Hcr = 24 %}. {2} Ikcau .. MCHC value, in most blood sampl•• are about 32- 36 gldL, th. Hgb numerical v:due typicaUy will b. about one_third of th. He! numeri",] ... Joe.

6. ROW a. Th. ROW i. a calcul.tal v:due (u",,,lly up ..... d :u a ptr""n~) that rdleccs th. amoum of vuiat;on in the erythrocyte volumes (Ott Morphologic Features of Erythrocym,

S<'CI.

VI).

b. I t typically repr ... nts the codlicie"t of vari.tion of the erythrocyte volumes that are u,a! to determine the MCV, and is cakulotal from the MCV and ,t;;ondord devitio,n o,fMCV -t- MCV) X 100. c. inc",as.d ROW rdl«t, incrra...,j anisocytosis. 7. Th~ Bayer T«hnicon and ADVIA analyurs calculate the MCHC as just described but also measu", th~ [Hgb] of e;;och individual ..ythrocyt~ by analy.is o,f Hgb_ induced light ,c;m~r. This provid~s th~ following :additional erydlfogram ".u1n fo,r the ADVlA: a. CHat i. the avuag~ of th~ cdlular Hgb cone<:ntration measur~d in rach ~rythrocyu, with the same cone<:ntr>tion .nd units as the MCHC but deter_ min..! di=dy by light ,c;mu. {I} Th~ CHCM i, unaffect"! by mall of the .p«tral int~&"'n= that can cau"" a f. hdy increased MCHC. {2} H.inz bodi.. will faJ..,ly incre"", the CHCM because: o,f ahtio,n. it is the eo<:fIicient o,f vari.tion o,f the Hgb roncentration. mea.rur.d in e.ch erythro_ cyu.inc",ased HOW is alJ.d an;>lll"hroma,;a. F. nRBC. I. Th~ nRBCs o,bstrv.d during the examin.tio,n of. blood film are enumerat"! by counting the number of nRBCs seen while 100 (or mo",) leukocytes a" differenti.t"! and rounted. Th~ conventional method of recordiIll: the number of nud... t"! ~rythrocytes is number of nRBCsJlOO WBCs (e.g., 10 nRBCsJ 100 WBCs). 2. M.nual and some electronic method. det~rmine the [WBC] by rounting nud... t"! cdl, o,r nudei (either of leukocytes or nRBC.). By these methods, • _a,urd [WBC] "present' the sum of nud...t"! cdl cone<:ntrations (leuL:ocyt~' plll'l nRBC.), and cor=tion o,f the m~asur~d [WBC] is na:essa,y to obtain a more .ccurate [WBC]. 3. A [nRBC] an be calculated from the m.. mr"! [WBC], th~ corrected [WBC], or both.

3/ ERYTHROCYTES Th~

{I}

123 most di=:t

m~thod

[nRBC] = m
is shown in Eq. 3.2:1..

I' nRBC / IOOWBC 100 + I' nRBC/ lOOWBC

{3.2a.}

m=
'""

[nRBC] = 20,ooo/ J.lL X -

= 4,OOO/ J.lL

[nRBC] = cor=tw [WBC]

x,:,,,,,,,,, I' nRBC

{3.2b.}

100 WBC

Exampl<: corr« tw [WBC 1= 16, OOO/J.lL, nRBC = 25/100 WBC 25 nRBC 100 WBC [nRBC 1=

16,000 WBC .'~5, oRB ~C:o x -;-: J.lL 100 WBC

[nRBC] =

m~asura!

4,<XXl nRBC

,L

[WBC] - corra::tw [WBC]

{3.2c.}

{2} A [nRBC] can be: co.kul.t~d from tbe corra::tw [WBC] (Itt Eq. 2.1) by using Eq.3.2b. {3} If the corr«tw [WBC] and m erythrocytes, then tb. RP is 1.0 'III. The RP i. also calla! the reticulocyte count. c. Th. rgrrnud micllu,cyu pn-crnlllgr (C RP) i, a colcul ata! pera:ntagc: that repr... ,.nts the RP if th~ .nimal we .. not .nemic but had the s:nn. RC (s.,. the following .=t. 1ll.K).

124

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

III.

Methods A. Microh~malocril l. On~ of Ih~ ~asim, moU accunl~, and mosl reproducibl~ m.thod. of asseuing [.rythrocyt~] in blood i. by Ih. "",ntrifug.,j microh~malocrit. 2. Wh~n fim describetl, th~ t~rm h",,,,,,,crit (Hct ) was Ih. nam~ of th~ proc.,jur~ us.,j 10 stl'''.r~ blood illlo its major compon.nu: paek.,j ~rythrocytes. buffy """ I'yc:r, and plasma. Wilh common us< of Ihe proa. 3. For mosl umples, a propc:rly adjusted .nd calibrated impc:dana: a:ll coulll~r atn provide reli.bl~ data for th"", a:lls if clots .nd plaldet clumps a.. not pr""'I11.' ~"

a. For conin~ blood, moS{ coulll~rs am rypicolly difftr~lIIiau erythrocytes :md plotelets and Ihll'l atn provide reli. ble erythrocyte .nd platelet dal • . Ho~r, num~roU! large platelets or small ~rythrocytes (e.g., in Fe d~fici~ncy) con resull in fal .. dala.

3/ ERYTHROCYTES

c.

125

b. For fdin~ blood. most rount~r> connO{ rdi. bly diff. th~ degr.., of inaccuracy in th~ ~rythrocyte data i. typically low o:erpt wh~n th k.g., CELL_DYN. Bay'" Technicon HI, ADVIA, and Sy.ma) I. Basic principles (0« Chapter 2, especially Fig. 2.5). The flow cytom..cric .. prcu of th~ CEll_DYN ar~ u.std in addition to impfflaner to """l'"'t~ l~ukocyt .. , whe<~as th~ ADVIA UstS only How cytomHric m~thods to evaluat~ leukocyt~., ~rythrocyt~., and pJ.tdm. 2. The ADVIA u... unique optical methods of evaluating erythrocytes .nd miculo_ cytes. With. special diluent, erythrocytes are isovolumHrically sphe=! ]"'£0", they pass through. l...,r ]",am in ",..hich they =n~r light. Spher«i erlls =n~r light in • more predict.ble ",..ay. The instrum~nt :as= propenies of each erythrocyte to mea.ure or calculate """,It •. a. As.n erythrocyte or platdet passes through the lastr beam, ito pr..~nct i. detect«i by light ""tte<. The low light =n~r (2- 3°) is us«! to dete<min~ a cd]". volume, wher~as high light scatter (5-15°) is u.std to dHcti"" indices th.n erythrocytes) and to det~rmine the Hgb conerntr>tion in individual erythrocytes (a gre.triation of the individual ~rythrocyte volumes. {4} fRBC] is the num]",r of erythrocytes dHt"CI«i in • given blood volume. {5} Het is the percentage of blood volume occupied by erythrocytes :as dH
126

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

<:

i

11'-------+

"

I Feline erythrocyte cytogram

Canine erythrocyte cytogram

e>'

Fig. j.6. Erythrocyt. cyt<>gntru (ADVIA) from 1 h..Jthy :md :on ..,.",ic dog: Th. yolUJI>< (V) rnd Hgb cODcen,mion {I-Iq of ..ch. ~rythrocyt< (RBC) is gr>phicolly di,£>lay«! in rn RBC VHC (vo"'"", hemogLobin oonCffitr:otion) grid (riCou.,._ displ.y). In , ..... cytogram<, molt . M>«."", irnm.rur. orytbrocyteo "'" hypochromic m. crocyt"'. no. min auy "" noted prior to changes in MCV, MCHe, mel CHCM b.<:aw. those v:tlu .. «fIe<:t the mun v:;d""" of tbe erythrocytes. TItis .,..mic dog b. d • mildly incuased MCV. mildly d.c .. ased MCHC:uxl CHCM . ..,d mod...",ly incnased ROW:uxl HOW. Differ..,,,,,, in grid phc<ment rd\«t diff<"'nt upect«l erythrocyte indices for c. '" oompared to dogs.

a visual repr=ntation of the morphologic classificotion of erythrocytes (5« Anemia, =t. I1.B). d. A dye (oxazine 750) dut .uins cellular RNA con be used with the sp
{2} MCVr i, the anrage of the individu..tly m... sur
3/ ERYTHROCYTES

127

{5} HDWr is the standard devi. tion ofHgb ooncemration, in individual miculocytes. {6} CHr is the .""rat<' amount of Hgb in reticulocytes. {7} CHDWr i. the dinribution width of Hgb con""'ntration in individual miculocytes {..lues usn! for calculating CHr}. e. Lit.:. the oth" instrumenu. the ADViA ly= erythrocytes and uses the cyanmet_ hemoglobin {or alternate chemical} method to d"I
128

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY E. [RBC) by th~ h.mocytomew mffhod l. A h.mocytomff.r Gm bt u=i to measure :I [RBC] in blood. Blood is diluted {mually in :I Unopeue ,y,{tocytom"".r m""hod i. typically imp,,",,i.. and thus js not ,«:ommend.d for routine :os.=sm.m of [RBC}. F. Hcr and blood [Hgb] d.t.rminal by conductivity l. Some dectroch.mical instrumenu that ... designal to m.... su.. blood g.. . nalytes {e.g., Po" Peo" :md pH; .... Tabl. 10.1 for .bb=i.tions} or dttlrolyus (e.g., N • ., K+, .nd will :0/'0 g.n .. a Hcr and blood [Hgb] as !"III of th,i, profile

en

·.t.

results.

2. Th. Hct is dormninal by m.asuring th. dectrica/ ronductivity of the blood and then ronnning th.t value to a Hct, bastrked hypoproteinemia yidds. falsdy decreased Hct. G. Blood [Hgb) l. Blood [Hgb) has bttn measu=i by photometric method. for many years. [n • variety of current blood analyurs, the blood [Hgb) i. typically mrasu=i by a cyanmethemoglobin =y using spectrophotometry. Bea"",,, of concerns of working with or di'poling of minute .mounts of O

129

3/ ERYTHROCYTES

Table 3.3. Potential efFc.:ts of ..,lccled . ample and palienl conditions on hemalocril (H eI) and values u....! 10 calculale H CI Condition

S~un

[noo:l«\"",r mixing Exces.s EDTA shrink. RBCs in blood Clots in sampl~ [n vitro h.molY'is [noo:l«\"",r "",mrifugation Agglutiruotion V rry IDull ~rythrocyt~. Hyponatrrmia Hyp",natremia Leukocytosis Thrombocytosis (marked) HYP"rprouin~mia (m ..kedJ

U

U

U

"" " ";

Hypoprol.in~mia

" " ;

Hcr"

Calc. HeI'

NN

U

;

MCV' •

;'

[RBC]

Cond Hc!"

U

U

"" "" " " NA

NA

; ; ;

;

"" "

NA NA

;

" ; ; ;

"

• Spun Het i.s the Het d,".rmincd by =trifug..ion, .ithrr the microh ......ocri. metbod 0' tl>< QBC _<W. • Cook, Het i.s .... Het c:dcuhtrd by imprd....., «Jl counter> after "",.mring MCV and IRBC], < MCV m< T oehoiron analyzer, The eflN:t is not ... n with molt o .... r instru1n
3, Th. Lasrreyt. H~matology An:dyur det~rmin .. blood [!-1gb] by me .. uring th~ transminan"", of light from two ligh,..,mitting diode" one for wholr blood and one for hemolyzrd blood, H, Diff~rrn= in erythrocyte dau drt"'mined by"",ntrifug.cion, im""""n""" opticol, .nd conductivity mrthod. J. A H ct pro",rly det"'mined by "",mrifugation of microhematocrit tubes is consid· ",ed th~ best mrthod of a<ses.!ing blood [.rythrocyte] in domenic animal" As. quality """ran"", m~thod, the ""'ntrifugr Hel (often colled 'pun Hct) con be compared to th~ Hel calcul.ced by im""""n"" counte" {calculated Hcrl or by conductivity methods {conductivity Hcrl , Di"""'panci.. among H ct valu .. con be coused by a v..iety of factors (Table 3,3), As a rule of thumb, calculated Hcr and spun Hcr should "l:rec with ..ch other within 3 prrcrntagr points; for example, if • spun Hcr i. 38 %, • calcul ated H ct of 35-41 % should be obtained, 2, [nad«\u ate mixing of blood prior to filling . microh.matocrit tube con co",", an ",ronams HeI, Erythrocyt.. tend to srttle if the antiroagulated blood is l~ft standing in • tube rack, .. pecially with pronoun"",d rouleaux, ., Filli"i: the microhem.cocrit tube with the top ponion of th.c blood will yield. falsely drcreastd Hel by any mrthod, wh", . .. filling with the bottom portion will yield. falsely inc", ..ed Hcr,

130

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

b.

Hc{ valu~, may diff~r bttWttD method, mixffl prior to one """y but not Ih. olh",

Th~

b«:aUst {h~

blood w;o.

pro~rly

3. Coll«ting or transr.rring >II inadaJuate amount of blood into an EDTA tub. (•. g., 0.5 mL of blood into" tub. d.. igoal for 3 mL of blood) "mJu in a ,dative excess amount of EDTA Th. excess EDTA cr"al., h~r{onic pl:uma and erythrocyus shrink kcau .. of osmosi.; the sm:.!l., u ythrocytes occupy Jess volume. and thus a spun Hct is f:olsdy d,"",rasaj. How....., when these erythrocytes are susp. gom. and young ho ..... ) =y ~ too small to ~ =ngni=l as ~rythrocytes by im~dan"" . If th~ small~r . rythrocytes ar~ not includ..d in th. MCV d<:nding on which of th. m.... mred values h.., th~ gr~at~n .. ror. 9. Wh~n an animal h•• a ~",isunt and marked d""r ...... in plasma o.molality (~u .. of hyponat"'mia and hypochlorrmia). th~ osmolality within . rythrocytes also d"".......,.. Wh~n rhos. ~rythrocytes ar~ placnl in an isotonic dilu.nt prior to impffian"" counting. th~ ~rythrocyt~. shrink as H,O mo,,",s from th~ rells vi. o.mosi •. " Thu •• th~ MCV m.... ur..d i. f.lsdy deer • ..,..d. and thus th~ Hct calculat..d is fal ..ly d""r~astd. Th. opposit~ osmosis occurs when an animal has a

3/ ERYTHROCYTES

131

ptrsist~nt

10.

II.

12. 13.

and markffl plas/IU hyperosmolality and thm th. cakulat.d Her is fal..,ly incr""sffi." Th. conductivity Hct /lUy also be, aff.cral by markal chang<. in pla.ma [N.+] and [Cl-], although analyurs may be, d..ij:nal to corr«:t for ouch abnormaliti.s; that is, hypon>l"'mic and hypochlo"'mic 5amples may han I... conductivity, which r~sults in a gr~a ,.r mnductivity Hct. Autologoll'l blood transfusions m"}" cau... falsdy d",r""""d conductivity Hct. In a study using an infu5au mntaining ",.,.shal pati~nt', ~rythrocytes in 5alin~, the infusat.', gr.... t~r d.crrolyu conantratioru and lower prouin conantr.uiom (romp.=' to plas/IU) incr""..,j its conductivity and thll'l =h.d in a fal,.,[y low conductivity Hct; th. infu.. t~' •• pun Hct "'.,.s n""r 5 I %, but the infusate's conductivity Hct was n.ar 37 %." If a larg< portion of a patient', blood volume is from .dmininered infusau, th~n a pati~nt'. conductivity Hct could be, falsely d",rrased. Th~ p .... nce of markal leukocytosi., /lUrked thrombocytosi" or marked hyper_ prot~inemia can redua th~ conductivity of blood and thll'l cau .. a f.lsely ine",ased conductivity Hct. If th~ impedana aU count~r cannot differ~ntiat~ pJ.tdm from .rythrocyt.. and thrombocytosis is markffl, the m""su=' [RBC] will be, fal..,ly incr""..,j and probably the Mev will be, falsdy d.cr......d. If the ~rror in the [RBC] is gr""t~r than th~ Mev .rror, th.n th~ calculated Hct will be, falsely incr""sffi. Marked hypoprotein.mia will incr.... th~ mnductivity of blood, and thus the conductivity Hct wiU be, f.hely d",,,,asaj. Th. Mev may be, d",,,,ased by in vitro ",.nU or ph.nom~na in .ddition to the afo .. m~ntional df«:t, of osmolality. a. A faJ..,ly d",,,,asaj Mev may occur wh.n .n d"'tronic all mumer cannot differ~miat.large platd~ts from .rythrocytes. This is pri/IUrily a probl~m wh~n there is • /lUrked thrombocytosi. and. concur",nt markffl an~mia."

I.

b. Such erronams ..",It, can bt .usp«ted or recognized by other findings. {I} Th. red ceU di
RC = RP X [RBC]

(3.J a.)

pat ient', Het CRP = RP X averag~ Hct for 'peci..

(3.3b.)

C Rl' RPI = - RMT

(J.3c.)

=c""'''''i':''':=

132

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

J.

R~ticulocyte

pge {RP} (som~im .. calla! m;cul~rylr munt) I. Th~ RP is th~ prr""ncag.. of ~rythrocytes th.t "'~ r~ticulocyt ... For a.mpl~. if th,,~ are 10 r~iculocytes prr ](XXl ~rydlfOCyt ... the RP is 1.0 'lb. 2. It is usal to calculate RC or CRP. It indicot~, nonr~nuativ. an~mia if not incrrasa! and ]><»Sibl~ re!:"n~ntin .n~mia if incr""'! 3. Pe=nt>ges may ~ :of&cta! by agglutination. 4. Basics of pro=:lures a. Manual (nticroJropic) procedute {I} Equal volumes of blood and NMB , rain are mixa! . nd krpt at room tempg' of .rythrocytes with th. light_ scottering prop
V".

Table 3.4. Evaluation of fCticuiocyte data R~~ren'" Ol.llin~

Units

int~rv;;oJ,

Do~

37- 55 0.0-1.0 0.0-1.0 5.5-8.5 0-'0

"

Hcr

%

RP CRP

% %

[RBC]

X lO' IJ.lL X 10001J.lL

RC

0

1

'.0 0.7 '.0 40

R~f~ren'"

Do~ 2'

"

9.0 3.0 '.0

'"

Do~

3<

"

0.0 0.0 '.0 0

Fdine

Units

int~rv;;oJ,

Cot I"

Cat 2'

Cat 3'

Hcr

%

RP (~regat~) RP (punct>tr) CRP (~regate) CRP (punctou)

% % %

"

"

"

[RBC] RC (~rq:.te)·

X lo'lJ.ll X 10001J.lL X 10001J.ll

24--45 0.0-1.0 0.0-10.0 0.0-1.0 0.0-10.0 5.0-10.0 0-"0 5- 500

%

6.0 30.0 '.0 10.0 '.0

'.0 30.0 0.7 10.0 '.0 40 600

0.6 30.0

"

U

6.0

OA

OA

20.0 4.0

'.0 '.0

" '"'' Evidmc< of incr.... d erytbrocyte prodoction is not pc... nt. If th.e

RC (punctate)'

'" 600

c;" ~

'""

Cot 5"

"

0.' '.0 <0.1 0.3 '.0

,

"

o Dog I: IJUlfOW is curr~tly ""pond. ing to th.e .... mi., it is not ",/l«ted by tb. blood miru~. Tb. RP is in"' • ...d b.oc ..... few.r . ryttuo.. cytes ...thor than more "'ticulocytes. or. in tho blood: tlut i1, RP is inc",ased t..c. .... th.e [r .. irulocyto [ is WRI ond tho [RBq is docr....d. • Dog 2: Th..., i, ...ide""" of in"' • ...d.rythrocyt<: prodoction. and tb. rruorro.... is runently =ponding to tho """mi •. Tbe RP is incr""'! beau.. fewer .rythrocytes ond more "'tirulocyt.. .,. in tb. blood tbon thor. or. in b.alth: tlut is. tbe RP is increosed beau.. tho [reticulocyt.] is increased and tbe [ROC] is dec .. ased. < Dog 3: Tb.", is not ...idmce of incr""'! erythrocyto prodoction. k n:tirulocyte.t """. not >eon. dec",ased production of .rythrocyt'" m.y b. 0 ... re...,n for the anemi •. Or. it could be too •.,ly (< 3-A d) fo, • ,eticulocyt. ""po ...... • c..t I: n..: .. is .viden"" of increased erythrocyte production. Tb. aggt"ll"'" ,.tirul<>g< .00 tbw difficult to in"'rpr" by itself. , Cat 2: Th. du .. tion of th.e anemu must be lrnmon to interpret tlw: , befo.. on >gg«'gat.,'" that tbe", is no longer." in"",...d rete...: of aggtegat. r.. irulocytes. • Cat 4: Evidmc< of inc", ... d erytbrocyte prodoction is not pre>
133

134

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

cyt .. and the rd.tionship. ktwetn marrow stimulus .nd marrow ,espon= (Ott

Physiologic Proce:s=;, .=1. VI) n=! to be, coruid.ral when blood reticuJocyu are ;merp.. t.,J. 6. Microscopic determination of RP has rdativdy poor analytical p=:;sion. Th.""fo.. , RP should be, consid.,..! an estimate. Values that are ",kulatal using the micro_ scopic RP (i .•.• Re, eRP, and RPI) should:.!so be, considerM mim>! ... 7. The d~tt ofincreasffi polychro/IUSi. mould correspond to the inerr• .., in RP {"'''''pl for fdine punctate r"t;cuJocyt ..j. K. Corr«tal ",ticulocytr p<'ctnt:age {eRP} {aho calbl nlno!u" micu/Nyu ptram .nd aixou.1r % mUll"',," rount} l. Cakulotion ofeRP oonv-ns RP to .. P"=IIt"l:' thot would estimate th. RP jf th. animal w". not anemic. This is n~ssary for "Pprop,i
3/ ERYTHROCYTES

135

3. Ulllil th~ validity and clillical value of th~ RPI i. esublish..:!. th~ CRP or RC should b. <=
A....essm.lII: Morphologic f..tures of uythrocytes ar~ ....atuat..:! .. P"fi of a routill' CBC or as a "p"rat. prO«dur~. For .bnormalities ill siR, shal"', .nd color, rdatin quantities of •• ch abllorm. lity p
136 II.

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY G~n~r:ll tntur~.

A DU('gQlrr. M.tur~ ~rythrocyt .. of ~~ch domestic "",mmalian s~i .. are disks with diff~ .. nt dq;"" " of biconcavity that crrates • a.ntral pallor (T.ble 3.2). When the .. ~ryd"ocyt .. han normal volumes, thq ..e coll<"!e 3A [for all plat.., ..., the color sa:tion of this book]) l. Common in some s~i .. (es~ally hor.... nd cors) 2. lu formation involv.. cM,!:e intenctiofil bc:twn:n erythrocyte memb .. n~s and plasma macromolecules and is therefore .ffected by erythrocyte facton (m ape and memb .. n~ composition), albumin factors (glyanion), globulin factors (charge, .ize:, and numbc:r), lipid content of plas"", .nd perhaps ~rythrocyte membran.., pH {affect. a.1I and protein CM'"C<"}, .nd exo!:"nous macromolecules {dextram)." 3. Inc",as.-d roul~aux tend to occur if the .. i. hyperglobulinemia or hyperfibrinogtn_ emi •. An incrra.. in roulraux is ",ident when they extend to near the feath ..<"<1 <".,ionally in animal. without evidena. of hemol",i •. The "gglutinin (a mMana. causing the au/utination) it typically a rold antib~:r. that is, an antibody that has maximal activity at 4- 20"C. 2. The erythrocyte dusters formed by autoagglutination must bc: difftrentiated from lOuiraux, which dassicolly appear as .racks of erythrocytes but con appear as piles or fallen .racks of coins. Wh~n examined macroscopically in • tubc: or as a drop on • slid~, blood containing the au/utin>!ed erythrocytes or rouleaux has • fine to co .... granular .ppearance. 3. One method to difftrentiate au/utination from rouleaux is the saline dilution (di.p<:rsion) te", in which blood i. diluted with saline .nd a wc:t P"'Paration of the diluted blood is examined (Plate 7A- C). Microscopic examination may lrad to the false condusion that au/utination i. present. a. This t~" is appropriate wh~n dumping is 'pparent, but it is not indicated to look for agglutination when clumping i, not apparent. b. Rouleaux .hould di,p<:"" into individual erythrocytes wh~n the pia."", [total protein] i. lowered by dilution of blood with ..line. A dilution of I p.n blood to I p.n saline {1:2 dilution} will olien disper... rouleaux, but ocauionally grrater dilutions {I pan blood to 9 parts .aline} a.. nttded to disperse roulraux. At least I pan blood to 3 parts saline ( 1:4 dilution ) i. recommended as a starting point. c. The erythrocyt~ dusters of autoagglutination do not disperse: with saline dilution {but the .. m.y bc: f~·~r dusters because of dilution}. 4. Hq...in m.y indua. agglutination of equine erythrocyt... 5. Agglutination may interf... with the dectronic or opticol evalu ation of erythrocytes when group' of ~rythrocyt .. pass through the counting chambc:r as "Ia,!:e crib." In such aues, the m~asured MCV .nd [erythrocyte] are erroneous (unle" proglOlms exclude outlier values) , as a", th~ values calculated from those meamred valu ... Automated RC. may al"" bc: unreliable. D. Rubricytosis {ailO known as metarubricytosi, or normoblastemia} (Plate 3C)

3/ ERYTHROCYTES

137

l. RubritytOlu is ~n inc"""",d oonctntration of nRBC. in blood. Um:dly. most :are

metuubricyt ... a few may b. rubricytes. and younger p=ursors:are r:ardy ..en. 2. Rubricytosis is oommon in ""gener>tin anemias {i.~. appropriate rubricytosis} but may b. ~n al"" in nonr~neratin anemias . nd in nonanemic ~nimah without ~iculocytosis (inappropriate rubricytosis). The""forr. rubricytosis should not I>. ooll!id"rd a ronsisuntly rdi. ble indicator of a responsive marrow. .. Approprillti rubriryro'is i. rubricytosis concur""nr with regenemin anemia {with miculocyto.is}. The nRBG arr rdea""" .., a respon .. to incr=d Epo stimulus. {I} It occurs during Kederatrd erythropoi.. is. Not only is the rde= of miculocytes incr ...""". but so i. the rd...,., of nRBG to blood. {2} It is =n in regenemin anemi.., of dogs. cau. cattle •• nd pig,. and •..,n occasionally in hone •. b. Inllpproprillti rubrirytotis it rubricytosis in the abs.nct of rrtirulocytmj,; for nample. concur""nt with non"generati"" anemia or in the abs.nc:r of .nemia. {I} It occurs primarily when th"e is • loss of the !indy oontrollrd rd...,., of nRBC. from nurrow or othrr erythropoi..is .it..; nRBC. from nurrow or othrr erythropoiesis sit.. without nucl ... r ntrmion and I>.fol< maturing to rrticulocytes. {2} Disorden or conditiom that cau"" inappropriate rubricytosis (a) Marrow dunagffl by n«:tosis. infLommation. endotonmia. hemic or nonhemic nroplasi •• or hypoxia; Nucle.trd erythrocytes gain entranc:r imo m:arrow .inu.., through damagrd sinu""idal endothdium. {b} Extramrdullory hematopoi..i, {esJ>"Cially splenic}; Thi. may :dlow rd.... of ctlls b.fore nuclear extrusion. {c} Splenic contraction; Splenic blood ronuins nude.trd erythrocytes that are oompleting maturation. {d} Splenrctomy; The few nRBCs that are normally rd.....d from marrow at< not· rought" by the spl..,n. (e) In.d poiwning in dogs. ""rhap' the result of damage to marrow "nus.. (/) Bone marrow dpcrasi. in poodles with macrocytmi. (..., Oth.. Nonnropl ..nic Leukocyte Diwrdm. Ket. II. in Chapt" 21"

=""

III.

Erythrocyte oolor A. emlr"{ pallor refers to the pale ""ntr:d region of an erythrocyte that is due to the rdati"" thinness of the area cr ... trd by the cd]", bironcave ,ha"". l. Inc"""",d ""ntral p:dlor is us,",lly indicative ofhypochrom.. ia. 2. Drc""asal ""ntral p:dlor m,",lly indicat.. abnormally .m.ped erythrocyt.. {poikilocytes. including spherocytes}. It is:d"" commonly =n near a blood film·, feath"rd rdge b.ao.us.- of anifact,",l distortion of erythrocyte .ha"". B. A ~if all i, an extremdy pate_staining erythrocyte consining primarily of cdl mem_ brane with only a .mall amount of r.. id,",l ""ripheral cytoplasmic Hgb (Plate 3D). l. Ghost cdls . 1< usually formrd during complement_mrdiatrd intrava5Cular hemolysis. Membrane acrack complexes form membrane por.. through which Hgb leaks out. 2. Ghost cdls may form in vitro as a result of smearing trauma. Thest artifactual ghon cdl. arr often distoned. C. A hypcchromk ")'t/n-gryti i. a poikilocyte with incr ...srd central paUor and more faintly stainrd Hgb than u,,",l {Plate 3E}.

138

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY l. HYJ!«"""",,i/J j. an inc,..,....:! numbtr of hypochromic uythrocytes, which ffi"}' bt

",f\«1ed by a d«r=d MCHC and CHCM if the hypochromic population is luge enough. 2. Hypochromic erythrocytes remit from a da:, raonj intncdJuJ:.r Hgb ronumration. When visually rvid.nt, th~ usually are ..,5Oci ated with F. deficiency. How~.r. hypochrom"'", b>=l on th. MCHC or CHCM alone (without microscopically ,pp",nt hypochromic erythrocyt..) is usutt.rn of the RNA. 2. A reticulocyte', cytoplasmic RNA may also ~ vilUaliud aft .. .raining with Wright stains. in which ca.. reticulocytes will appear as polychromatophilic erythrocytes. All polychromatophilic .rythrocytes are reticulocytes. but not all reticulocytes have enough RNA to appear .. polychromatophilic erythrocyte>. 3. & rirulocytolu {increased blood Re). like increased polychromasia. i. an imponant indicator of acederat"'" erythropoiesi •. [yo

Erythrocyte organisms A. IdentiJYing features are list"'" in Tabl. 3.5. B. Major .. peets of th. anemias or disord.rs caused by organisms.", includ"'" in the Nonr'l:"n ... tive An.mia and the Hemolytic An.mias KCtions.

v.

Inclusions other than organisms crable 3.6) A. BalOphilic stippling {puncute basophili.} {Pl ate 4GJ l. Basophilir ftippling is the presen"", of fine to roar... blue to dark purple don of aggregat"'" ribosome! (RNA) dispersed within the erythrocyte cytoplasm. Basophilic

'1

,11 1

t t

II

L

II

'1

J l{11

11!1 jf,!· ,!!jl

j

~ Ii

Iillh!

dtlt i i j j"]} j' j' ]'1 1!,,"'1l I!, l'ljc Jii l1 l" l l, l'l'lj 1',l l, " " .~

£1 1

.• 1 I ~-:c! ... ~I

1 · ~"···'I"IJ" ~~~ '! !u ; ,,1~~ -i! ;~i!"~~_~~! ;. ii.~gj • ,

,f

.g

!

t,

~

,

~ ;It~l i1~i:i~1~r ~ 1~~1j! ~ r 'I ,£, "1,""-·' "J! :f· 1 '!..!;~m ~~ "lR.·.Pi ~.P 1 1 ~Il r ~;~ia "IF", ~ .gjll! ~;:: II: ,• ",J~8 ; . f~"I ~~ "t"~1i . ~~. ~! R a_ lt~"e lyl- ;• 1

r

I ,.

".

.,~

1l5~~.i: !r-~ i~~1i"'-li"-!3

8~'1!~_.::L

:l

"~f"h~il·dii!fh"'jt'!H1 ~t i.i J-i h ~~1t P

~6

11

,Ij}l,jll] 11]1 !Jll~!l!J!. ,! f! :5

• 1

i

•

,~

.t

•t J

:t

.

f

•

0..

1, I ']. 'tJ:;p l'! §:

]jjlH

1I ~ ~ ~

1'/

l

1 1

1

I

J

1!

1

mli

.. . , .

,.

Ij IE:

11 .

t

l.

.

., il , p.

!

jI,

t

i!!

h J ,',

t'"

i!l

§.i j

Hli Ji lJ!~'" ~tl

~g~

g

r iJ

-I'

llt5-ti

z.t

j 11 ,

i ,.

J 1 ,.

i~}~

,I , •.

I

! .t

i 1t

Ji~

li~

. .

!. ,

H

"

1

1.lh t'' ,.

i;

~

,

!

t .1,.l H

'

1q Ii ,j '

1't I

'j'

j

. -

I

,.1 'f"

j

.

11

11

l!l~ 1 11i, 11l {l - "-1 " •• 'J,t ' " I 'j ljiiP lli~ ~lijl!j!hHt }l . . ." . . . l~t -.'"'j·.'l . . 1·j _j~.i,~ttit • 'Jolt!l' i~ iljH! l.l1tglj~rJl~i~ I ' j' I f -i I• , ! ;!i fj i f 1 ' II j:." .. flJi

;

§!

" .

,.

flfl

Jl

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.

n

3/ ERYTHROCYTES

B,

C.

D,

E,

141

stippling must b. difftr~ntiatw from sid~rotic gr:mul.., which a.. mu:dly located in duners. 2, ~philic lIippling is 5«n with r~nemi"" an~mias, esp«iIlly in canl~, but :dso in dogs .nd en" 3, When seen without corresponding polycluom:lSia or reticulocytosis, or in nonane_ mic .nim:d" plumbism is a common cause, eo;pecially in dogs, Lead inhibits the pyrimidine S'_nud..,tid.... cru.t hel", degrade nud..,tid .. in RNA Heinz bodi.. (Plate 4H :md I) ], Hrinz bodin a.. ~regates of d~n.tured Hgb cau...! by oxidatin dam:.ge, 2, H~inz bodi.. are visu:diud with NMB min :lS pal~ blu~, protruding, rounded structu.., associ.tw with ~rythrocyu membran.. , In Wright_stained films, H~inz bodi.. han nearly th~ s;om~ suining featur.. as norm:d Hgb but appear as slightly p:de structures th. t c",at~ membr:me defects or protrud~, Heinz bodi.. nuy deuch from erythrocytes .nd OCCut as free bodi.. in • blood film, 3, Exc;.,pt in cal>, the preseno: of H~inz bodi.. in :m :mim:d with. hemolytic anemia indicates Heinz body hemolysis, Sm:dl single Heinz bodies {diam~t .. .. 0,5 ).lm; ..e the srruollen forms in Pl. te 41} can b. found in the erythrocytes of cats without dinical anemia or h~molysis, Hgb """,Ii (Plate 4J) L The.. are seen occasioruolly in domestic mamm:d erythrocyt.. (induding dogs and cats), but their significano: i, unknown, Some may form in vitro becau .. of s;omple storage ronditions, 2, Hgb electrophoresis has failed to demonstrate abnorm:d Hgb molecul.. in domestic numJrulh that have ru.d Hgb cryllals, Howell-Jolly bodi.. {Pt.", 4K . nd L} L A H~UKll-J~11y IMdy i, a nucl .. r remnant th . t h •• remained free in the cytoplasm after mitmis of.n erythrocyte precursor, The Howell-Jolly body is nuclear materi:d tru.t W:lS not incorporated into. new nudeus, 2, Howell-Jolly bodies can b. found in healthy Jrulmm:d., frequently in cal> and occasion:dly in dogs :md hor ... , The numb.r of Howell-Jolly bodies in blood incr..... during .cederated erythropoiesis .nd also m.y incr.... in Jrulmm:d, with decr .....! splenic function {including aft .. splenectomy}, Refr.ctile anifacts {Pute SA} L Erythrocyte ..fractile anifacts .'" frequently found in stained blood film" Objects are rrfoutik when they chang. from dark to shiny as the focal plane is ch:mgffi; refractile anif.ctl a.. recognized by fomsing up:md down :md :use:ssing for this pro!",ny, They may appear as cre=nts or as sm:dl to brge, irregular sh'pes within the erythrocyt.. , Erythrocyte refractile structu... in blood films suined with Romanow.ky_type stains .re :dways .nif.ct" The d.fect that creat.. the refractile structure devel.o", during the drying or suining of the erythrocytes. 2, When refraclile .nifacts .'" in a pl. ne of focm that Jrulke. Ihem r..emble bl. ck structure!, Ihey can b. contu...! wilh erythrocyte indu.ions or """,,ites, 3, Refractil. arlifacts are different from erythrocyte refractile bodi.. :lS described by Sch:dm!' Erythrocyte refractile bodies are Heinz bodies seen on air-dried blood films by using. weI NMB sl. in under a cover glass, In thest prq>.ralions, Heinz bodi.. appear :lS erythrocyte refraclile bodies in erythrocytes; the bodies are dark foci in one focal plane but become ..fraclile when slightly out of focus,

142

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY H~inz

bodi..... not .. fracta~ in films suinffl with. Rom:mowsky_typ< stain

Wrij:ht,

(~.g..

Wright_Gi~msa,

or Wrij:ht_uishmon). F. Siderotic I:r.;mul•• (p. pp..11 :woci~tM with chlonmph,"irol th.rapy ill dogs. Sid..ocytosis alld sideroblastosis may bt rd.cM to F. onrl""d .lId :oIso h."" 0011 rq>
Abllormal erythrocyte volume A. Erythrocyt.. ap~ar two.dimeIlSiollal 011 • Wright_sraillM blood film, alld thus a cell·s diamet .. is &equ.mly oo",id..w to rdl«t its sa.. Howevrr, it is impon:mt to r«<>g_ 1Ia. thot two cells with the urn. di.m".., but with diff... m ,hidrn.= , h."" diff..em volumes. An .rythrocyu·s thid",e" is rdl«tw by the cdrs .raillillg im.",ity. A thill cell will bt lightly staillw (hypochromic), wh ....... a thid: cdl will bt sraillw mo .. im,"sdy (hy~rchromic). B. AnisKJ""is i. var;"tioll ill the volumes of erythrocytes l. Alliwcytosi. call bt a1usa! by macrocytes, microcytes, or both. Ikcau.. of th.ir d«rra"'! diam"."" sph..ocytes may produa. .ppa.. m allisocytosis """" if the sph..ocyt. volum .. are 1I0t d=•• ...!. 2. lu diagllostic signifialllce d'~lIds 011 the cells thot ... c",acillg the allisocytosis {s.. the lIext 1«tions on macrocyt.. alld microcyt..}. It i. oommollly .ssoc;"ta! with m.crocytOli, alld thus reg.lI.rati"" :memi... 3. RDW( ... Allalytical Prillciples:md M.rhod , ""'I. II.E.6} is:m . utomata! mrasu", of anisocytosis b...d 011 volumes, not 011 the microsropic a"essmem of cdl diam.r .... C. A mlUTtJryu is .11 erythrocyte that has inn......! volume (Plale 5C). l. MIlC1"Orytolis is .11 illc", ... d OOllcem""ioll of m.crocytes ill ~ripheral blood, which alII bt rdl.aa! by a shift ill Ihe erythrocyte cytogram or by an illcr.......i MCV. If 1I0rmocytes or microcytes are :oIso pres
3/ ERYTHROCYTES

143

D. A microryrr is an ~rythrocyu that h"" deer..,....:! volum~ {pJ.tes 5D and E ~nd 6B}. l. Mirnxy""iJ i. an incr~aKd con""ntration of microcytes in peripheral blood. which can b. rrfla:tM by a d=""sM MCV. If normocyt~s or mocrocytes ar~..!so pr..ent. th ..~ will b. anisocytmis :md th. ROW will b. incr! ~nough to inhibit mitosis. A microcyte con han a normal di.mH .. but incr""...:! ""ntral pallor b.a...,., of in thinn ... (a hyptKlJrgmir mimxyu and leptocyt.). 4. Dog. in ",me b=ds (~.g., Akita.!, Shibas, :md possibly Jind"" chow-choWll, .nd shar_peis) may han ~rythrocytes who .. MCV. a", 50-60 fl, though most b=d. han Mev. of 6O-n fl.'" Young ho""s (up to 6 mo of ag..) h.Y< low.. Mev. than mature hors",." Young kittens ha"" lower MCVs than matur~ cou." 5. Sph..ocyt~s may microscopicolly 'pp"ar microcytic b.c.u.., of deer.,.K, but th~ir volumes are typically WRl, and Mev. for the .. mple m.y b. WRl or incr"",."j b.a...,., of a regtneratin =pon,.. 6. Sid.roblastic .nemias occurring in association with other dis, especially .plenic hemangiosarcoma and other infilmuiv. splenic disorde". Why ~canthocytes form in th~ .. disorde .. is not known. 2. Poikilocyt.. that have the microsropic f..,.tures of aconthocytes (Stt T abl. 3.7) have bttn...,n concur",nily with h ratocytes .nd schizocyt.. in cases of lymphoma,

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3/ ERYTHROCYTES

147

h~mangiosarcoma,

C. D.

E.

F. G.

H.

I.

cirrhosis, panc""atitis, intr:lv:lSCIllar co'4:"lopathy, and glom~rulo_ nre..,nt anoth" form of poikilocyte formal by intr:lv;u cular trauma.'" 3. In people, acanthocytic change is ronsid~red the =It of abnormal lipid composi_ tion (high cholesterol to phospholipid ratio ) acquired within an erythrocyt~·. membran~ during circulation. Simil ar finding. have not bttn ducribal for domestic nunun"l a.canthocytes. 4. Some acmthocytes may k difficult to distinguish from echinocytes. Blister ceU (Ott =ntrocyte .nd keratocyte in the following sects. H and K) &rr un i, common nam~ for many spiculated erythrocytes. I. Echinocyte. , acanthocytes, .nd oth~r spiculated erythrocytes (tho.., with m~mbrane projections) """ callrd burr cdl, by diff.""nt people. 2. Breau.. the term may refer to ieVeral t)'pt, of poikilocyte" its u .. may lead to confusion and thus its clinical value i, limited. Codocyte (carget cell or Maian h. t cdl) (Plate 5H) I. A codocyte', results from. central bul~ in the cdl caused by an incrra.e
,ha""

'48

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY 2 Echinocyus ... thought to form wh~n th. surfa"" area of Ih. ouUr lipid Jay'" of Ih. ""U memb"m. incre>ses relal;"" to cru.t of Ih. inn., lipid loyer b«:au.. of in .. rtion of lipid. or amphipathic drugs. Th.y Im}' ""0 form KCOndary to inc,,,,,..,. in pH. nythrocyt. ATP dq.l.tion, dam~ by phospholipases, and <:dlula. dehydration. 3. P>thologic minocyto,j, h", bttn :rn<>Ci
hor=J .. ·.. b. Strenuou. eRrci.. (in racing hones)" c. Doxorubicin toxicosi," d. Rl~tion or inc..=<:! plasma lysolecithin fortrultion}." 5. Diffe,..,ntiation of artifacrual and p"thologic ..rninocyte. con be difficult. These thrtt methods might assist with the challeng~: a. Mak~ :mother blood film .nd u.sc:. hair drier or oth~r means to dry the blood quickly. b. Examine blood film. p""p"r«i on plastic slid.. or oovc:rslips. Cr~nation is less likdy to occur on plastic. c. Examine erythrocyte. in a wet mount of blood on a slid~. If echinocytes ..~ not present, th05e =n in th~ suinffi film con be oonsid~r«i artifacu. J. Elliptocyte («< ovalocyt~ in th~ following sect. M ) l. Elliptocytosis {ovalocytosis} in domenic mammals am be cou.«i by either acquir«i or cong..niul disorders. 2. Ovau,cytOli, and rllipfilcytO';' are typically oonsider~d 'Y"0nyms. Som~ prd"~r one term ovc:r th~ other, using ovalocyte for plumper cdls .nd dliptocyte for more dongatffi or dliptical ""lis. K. Keratocyt~ {hdmet ""ll} {plat~ GA} l. K<:ratocyto.i. truly be caus.d by trautrul to erythrocyte. within the VlIOCul... yst~m; the same pr""""'" truly creote .roizocytes. Oth~r m..rnanisms may also be involved. K<:ratocyt~s .nd !Chizocyt.. ar~ both « in fdin~ blood rollect«i in EDTA, but the mm.ni,m was not provid«i." 2 An interm«iiate form sometime. callffi a p"kmlllK)'tr {blister cdl} h:u a cytoplasmic dear {blister} th at may r~pru~nt • vacuole or a hole through the c;.,ll. Others suggest it "'presenu fused m~mbran~'. Scanning dectron micro_ scopic assessment of th~ .. cdls has r"""alffi hoI.. , not vacuole. or fused m~mbran .. (MAS. unpublishffi observations). The m~ch:mism' by which these cdls form ar~ not ""n.in.

'''''0

'I"'''''

3/ ERYTHROCYTES

L

M.

N.

o.

P.

149

3. Some form, ofk.ratocytes. such as tho.., with one horn. han al,o bttn classifi..:!.., buddifll: fragmentuion. aconthocytes. or dacryocytes. uptocyt< {plot< 6B} l. Some rodocyte. and most hypochromic erythrocytes "e leptocytes (... rodocytes and hypochromic erythrocytes. in Morphologic Features of Erythrocytes. "",u. IIl.C and Vll.E). 2. Some people consider leptocyte a .ynonym for rodocyte or a t"m for a hypochro_ mic erythrocyte. Codocytes and hypochromic erythrocytes may be leptocytes. but not allleptocytes "e rodocytes or hypochromic erythrocytes. Aho. immature erythrocytes may be hypochromic, b..,..:! on the CHCM or MCHC {but not microscopically}. or rodocytic, without being leptocyt... Ovalocyte (elliptocytr) {Plate 6C} I. Acquir":! ovalocytmjs i. lttn in dogs with mydofibro,i." .nd in animal, with F.... deficiency .nemias {along with oth" abnormal erythrocyte ft.atur ..}. 2. Ovalocytmis has bttn found in cots with hepatic lipido,j •• " portooystemic shunts.'" and doxorubicin toxicity." Ovalocytoois is occ..ionally...,n in blood film. of cat, with oth" disorders. Etydlfocyt. membrane analy.i, h.., failal to detect qualitati"" or quantitatin defect, (unpublish..:! repom). 3. Two types of canine hereditary ovalocyto,j. (dliptocytosi.) have bttn reportal: one :woeiat":! with • protein band 4.1 deficiency" and one with mutant membrane spectrin." Clinical aspects of the .. di!Orders "e p ....,ntal in thi. m'pt" (Oth" Erythrocyte DilOrders. srets. VI and VII). 4. Elliptocyt.. ha"" bttn subclassified: Type I i. neorly circular. typ<: II is oval. and type III i, mo .. d0fll:>l..:!.'" 5. Healthy cantdid•• vi.n. reptilian. and amphibian species have ovalocyt.. as the ""pectal erythrocytes. Round discocyt .. would be poikilocyt.. in the .. species. Pincered cdl (Plate 6D) l. Pincer":! cells havt bttn associ>lal with erythrocyte trauma and PK deficiency in a Cairn terrier (unpublish..:! case report) but i, rarely report":!. 2. In people. pincered edls h.vt bttn associat":! with erythrocyte fragmentation. h"alitaty spherocytosis." .nd erythroleukemia" Pyknocyte {irrq;ularly cont"'ct..:! cdl} {Pl.te 6E .nd F} I. Pyknocytosis is lttn concurrently with =ntrocyto.is in dogs and horses :md probably will be ...,n in other animal,. Pyknocytes likely form from =ntrocytes. but oxid atin damage might cause: both directly. 2. Pyknocytes suin more intensdy with NMB nain than do di=x:yt.. or spherocytes. at I..." in ho ..... 3. Vi. light microocopy. !Orne pyknocyt.. look like sph"ocytes. However. the.., spheroid pyknocytes a.. usu.lly .crompanial bye=ntrocytes :md pyknocyt.. with memb",ne tags. so the pathologic mange:. can be recognized. Via dectron microscopy. pylmocytes had memb",ne irregularities or t"i:s :md w"e not pc:rfrct sph"es.''' Smizocyte (.rni!locyte or RBC fngment) (Pl.te 6G) l. Schiwcytosis occurs when rigid structu.., or rheologic forces traumatize erythrocytes. 2. Pathologic sum associat":! with ,mizocyto,i. indude int"'VlIOCUl" ooagulation. va",utiti, induding glomerulonq.hritis and hemolytic uremic syndrome. hem' fIl:iosarooma. caval .yndrome of dirofil.ri..,i,. endocarditis. liv" di ....... heart failure.

".

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

Q

h~mophagocytjc hisliocytic dj""rd~n, a.cquir
ob .. rvations}. 2. Th.ir formation is d.K,ibffl .. a cwo_nq> prOC<:Sll: {I}. hoi. is formand 3 deficiency in Japanese black cottl~,'" and with dyserythropoie.is in Englim .pringer sp.nid,." 4. Spherocyto.i. may k fal,dy detect«i if one a"eues cell, too n~.r the futher«i «ig~ of. ,meor wh~", mif.ctual di,tortion of ~rythrocytes make. erythrocytes lack central paUor. Pylmocytes may k faJ,dy ronsideral spherocytes. 5. Procuses that produce sph~rocytes may al", produce spheroid cells that are not p_ plas"" Dr~nt'" patrij,hond. with roncurr~nt hyp
3/ ERYTHROCYTES

151

in its p
General information A Annnk> is ~ d"'r ......! [RBC], ~ d",r..asN blood [Hgb], or a decre~...! Hct, B, A blood', Hct, blood [Hgb], and [RBC] g
E, The major physical enmination finding is pale mucous membranes (gingival, conjunctival, or vulvar) due to .n.mic blood in capillnies, With. mark.d ~nemia, blood berom.. less vi"x>us .nd m.y cau.. ~ systolic h...rt murmur, [I.

Classific;;otions of anemi., Th"e are three common dassific;;otion syst.ms, e~ch with its .dvanug.. and limitations in certain clinical situations, A, Classific;;otion by IIUrrow ... ponsiveness L This dnsification syst.m is primarily b=<:! on th. pr... nce or abs.nce of reticulocy_ tosis in blood, but oth" blood film and marrow findings IIUy influence the classific;;otion, a. R(gm,,"dw Iln(m;Il i. anem;" with a concurrent reticulocytosis, Marrow may be respomi"" prior to ~ reticulocytosis (p .. regenerati"" anemia), Until th"e it a reticulocytosis (or progr=ive Hct incr. . ..s), one cannot be certain that erythropoiesis will k effecti... and th. ~n.mi. will be r
.Ii"

152

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

rrticulocyt<>!is p'.kal 9 d .fl~r

3,

4,

5,

6,

.nu blood loss and did nol mum 10 basdin~ until

day 21."

c. Animal, in ram sp«i .. vary in th~jr ability to prod""" a r~{jculocyto,i,. {I} Dog. han a g'''''{ .bility. RC or eRP our incr~,..., sixfold to eightfuld in ""'pon.. to ~.r. anemi •. {2} Cau h""e modem. ability {mark thrttfold 10 fivefold}. {3} Canl. han mild ability. Increasffl ""lyrorom"'", is fr«iu.ntly aerom"""ial by erythrocytes with Nwphilic stippling. (4) Ho .......'1 rudy rd.... polychromatophilic erythnxyt.. from mmow, 50 allempling to establish p<.iph,,'" blood miculocytOlis h.., nol bttn ""Juab]•. However. hono; with.n erythropoietic stimulus may rd..,... miculocytel that are d.ta:ubl. by automated .nalyurs {• .g., ADVIA l20}: Re. w"". 5- 10 X 1001IlL .fte, phldlO{omy indu<"nl anemia ill horses,'· .nd the RC was 57 X IO'lj.IL in a hors< with a hemolytic anemia." The pr~""~ of maCJocyr~. ill aJuill~ blood sugge>ls, but does 1I0t pron, marrow respon,i",,"~ss to Epo ill mon clinical .ituations, BoII~ marrow namillatioll may provid~ ~dffi"" for marrow Epo r~.po",inll~" in hot'llel, Similarly, .... rly marrow =pon ... may be, 1I0t~d ill othu ~i .. prior to miculocytosi., Th~ following ~rythroc~ wlIOrmalities would support a ~n=ti"" status, but .... cb may also b. found ill 1I01l~1I"'lIiv~ annnias: m3oCl'OCytic ,"d1or hypochromic illdi...., anisocytosis, Howdl-Jolly bodi.. , rubri!is, or b....,philic >tippling, Mild to mod~me bon~ marrow ~rythroid hyptrplasia without a reticulocytosi. may ",flet:t a p""di"g rq;~II~ratin all~mia, particularly wh~n th~ ~rythroid .. ri~. is l
7,

Noor~n~ratin .n~mia

{nom.. ponsin an~mia} a, This occun diua .... that diret:tly or indiret:tly caUst d~f<'CIi"" or reducal ~rythro_ cyt~ production, (During th~ fint f~ days after h~molrsi. or blood loss, .n .n~mi. will be, classified as nonreg~lIer;Uiv~ !,.,caw.. th~ marrow h.. not had tim~ to produce. miculocytosi,, )

3/ ERYTHROCYTES

153

b. A p<",in~nt nonreg~n~rativt status indicates that bon~ marrow is not rrg~n_ ~rating a "'pla""m~nt population of ~rythrocytes. A severe, nonr~nerativt anemia typically r.fleeu =.rr and prolonged da~ to erythroid cdl pr<"Cunon. c. Finding. in bon~ marrow examinations include erythroid hypoplasi >, marrow .plasia, =i cdl apIa, i., mydofibro,is, myelitis. mydophthisis, rdativtly normal erythroid Jeri .. (hypoplas", may bt too mild to det<"Ct), mild to moderate erythroid hyp<rpl",", in tarly respomivt anemias, or Jrulrked erythroid hyp"'pJ:.sia with or withom maturation arrest in conditions of indf<"Ctivt erythropoiesis. d. Most nonregenerativ~ anem"" a.. normocytic normochromic an~mias withom poikilocytosis or other erythrocyte .bnorJrullities. How<'V~r. blood may conuin the following erythrocyt~ abnormalities ",hted to th~ underlying di ...... proa..: Howell-Jolly lxxIies, rubricytOJis, oodocytosi., basophilic stippling. macrocytes or microcyt .. , or hypochromic erythrocytes. B. Classific;nion by erythrocyte indict. (morphologic cla.. ification) l. This clas.sification system is b..ed on MCV and MCHC (or CHCM) (f.ble 3.8). CI...ification should bt funher characterized by rxamining erythrocytes on • Wright_suined blood film. [n the original cla.. ification .yn~m, ther~ was not a category for blood .amples with increastd MCHC values btcause such values were comide=i to bt erroneous. How<'Ver, an incre• .ed MCHC or CHCM may bt valid and rdl<"Ct a pathologic stat~: thu.s wr havt added th~m to the .yn~m. {Note: The MCV .nd MCHC or CHCM values are used mostly to m.racl
154

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

Table 3.8. Causes of anemias cl... ified by erythrocyte indices (MCV and MCHC or C H CM) DiKlrd~rs

An~mia

cla"ification

Normocytic normochromic

Mev

MCHC or CHCM

WRI

WRl

or condition! that em",

th. anemia If ptf,iSlCllt, chen {ypi""'ly diso,d .., that rMua: erythropoiesis; most .nemias bq;in as normocytic normochromic

M.crocytic hypochromic

i

,t.

Regc"".{j"" =pon.. aftu blood loss or hemolysis

M.crocytic normochromic

i

WRl

Regenem;"" =pon.. aftc, blood 105S or hemolysis; OCClSion<>JJy due to d.f=i"" uythropoies;s (hLV indu=l, poodle

Microcytic hypochromic Microcytic normochromic

Normocytic hypochromic Normocytic hyptrchromic" Macrocytic hyptrchromic" Microcytic hypuchromic"

"" " ";

WRl

WRl WRl

;

1 " ;

;

macrocytosis); in vitro changts" F. ddicicncy, pyridoxine ddicicn
including portosystcmic ,hunn, in vitro changt." Rardy =n, susl'ffi error

MCHC

or

CHCM may

~

factitiously incr......d or a pathologic state may au.. tru~ inc=se:s {= th~ text for nplanation}

• &. th. tat for tt.. co .... of in vi'ro cbaag .. tho< an produe< bigher OJ lower MCV v:ol..... • Tb. hJjHr
'0

c. Becaust MCV .nd MCHC or CHCM are a""rag.', erythrocyt~ cytogums or blood film exoomiruo,ion. are typically more .. n,i,ive m.. hods of d....cring manocytic, microcytic, or hypochromic cdls. With .ith~r m.. hod, it i, possible to have a normocytic normochromic an~mia with detectable macrocytic, microcytic, or hypochromic popul.cions. 3. Normocytic normochromic .nemia, a. Blood film findings: Erythrocytes are typically uniform but =yoccasion.lly hav~ morphologic abnorm.li,i ... b. Most .nemia, btgin as normocytic normochromic anemias. Wh~n marrow rd~..... many larger or smaller ~rythrocyte. wi,h normal or d,""reaonl Hgb conce"'rn.tions, ,hen MCV or MCHC (and CHCM) will chan~. MCV or MCHC {and CHCM} mu", be ou"id~ of refe .. nce int~rval, btfore th. morphologic dassifico'ion changes.

155

3/ ERYTHROCYTES c.

P~rsin~nt

normocytic normochromic

an~mias ar~ n~«i

to b.

noru~generative.

d. Most ~nemias in horse. at< normocytic normochromic because their trulrrows rde ... few r~ticulocyt ... [f sufficient macrocyt.. a.. rdused. th~ anemia will b.com~ macrocytic. 4. Macrocytic hypochromic .nemias a. Blood film findings: One <= ""p«t polyffiromas", (""""pt in hor=), macrocytosis, and ~ni1OC)"losi •. Visual hypochromas", is not n~«i becau.sc: retirulocytes are large and contain. normal mass of Hgb {MCH is not da:=sedl; thq do not spr~.d thin enough to have .n increas«i ""ntral paUor. b. Concum:nt m. crocyto!is and hypochrotrul,i. support the pr..~n"" of imtrultllre erythrocytes, and thm the anem", is probably due to blood lOll or h~molysi •. c. Dogs (e.g., selmau,.,rs) with nom>tocytosis may have macrocytic hypochromic cell •. " d. [n autotrulted h~matologic instrum~nts, th~ MCHC is cakulat«i from the measur«i blood [Hgb] ~nd a Hct that i, calculat«i nom. measu=' MCV.nd [RBC]. [f the [RBC] is .ccurate .nd the MCV i, fal!dy inc..ased (see the foUowing sect:. S.c), th~n the cakulat«i MCHC will b. falsely da:reastd. 5. Macrocytic normochromic an.mw a. Blood film findings: On. <= ""p«t polychromasi., macrocytosis, and .nisocytOJis. b. Disorders or conditio", {I} Thq a.. common in rq;enerati"" anemias beau.. of blood loss or hemolysis. {2} Thq a.. lOmetimes associated with defective erythropoi.,is. (a) F~lV_infect«i cats may have defective ~rythroid trultllration that yidds megalobl~nic cells {Pl.t~ 9LJ with d~fective DNA synthesis and thu. decreased milO"': m.galoblastic cd], trultll .. to macrocytes. {b} Folic acid and cob.lamin (vitamin B,,) deficiencies couse defective nucl~ic acid meubolism that could couse trulcrocytOJis (po1!ible, but rardy document«iJ. C~tcle that gra:z.e • col>alt_deficient paSlur. truly have. normocytic or m.crocytic anemia due to a rol>alamin defi_ ci~ncy." Cobalt is an .... ntial compon~nt of cobalamin. Cobalamin deficiency depre,s,,. the activity of. methyltunsfc:r= that blocks folate metaboli,m by trapping a methyl group in 5_methyltetrahydro_ folat~. Thu" in the .bs.nce of cobalamin, • functional folat~ defi_ ci~ncy truly niSI n-en though the ..,rum [fola.. ] truly b. WRI. {S_Methyltetrahydrofolate is detect«i in fol. te .....1'; ,ee the FoJ.t~ Concentration in Dogs .nd Cats ..ction in Ch.pt~r IS.} (c) Poodles with the poodle trulrrow dyscrasia (see Other Nonneoplastic L=kocyt~ Di!Ord~rs, sect. II, in ChapUt 2) will have a macrocytosis and may have .n anem", due to .nother pathologic prOC<:1s. Th. pathogen.. is of the macrocytosis is not ~sublish«i. {d} Erythroleukemi a {e} Congeniul dyserythropoiesis and progr.... i"" alop«ia of poll«i H..~ford calves"

'"

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY c. Th~ MCV may bt jncr~as
b. ignorw. {2} Cd] swdli"g during stoug. I> most fr«J.u.ntly with mail.in .. ruple., Th. MCHC (and CHCM) =y b. drc,"""'!' {3} [n vivo hyperosmolar sUUs (e.g., hYp'rnmemia) con [
intncdJul .. osmol.lity. When the blood within the .n:.!yu, is dilut.d by fluid of lower osmol.lity {.pproxim.tdy isoosmotic with normal plasma}, H,O mo~. into th. cdl, and cause. acut ......dli"g. Th. MCHC (.nd

CHCM) may ~ daor......d." Ex~ss dipotassium_EDTA .ntic=gulant may c;;oUst .rythrocyt~ sw~lling and d.cr•• '" the MCHC (CHCM) with the Bay.. T.chnicon instrum.nt wh.n the ~Ils mix with analyzer dilu.nt. EI<~S tripoussium_EDTA did not have the ",m. df.a.""n 6. Microcytic hypochromic an.mi", a. Blood film finding.: Expect microcytmis. lq>tocytosi •• oodocytosi •• hypochro_ masia. and ani5OC)'lmi s. Oth.. poikilocytes may indud. ovalocyt... ..rnirocyt... • nd foldal ~rythrocytes. Polychromasi. may ~ pr=nt but I.... than expectrd for the "",..ity of the an~mia. b. Microcytosis .nd hypochromas;' may ~ du~ to d~frctin Hgb .ynthesis amstd by th~ following: {I} F~ d~fici~ncy (Ke Blood Los! An~mias ...ct. II.B) {2} Copper deficiency in dogs may c;;ou "" a microcytic hypochromic an.mi.!' How...... an experimental Cu d~fici~ncy in dogs producal a normocytic normochromic .n~mia!' (3) Potentially. vitamin B, (pyridoxine) ddici~ncy c. H.patic failur~ {rard,... mo,", likdy microcytic normochromic} d. Th~ MCV may ~ decreasrd by ~n.in "'mpl. or patient conditions (s.., T.bl. 3.3) .nd microcytic hyperchromic stat~. (= th~ following Ket. II). 7. Microcytic normochromic an~mi.., a. Blood film finding' vary from thOst Ken in microcytic hypochromic anemia to normocytic normochromic :memia. b. C.Ust, of microcytosis {I} F~ d~fici~ncy (e.rly or mild): Prior to c;;oming. microcytic hypochromic an.mia. Fe deficiency m.y produa. a microcytic normochromic anemia. but th. MCH is daor..... d. {2} Hq>.tic failure due to hq>atic di ..... or ponosynemic munu: The c;;ou", of microcyto,is is not known. but data su~t • d.f.a in F~ tramport to .rythrocyt~ prrcursors. MCH i, decreasrd. but th~ MCHC (C HCM) typicaUy r~mains WRI. {3} Dystrythropoiesi. in English spring.. ,,,,,nids" {4} Som. healthy Mitas and Shilw han lo_r MCV value, (in the 50-60 fl ran",,) than do dog. of oth.. breeds. Th~ 5am~ may ~ tru~ of some dogs ~longing to oth~r Asian brttd. (e.g .• Jindos. chow-chows. and ,har_pei.). {4}

3/ ERYTHROCYTES

8,

9, ]0,

IL

12,

157

Also, foals .nd kimns h. ve low.r Mev values th. n do adult animals of the respective .pecies,"'" Normocytic hypochromic an.mias a, Th... n. uncommon, If fOund, one must consider that the data may be inaCC\lr.lte or that the reference intervals may be inappropri.te, b, They cm be found when erythrocytes n. hypochromic (bemuse of immaturity or Fe deficiency) and the Mev ill.. not changed enough to be out,ide of refer.nce interval. Examination of blood film may r",eol nurked .nisocytosis, but overall there w.re not enough ma.crocyt .. or microcyt .. to inc..... or dec=se the MCV, Macrocytic hyperchromic :memias: Typically, the MCHC is fal ..ly increased (>« the following sect, 12), Compne to the CHCM, if .vailabl., Normocytic hyperchromic an.mi:as: Typically, the MCHC is falsdy increased {see the following sect, 12}, Compne to the CHCM if it is av.ilable, Microcytic hyperchromic anemias a, Falsdy low Mev and high MCHC (CHCM) may be produced when erythro_ cyt.. are in hypoosmolal plasma,"" Erythrocytes adjust in vivo to the hypo_ osmoW.nvironment mu.ed by hypon . tremia and hypochloremia by h. vi"i: decreased cytoplasmic osmolality, When placed in • diluent prior to counting, osmo,is results in H,O leoving the erythrocyt.. and thus decreasing volume of .rythrocytes, b, If the MCHC {or CHCM} is falsdy increased for other reasono, then potenti.l mu"", of a pathologic microcytosi. mould be considered, Inc..ased MCHC or CHCM a, In theory, it is not physiologimlly possible to produce hyperchromic erythro_ cyt.. beame Hgb synthesis stops in . n erythrocyte precursor when an optimal [Hgb] is reached within its cytopl:asm, b, Most increased MCHCs n. falsdy incr....ed, and the blood "'mpl.,.' MCH value> also are falsely incrrased CHCM. are more reliable but mn .lso be falsdy increased, c.u..s of falsdy inc ..... ed MCHC, CHCM, and MCH include the fOllowi"i:: {I} P.thologic hemoglobinemia: Blood [!-1gb] is used to calculate MCHC and MCH, and it would indude Hgb from .rythrocytes and the Hgb in pia. rna, The CHCM would not be . ffected A more acrurat. MCHC amId be calculated by correcti"i: the blood [Hgb] by usi"i: • value for Hgb....., {2} Oxyglobin: Th. free Hgb &om theraJ><'utic...., of Hgb_based 0, mrrie" mus •• an ove""tim. tion ofintracdlular Hgb and f.lsely incr ..."", the MCHC, The CHCM is not .ffect.d. A more .ccurate MCHC could be mkulated by correcting the blood [Hgb] by ming • value for Hgb ...., {3} In vitro hemolysis: Blood [Hgb] is u=i to calcul.te the MCHC, and it truly represc:nts the blood [Hgb], but the Hct and [RBC] for the sample ne falsdy decr.ased and thu. the MCHC :md MCH are falsely increased {4} Spectral interferences in the blood Hgb .....y: Interferences that produce a falsely incrrased blood [H gb] include lipid droplets in grossly lip<mic ",mpl.., pigmenu in markedly icteric "'-mples, nudei or intact WBCs in "'mpl.. with .xtreme leukocyto.is, Heinz bodi.. (due to incomplete erythrocyte lysis), .nd precipitates of immunoglobulin. {e,g" immuno_

158

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY globulin A}." This would yidd. f:'!stlr incr.,.snl MCHC. Th~ CHCM Im}'..!so ~ falsdy inCf~~.ffl when many H"inz bodies ar. prestnt ~""'" Hrinz bodi .. ..It., Ih. light_K;O[{oring propeni.. of .ff'""tal .rythrocyta" {5} As discussN in Ih. pr«:aling Sffiion II, th. MCHC and CHCM may ~

f. 1stlr inc,.,....,j ~"'" of cdl shrink3i:" ,dartN and Ih. CHCM is not, Ih." ch. MCHC is probably f:.!,dy inc..OiSN b«:.Ust of Hgb in p[",ma or ~""'" of a sp«:tral interf... nct in the blood [HgbJ =y. {2} If both the MCHC and CHCM .'" increa=l, then th.re may truly k a hyptrrnromic sUte. d. Pathologic conditio", that con caus. true increaso; in MCHC {~nd CHCM}~" ra ... {I} Blood with .=ntrocyt05i, .nd pyknocyto'i' .om.-tim.. ha, an incrra,.d MCHC ba::.u", oxidati", oond.nsation ofHgb and fu,ion of cdl m.m_ bran.. cou", a loss of ""U volume without .. proportio .... u loss of cdl Hgb ....,. Thes. may aho b. microcytic. {2} Sph..ocyt. populations with incrras.d MCHC and CHCM may poun_ tially form in some .ph.rocytic an.m;", if the ,ph..ocytic proc.., couses loss of ""J] mlum. in excess of Hgb. Th... ""It. moyo.lso b. microcytic. G..nera.lly. how....... sph.rocytes in domestic spn;ies only appmr to b. hYP'rchromic .nd micr<»ropically small ba::.u.. of th.ir thickn .... and th.ir MCHCs. CHCM, •• nd MCV••" WRl. C. Pathophysiologic classificotion I. It i, b...d on the p>thologic m.rnani,m or process thot produ=:l the ""emi •. Multiple pathologic prO<:e!.'l<' may oontribut. to an .nemi .. a. Blood 10... an.mi ... con b. amte {hour:! to days} to chronic (weeks to month,). {I} In o:tn7J;l/ blood lou annniill • • rythrocytes are Ion from the body or lost into the alim.nury or urinary tract. {2} In intn7lal bu,,,J u,u an(mids. eryduocyt.. mo", from the intraVllOCular to the .xtravascul.. 'pa"" {typically into ~ritonffi or pl .... ral cavities}. b. Hemolytic anemi.., {I} In ~vaJi"ufilr hnnolyris. ther. i, erythrocyte ly.is ouuide of blood ve,set. {in mocrop.}. It d~ not indud. hemorrh>ge. {2} In intraWlscu fil r "'m~Iy'i'. the .. is .rythrocyte lysi' within the blood vascu.t.r ')"lum. It dot, not indude ph>gocyt05i' by ti,me macroph>g" while they pass through the .inuse, of the spl..,n.liver. or bone marrow. c. An.mi ... co"""" by d.crras.d .rythrocyte production {I} InHammatory disra.!es {2} Re .... l di"'.... {3} Marrow hypopla,;" or .plas;" {4} Erythroid hypoplasia or indfecti", erythropoiesis 2 The p:uhoph)"liologic classificotion ')"lum is f"
""U,

3/ ERYTHROCYTES

159 j He!, j [H gb[, or j [RBG[

I

Raticulocylosis or

incroased poIychmmao.ia

I P.......nl

,

...

, -"'

Rogooe
,..,

."".

Blood I""" or hemotvsis < 34 d

• Exl.. "",1

.

• Chronic • Inl.. mal

.""

• Chronic

H&mOIysis • Exlrwascula, • InlrllVascuiar Erylhroid noopIasia ("",.. )

Reducod erytlvopoiosis

• Decruased ery1hmpoiotin • Refractory 10 &ry1IYopoioIin • Bone marmwdam.mplacamoo1 looffIIc1iYo 8')'thropoiesis • o...1N<:tion 01 ory1lYoid p<9
• abner .....1ory1IY<>id "",1u'alioo • doIoc1Mo nucluic Itcid motabolism

Fig. 3.7. An "Pproocb to probJ.m...oMng .,..mi .. : Aft.,. an<mia b.. b.en , then it is due to reduced or in
a, It

,="', .. a diff~..ntial diagnmis Ii" or to anSW~r qu. "iofl! mch .. "What ar~

th~ b.,ic cau .., of an~mi .. ?" (Fig. 3,7) b, It is usal to group s~fic dis.as .. basal on th~ m.. hod or method, by which they cau.. ""~m;"

NO NREGENERATIYE ANEMIAS L

Gen~ral

roncq'" A, Th. major =son fOr a p"rsist~nt nonregtn~ratin ""~mia i, d",,,,asM uylhrocyt~ production; d~f"'ti", uythropoiesis can also rontribuu, Sin"" ~rythrocyt~ lif. spans of dom~stic ""imals ar. !:"n~rally 2- 5 mo, an an~mia will tak~ ..,.."ral w.do, to months to devdop if il i, causal only by d",rease 50 d old and b.lf . '" < 50 d old, [f a di ..... stopped .rythropoiesis completely and did not alt~r uylhrocyt~ life 'P"", il would tah 25 d for th~ dog'. Hel to drop from 40 % to 30 %, ""d . bout 50 d to drop from 40 % to 20 %, B<e;,use cat ~rythrocytes Ita", shomr life span. (approximately 70 d), such an. mw would develop quichr, lik~i .., prodUClion.f.jJur~ an~mia would develop slower in hors .. and calll~ becau .. Ih~ir .rythrocytes Ita",lon!:"r lif. 'P"'" (approximately [50 d),

'01

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY B. Mon di..,ase; do not ,top ~rythrocyt< production completdy but only d,"""""", {h~ nt~ of production. Th~r~for~. nonreg~n~"'tiv. anemw =1 uh nell Jon~r to drvdop. How"""., many di...,...,. t .... t rfflllCt erythropoiesis .1.0 ,horten erythrocyu life 'pan, and thus .nemi. may devdop quicker than ex~al from 'MUen! erythropoiesis .Jone. C. Mon anim:.!, with nonr~nua{in anemias h.ve betn anemic for ~ral w,""h I>
[I.

Disorders that cause nonreg~n~r.otive an~mi"" (T.bl~ 3.9) A. InH.mJrultory di ..ase l. [nHamJrultion causes AID (also called Ilnrmia of chnnic dmll~ .nd IllVnlia of ch,."nic injlllmmll,ion). It is the most common nonreg~ner.ti"" .n~mi. of domenic Jrulmm.b and v.ries &om mild to mod~r.ot~ ..verity. Typically. it i. a normocytic normochromic anemia but. r ..dy, is microcytic.

Table 3.9, Di.oro.,"" and conditions that cause nonregcncrative anemi ..

Rcdu=:l erythropoiesis [nH.mJrultory diseases (primarily chronic) '[nfectious: b.cterial. fungal, viral, protozoal, p",,,,,itic 'Noninfectious 'Refl<01 di ..ase (chronic) Diseases causing marrow hypoplas;" or . plas;" Infectious agents: b.cterial, fungal, viral, protozoal Toxicosis: chemoth~ra~utic agent., cnrog..n, bracken fern, phenylbutazone [rradi.tion: whole body or ~nvironm~ntal Marrow neopl"'i. or replaa:ment: n<"Oplas;", mydofibrosis, oneopctrosis Diseases causing ..lective erythroid hypoplas;" or . plas;" Pure red cdl apl""i. (including immune.medi.t«i mechanisms) • FeL V.indu=:l erythroid hypoplasi., 'Endocrine: hypothyroidism, hypo.drenoconicism, hypo.ndrogenism 'liver di ..... or failur~ (including portosyst~mic shunt.) In~ffcctive erythropoi .. is Nutritiofl
3/ ERYTHROCYTES

161

2. Al D is of rdativdy litd~ clinicol signif]cona. .ner ir i, r«:<>gni=:i. Most di<>gnonic drom a.. direcral toward the priJrulry di",= and not the strondary .bnormalities caus.d by the infLommatory di",=. 3. Almost any rnronic di50rd~r with an inflamJrultory component will initiate th~ p""""s.., that co"",,, the anemia. a. Chronic inf~tion" bacterial (including .napl""mal). fungal. viral. and prot()7.()al b. Noninf~tious disorders: immune. toxic, and neopl""tic (mually a malignant neoplasm that cau.., n~ro";, and/or infLommation around or within the neoplasm) 4. P.thogenesis of the anemia involves the", thr... roncurrent m~hanisms initi.ted by infl.mmation:" a. Shortened erythrocy" survival {I} Pathologic n-ents a.. not understood entirely but . ", associated with incr ......! [11..-1 ]. {2} Oxidant damage to erythrocyte membranes and subsequent binding of immunoglobulin molecul .. Jruly .ccder.te the ",moval of erythrocyt..! ' b. Impaired Fe mobili7.;Jtion or utilization {I} Hepcidin production by hepatocytes is stimulated by IL-6. and the binding ofhepcidin to ferroportin in cdl membranes internalizes ferroponin. Without membrane ferroponin. the Jrulcropru.ges cannot expon Fe. ' {2} Alt~rations in ferritin production .nd alterations in transftrrin =eptors incr ..... Fe storage and therefo .. decr ..... the a..... il. bility of Fe for Hgb synthesis. {3} Cytokines involved in .ltered Fe kinetics include 11..-1. 11..-6. int~rftron. and TNF. c. Impaired erythrocyte production {I} Erythroid cdls become refractory (nonr .. ponsive) to incre""ed Epo becau", of the df~cu of infl.mmatory cytokines (lL-I. interferon. and lNF) on precunors. {2} Bluntal Epo r.,pofist to anemia {Epo production is incr ......! but not as mum ., expected} i, due to action, of IL_I. TNF. and tumor growth factor

p. 5. Thestlaboratory findings support the rondmion that an .nimal h"" AID: a. Mild to mod~rate normocytic normochromic anemia with little to no poikilocytosis b. Chronic inflamJrultory leukogram: m.ture neutrophili •• lymphocytosis. or monocytosi, c. Hyperproteinemia due to increased concentrations of1-globulins or positive acute_ph... proteins d. Marrow that oonuins =ntially normal to mildly reduced erythroid population. mild to mod~rate granulocytic hyperplasia. pos.Jibly plasmacytmis. and abundant hemosid~rin (except in fdine marrow. wh~ .. it is not exJlft'led) e. Hypoferremia... rum [ferritin] WRI to increased. and adequate to increa...! suin.ble Fe in tissu .. (Jrulrrowexcept for spleen. or Jj""r) B. Renal di ..... {chronic} l. Mon patients with rnronic ",nal di ..... a.. anemic. The anemia! a.. slight to modera" in ....erity •• nd essentially all are normocytic normochromic. 2. P.thogenesis of anemia

CO".

162

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY a. In.d"'luat~ Epo production: Chronic reD:.! di ....... domage; th~ kjdn~ suffi_ ci~ndy so that Epo production d,""reast., and thus the stimul.tion of erythrocyte production it j""dscuJ.r damage: poor nutritioruol st;;otus 3. uboratory finding. a. Normocyti, normochromic, nonrege"."'tiv. anemia b. Evid.nct of mronic renal dise;;o.., or dy.function, such as not.mi., utine .~ific gravity in the isonhenuric range .• nd d.ctrolyt. disturb:mces C. Dj ....... , causing marrow hypoplasia or .plasia of multipl. cd] lineage. l. Major con~pts a. Dam"l:' can ~ to one or more of the rompon.nts of the marrow', micr""nvi· rooment: blood vessels and/or ,inusoids, r~ticular adventitial cell" marrow stroma (fat cells or fibrocyt~.) , or h~matopoiHic n~m cell.. Th~ r~"Ilting marrow will bt hypoplastic or aplastic, b, Dam"i:~ may bt irrrversibl~ or reversibl~ and may cause: aplastic an~mia (hypo_ planic pancytopenia), 2, Disorder> a, Marrow hypoplasia or aplasia in domestic animals ofi~n has an unprov~n disord~r (idiopathic), [n peopl~, aplastic an~mia is usually immun~ m"!iatM. b, Inf,""tiollS ag<:nts may SlIppr~"" hematopoiesi. through direct hemic ceU inf'""tion, myditis, or sc:condary ~ffects mch as immun~ ",actions, ( I) Suppression may occur with bact~ri:d stpticemias, ~hrlichi05is, di""eminat..! my= (e,g" hinopla'mosis), viral infn:tions (~,g., EIAVor F. LV infH:tion), or protozoal infn:tions (~,g" l~ishmaniasi, or cytauxroono,is) {2} [nf=ions with C)'UIIIXZODn frlis are som..what uniqu~ among th= inf,""tious ~nts ba::.use: th~ ~nt has piroplasms in ~rythrocyt .. and .chironts in macroph~.," Clinically, cytaux7.00nosis is oft~n a npid and highly fatal dise:... ," but a f<'W caU do survive: natural inf'""tions," (a) Wh~n blood erythrocytes contain the piroplasms, cats mayor may not bt an. mic, Wh~n pr..~nt, an~mias can rang<: from mild to sc:vc:r~, ar~ nonr.g<:n~rative:, and probably ar~ caus..! by AID and do~ to marrow, spl«n, and liver, {b} F..v.. and iClusffl by hepatic dam"i:~ and cholestasis, Erythrophages are found in many organs, but h~molysi. umally is ronsid~red a minor contributor to th~ anemia," {cl Oth~r lahomory findings may include the following: thrombocytopc:nia, leukopc:nia {som",im.. with toxic chang~s}, larg~ m3oCroph"i:~s laden with .chizonts in th~ blood film. (rardy), and oth~r hematologic and clinical chemistry abnormalities c>uKd by h~p"tic, 'pl~nic, lymph nod~, and marrow dam"i:~ as.sociated with th~ schizont stag\' of th~ parasitic inf'""tion,

3/ ERYTHROCYTES

163

c. Toxirosts involving compound, such '" ch~moth".ptutic "l:~nts. estrog~n.·~" ph~nylbut:IWn~."'" and ch~micals in brachn f~rn (Ptiridium "qu;{inum) d. Ir",diation donuge prodUcffl by whol~ body th~"'pnllic or ~nvironm~ntal «POSU", to X_rays. gamma irr>diation. or bota irradiation e. Marrow r~pla~m~nt ( I) Di",ases may co"",,, anemia and oth~r cytoptnias by r~placing hematopoietic cdls in th~ marrow. Such anemia. a", commonly colled mylophthi.ir

"mmill,. (2)

Di50rd~n

that may cou", mydophthisis (myrlo- "marrow" plus _phthisis

"waning") (a) Myeloproli~mi"" di",a...: gr.nulocytic. monocytic. erythroid. or m~a,}"oc}"tic nroplasia (b) Lymphoprolifemi"" nroplasi:a: lymphoid and pl",ma cdl nroplasia (c) Memtatic nroplasi. (i) Lymphoprolifc:mi"" nroplasi. (primary in lymph nod ... 'pl..,n. or oth.. ti ....."') (ii) Man cdl nropl"'ia (iii) Carcinomas and nonh~mic s;oroorrta..! can m~tastasiu to marrow. but such lesions ar~ not «ptctrd to cou", sufficient marrow damage to produ~ an~mi .. (d) Nonneoplomic ~Jl prolif~ration (il Myelofibrosis is fibrou, ti.lU~ prolifemion. usuaUy for unknown r~awns. It may occur afi~r inflammation and/or nrerosi •• with myeloprolife"'ti"" di"'.... or with a chronic ~rythropoietic nimulus. (ii) Osteopttrosi" bone proJjf~ration into medulla,y spa~ D. Diseasrs cousing sdreti"" ~rythroid hypoplasia or indfreti"" erythropoiesis (without gen~ralized marrow hypoplasia) I. Pure rrd ~Jl .plasia a. Purr rrd aU "p!mill i, a descripti"" term for di50rd~fll in which a nonreg~nerati"" .n~mi a is cou..d by markrd erythroid hypoplasia or apl",ia. but oth~r hemato_ poietic ceU lines ar~ not dd«ti"". It ha. been rSSQCiatrd with T_lymphocyte donal di50rd .... Antibodi .. m.y ~n be directrd ag.inst Epo. (2) Some dogs have h:>d • demonstrable ",rum SIlbsun~ (probably .ntibody) that inhibits ~rythropoi ...i, in vitro. but oth~r dogs do not."' c. The disord~r may b. ... ponsi"" to immun~_SlIppr... i"" dosage> of glucoconicoid compounds or oth.. immunosupp=sive th~rapy. but the",py may tak~ 2 wk or longer bofo.. .vidence of respon'" (~.g .• reticulocytosi,). Some dogs =Iuire long_ t~rm th~rapy to pr~nt r~cur .. nce of an .nemia. d. Loboratory findings (I) Typieolly. thry indud~ normocytic normochromic anemia and sometimes spherocytic an~mia.

164

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY {2} Nonregtn~ra{in .n~mia {3} A Coombs' un might yidd • positin mult. (4) Mmow aoomin<>tion ~n.J, markffi hypopluia or aplasia (.b",nct ) of the erythroid a:l.lliorag<'. with presmmion of the other [in. :og ••. 2. Immun._maliatw nonreg.n.rativ. anemi. " a. i",munr.mrdwml nonTffP'""dw (mtmin is simil" to pur. ral cdl 'pla,i. with Ih. a~p{jon dUI the erythroid st,i.. i. pre.. nt in marrow and rn..r:ocuriud by either. ld't mift and matur>tion .rrest or by ~",iS{.nt ''Yduoid hYP"rpla,i. concu,,,,nc with anemia. b. The disord" may ..spond to immunosuppressive therapy c. Loooratory finding> {I } Typically. thry include normocytic normochromic anemia and ",melimo. spherocytic anemia. {2} Nonregrneratin anemia, ofte" SNore {3} A Coombs' ten might yidd • positi'" mult. (4) Mmow ex;;omin<>tion "'''",lions from erythroid hypoplas;" with an inromplm l.ft_shiftal stries (m. turation ar=t) to erythroid h)'P"rpl.. i. , oft~n with subtle but d~.a .bl~ phagocytosi, of intact erythroid pr«:llrsors at Ihe latesl stage of ordtrly d""dopment (plat~ 9G). Th~ production of oth~r cdllines i, effecti"". 3. F~LV_ induc:ffi ~rythroid hypopl .. i. a. F.LV nuy sd.aivdy damage .rythroid ""II, to QlU,. erythroid hypopla'ia or lransform a cdl into a neopla'lic cdllin•. b. Palhog<'nesis of an.m;" (I l If .rythroid precursors a", ..l.ctivdy damagffi, th.n erythroid hypopla'ia or aplasia d""dop. and Ihus erythrocyte production decrra..,.. {2} If erythroid ""II, undergo neopJ:.stic lransformation, the proliftmion of cd], Jruly ~ markrd, but th~ir funclion, cdl metaboli,m, and maturation will ~ def.ai"". Ac:rordingly, th~ ""II, may not Jrultu .. or Ih.y IIU)' die ~fo .. m. turing to .rythrocytes. and thus anemia develop. ba::au .. of d.er•• ...:! .ffecti"" erythropoiesi,. c. Laboratory finding> {I} Th..~ Jruly ~ mild to srvtr~, nomeg.nerali"" an.mia; .ilhtr normocytic normochromic or ma.crocytic normochromic. {2} Th. an.mia may ha"" inapproprial~ rubricytosis, "p"'ially in mydodyspl .... lic syndrome with .rythroid prrdominance (MDS_Er). {3} Marrow findings may vary from ~rythroid hypoplasia to nropl .. ;.. of any marrow cd l linrag<'. {4} Meg. loblmic ~rythroid cd], Jruly ~ found in blood or marrow (~lobfm_ tk IIntrniIJ) (Plat. 9L). Meg. loblmic cdls hav~ asynchronous matu"'tion of nucl.i and cytoplasm.: CytoplamIS mature but nucl~ar Jrulturalion is inrompleu. Th. def.ai"" Jrulturalion produces larg...rythroid p=urson with atypirnlly large nucl.i for Ihe degr.. of cytoplasmic Jrultumion. 4. N utri.nt deficiencies a. F. d.fici.ncy {I 1 Thi, ocrurs ba::au.. of chronic ",Iunal blood loss {•. g., alim.ntary tract blood loss ba::aUst of ul""" or parasil" or cutanrous blood loss ba::aUst of H~as or licks} or in. drqual. di
,,,,,,.Is

3/ ERYTHROCYTES

diagnOMd, th~ .n~mi. is d .... ically microcytic hypochromic but IIU)' bt microcytic normochromic {for iu pathog~n ..is, ~e Blood Lo.. An~mi." sa:t.II.B}. b. Cop~r ddiciency {I} This is uncommon in dom .. tic m.mmals but h •• bttn .. pon.d in pigs . nd in dogs. Erythrocyt~ . bnorm. liti.. d~dop btau"" of d~f~ctive F~ t.. nsport. {a} vrulopI:.smin {~rroxid...,} and h~pb. .. tin ..~ rdat.d Fe oxidas.. th.r conuin cop~r and promot~ th~ oonnuion ofF~" to F~ during Fe""' t"lIIspon out of ma.cropru.g.. •• nd em~rocyt .., respffiivdy. {b} [fan animal i. ddiciem in cop~r, th~ .. is less fwic oxida..., activity, less F~" .bsorption from the intestine, l~ .. rd....., &om m. cropru.g.., less F~" .vailabl~ for h~me symh~sis, and thus def«tivt Hgb symh .. is. B«>u.. of th~ oop~r ddici~ncy, the .. i. a functional F~ ddiciency, and thu. a microcytic hypochromic an~mia an d~dop. s.rum Fe con"",n_ trations should not b. d« .....d. {2} [n >II ap..imenul study, copper-ddici~m dogs de-veloped a normocytic normochromic .n~mia!' Th~ pathogenesis of the an~mi. was not mabli,h.d. {3} An iarro!:"nic cop~r ddici~ncy WlI.! creat.d wh~n tri~ntin~ hydrochloride was us.d to treat copper stonge di ...... 71 A microcytic hypochromic .n~mia persist.d aft~r t..atm~m for an Fe deficiency. The >II~mia resolv.d .nd th~ erythrocyt~ indices improvtd when th~ th~ .. py for copper nO"'1:e dis ...... "",...d. [m~rpreution of th~ = dar. WlI.! compliatal by ~id~nce of hq.. tic dysfunction. {4} Microcytic hypochromic anemi", do d~lop in copper-ddicient pigs. c. Folat~ or oobalamin (vitamin B,~ d~fici~ncy {I} Folate . nd cobalamin . .. requir.d for DNA synthesis, .nd thus d~ficiencies might au.., .bnormal ~rythrocyt~ d~dopm~m. Folat~ and cobal.min deficienci •• may cause a macrocytic .n~mia in people but .. rdy a.. such disord~rs found in domestic mammals. {2} Cau with aperimental folate deficiency hood m~oblastic marrow erythroid cdls but n~ith~r macrocyto1is nor an~mia." A at with a congenital coJ..la_ min defici~ncy b.d normocytic erythrocytes." {3} Giant .mnauurs with .n inherit.d malabsorption of cobalamin had a robalamin deficiency and . normocytic nonr~gene ..tiv. anemi • . M. rrow sampl.. cont:nn.d megalobl.,tic erythroid cells, .nd macrocytes and ovalocytes we .. found in blood films. Rq>ort.dly, an increasffl MCV WlI.! not p..",m btau.. of roncurrem microcytosis; an aplanation of th~ microcytosis v..as not provid.d.'" Dy.plastic ch anges in the mydoid cells indudal hYP"~ent.d n.... trophil. and giant neutrophil .. M",hylmalonic a.ciduria WlI.! also present. {4} A Bord~r coUie with inh..it.d malabsorption of cobalamin had a normo_ cytic normochromic anemi .. " {S} Cattle that d~dop a oobo.lamin deficiency &om gm.ing on cobalt-deficiem soil may d~dop a normocytic normochromic anemia." d. Pyridoxine {viumin BJ d~fici~ncy: A di=ry pyridoxine deficiency in growing kitt~m ..",h.d in .nemia, but th~ features and pathogen.. i. of the anemia w~'" not deseribtd." {2}

Wh~n

165

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

'" 5.

Endocrin~ di""rd~rs

a. Hypothyroidism {I} Sttn primarily in docs, chi, causes a mild normocytic normochromic an~m

{2}

....

P.thogtn~sis

ofch. anemi., D""""asffi [toul thyroxine] and [total triiodo_ in a d=eaIM metabolic "Ie and thus • d""r~ n~ for thyronine] 0, in "",iph.",] {;ssu... Th. decr.,.,ffi lI,""d for 0, leads to decreasal Epo production and thll'l I... ''Yduocyre production. A II<'W homeonas;, d""dol" in which mffabolic n=ls for 0, ."" mff by a lowe, blood [RBC]. {3} Loboratory finding. (a) Mild normocytic normochromic, nonregtneratin .nemia (b) Evid.n~ of thyroid dysfunction, such as d=.aKd [total thyroxine], d""""asal [fr.., thyroxine] .• nd incr.a=! [thyroid_stimulating hormone] b. Hypood .. nororticilfll {I} Sttn primarily in docs, thi, may em", a mild to mod.rate normocytic

,,,,,,,It

normochromic .nemi•. {2} Pathogen •• ;. of th~ .nemi. is not establishal, but glucoronicoids h."" b«n repon.d to S{imulat~ rrythropoiesis in vitro, so th~ir ab",n"" may bt rdativdy marrow suppressi"". G •.mointesti,",l blood loss may enh:on"" th. :onem]a. {3} Loboratory finding> {a} Mild normocytic normochromic anemi., which may bt maskal by hemocon""ntration ","ustd by hypovolemia {b} Eviden"" of .drenal dysfunction, .uch '" hyponm.mia, h~rkalemia, azotemia, hypoconiKllemia, lymphocytmis, and eosinophilia c. H}'J>"=trogeni,m {I} Blood [emo!:"n] may bt inc",astd btau.. of excessive production by. neoplasm {e.g., S.noli cdl tumor or gr:onulosa ""II tumor} or by the administration of euro!:"n compounds. {2} Besides developing clinical signs of feminization, manifestations of hy~ ..... trogenism in m.mmals (espa;ially dogs and ferr~') may include a "",ere non..-gtnerative anemia as p.n of the pancyto~nia of estrogtn toxicosi •. 6. Liver di",..., or inmfficiency (including portosystemic shun u) a. Mammals with chronic and usually prog"""ive Ii""r di.ora.!e or ponosystemic shunt. f~uently h.ve mild to moderate anemia. Typically, the .nemias . ", normocytic normochromic, but decre ...d MCHC values ha"" "'en reponal. When the di ...... Gl.use. hep.tic insufficiency in dogs, some will have a micro_ cytic normochromic :onemia. b. p. tho!:"nesis of :onemia {I} Th. normocytic normochromic anemia could bt an AID in Klme ","s,,,. {2} Other potential mechanisms include defecti"" amino acid .nd protein synthesis .nd .bnormallipid m~aboli,m, which .ffect erythrocyte lipid content .nd life 'p:on. {3} [n dog. with hepatic insufficiency, the microcytosi. i. not am..d by total body Fe deficiency. Howev.., defective protein synthesis may crrate • functional F. deficiency beau.. of defectiv~ Fe tr:on'pon. c. Loboratory finding. {I} Mild to mod ... te normocytic or microcytic normochromic (or tardy hypochromic) anemia

3/ ERYTHROCYTES

167

{2} Evidence of liver di......., {e.g., increased .. rum hepatic enzyme ""tivities} or h.".ric dysfunction (e.g., dec",=<:! .. rum [u=]. hypoproteinem;", hYJ>mmonemia, or ammonium biurate crynaUuria) 7. DY'.,rythropoi~i, in English .pringer .p.nid." a. Thi.< multisystemic disorder is cha..ct.,rized by dysuythropoiesis, polymyopathy with mq;aesophagu., and v.rying dq;r..,s of cardiomegaly. The clinical findings in the young dogs includal regurgitation and d=.".K..
I.

Caus.. of blood 10Sl A. Hemorrhage I. Blood vess.h dam~ by traum., ulce .. tion, neopl",i. , or oth.,r meam 2. Acquired or congenital coagulation f""tor d.,fici.,ncies or von WilJeb .. nd di ..a.., 3. Thrombocytopenia (marked) B. P. rasitism: hookworms .nd whipworms (docs), haemoncho.i.< and ostenag;"'i. (rumi. nants), coccidiosis, tick.., bloodsucking lire, .nd II ..... (dog., cats, and calves)" C. Removal of blood that i. to be, used for a trannusion

II.

Classific;otions baKd on dumion and locotion A. Acute blood loss anemia l. Thi. occurs when blood i.< lost from th., vessel, in • few hours. Anemia results nom the dilution of erythrocytes that remain in ves.sd. (Fig. 3.8).

' 68

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

A. Sudden loss 01 blood from - ' cmalBs hyp<M>Iomi~

B. Shift 01 ECF inlo "" ........ dilut... ~rylhrocyle. and """ .... anemia: spIonic conlntelion ,edJca • • evmity of 8 .... mi8

Fig. .}.8. Erythrocyte lWmia of ,",ute blood 10... A. lVOJ.mi. stimulates thirst to "'pJ.nish ELF voJwne :uxl induces mew.."..,' of ECF from tb. e:xv;uruJar 'P" " to th. intun.rul", _pa'<, thus .. panding blood ",lum<. Th. /Iuid ,bift dilutes erythrocytes (rnd plasma proteins),:md es ond dogs) .

•. Th. 5n'
~'Yd"ocyt~.

are

~bsorbed (autotr~fl!fi"ion)

or destroyed .nd

th~ F~

is

.. utilized, b, Sudden anemia crntes ti ... u~ hypoxia th.t stimulates Epo production, If marrow is r.. ponsin, miculocyto. wh.n blood is lost from th~ body {includifii: into the gastrointestinal tract or urinary tract} over several w..,h to months, Anemia results from. combination of f.ctors but is primarily the r~SlI1t of Fe ddici.ncy (Fig, 3,9),

3/ ERYTHROCYTES

169

T...... .

Spleen

8. A.

'0

'''' c.

When No

t,

erythropoie'" mk:rocytic hypoclw'omic srythrocyto •.

Fig. .}.9. Erythrocyte kinotia of duonic blood loss tlut <=lit. in

defici..,cy. A. lniti.uy. thore is • continow 1o" of .moll quantities of blood ov ......ks to months. Anomi. <Joe, not develop .. long .. componsotory inc .....,J erythropoiesis (wing storod Fe) '"Pt.= lost erythrocytes. B. Afte, prolong. def
a. Oner F. ddici.ncy i. pl"nltnt, maturation and rei"" .. of ~'Y'hrocytes .. ~ imp.irM sum that tho .. king lost connot k replac..d rapidly enough d~.pite erythroid hyp<rpl .. i•. Th. ongoing blood loss in the pr~ .. n'" of F~ ddiciency also contributes to the anem;", but the amount of blood loss itsdf typically is not the major funor. b. During Fe deficiency, the erythrocytes krom. mo", f""l:ile and I... d.formabl.;" th... changes dec=>< ~'Y'hrocyt~ lift 'I"'n. The reduc..d d.formability was

'"

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY mribulffl to increasnj m~mbrall' rigidity, but Ih. ",awn for Ih. rigidity was not aplainal." Th. pr... n", of hratocytes .nd schiwcytes in blood of an Fr_ ddici.nt animal is microscopic tion. " 3. Clinic] data supporting a conclusion that an .nemia j. due to chronic atJ blood Joss include the following: a. One may find tony t«:es {md.n.}, urine or f= may"" heme positi ... , and frank h<JIlOrrhage is not o~rvai If the .nemia is COU.ffl by iti,m, on. may find intestinal neJrultod. ova, or ocher p.=it... b. Poorly rq:.n.r.tivt anem;" or nonrege".rativ. an.m;" c. Microcytic normochromic to microcytic hypochromic ~n~mia d. D=~asM CHr and MCV'" ~. Erythroid hyptrplas", in marrow but indf
fl.""

p."..

HEMOLYTIC ANEMIAS I.

Conapts and clauifiallions A. HmtD/y,iJ {rrytltrDlytiJ} is erythrocyt~ n«rosis and occurs at the ~nd of ~ry erythro_ cyte. lik Wh~n th~ rate of in vivo hemolysis incr~a.es, th~n it is ~ pathologic stat~. P"thou,gk hrmofJlu may k definM .. ~n inc",asal rat~ of ~rythrocyt~ destrucrion that d«"'.... ~rythrocyt~ life span. B. Extravascular vtrsus intravascular hemolysis l. M~jor diff..~nce, a. IntraVll.lCular h~molysis {I} Erythrocyt~ destruction occurs in th~ blood within blood v....,h or h""rt, not induding phagocytoli, by ti",,~ mocrop. while erythrocyt.. pass through linus.. of the .plttn, liv.., or bone morrow. {2} Intravaocular h~molysi. i. dinicaUy r«ognized when it COUS<::'l h~mo_ globinemia and h~moglobinuria (or, if m=rM, d=e .. M .. rum [haptoglobin]). b. Extrav..cular hemoly.is {I} Erythrocyt~ d.. truction occurs outsid~ of th~ an.r",l-capillary_nnous ,ysum and is unrd . t«l to h~morrh~. It has bttn call«l in'rIlt'tUuklr hnrwlym ka"", destruction occurs in m.croph~. n~.. venular linus .. of th~ sple~n, linr, and bone marrow. Spl~nic ma.crophage, ru.vt gr~aten contact with ~rythrocyt .. in th~ r«l pulp (except in caul. Macrophage. also con allarn to ~rythrocyt .. within blood by reaching through noncontinuous capillary walls and then binding, ~ngulfing, and lysing th~m. {2} Extravascular hemolysis does not co"", h~moglobinem", or hemoglobinuria.

3/ ERYTHROCYTES

111

2. Why diff~ .. ntiat~ intnva!Culu h~molysis nom utraVll.'lCul.. h~molysi,? a. Establishing a mojor site of uythrocyt~ d~nruction may be a diagnonic du..-, tru.t is, cuuin di,.,~ typiC;;O[ly callS< utravascular h~molY'is, whu= oth~n typiC;;O[ly cau.. intr:lv:ascular h~molysis (Tabl~ 3. to). b. Diff~ .. nti.tion may be hdpful in d~t~rmining progl\OSis .nd t... tm~nt. Intrav . ... cular h~molY'is usually occurs with li~_thr .... t~ning disrases, so iu pr... n"" sugge;ts a poor~r prognosis, and imm.diat~ tr~.tm~nt and monagtm~nt of th~ """ ... indicat.,j. 3. Probl~m. with d."ification .yst~m a. "Di.......,." don·t r....d th. book; th at is, • disordu m.y be described as cau.ing utravascular hemoly.is, but your ca.. may be th. uncommon ucq>tion with intrav:ascular h~molysis tru.t was not m~ntion.d. b. Di ..~ may cau.. an~mia by both intnvascul.. and extravascular h~moly.is. Extravascular hemoly.is typiC;;O[ly .crompanies intr:ln.ICular h~molysis. c. Disord.rs may switch from on~ to .noth~r (i .•. , .n intnn.ICular h~molytic crisis can d~dop in .n anima! tru.t has a mild extravascular hemolytic disord~r). 4. M.jor f~aturr:s of th. h~molytic di!Ord~rs ... listal in T .bl~ 3. II. C. Thoro~h u amination of ~rythrocyt .. in • blood film i. an e=mial diagnostic proc.du.. for suspa;t.d or oonfirm.d h~molytic .n~mias. A well_mad~ and wdl_nain.d blood UIl... r and a good microscop" with a IOOx oil obj.cri"" a.. nttd.d for such examinations. On. may ..e organisms or ddinite dues of a hemolytic pro",,,•. I. Organisnu; MyrDplimnll, A""plimnll, Bab";.,, .nd Thtikria 2. Clues of a h~molytic proe<ss; sphmxytes, H.inz Ixx!ies, =ntrocyt<s. pykn.ocyt~s, .chirocytes, ~rato(yu" and .canthocytes D. Hemolytic ict~rU! ijaundi""} {Fig. 3.10} I. Pathologic hemoly.is l~.ds to incr~as.d Hgb d.gr. dation, thus inc..as.d bilirubin formation, and p"rh ap" drvdopm~nt of ict~rus. Jct~rus may d~velop in .nimah with eith~r imravascu.l .. or utraVll.'lCular h~molytic diKlrd~rs. [n both forms, Hgb degradation incr~~, but the ,;tes of ~rythrocyt~ destruction diff~r. Jct~rus may drvdop in animals with intnva!Cular h~molysis beau.. of concurr.nt utravascular h~molysis.

2. H.molytic hy!",rbilirubin.mia occurs when Bu travels through th~ blood from tissue /lUCrophages to th~ linr. Excq>t for uncommon situations, th~ capacity for Bu uplah gHatly ucttds that of bilirubin uc..-tion, so upuh is not rat~ limiting. If Bu formation ucttds an .nima]"s .bility to acret~ it imo the bile ., Be, hyperbiliru_ bin~mia will d~dop. If th~ capacity of th. linr for Bu upuh, conjug.tion, .nd acretion {the rat._limiting st~p} ... not excttd.d, .. rum [bilirubin] moy .. moin WRI ~.n though pathologic h~molysis is pr~.. m. 3. Th~ rat~_limiting nep in bilirubin acretion is the transport of Bc to th. biliary s)"lum. On"" the transport maximum is r.-.chal, Be is "regurgitat.d" out of hepatocytes .nd imo plasma. 4. Bu and Be com!"'t. for the same = [Be]. In 10ni:"r_51anding h~molytic disord ... (I ~k or longu), th~ [Be] moy
Table 3. 10. Henmlytic disoNen and condition. lmmun~ h~molytic disord~f1

'Idiopathic' {includes autoimmune} Drug induced' V .ccin~ a,soeiaud Alloimmun~

Neonatal i""'rythroly'is" Blood tr:lnsfusion r....ctio"'· H~molysi, induccd by Mcurial and viral infn:tions"

'M}mpiP.""a 'PI'. 'AndpiP.,ma 'PI'. Lrp""pira '1'1'.' Cumridium 'PI'. coming ~rythrocyt~ m~mbr:lne dUllagt by pho'pholi!","" B.ociJJary h~moglobinur;" (Cumridium harmo/yricu", or C. nDvyit)' ydl" ..... laluL Ui't""d>< (C/",rrid;um pnj'rinx"", ITI'" A) Clostridial infection, in horses EIAY" F~LV' H~molY'i, a"""iatt
Erythrocytic metabolic d~fects (acqui..d or inh~ritt
Rheologic pro<:t::SSes Caval 'yndrom~ of dirofilariasi, Card;"c valvul .. di ..ase Hemolytic anem;" of other or unknown pathog<'neses Protowal infection, Balm;" 'Pp,d

Thdinkl 'PI'" TrypanDw"", 'PI'.' H~parin.induccd hemolysi, latrog<'nic hypommol .. hemoly,is" Ennnomation (,nak.., mid~f1, in...:tsl H~mophagocytic hie> with liver di""""...·

1

• A ,.ll1i""ly common dioe ... 0' condition 'H omoglobinuria or hemoglobtin<mu may b. p~nt boca"", of morhd intnv"",,1ar MmolyU" DtMr D>Khani,ml may . Iso contribu.. to an<mu in th.e.. infK:tion<,

n •

3/ ERYTHROCYTES

113

Table 3.11. Major featuccs of inlravascular and cnnvucular bcmolytic dioorocn Fnlure

CliniCl.I imra""",,ul.r hemol!,i,'

Site of hemolysis

Wilhin blood ve.sd. or hean

Degrtt of RBC damage dir«dyall1srd by Ihe hemolytic agem or process Sev.rity of anemi. On!el of illness Reliculocyto.i.

Marked

Hemoglobinemia

Yes. but may nol be. Crossly yj,ible

Hemoglobinuria H yperbil irub inemia

y"

Bilirubinuria

Marked or rapidly falling Hours 10 daY" U",ally :mer inilial presentalion

No or yeif

Prrdomin.ndy exlravaocul. r hemolysi.· M.aoph.gt. near blood sinl1Se'l of spleen. liver. or marrow Mild 10 markrd

Mild 10 m.rkrd Day:; 10 weeks Usually at initial presentation No No Usually at pre.ent.lion; Bu;> Be Usually at pre.enmion

NoorE • OinicaJ intr.v1K'Uw, homolysis is =ogniu
homoglobinuria. In mon oft ..... diso«kn. therr wiU be conaurrnt <'I".v ...... hr hrmoly.J, . • During tbe>< diso«kn. intw.=ubr bemoly>is rruy be occwring but not =«",ly enough to cwsr homoglobi ...mi. ot homoglobinuria. < Soon after the 0 ..... of intr>vucuW hrmol}"is. hY!"'rbilirubinrmi. and boilirubinuri • ..iU probably not be p, ... nt. Witb timr. incrrasris could contribu.. to tbe .. =s boi~rubin formation and thUll byprrbi~rubinrmi. or bi~rubinuria. HY!"'rbilirubinrmia and bi~rubi· nuri. would be mot. upectrd .. ben tI",rr is concurrent _ransrul .. hrmolysis of mf!icient du .. tion and _rrity.

2. Bu is H,O in.olubl. and i. bound wilh albumin in plasm>. so ""I}' little Bu p also ha"" a mild albuminuria. and thus Ihe bilirubin det«IN may be. Bu bound 10 albumin. 4. Bilirubinuria umally occur> be.fo.. clinical hyp
174

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

Fig. .}.IO. H ....olytic iet1mlS: Accdtto"'<"'i".ntly inc."""! ph""" [Bel, ther. may he in=n«l fornution of B5 (_ th. Bi~rubjn Co"""'tr.ltion ><'COon in Clupt .. 13). Bu/AIb, Bu :ISS<>ci. ted .. i,b 1lbumin: macroplugo: Sb, ".rcobiJin"ll"n: Ub, urobi~nogon: rnd U D P.G, widi ... diphoopboglucoroni
M ,.

2. Uriruu}' urobilino~n excmion inc,,"ascs becau.. :011 th~ p"thw>ys ou.soci<>,rd with th~ aCrHino ofh. m. drgudation products.", rnhancffi. G. Homolytic hrmoglobinomia and h.moglobinuria {Fig. 3.11} l. A ~ to r<=Jgnizillg intr>v:l5cul" h~molysis is diff~rentiatillg the pathologic cou ... of red, brown, or bbck urin~, Thes< are primarily h~moglobinuri<>, hematuri<>, and myoglobinuria. TIme pigm~nturias call typically be classified by crit~ria listed ill T abl~ 3, 12, Other comes of pigmenturia are described in Physical Examination of Urin~, Sect, Le., ill Chapter 8, 2, Hgb nephropathy is . pathologic state Jttn in intravascular h~molytic di",rders clu racteriud by proxim:d tubular deg<'neration that =y remh in acur. ","al inrufficiency, Hgb casts may be pres
-----9 ---------9--Primary

Secondary

-B~

Hgb

Hgb

/'-..

Hgt> d .......

T\

--- <;> ---

~

HPI~

Hgb

I

r

Hgb dime< Hgb dime< '

HI*

,

Me~

HpIIHgb dmoors

. "- -. \.

--- -- ------

,

,

Glomerular

capill

HptlHgb dimers

I

'"\.

, ,

/'-..

""'!hem..

d.......

Hgb dimer

Proximal tubu epHhelial cell Hgb dime.

I ..,. I

"

'"I

Hepatocyte

"

---!---Hgbdn-

I

Hgbdimer

in uri....

--------' . _ .. _ .--

Fis. j. 11.

Padtog ...... is of ... moglobi .... mia and h<mogIobinuri. duri", imnY2&CUlar h<m<>lyoH. f;e ~Ia>«l du.in, intnyaxular hemolysis an be divided into primary IUld ...:onduy 'l""' ..... Thor 1)"''''''''' ar~ no< .. rurat«i durin, .... ltk , and th", be"'ocIobi....rn. .. nor _n in heoIth. In pathologic tion of the prirnory and S«JOndary O)"temoglobinuria (1001 ofH~ and Fe). • The primary . J"k1D aFe ~tion (the "'_ UnportlUlt, which mor boco"", ....... «<1 01

• The no •...w physio6ogical procasa ,h>< c:onoc ......

_..........,

.tat.., .......

• In' ........ b. nythrocyw dam.V o. darh au ... .. Ie ... ofH~ to pI ....... n.. ul"dtoblt: Hgb mnnw:. 'I'~tJ in' o di....... and il1lIDOd.i. tdy binds H,r, (if ....,.jlabl~). n.. HptfHgb d ...... <:oITlf>Ian ore de>=l from plas ..... pnmarily by hepatocyte" 'which ckgnde Hgb dimrn t o Bu, F...., and omino acidJ. I:IUII n..~ .. tyidmoo tho, m""roph'ga have . rKIOpwr for the H p", he..,.,.;ooi.... mi. is rKOgnilOd. PWma..-ill appear pink wlien plasma IH,b] .. 10..... SO mcIdL. • The ~ 'l""'m of f;e "",,--","arion (boco""" more impomnt .ft.. plasma IHp') dec..".... ) • Continuod or lDOIe _..., intml methemoclobin, which dio:.xi.... ."d rdnoa moth...... and globin. M .. be .... bind. to HI'" to fixm a methetoelHpa:
'0

F.....~' • In pooplt: ond nonhuman prima_. m..beme:alto binds to :albumin. and the tocthemei:albumin complt:. ..,"'n hepOto<.")'b'110 dur F. can he ~. H own.. , albumin moIrcula of dog" cattt., ........ , and od.... do..,..tic mammal! do "'" bind """ho...... and thus this pa,hW3)' is DOl functional in

....... :animals."" • O...nlow (whicb DC<>ln..n..n F. ~tion 'Y"""'" _ u tu .. o.d and multi in mukr
prom,,"

n..

15OavdL.

• H gb di ....... pal 'krouch the gIomnul ... fil"otion barri .. IUld onexcm«i in tho uri... (hemoglobinuria). Prw;im:al tubule opi,he~:al oou. b""" ""toe .bUity to JeIOIb Hgb dimen and ~ tft..., II> Bu fix conj ugation and urinary uc",tion. Hpt, b>ptoclobin; and Hp .. htmopeUn.

175

116

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

T able 3. 12. D ifferential feature. of hematuria, hemoglobinuria, and myoglobinuria Ho P[",ma color Urine oolor

H~maturi.

H~mo~obinuri.

Mro~obinuri.

WRI" Not pink to r~d' Pink to rcd'

D,""",,,,,,,d Pink to fffl Pink to =t' Posit;""

WRI

Urine heme .. "",ion'" Positi"" RBC. in urine wimcnt Yo" No' Information to su~~r{ mUld. d . mW No No • UnLess ,b.", is :on :woci. «
Not pink to Pink to rro< Pm;ti"" No' Yo.

='

• Unless Ib.« is oonru,omt in vivo or in vitro h<mol";' < H.me typicilly is r«l but m ay b.com< Ixo.. n to bhd (with degr.o<:after th.y ente, uri"" (~i.Jly in dilut< uri",,) ond th"" auy not b. _n in > wi ... >«Ii""", ",,:urUn>.tion. 'The« could b. concu,,,,nt bmutu,i. bee ..... of anotb.r pathologic P'''''''' or to '""'pJ. colJ.ction. 'Infornutioa auy incJud. hiltorical or phyo;c:d ffid.nc< of muscJ. d:omoge (~.g .• "jJJ..... 0' tnwna) 0'

incr<...d >
II.

H~molytic

disorom and di",,"sN (T.bl.. 3.10 .nd 3.13) A. Immune h~molytic .n~mia, (noc associatM wich inf.aions) J. Con""pts a. Immune hemolytic anemi", occur when an animal·, immune .y"~m produ"",, .ntibodies thac bind di=:dy or indir.aly co its own erythrocytes (ESAlg) .nd l.-ad to erythrocyte descruction. The p~ may k inic;"cM by. defecti"" immune syscem. defective erythrocytes. or adsorbed .nti~m from drug•• infectiou. "l:enu, or neoplasm,. Facto", chat init ;"te the p"""",ss.,,, usually noC known. b. Hemolysi, may occur through three major processes '" deocrikd in Fig. 3.12. c. Clinic;;o[ rnden"" may suggest th~ presen"" of intraVll.lCular hemolysis, extravascu_ lar hemolysis, or both. ESAlg molecul .. of immune hemoly.is may be IgG, IgM, or 19A. ,ouo, ExtraVllOClllar hemolysi, occu", when erythrocytes cootM with immunoglobulim are engulfM by mxrophag ... If the immunoglobulin, fix compl~m~nt, the membrane atta.ck complex (C5b-9) may form .nd CUK intravascular h~molysi,. Thi, complex consi", of complement protein. C5b. C6, C7, C8, and muhipl~ copies ofC9, which form a ring in th~ membr;m •• nd enable free diffu.ion of molecules into and out of the ""ll. V.riations in .ntibody involvement cr~ate ... r;"tions in clinical m.niftst.tion,. d. Direct .ntiglobulin tests (Coombs' te" or How cytometric methods) may be u...:! to d"'ect the pr.sen"" of ESAlg or romplem.nt on • patient', erythrocytes (see the ""'tion on M",hod, for Detectifll: Erythrocyt. Surfa.ce Antibody or Complement). e. To understand some of the clinical manifostation, .nd laboratory finding' in immun~ hemolytic anemias, the m.jor f~.tures of warm .nd cold antibodies need to k undemood.

3/ ERYTHROCYTES T able 3. 13.

117

Rcoognize
Trpeofan~m",

Dog,

Cat'

Horst.

Cml~

[mmun", m«iiat«i (not infr<:tious)

IMHA Drug_inducffl N[ T ran,fu,ion MJrop!",,,,., Balmia Ehrumiil

IMHA N[ Tran,fusion

[MHA Drug_inductd NI T ran,fusion Bab"iil L(p"'rpirtf' Equine infn:tious

IMHA Tr:m,fusion

[nf~tiou,

MJi'Op/m",., F~LV'

Ehrlkhilf' C}tauxzom"

an~m ",

M=bolic

H~inz

lxxIy E'.cctntrocytic PK ddici~ncy PFK ddici~ncy

H~inz

lxxIy PK deficiency Erythropoietic porphyria

Ehru,hilf' cu,,"idium Heinz lxxIy E'.cctntrocytic

Angiopalhy Envtnomation H~mophagocytic

Angiopathy Idiopathic (T fragility)

T'lfrmo",,,,., H~inz lxxIy Hypopho.ph at~mi.

Congenit:d erythropoietic porphyria

Hypophosphal~mi.

Traumatic Other

L,p""pi,,, ctlJltridium

Angiopalhy Heparin_inducffl Liver disuse

Hypoosmolar

hisliocytic

.==

Hereditary non,pherocytic • Firm rudence of hemolytic :anemia w>s not fou nd by tbe authon . • Homoly>is moy [,. a minor contributor to the """mi. (... Noruog..,.,mi"" Anemi ... =:t. 11.C.2).

( I) Warm antilxxlies (usuaUy [gG) (a) Maximal binding activiry al 37- 39·C (b) Warm antibodies ar~ mo,", common Ihan cold antilxxli ••. Warm antilxxlies may bind submaximally to ~rythrocytes al room temptrarum; and thus am cause .utoogglutination in samples al routin~ handling ICmptraru.... (2) Cold amilxxli .. (usually IgM) (a) Maxim:d binding aCliviry'l 4- 20·C (b) Cold .milxxlies may be able to bind in vivo while passing through 0001 exrr~mities, :md Ihm Ihey c:m a.ctiv.t~ complement :md initi'l~ immune hemolysis. They typically will cau.. autoaWurination as erythrocytes 0001 during or after coll~lion. NOI all cold antilxxlies " e pathologic in vivo, evtn if they cou.., autoagglutination in vilro. 2.

Disord~"

a. IMHA (also called idio/",mk immun( fmnqlytic "n(mia or llHA) (I) [MHA i. the JI\05I common hemolytic .nemia of dogs. [t i. also occasion_ ally found in can, horse" and c;mk

Blood

,,,

.t=lnt""'~'CUla' oomolys;"

Spleen

'"

h.

V·' •

V

;::

"'Y~,

BuiAlb

,-- ---- -- -- --.

Fig. .}.12. P. thogonesis of immune ""molysi~ • EJytbr<>v:osru.br bemol)"is in macroph:.ges. • EJytbrocyt<. ""'ted with ESAlg andlor C3 at< converted to 'l'berocyte< by IIUcropb.g .. "'moving the ~hrocyte membnn<. Spbp«:tivmponent of compJ.m
'"

3/ ERYTHROCYTES

179

{2} IMHA i, commonly rd'trr.d to :as ""Nlimmunr h(m~lytic "nrmill WHA), although tht f""rors Ih.t initial.d Iht immunt htmolysi, >rt unknown. Typieo]Jy, it i, ~ ..... m.d that tht htmoly.is i. an autoimmunt process kcou .. known eo<= of immunt htmolysi, arr not found. Ho~r, conditions rrftrr.d to :as IMHA or AIHA h. vt bttn associat.d with inftctions, ntopla_ ,ia, and vaccinations .nd m. y not bt . utoimmunt. Autoimmunt cdl dt"ruction rtsult. from immunt rt.ponses to normally occurring .df tpitop... Whtn tht tpitopt. art from forrign antig<'Dl or alloantigen., tht body'. own ctn, >rt destroyrd, but tht proctSS is nol ",iuly.pt. king autoimmunt in pathogrnesi •. {3} Classic l. boratory f....tur.. oflMHA: rq;entrativt antm", {mild to SNtrt}, icttru,. possibly htmoglobinur",. sphtrocytoois (difficult 10 r=>gnizc: in sptci". oth.. than dog.), politivt Coomb.' test, positivt flow cytomtlric methods for ESAIg. inflammalory Itukogram including n.... trophili. with regentrativt Itft shift and monocytosi" and .bstnct of .vidtnce of other immune hemolytic antm",. {4} Exctptions ro classic f....tures {a} Antmia may bt nonregc:nerativt if it l:as{, < 2- 3 d, if . ntibodi .. also attack marrow erythrocyte prtc\lrsors {t.g., purr ral cdl aplasia}, or if another di",= process inttrft= with trythropoiesi,. {b} Evidenct of utravaocwar hemolysi, {icterm, bilirubinuria, etc.} may not bt presc:nt if the rale of trythrocytt destruction i, only .lighdy incre.ttd or if accderat.d hemolyti. i. brief. (c) Sphtrocyto.is may not bt p.."'nt bteoUst sphtrocytes Im}' not accumu_ late in blood if thtir ralt offormation is less th.n tht ra .. of thtir removal. {d} The ..sub. of the Coombs' tt" may bt nrgativt due to a low dtn,ity of ESAlg or t.ronical problem" including prozont rt""tion, poor "'agtnt., or poor ... mple. {5} Oth.. l. boratory or diagnostic fe. tures of !lOmt IMHA =: trythrocytt autoagglutination, ghost erythrocytes, thrombocytoptni. , .nd .pltnomegaly b. Drug_inducal immunt hemolytic anemias {I} Drug,.rt thought to induct immunt htmolysi, through tht'" thr.,. IIUjor mtchanisms:'·' (a) Drug . dKlrption: A drug {t.g., ptnicilJin} bind, covaltndy to erythro_ cytt membran.. and "imwates hapttn..dtptndent antibodies. Such antibodies still bind ro trythrocytes aft.. the trythrocyt .. havt bttn upostd to the drug and then wash.d. {b} Autoantibody induction: A drug (e.g., mtlhyldopa and procainamidt in ~plt) inducts forllUtion of autoantibodies for trythrocyte mtm_ brant proltin,. Antibodies bind to normal trythrocyt .. in tht absc:nce of drug. (c) Drug..dtptndent antibody induction: A drug (e.g., quinidint in prople) induct. antibodies that bind to erythrocytes only when Kllublt drug i. p....nt. (2) Drug, rtpon.d to initiate IMHA in domestic mamllUl, include ptnicillin in horses,'''''·' propylthiouracil in eo",'" ceph. losporins in dog. (supraph.._

100

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY marologic doses),'''' trimethoprim .. ,ulf'ffiethoxnol. ill horses,''' J~.misol. in dogs,''' and pirimicarb in dogs."o Many drug. are Iq>Ontd to au.. IMHA in pwpJ •. '" c. V.ccin •• inducW immune hemolytic anemia {I} Some clinicians ha"" associatffi the onset ofimmune_ffiffli. ta! anemia in dogs with r«:tnt vaccination., but little d ato h."" bttn gtnerattd to suppon

a ca"",-..nd ..dfa:! rd.lionship. One

re{ro,~i",

nudy provided

~id.na:

10

suppon Ih. linkaj:•. "' {2} An immun...ndi>lffl sph.rocytic anem;. W :l'l found in a cow aft" it had bttn vaccinalal with. polyvalent botulism vaccine.'" d. Alloimmune hemolysis {I} Nl (neonatal j,orrythrolysi., also known as hemolytic di>e;>st of the n~rn)

(a) [n NI, ingesta! m aternal colostral alJoantibo
d.rived erythrocyte surf~e. .nti!:"ns. The ~ntibody-ro.ted erythrocyt.. a.. then ly>ed by macroph.!:", or romplement. {b} In cau , NI occurs when. qu.. n with typ< B blood passes h.. anti_A alloantibodi.. to h.. ty~ A or tJ'P" AB kitten! via colostrum. TJ'P" B eryduocyt.. are uncommon in American domestic shonhair cats (< S %), and the high .. t incidene. i. found in British shonhair {41 %}, ~on rex {40 %}, .nd Corni,h rex (34 %) cots.'" Anti_B allo are not known to han clinically ,ignificant naturally occurring alloantibodi.. to DEA 1.1. and other .cquired alloantibodies probably do not cau.. hemolysi •. "' {2} IMHA ..rondary to incompatible blood tr'n!fll'lion! {a} Tran,fused donor's
3/ ERYTHROCYTES

181

antig.ns on th.ir .rythrocyu m.mbranes. Th... at< allo.nti~ns causing alloimmun. h.molytic an.mia. {b} Th. ~ll"'mtibodies th~t cau.. h.molpis at< the same as tho.., th.t cau.. Nl. In doC' and hor.... the alloantibodi.. "'" :ocquir.d . ntibodies that devdop during pregn. ncy. parturition. or after whole blood transfu_ .Yons. In cau. the antibodies a", natural .llo. ntibodies. {c} To r.du"," the po"ibility of a hemolytic transfusion r....ction. blood typing or crm.smatrning Jruly be used (..., the Blood Typing and Cro ... matrning section). {3} Early . ttempts to develop. v. ccine for bovine anapl .. mosis ","uhed in production of anti ... rythrocyte antibodi .. breau.. of bovine erythrocyte stroJrul within the vaccine.'" These antibodies we .. passed in colostrum and led to NI. '''' Purification of blood-deri'lffi vaccines reduced the moJrul problem. but the vaccin.. breame un.vailable during the 1990•. '" B. Inf«tious h.molytic an.mi", l. Hemotropic mycoplmru, specie. a. Fdine hemic Myropliuma 'pp. (basonym. HIl(mobllrro~l/a spp.}l>'-llJ causing fdin. inf'«tiollS anemia {I} M}rop/a'ma ha(mofilis: This org.nism was originally called the Ohio or Aorida strain of HIl(mONrtOnd/a filis and is mo .. pathogenic and lorger than the oth.. fdine hemotropic ~plm"", spp. {2} "umdidaru, Mycopl"'ma haemominutum": This org. nism v..as call.d the small.. form or CoJifornia .pecies of Ha(mobammdJafilis. It is I.... patho_ gc:nic than the M. ha(mofilis. and typically is comid..ed an opponunist. {3} Another hemotropic Myrop/a""a sp. that r... mbled the rodent hemotropic myropl .. mal org.nisms w .. id.ntified in a cat in Switzerland.l>· {4} P~rasitemi. is usuaUy pr... nt during h.molysis. but it may sudd.nly disappear (within hours). Abo. organisms will falloff erythrocytes in vitro and thus it is advisable to =omin. blood films made &om fresh blood. b. Conin. hemic Myroplanna .pp. {I} M}rop/a""a ha(mot"llnis {n.,,,,nym. HIl(mobllrtondJa ranis} (a) P....it.mia i. usually...,n in .plen«tomized and in immunologically compromised dogs. {b} Like the fdine hemotropic mycoplasmas. M. ha(molilnil Jruly d=rn from .rythrocytes '" the umple ages and it i, difficult to identify the organisms in pl.. JruI. {2} "umdidaru, Mycopl"'ma haeJrultop.rvum" {a} This small org.ni,m was found in a spl.necromized dog.'" {b} Genetic a""lysi. indicates the organism is more do..,ly rdated to "umdidllruJ Mycoplasma h •• mominutum" than to M}rop/a'ma ha(moranis. u , c. Oth.. hemic Myropliuma .pp. {all previously called EptryffnW;Mn} {I} M}rop/a""a ha(mo.ui, . nd M. paroum in pigs {2} M}roplmma W('9'onii in catde {3} "umdidAtul M. h .. molamae" in llam", and .lpacas d. P. thogc:n .... of the.. hemolytic anemia.! a.. thought to involve immune_ mediated mechanisms. Antibodies bind to the p.rasitized .rythrocytes eith.. beam.. of bound parasite ~nti~ns or anti~ns exposed on . lter.d m.mbranes.

182

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

•. Major l.boratory findings

{l} Thest include Myrop/a'mll 'pp. on uythrocytes in films of fresh blood. P"..,it.. are most numuous when He! is falling. Organism. =y detach from erythrocyte, in vitro, so th. kn crun". to det'"'" and id.mity organ_ isms is in blood films mad. imm«iiatdy aft" blood colb:{ion. {2} Loboratory finding> also include moderate to ~r. :memi., reticulocytosis and polycluom.'"" mild to mod". te hyp
2. Andp/d,ma 'pp. (..ythrocytic 'pm") a. Anap/a",,,, marginal, (cattle), A. ovh (sh,""p and goau), and A. emtr"/' (em). of South Ameria, Africa, and the Middl. Em) b. Anap/mnw ""'TM/'.", most numerous when He! is falling: 4- 5 d lat~r, it i, difficult to find org.nism., Via light microscopy, th~ m:orginal body IIUy .ppe:!r to protrud~ from th~ ~rythrocyt~ m~mbran~, but th~ organilITl i. an intra""Uular path~n,

c, Pathogenesis of anemia is mostly immune mediated, Antibodies bind to erythro_ cyt.. dam"l:ed by the anaplasrrud inf..aion and cau.. ext",v:oscular hemolysis,m .. ,. d Major laboratory finding, {I} Organisms (marginal bodi..) found ... rly in dis ..... {2} Mod~rat. to seve~ an~mia, reticulocytosis, incr.....d polychromasia, b""'philic,tippling. mild to marked hYP"rbilirubinemia and bilirubinuria, and PCR positiviry for AlMpfMma 'pp, J, Lrptolpira 'pp, a, Lrp""pira imrrrogam .. ro,,:us pomolM and imrohmwrrhagica do not infect erythrocytes, but ""oculiti, and infrction of kidn"Y' or liver em l~ad to a hemolytic sUte, b, Lrp",spira 'PP, infrct most domestic animals .nd IIUny wild animal.,'" but the hemolytic st.te is .",n prim:orily in caIns, lambs, and pigs,'''''''' Toxi", of Lrp",spira 'PP, are not known to b. in "ivo hemolysi", in dogs and cats, c, P.thogenesis of the anemia is not established but IIUy involn an immunologic ..spon...MOCi.ta! with an IgM cold "l:glminin'"'''' or leptospiral phospholipast activity,' ..... '" d Major laboratory findings: mode..te to ..vere anemia, hemoglobinemia and hemoglobinuria, hyperbilirubinemia .nd bilirubinuria, neutrophilia, leptospiral spirochetes detected in urine or oth~r Huid, by direct dark_fidd microscopic examination, .t le:ost a fourfold incr.... in ronvales""'nt tit~r to eith.. pomona or ictm>hrmorrhagira .. rotype., PCR positivity for Lrp""pi,a spp" .nd Lrprorpira not found in blood film., 4, Cwtridium 'pp, a, Clwridium harmo/yricum and C novyii type D {I} Disease: bacillary hemoglobinuria in cartle and sh"'P {red warer disease and Nevw ra! water} {2} Cu,rtridium harmo/yricum .nd C novyii type D a.. normal inhabitants of soil .nd may remain dormant in cattle until an ... rohic conditio", promote their growth, In th~ United States, bacillary hemoglobinuria is ...,n prillUr_

3/ ERYTHROCYTES

183

ily in poorly dnining ."".. of GulfCoasl and Wm~rn states, :md th«~ is • rq>On«l >SSOciation with liver flukes, {3} Pathogenesis of anemia: Erythrocyu damage is caus«! primarily by a p_toxin {produced by either C h,,,,,"oryricum or C /Wry;; type D} that has phmpho_ lip... C or lecithinase activity, The p_toxin thus degrades membrane lecithin :md cau..s lysis of erythrocytes, Oth~r rdativdy minor hemolysillS {lipa.., proteinase, and .noth~r lecithinase} h.ve .lso bttn .. ported as products of C hannolyticum,IJO {4} M.jor labomory findings: acut. seve...n~mia, h.moglobinemia, and h~moglobinuria

{5} Confirmatory diagnostic finding.: postmon~m l.,ion., Gram_positive baciUi in tissues (liver :md 'Plttn) or fluids {blood .nd peritoneal fluid}, C hannolyticum cultur«l from liver, and ph<>spholip= C activity (p_toxin) detected in tissues b, Ch>rtridium pnftingrm type A (C _khit) in rumin:mts {I} Di... ",: yeUow lamb di",= in cal""" and lamb. (also know as emerotox_ emi" jaundi"", or the ydlows) {2} CIo>tri4ium pnftingnu type A i. part of normal im<::'ltinal flora in animals, but multiplication has bttn auociat«l with highly prouinaCNus dim, overftt in spring nursing lamb. in nonhern o.lifornia . nd in Oregon.''' It also occur> in lambs :md calv." of oth« region. (e,g" Canada, Australia, N= ualand, and South Africa), {3} Pathogenesis of anemia: An a_toxin th.t ill.. phospholip... C .ctivity .nd i. produced by C pnftingnu type A came. hydrolysis of membrane phospho_ lipid. and thus lysis of .rythrocytes, leukocytes, plotdm, endothelial cdls, :md myocyt ..,'" {4} Major labomory findings (a) Acut ........ cases: anemia, hemoglobinemia, hemoglobinuria, and icterus {b} Le ......... form.: an.mi., polychromasia, basophilic stippling. rubricy_ tosis, .nd leukocytosi, c, Ch>stridium pnftingrn, type A {C uxkhil1 in hor... {I} A horse with a clostridial aoo.. had. Coombs' _positive . nemia with sphero«hinocyt<::'l .nd autoagglutin. tion, lktrosp«tively, other hors .. with clostridial myositis wen: .lso found to be .nemic," {2} The n.:Ict ..lotionship bet""",n the clostridial infection .nd the immun.... m«liat«l h.molysis has not bec:n <:stablimed but probably is rdat«l to erythrocyte membrane injury, {3} Acute imravaocwar 'Pherocytic :memia caus«l by a C pnftingnu infection occurs in ra'ple,'" 5, EIAV a, EIAV i. a retrovirus that infects cell, of the mononude .. phagocytic syst.ms of ho .... , ponies, donkeys, :md mules, The disea!e is called rqui~ infwioul annniA {also know .... ''''''mp frv«, equine molarial frver, moumain f.ver, and slow frver}, b, P.rsin~m replication of EIAV in macrophage> and . ho ... ', respon .. to the infection create the pathologic srat.. that cau", dinical di"""",,'''''''' The clinical

184

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

di",,,,e may

~

.n acute hemolytic stat. or a chronic debilitating dis ...... causal

by the rn::u,rem .pi,ode!. c. Anemia pathogen.. ;': both da:,..asnIerythrocyu production and h.molysi, {I} Production d.c, .... oe< ba::;,,,,,, ofTNF .nd other cytokines th at inhibit erythroid p.«llrsOIS. {2} Hemolpj, i. due to immune rompl.",. or complement fngmem, .dh.,M to erythrocyt.., which ... then d.stroyed by ma.crophage,. d. Major J.bor.uory findings {I} Anemia =y ap~.r as >II acute imravascuJ., hemolytic .nemia in :lCIIte '["l:'" Mor< commonly. it is • chronic extra ..... "'ula' hemolytic anemia or .. chronic normocytic normochromic anemia. {2} Ocher finding" hemoglobin.m;" {acute}, peth.!", anisocytosis and Dltffi with F~L Y inf«tiollS ar~ caUS<"d by d«reol,ffl ~rythro_ cyt. production.'" On~ study providffi data "Iggestiv. of h~molytic .n~mia. but most data in th~ study could have "1,,",,,,ntal indf«tiv. ~rythropoiesi, (~ry_ throid hJ'P'rplasia) and dysrrythropoiesis {mxrocytosi. and rubricytmis).'"" b. A FeLY inf~ction truly pralispo.., ~ cat to ""flain inf«tions (~.g .• Myr()p"''''''' spp.) or ~ :woci.tal with immun~ hemolytic p~. 7. Bab"ill 'pp. a. Th~ exptltal num~r of piropl..ms per inf«trd ~rythrocyt~ is notrd :ofter.-a.ch species and its hon: B. higrmina. canl~ (l or 2); B. whaW. horse (2 or 4); B. canil. dog (I, 2, 4, or 8), B. gihHni, dog (lor 2). Another small canin~ &/mill has not been namffi.'" Bab,,;.. canis h., th..., =gniud ,urupeci.. (B. canil vugrli, B. ,"nis ,"nil, and B. canis rtJu,), and the proposed nam~ for anoth~r organism found in cats in lsrad is B. wni, P"'nlt;;.'" Other ,mall babesi<>l species al", occur in caU {B. ftlil and B. '"").'" b. Sizes: &/mill gihumi and other smaU &b,,;.. spp.• r~ < 21lm, whrrn.! others are 2-4).1m long. c. P.rasit~mia: It may ~ difficult to find organisms in chronic forms of di",ase. Th~ ~re mo,", fr~uemly fOllnd in capill.ry blood ear stich, toenail clips, or in buffY coat preparations.'" B.besial org:mi,ms can induce ~rythrocyte surface adhesion molecules that bind to ~ndothdial cells. The binding truly con""ntrat~ cdt. in capillary brds .nd also sequester them .way from the .nimal·, mono_ nucl~.. ph:ogocytic synem.'" d. Pathog<'nesis of an~mia appe ... to involv. nonh~molytic and hemolytic mm._ ni,ms.'" Hemoly.is truly involv. protu..,s produced by the invading para,ite, an immune r~ction to par.,itizrd cells, .nd/or oxidativ. dam~ to erythrocyte>. e. Major laboratory findings (I) Chronic form: few to rare organisms in blood, mild an~mi<>, mild lympho_ cytosis {due to chronic .ntig~nic nimulwJ, seropo.itivity to &b,,;.. spp., and PCR positivity for &b,,;.. 'pp. (2) [n .rute or subacute forms: many piropl.,,,,, in blood, mod~rate to =er~ an~mia, retirulocytOlis, increased polychrom .. i<>, macrocytosis. hJ'P'rbilirubinemia, bilirubinuria, possibly h~moglobinuria, ,ometimes sphrrocytosis, and occasionally =ntrocytosi,

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8. "Iki/ma spp. a. Erythrocyt~ piroplasms .'" intr.ocdlulor and highly pleomorphic, d~~nding on spa;ie! and Slag~ of par.olitemia. Many form. resembl~ CJlaUXZJIQ'" but oth~" r..~mbl. "nan b.be.ia[ piropwms. b. "Ikikr;a rqui (ba""nym, Bab"ia ~qul)''' infects horses and is ~ndemic in many tropical and subtropical ar .... Th~ infectod ho"" may han a subclinical di",,,,,, or may han pathologic SUt .. , including intr.ovascular h~molytic anemia, ict~rus. hepatom~y, and splenom~y."· c. In th~ United States {Mi1SOuri, Texas, North C.rolina, and Michigan}, bovin~ case:s of theil~riosis hav~ i>ttn caused by T. bujfiu.' ....'" Num..ou. piroplasm' w~re pr=nt in clinically ill row,; piroplasm. we", . .", in subclinicol cases. P.thogenesis of the an~mia is not esublished and may includ~ both d=eased erythrocyte production and decreased ~rythrocyt~ lif~ span (becau.. of .ith~r iDUIlunologic or oxid atin da~). Ntojor laboratory finding. ar. piroplasms in erythrocytes. macrocytosis, polychromasia, basophilic stippling, lymphocyt<>lis, and hyperbilirubinemia and bilirubinuria. d. In other oountri.. , many lbritnu. spp.•'" ra:ogniud .. causing h~molytic .n~mi .. in canl~ and oth~r ungulates. Erythrocytic piroplasms are th~ major pathogtnic forms in T. mutant, T. orirnta!iJ. and T. "rgmti. Th~ m.jor patho. genic s~ of T. parva i, in th~ intralymphocytic schizont, wh~r..... ~rythrocytic .nd lymphocytic form. are romiderod imporunt for T. an" ufllta.''''·''' A Thn~. ria sp. wa. found in SwiM cotd~ that w~re roncut",ndy infectod with la~ &Imid .p., ANJplmmll m'''-giNJ!'. Myoplmma wm}unii, .nd A. phagoryrQphiu.m.'" •. In Spain, 21 dog. wer. "'ported to be infected with B. mi""ti.liu organisms, but .nalysis of ribo""mal RNA from organi.ms in thIN dogs indicoted that the org.nism. w~r. T. anna~. t. . The dogs wer~ anemic .nd had ""id~nce of a prot.in. losing glomerulop.thy. f. PCR testing can be used to diffe .. ntiat~ .imilar org.nisms. 9. TrypalUlwma spp. (Pl.te 8K and L) a. The.. are Hag~U atal protozoa th.t occur as fIN.living Hagdlated trypomanigot.. in blood and .. amanigot .. in J>S"udoc)",,, or macroph"l:es in oth~r tis.!ues. The p.thogenicity of th~ par..itic spa;i.. varies, and th~ host .pa;ificity is minimal. t" b. In th~ United Stat~s, T. ~i/m (b ..onym, T. amnkanum) is found in cotd~ .nd typically i. not rollSid~rod a pathogen. Th~r. are repom of finding it in Kansu, Wyoming, Oklahoma, Louisiana, Mis.iOuri, llJinoi" P~nnsylvania, and N= York. It is .1"" found in oth~r rountrie!. c. TrypanD"""a cruz; infects dog. and cou in South .nd Central Am~rico and in the south~rn Unit~d States of America. Trypomastigotes.", found in blood in the acute stagts, but mon lesiollJ involve amastigote! in nonhemic tissue! (h~art, brain, .nd lymph node), .nd h~molytic anemia is not a f",cur. of th~ disorder"" TrypanD"'''''' cruzi caus.. Chag.. di .."", in people. d. Major Mricon trypanosom .. of v"'~rinary significonce are T. crmgu!mu, T. vivax, T. brucri, and T. ,imilU. A sub.pa;ie! of T. b,uc,; caus.. African slerping sickness in people. {I} In the .cute nages of T. vivax infections in canl~, the .nimah may have an ""ute hemolytic an. mia, l.uko!",nia, .nd thrombocyto~nia. Phagocytmis of

' 0;

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY h~mic pr«ursors ~nd pJatd~ts is • promjn~nt finding in IIUrrow, :md Ihm indfa:t;", h~matopoie;j, comributes to ch~ p"lIcytop
{2} Trypano",,,,,, CDnptmw jnf~jons in cattl. and .h~ have ... "hM in th. coating of erythrocyu •• nd leukocyte. with nyp"no50mal .ntigen_antilxxly compJexa '«"6> C. Erythrocytic metabolic d.f.en (acquirffi or inh,,;uC" 'pont>n~l1' conformational chang .. to form

hemicl"om.. (Hgb_F .... with unique binding of Fe to nitrogenous bases of g1obins) or to form a h~me-dqJl~t..d Hgb. {cl H.michrom~s or h.mMqJl.t..d Hgb mol«ul .. pr«ipitat~ .nd aggr<'gat~ to form H~inz bodi~ •. Oxidation of sulfhydryl groups and rorm. _ tion of di1Ulfid~ bond, occur during th. pr«ipitation proces.s. Th~ H.inz bodies are fr.<SOCiat..d with th. ~rythrocyt~ m~mbran. through hydrophobic bond,. {2} Many oxidanu or substances ronuining oxidant, are repon..d to cause H~inz body for""'tion in dom~stic mam"",l,. (a) Dog" acetaminoph~n. b.n7.OGl.in~. hydrog~n peroxid~. onions {wdium n_propyhhiosulfat~. n_propyl di,ulfid~. and other sulfid.,}.,.,..... vitamin K, (phyton. diond. vitamin K, ( m.nadion~). naphthal~n~. ph~nylhydu_ zin~. prn.sibly zinc.'" garlic (Allium kltil1llm). Chin= chin (AUium rubaosum}.'" and prn.sibly .kunk spray'n {b} Cats: acetaminoph~n. b.JI7.Oatin~. m.thionin~. m..rhyl~n. blu. (in old urinary antisq>tia). onion, {Allium 'pp.}. ph.nazopyridin•. proporol. and propyl.n~ glycol'" {cl Horses: onions. ph~nothiazin•. wilt..d or dry r..d mapl~ (Aur ,.,.b,.,.m) and red ""'pl. hybrid l~aves {gallic .cid and oth~r oxidanu).'" possibly other ""'pIe leaves. and g.rlic (Allium Ultivumj'" (d) Ruminants: Bra"k" spp. (kal •• nd rape). cop!",r. hydrogen !",roxid~ (intrav.nous). onions •• nd ryegrass (red mapl~ leaves in alpaeu) {3} Can are ""ry sUKeptible to acetaminoph.n bea.... they lack a glururonyl transferast that is usn! by most .nimals to conjugat~ .ceuminoph~n. Without th. conjugation •• ceuminoph.n is connrted to """'tin m.tabo_ lites that dqJlet~ gluu thione concentrations and th~refore d«re... prot«_ tion from oxidatin injury. {4} Pathogenesis of the anemia may inml"" multiple mechanisms'" (a) Erythrocyt.. oontaini"l: Heinz bodies are less d.formable and ar~ trapped and lysn! in the spl..,n. {b} Structural dam"i:" em=' by oxidation of membrane prot~ins or binding of Heinz body to erythrocyte m.mbrane le.ds to fr:li:il. cells that ""'Y Irs<' within ""....,1, or .inusoid.. {c} Binding of h~michromes to band 3 proteins of the erythrocyt~ mem_ br""e em.s es. redinribution of band 3 prot~in. and formation of an

3/ ERYTHROCYTES

187

antigen that is r«ngniud by autologous antibodies. Aft" antibody binding. the def=i", erythrocyu i, ",moval by splenic or h"".tic mocropmg. •. {5} M:ljor labomory featu .." mild to ..,n", anemi., polychrom..ia and miculocytosis, Heinz bodies in p..ipheral blood (NMB or .n oth.. vital stain may hdp confirm the pr... n"" of Heinz bodies), auntrocytosi., h)'ptrbilirubinemia and bilirubinuria, hemoglobinemia and hemoglobinuria in acure ON. . . cases, and ptrhap.s methemoglobinem", (dark or chooolau. colora! blood) {6} Fdine Heinz bodies (a) Clinically healthy, nonanemic alU and alU with nonhemolytic anem"" fr"luendy ha"" circulating .rythrocytes that ronuin 'ingle .m.ll Heinz bodies {diamcol plane but h«:ame ..f..ctile when the focal plan. was .djuna!. {Do not oonfu", with ..fractile anifact on Wriglll.lIaina! sm...",.} {cl Th.",ries that atumpt to expl ain why h.althy cats may han circulating Heinz bodies (i) Fdin. erythrocytes ronu ining Heinz bodies a.. prob.bly not .. mova! from the circulation as rapidly", in oth.. sptcies h«:au .. the cot's splttn h .. a closal circul.tion. Th...fo.. , erythrocytes do not flow through the ..d pulp in which pitting of Heinz bodies is thought to occur in oth... ptcies. (ii) Bea.... fdin. Hgb has mo .. sulfhydryl groUp' than other sptcies, it m.y b. prone to form mo .. disulfide bridg.. and thus mo .. denatural Hgb. (iii) Feline erythrocytes may han l.so ra!uctin cop.city. Cd} Cats th at .at semimoist food containing propylen. glycol ha"" mo", Heinz bodies, and their erythrocyt.. han shomr life spans, but clinicaUy .ignificont :mem", is not exptcta!. {el Cats with. variety of disorde .. (e.g., d",b.te. mdlims, hyptnhyroidism. and lymphomo) may ha"" incr ......! ptr"'ntages of erythrocytes contain· ing Heinz bodies. (/) Diagnoli. of Heinz body hemolytic .nemia in cou "'I.ui... finding a rq;<'nerative anem"" .vidence of hemolysis {Ulually hyptrbilirubinem", or bilirubinurial, and demonstration of Heinz bodies in .rythrocytes. Known exposu.. to an oxidant is ""ry hdpfuJ. b. E=ntrocytic hemolytic anem"" {acquired or inherita!} {I} Pathogenesis of the anem", probably involves multiple m.roanisms (a) E=ntrocyt<::l at. mo", rigid .nd thu. Ie...ble to pass through splenic linusoids, whe .. thry are trapped .nd ",moval by macrophag.". {b} E=ntrocytes may b. more fragile h«:allSt of the damaga! membrane or cytoskd
'88

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY {2l Acquir~d stat.. (a) Oxi""!i ... dam3i:' to erythrocytes may COUM forJrultioll of =ntrocyte., H.inz bodies, or both (for r<=>gniud oxidants, ..., Hemolytic Disorders and Di",>=. Ket. c.l..). {b} Factors that determine th. outcome of oxidativ. d.nugr are not und.mood. Oxidant, .. ponM to cau .. Heinz bodies in one a!lim:.! =y ca".. =ntrocyto,i, or methemoglobinemia in .noth.r. {3} G6PD ddiciency (a) This X_link"'" d.f= is wmmon in ~J •• nd W:lS found in one Am.riCOD saddld"M colt ." The no",",n .. mutation ocrurrffi in the coh'. d.m'" .nd j. not a b,,",,d problem "'CO".. none of th. oolt'. mal. liblings li",d to produ~ progeny. One dog v,... reportM to h.ve

''Yduocyte. with d«r"1ffl G6PD activity (44 % of normal), but th. dog did not h.ve a dinical disorder ,d atal to th. d.f«t. m (b) Pathogenesi. of anem;": G6PD js the rate-romrolling .ir dam"l:~ coused by .pont:meou. oxidation; thu •• th~ ails.", pron~ to =ntrocyt~ .nd pyknocyt~ formotion .nd redu=llife spans. {c} M.jor l.boratory finding. in horses: pe .. ist~nt macrocytic normochro_ mic an~m;' {Hct nnr 20 %}. =ntrocytosi. and pyknocyto!is. m.crocytmis. :md persist~nt ict~rus (primarily inc",....d [Bu]) (d) Confirmatory di:.gnostic findings: grudy rrou=l erythrocyt~ G6PD activity. d«r~....d ~rythrocyte [GSH] :md [NADPH], and increased blood [Hgb-Fe1 {4} Erythrocyte FAD ddici~ncy {aj A def«ti", bioch~micol pathway in th~ ~rythrocyt .. of a Spanish musung mar~ c",. ta! a FAD defici~n
3/ ERYTHROCYTES

189

{3} '\hjor bbomory findings includ~ modeme an~mi. , moderate to extr~me miculocytosis (RP = 40--60 %), .nd mild to mod."'te icurus. Spher"",hi. nocyt .. a.. reponed to occur but .'" uncommon in canine case •. P.n+.transport d~fect with the alkaline.induced h~molys.is and incr~....d erythrocyte let) were not found. (b) D«......d 2.3·0PG ..sults in greater intracdlular let]. greater pH. and enhanced alkaline fragility (normal canine ~rythrocyt .. are gen~rally .. nsiti"" to alkalinity). The low 2,3·DPG decr ...... 0, ddinty to tislues ouch that Epo production is inc... ..d. This results in • comp"n. "'ted regenerative sUte. Wh~n hyperv~ntilation occurs bec.use of excitement or exercise. blood p.co, decreases and alkalemi. may lead to a hemolytic crisis. (3) The major laboratoty findings.re anemia, hemoglobinemia, .nd h~moglo. binuria after hyp"rventilation (producifii: respiratory alkalosis), and hemo. lytic icterus. When not in :m a.ctin intravascular hemolytic nate, dogs ha"" mild regenerati"" anemias or r..riculocytoses without an~mias (comp"nsated hemolysis). (4) Confirmatory finding.: PFK..deficient erythrocyt.., decreased erythrocyte [2,3·DPG), .nd increased let). peR tening can confinn PFK deficiencies. c. HypophOlph.temic hemolysis ( I) Postparturient hemoglobinutia in cottle'" (a) Occu.. 3-8 wk after calving (b) Pathogenesi. of anemia: Oefeain mobilization of phosphorus &om bone and increased pho,phoru. 10.. vi. milk couse a pronounced hypophosphat~mia. Without plasma phosphorus, the A11' production by erythrocytes i, d~fective, which results in A11' d~fici~n
""

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY {2} Th~", ... sporadic .. pom of concurr~nt hypopho'phat~mia :md =~'" h~molysi,

in ho=s, dogs. and cau.''''''' ~,,~ hypophmpru.tem;" =1 d"",,,,, .. erythrocyte ATP production .nd thus product unsubl. erythrocyte membra" .. and h.moJ".is. [n dogs, • low [2,3-DPGJ may aho playa roJ. by making ""lis more ,u'cq>tibJ. to . Jkoline_OWlOCi. ta! hemolysis,' {3} Bilirubin int.,f.renu Gm cau.. artifactual hypophosph atemi<> with some ...... y methods, 50 one must k ca",fuJ about interp .. ting the concurrent pr=o"" of hypophosphatemia and hmlO[ytic anemia. (4) Hyperinsulinism {primu y or =ndary to hypergl~mi.} may cau .. hypophosphatemia by promoting monm.1It of glum .. and phosphat. into ""US oth.r than erythrocyte!. (Glum .. transport into erythrocyt.. is not insulin d.~nd.nt.) Pm;,[ent hypophosphatemia may deplete erythrocyte phosphate, dan ..., ATP production, .nd ca<= h.,molysis. d. L_sorbost intoxication'''-''' (I) Dog. that han 10w_K+ .,rythrocyt.. (most brtts.nsitivity, and

3/ ERYTHROCYTES

191

an.mi. of v:uying ' .... rity. Th. disord" is found primarily in Holsuin,. but .lso in shonhorn and J:mlaiam cauk'" {b} Th~ porphyria i, caUsM by a h.=iiury ddici.ncy of uroporphyrinog.n [][ rosynth .... an .nzym. that cat:d)'"U! on. of the first porphyrin ..actions mown in Fig. 3.2. {2} F.tin•• ryduopoietic porphyria {a} Erythropoietic porphyria was di:ognosM in • female Siamose cat and two mal. offspring. The cats had photosensitivity. hemolyric anemia, .nd .. nal di ......''' {b} The 'pecific enzymatic defect wa, not esublished in the cau. c. Oth" h"editary erythropoietic porphyrw ha"" bttn di"l:no.>«! in cattle. P4:" and cats; how",,, .• nemia Wll.I not rq>Oned as • fe.tu .. of these form •. ''''''··''' Lead poisoning creat...n acquired porphyria becau.. lead inhibits enzym.. in the h.me synthetic pathway. An.mi. =y be pr..ent in plumbism but is not con!id"ed a hemolytic anemia caused by the porphyrin accumulation. 4. Hypo05mol .. h.molysis a. Rapid infusion of hypotonic Huid int..""nou.Jy {such as st..ile H,O} or the inge;tion of large quantiti .. of wat .. by calves {v.at" intoxication} can cause rapid intravaocular hemoly!is. b. Pathogenesis of th. anemia: Infusion of a hypotonic solution or .bsorption of l..~ quantiti .. of wat" c",at.. hypoosmol .. plasma. Rapid mo""ment of H,O into erythrocyt.. vi. o.m05i, causes .rythrocyte swdling and l,.-..i •. c. The major laboratory findings are an.mia. h.moglobinemia, .nd hemoglobinuria. the ...."iti.. of which d.pend on the ,,,,..ity of the hypoosmolar ,Urr. D. Erythrocyte fragmentation in blood c"'ating .roiwcyt... keratocyt ... or acanthocym l. Erythrocyte damage is thought to be due to trauma caused by rdatively rigid structu... (fibrin) or by rheologic forces {= T.bl. 3.10 for disorders}. but oth" f.cto .. =y be involved. 2. P.. hog.n.... of an.mia: Becau.. the .rythrocyte trauma i. a oon"'lu.na: of oth.. pathologic starr,. proa:.... that cause: the .nemia may be multifaa:ted. a. Erythrocyte trauma .ith" directly causes ly.is or creat.. poikilocyte> that have • ,hort.ned life .p.n. Acanthocyt.. =y form in the circulation became of memb",n. lipid chang.. rath" than mech.nical or rheologic for=. but th... for= may contribut. to the .ced.rated fragmentation of .canthocyt.. (budding fragmentation). b. Primary di ....... ar<: frequently infectiou.s or noninfectious inflammatory disor_ d .... and thus the inflammatory starr may be producing anemia {= Nonregen_ e"'tive Anemias. sect. II.A}. 3. Major laboratory findings may include mild to moderate an.mia with no .. ticulocy_ tosi, or mode"'te reticulocytosi. and polychromasia • .roilOCytosi,. keratocytosi,. ac;onthocytosi •• and thrombocytopenia. The.. =y be other evid.na: of a oonsump_ ti"" coagulopathy. Th..e may be evidena: of renal failure with hemolytic u.. mic syndrome. E. H.molytic disorde .. of oth.. or unknown pathogen...s l. Heparin_induced hemolysis a. Heparin .nticoagulant therapy in some hor ... can cau.. erythrocyte agglutina_ tion. mild to mod.rate anemia. and increased biliary bilirubin exc.. tion that rdlecu increa,ed Hgb degradation.

192

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY {I} P>thogtnesis of th. d",r.asgg/utin.tal cdl. i, comid..al on. I..gt cdl (a panid.). Th... fo .. , th. fahdy d", ..as'rim.mal nudies, 'pun Hct (microh.matocrit) d",r.....d from n... r 30 % to n<ar 20 % within 6-8 h .ft.. h.parin t... tm.rin tr ... tm.m.2.

En~nomation

a. V.noms from wm• • nimah (•. g., makes, spid."" .nd ins.cu) c;o<= h.molysis. M.roanisms indud. compl. m.m activ.tion with sub..qu.m d. nruction (•.g.. cobra v.nom factor) .nd direct h.molysis from h.molysim (induding phospholi_ p"" A, in ratcl.,nak. v.nom)." b. Sph..ocytic h.molytic an.mi., may occur aft .. btt sting•. Th. h.moly.'rplasia in marrow .. mpl .. , .hon.n
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193

5. H~molytic .yndrom~ in horses with livtr di ......?" a. Nin~ hors .. with ~id~n'" ofhep.tic or hepatobiliary di= had inttavascular hemolytic ID~mias. Fi", hors.. we~ considorw to ha", pyrrolizidine alkaloid toxiciry. but the cause, in the oth.r fOur w.~ not determinw. b. Th. cau.. of th. hemolytic nates v..as not detorminw.

ERITHROCITOS[S AND POLYC'ITHFlvIIA I.

T because of h.mocon"'ntration or splenic contraction. [t is . lso callw puu,u,_polycythmtill or tpuriou. /"'fycythrmia to .mpha,iu th at the ,tate is really not an .bsolut~ inc=sc: in erythrocyte mass. b. Ab",",u or tnJr po/yt:ythrmia: Erythrocytosis occurs because of incr ... sed erythro_ cyte /IUSS. :md th ... i. roncurr.nt .rythrocyto";s. Polycythemia ",ra is on. fOrm of this type but also includes secondary .rythrocytotic disord.". 3. [n this textbook. erythrocytosis and polycyth.mia a.. not consid..aI synonyms. Eryrhr«ytol;' is .n inc~ased [RBC) in blood. jun as • /ruktxyro.il i. an incr• • ..d [WBC) in blood. Polycyth~mi. will be used only in the rontext of the neoplmic stat. of polycyth.mia ""ta. 4. Som~ peopl~ ha", combinal terms and concepts to fOrm :moth.. cl ... ification system. a. RrllJ,iw nyrlmxyro'ir. Th. d.finition is the same as fOr "IIJtiw polycythrmia. b. Ab",/uu rrytbttK]ItJ'is: Erythrocytosis associat.d with incr.asw erythrocyte mass. D. Extreme .rythrocytosis may cam. Judging of blood and thus imp.ired blood flow.nd poor oxygenation of tismes. Rdatw clinical 'igm IIUy includ~ purpli,h mucous m~mbranes. rongtnw retinal blood =sds. :md sei7.U~'.

[[.

Erythrocytotic disord." .nd conditions (Tabl~ 3. (4) (Fig. 3. (3) A. Hemoron"'ntration l. D.hydration a. Th. most rommon cam. of .rythrocytosis in IIUmmals. dehydration occurs '" a mild to mod~rat•• rythrocytosi. of no direct ""thologic significance. b. Erythrocytmi, i, present. but it is not due to :m inn....d m... of .rythroid cdt. in the body. c. Pathogenesis of the erythrocytmis of hemocon"'nttation is shown in Fig. 3. 14.

Table 3.14. Disorders and coodition.

mat ca u"" erythrocytosis

H~moconctnlr:uion

' Dehydration EndoIOxic !hod: 'Spl~nic contraction S«ooo..ry appropriau ~ryt:hrocyt:olic disorckn Righl.lo-ldt ,hllnu, convni!al or ocqui.od C hronic PlIlmorury di~ Hypenh)-roKI ism S«ondary inapproprial~ ~ryt:hroq10Iic diso.dm R..nal nalplasrru. cpu, or di.......... OIh.. nalp/asm. (h~Jl'I[om2,) Prinury crythrocyto{ic disol"tkrs Pr imary eryt:hrocytosis PlilyCYl[,~mi~ ...... ~ (pul},;,.t1[e",i~ [u~r~ y . . ~) Idiopalhic ~rythrocytosi' • A rd.rivdy mmmon di ..... "" ....,.jiti"" NOI ~:

Animal. dw 1M a, hip a1.i",deo o.

p..tupo ,110.. tho,

II.... b«n phpially tnined (•. ,~

raci", arumah) '-" an inanoed netd for Hgb 10 ttVdf'Ol' 0, 10 0.. .... and may ptodo>oe mo.. orythrocytts. In addition, cen.in breodo of dog. and bot.... h.v. , ....... Ha v:olua.h.an om. • .rumdo of m. urn< 'f"'Cios (_ Iisr in &ytbrocytoois:ood Poly
t

Hcl ,

t IHgb~ (0" t (ROC1

J I

PrimItry eoylhrccylo!ia Erythroid .... , 1"-;. • ~ia .......

Seconda
I

s.coodafy awopIi.lfI

u>

I

I

Secondeoy irIappropNhI

k}1hnx:yIC:U

wyl\rocy1oli l

(Il)pOOI< . ,••

RIInIII neopl ........

uc,_l

--

Cardiacl. . . . R
Fig. J" I). An opptoaell

~....

•

fIkn.mnaI

problem...oMns aythfOC}'U>Ml'},fu. OfY'Nocy.OIIU h.. IMen

neopI~5m!I

d.oooa~

o.

confiaoed, m. .rumal is ",omiD«i ro....id.moo of tho "'".. COm""'" co""",: I>nnoconcrntnrion '" ",Ionic contr>
194

Secondorr erythrocytosis I~II' .).14. Pathogmaa of the .. ythtocymoa. • Erytbrocymsis of ~nt<:ltion: Dehydntioo (d..cr..-l total body _ , ) mull> in a d..c=-!. ECF vol""", and ,bus. dccrnotd plaun.. volume. With. doc=-!. pi..... vol ...... bu."., c""'~ in n"",be. of blood nytht'OCJ'l<S. th .. [RBC ) is in=-d. Lo. of pI ....... H,O beea ..... of inc..-d vucuI.r pc ........ ab.iIity in ..ndotomic .bod: abo "'.,... an ~ocytosi • .,.;. h.nnooonat:.tr::l.tJon. • Erytbrocymsis d .... to .plcnic conaaaion (pby>ioIopc: erytbrocytOlis): The ¥t-o<.ftjght raponH 0< a..u.. ClW<S """ rd..... of laoi. and aytbrocytoois aoooci .. od with an ~ in total body aythrocyto m ... [~Epo plOducrion may be opptopriaw (if .imubtod by Ii..... hJ'l""'i.t) Of i""ppropria", (if not "imulaood by ........ hypoxia). &.-K:id <Wy.-rythrocytoo.io (_ &ythrocytooio and Poly
admin,,,,,,,;""

and 0). • Primary nythf<>CJ'looio: Thtre i• • p«>lifc ....tion of aytluoid ulb in the abMnoo ofina..-t Epo p~ ,;..., th" cam.. an orythrocyto<.is and inc~ toW body «ytltrocyt< lJUIL lltis lD2)' k ~[."ic or notIntOplastic. Nonneoplaaic primary erytb,OC) ...... causoed by ddioccivc Epo ,eupton hu bHn '<J'Onod in poopJ.. If aythrocytoois io OOlXUI'r
195

".

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

d. Loboratory finding. that suppon a condusion that ~rythrocytosj, is causal by d.hydmion {I} Hyperprot.in.m;" with poslibJy a conrurr.nt hyp may berom. h.moroncentr>ta! b«:.Ust of the .hjft of H,O from imfav.scuh r to extnvaKUl" .p~. b. r . thog<'nesis of the nythrocyt05is: Endotoxin. dontag' endothdial cdls 50 that blood .,..,...,Is b«:ome mot. permrabJ. to proteins. Th. atrav:.scu.l u proteins th. oneolic pressure grndi.m, which promote. the shift of pWm. H,O from imrav>SCUJ .. to ext"","",ulor .pact, thu. ausing" d="".., in pw.m.. volume and an "ylhrocyt05;'. c. Loboratory finding. that suppon endoioxic ,hock ( I) Erythrocytosis is usu
.roUct

sp''''

3/ ERYTHROCYTES

197

d. In theory, a d~fw:ive Hgb mol~cul~ that h", d,""r~ ability to tromspon or rdeast 0, could cause an ~rythrocytmis, Acquirrd di!Ord~rs (such as cyanide poisoning, c;;orbon monoxide poisoning, and nitr>t~ poisoning) are typicaUy acute and thu.s of insuffici~nt duration to produ,,", in",~ased Hcr, Hereditary Hgb disorders (hmtcg/4b;nopathks) ..e not docum~nted in domncic animals, 4, Physiologic proce ....s a, Mammals that move to high altitud .. may drvdop ti"",e hypoxia, Th~ hypoxia stimulm:s Epo producrion and ~rythropoi .. is, resulting in erythroid hyperpl"'ia and ~rythnxytosis that I'=>lve the tiMl1~ hypoxia. Mammals that Ii"" at high altitudes are ""peeted to h:IVe creater Hct value, than mammals that live at ,... 1"",1, but without tissue hypoxia or pathologic con~uence, Such animals really do not have erythrocytose1; .. ther, reference intervals for high altitud .. are g",.ter than thOle for low altitudes, However, one study involvinc dogs indicates that the erythrocytosis one upecu doe, not . lwaY" occur,'" b, Prolonged uercise training in horses has produced differing results, In some studi.. the trained horses had greater Hct values but in other studi.. they did not, Incr..... d 0, dem. nd dutinc training miCht stimulate erythropoi"is, but other mechanisms may contribute,-c, Some breeds have greater erythrocyte concent.. tions than others, {I} Thoroughbred., Standordbreds, and quarter horses have greater Hcr. than draft horses, {2} Greyhounds, Afghan hound., ",Iukis, and whippets have greater Hcts than moll other dogs. The Hcr. of the former ave"'ge in the high 50s to low 60s,". whe",... most other dogs have average Hcr. in the 40s in health, Poodles, German shepherds, bonrs, beatles, dachshunds, and Chihuahuas are also mentioned . , having Has creater than tho .. of most dogs,m 5, Pathogenesis; Hyponmia or increased tissue 0, consumption (hYP"nhyroidism) caus.. mstained tiMl1e {remol} hypoxia, which leads to incr"""! Epo production, incr .... sed erythropoiesis via erythroid hyperplasia, and, with time, erythrocytosis, 6, Clinical data th . t would support secondary appropri. te erythrocytosis a, Clinical history or evidence of a right to left shunt, chronic pulmonary disease, or hYP"nhyroidism b, Dw: .... sed P.o, (mongly if < 70 mmHg1l1 or < 60 mmHg"') c, H~mogIobinopathy {rare} D, Sa:ondory inappropriate erythrocytosi. (Fig. 3, 14) I, This state is caUed "rondA" becau.. erythroid cdl prolife"'tion is stimul. ted by Epo rather than being autonomou.s, It is caJlrd ;'Wpp,."prilllr because the increased Epo production is autonomous rather than being caused by systemic hypoxia. 2, Disorders in thi, erythrocytotic croup can cause mild to markrd erythrocytosis and erythroid hyperplasia, Marked erythrocytmis may cause sluggish. blood that leads to poor tissue perfiuion, 3, P. thologic causes (both "'.. or tardy =Cniud) a, In. ppropri. te Epo production by renal tissue becau.. of renal cyst<, renal neoplasm., and, ",rdy, other renal disease b, lruoppropri.te Epo production by other benign or malignant neoplasm, (e,g" hepatoma, hep. tobl .. toma, sffi".annoma, or leiomy",ucoma) 4, Clinical data th . t would support the conclusion th . t • persistent erythrocytmi, i, =ndory and inappropriate

198

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

a. Known associa!M pathologic di""rdw; ... found. b. ~rum [total protein] ~nd p.o.." WRI. E. Primo'Y uyduocytosis {Fig. 3. 14} I. This ,me is mUM primary kc.u,. erythroid cdl proliferation is ~utonomous rather than ~nd .ry to Epo. 2 Disorder> in thi, .rythrocytotic group can <:aust mild to markffl.rythrocyto,i, that may cau.. sluggish blood, poor {issue ~rfusjon, .nd th.",fore strolldui]y incrrasN Epo production. 3. Disorder> a. P,imJry rryr/trory""u j, eith., a lleopJ:.,{ic or nonneopi>S{ic condition in which th. toul num~r of matu .. uythrocytes in the body.nd blood is inc",a.=! without incrraoN production of Epo. b. Polycyrlmllm vml is neoplasia of erythroid, myeloid, and m.-ga\tion that would .uppon th= disordw; a. Ab .. n", of oth.. G1US . . of .rythrocytosis b. With polycyth.m;" ver .. bon. marrow with too many .rythroid cd!. mydoid cdl. and megabryot he .. is littl. or no stimulus for inc"",=' Epo production at th. time of c.sting. F. Jdiuparhk "JthrtXJl'»U is th. classification for patients who .. erythrocytosi. cannot b. cl""ifi
Methemoglobinem;" A. Muhnn"t;lobinmtill is the condition in which Hgb. Fe» .ccumulat.. in erythrocyt... It m.y b. h..«liury or a.cquir
3/ ERYTHROCYTES

199

{I} A v~rir'ly of toxicants . '" oxidants th. t ov~rwhdm the r.ductive capacity of .,ythrocyt~ pathw>ys and thus can l~ad to connrsion of Hgb to Hgb_ F~><-. {2} M:my of the.., oxidant. ~lso da~ th~ Hgb mola::ul~ ~nd lead to th~ furmation of Heinz bodies {~ H~molytic Disorders and Di.uleS. =t. c.l.a}. B. Diagnosi. of mr'lhemoglobin~m", l. Spur r{tf. A drop of blood is pia=! on whit~ filt~r paper. Mr'lh~moglobin~mic blood has ~ brown disooloration compared to blood conuining primorily oxyh~moglobin. 2. CO-axim(/rr. Thi. instrum~nt use. polychromatic light to dr'ln:1 oxyh~moglobin, deoxyhemoglobin, cuboxyhemoglobin, and Hgb_Fe+ by m~.ns of th~ir diff~r~nt ab50rption .peetra. The =ults .'" express.d as ~ per"'nt~ of total Hgb; for example. 20 % Hgb_ F~><- (~ Analyticol Con",pts, =t. III, in Chapt~r 10). 3. Pulst oxim(/rr. Thi. devi", aho <= th~ . bsorption spectra to mnmre pe''''nt Hgb satumion with Hgb {Spo,J. Unfurtun . tdy, th~ pul.. oximet~r cannot diff~r~nt"'[~ Hgb_h><- from oxyhemoglobin, 50 the Spo, i. f.l..Jy inc",ased wh~n ntr'lhemoglo_ bin~mia i. present. Mr'lhemoglobin~m", should be consid.,ed if th~ Spo, is signifi_ contly grrat~r th.n th~ So, (per"'nt~ of Hgb saturat.d with 0,) dr'l~rmin.d by blood gas .nalysis (... th~ Analytical Con""pts S«tion in Ch' pt.. 10). [I.

Cytochrom .... b, r.duct .... (Cb,R) { NADH _mr'lh~moglobin rffluctaK} ddici~ncy'l""'" A. Cb,R cataly=; th~ primary rraction involved in the con""rsion of Hgb_F~><- to Hgb. It UIeS FAD as • cofactor {Fig. 3.5}. A Cb,R ddici~ncy allows Hgb_F..... to .ccumulate in erythrocytes. B. It i. a h~r.ditary disorder in peopl~. Th~ /:"netics .. ~ not known for dog and caU (r~ported in pur~br.d and mongrd dog.). C. Classic f~.tur~s l. Young and old dogs with dinical.igns of hypoxia beau.. of poor o,..cmying capacity of mr'lhemoglobin 2. Meth~moglobin~mia (dark to chooolat~ blood) ~nd u.=tlly H~inz bodies p.... nt 3. No .n~mi. or mild .n~mia; may be a compensatory reticulocytosi.

III.

F.mil",l meth~moglobin~mia associat.d with GR d~fici~ncy in • ho"",'" A. GR catalyze. the conversion of glutathion~ disulfid~ to GSH. It <= FAD as a cofactor (Fig. 3.5). A deficiency in GR can l~~d to • d=ulffl [GSH ] in erythrocytes, thus m:oking them more sUKeptible to oxidants. B. A young mare and h~r dam both we", ex~rcise intol~rant ~nd had similar ~bnormolities. C. Clinical fntures l. Meth~moglobin~mia

2. 3. 4. 5.

[v.

Mild anemia (Hct n~u 25 %) GR deficiency (~bout 30 % of .ctivity of hralthy horses) Redu=! [GSH ] (.bout 50 % of h~thy values) Cb,R activiry:md NADPH dehydrovna .. . ctivity w~re not decr..... d. methods gin false valu ...}"

{Not~ Som~

Heredita,y nomo{ocytosis A. H...ditary nom.{ocyto.~s have bttn ,ecognized in {hr.., canin~ br..,.j.: AJa.kan malamut~., Dr~nt .. ",,{,ijshonds, and schn. uulS. Th~ sp«ific ~rythrocyt~ d~fects not established; dinically, {he dogs probably h. ve diff~ .. nt disord~rs.

u~

200

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY A1:asbn m. J.mut .. , th~", is • concurrent short.limb dw.rfism."" Th~ ~rythro_ cyt~ disord~r i. clurac{~riud by mild .n~mia, mild to mod~r.u~ reticulocytosis, mild to moderate nOJrultocytos;s, .nd s.honenffi erythrocyu life 'P.n. e. [n the Drenes< patrijshond., the ooncurrem findings include hyptDrophic g.micis, polycystic renal di ..""" and regenemivt macrocytic hypochromic .nemia due to hemolysis." Studi.. of erythrocyte membranes and plasmas of ill'""tN docs indic;ote that abnornul fatty acid composition in plasm. phmpholipids l.ads to aboornul phospholipid composition in erythrocyte membranes.= D. Among the miniature and ,t;;ondard 5Chna~rs. those with Slom. tocytos;s w", not anemic, but th.ir uythrocytes w ... m:oc,ocytic .nd hypochromic." The amount of noma!;" {erythrocyte membrane protein 7.2b} in uythrocytes of standard schn.uu" with s{ODl
th~

exchang~."

v.

Heralitary band 3 ddici~ncy in Japan.... block cattle'" A. Homozygous cattle for this .utosomal domin. nt di50rd~r have a chronic h~molytic anemi. , spherocytosis, and m~ubolic a.cidosi,. They also mff~r from retard«i growth. B. Ery,}m"yu band 3 is a membran~ prouin that h", two major functions; {I} it serve; as an ion-achange prot~in for th~ rapid affiange ofCt and HCO,- to allow th. transpon of CO, from ti,mes to IUfll:s, and (2) it rontributes to the .rythrocyu cytoskd
VI.

Heralitary dliptocytoli. in dog. that i. c;mlffl by prouin band 4.1 ddici~ncy".n< A. Elliptocytosis is caus«i by qualitati"" and quantimin d~f~cu of "yffm"yu mmtbrtm~ prottin 4.1, a prouin th .. is n<ed«i CO stabiliu th~ cytoskd~ton of th~ ~rythrocyte m~mbran~ by providing :m anchor for the .p«:trin ..;mkyrin compl. x. B. Cl .... ic f~atur~s of canine disord~r l. Markal dliptocytosis is p.."'nt. 2. Th~", i• .light an.m;" but with moderate r~ticulocytosis {com~mat«i 'Mmia}. 3. In cases report«i so far, th~ disorder was not a mojor probl~m for th~ dog•.

VII. EUiptocytosi. in a mix«i .. br~«i dog with muUnt spectrin" A. Elliptocytosis w .. caUIffl by . mutation in th~ spectrin gen~ . nd W:lS ",soci..«i with . n inc",ased ratio of .p«:trin dim.", comparal to .p«:trin tet .. m~'" in erythrocyt. m~mb.. n~ •. B. Sputrin,. major protein of th • • rythrocyt~ cytoskdeton, is compos«i of het~rodim~'" of two prouins; et. ..spectrin .nd ~ ... pectrin. Th. ", proteins .df... """,;.,u to form "!r.me.... Mola::ulor d.f,""ts in ~ith .. Cl-sp«:trin or ~ .. sp«:trin an raluce thi, associa .. tion. and thus mo .. dim~" are p...... nt in th. m~mbrane. In peopl~, th. location of the muution in . ither u .. sp«:trin or p..sp«:trin produces ~Uiptocytosi. or spherocytosis.'" Elliptocytosis occu", when the", i. a def«:ei"" horiwntal int~ ..ction of membran~ pcouins. whe=.. sph~rocytosi. occu", when th~ nnical int.taction of membrane prouins is d~f~ctiv• . C. Clinical f~tures of th. canine disord .. l. Elliptocytosis; Many ~rythrocytes . '" involv«l but to a varying degr..,. Typical finding. are 40 % 'YP" I dliptocyt .. {n~arly circular cells}, 35 % trr< II elliptocytes (oval cell.), and 25 % 'YP" 1Il dliptocyt~s {dongat«i cells}.

3/ ERYTHROCYTES 2.

Th~ dog wa. clinically cal disorder.

201 h~:dthy.

not anemic. and thu, its dliptocyto.is

wa.

a mbdini_

VIII. Sp«trin ddiciency in Dutch golden rmi~ ..." A. Sp<elrin i,. membrane protein that i,. nujor component of.n uythrocyte', cytoskel_ eton. [n J>'Opl~. a sp<elrin ddiciency causes. disordu known '" hrrNiitary rpfm-ocyrotir. Concur",ndy. th ... J"Ople ha"" • hemolytic anem", and incr~aK _'" detected only in dogs diagnosed with immune_ mediated hemolytic anemia. A cau",-.md_dfect association b.cw.. n sp<elrin deficiency and hemolytic anemia was not ~sta bli,hed in these dog•. Paligr...nalysi, indicated an autosom:d dominant inheritance. [X.

Megaloblastic anemia A. A m~fqb/a;tic anrmiIJ has th~ concurrent findings of .nemia and mq;aloblastic erythroid precursors in bone marrow or blood. B. Megalobla.nic ~rythroid precursors form became of asynchronou, maturation of nudeus and cytoplasm (Plate 9l). N uclear maturation i, arrested. 10 nuclei.", largr. with exagg<' .. tal .... chromatin regions that !",rsi!l to lat~ rubricyu stage •. Cytoplasmic maturation is rdativdy mor~ complete. 10 Hgb is appar~nt in ceU, with rdativdy immature nuclei. The edl, ha"" relatively abundmt cytoplasm and thu.s .'" largu than would b. exp«ted for the maturity of the nucleus. C. Causes l. Th~ ex.:Ict defect is rardy documented. [t may b. the r..... lt of neoplastic transforma_ tion of erythrocyt~ precursors. defecti"" nudeic acid metaboli,m cauK with erythro_ cyte diameters I Yr 2 times those of healthy cats 3. Bon~ marrow finding. in cats a. Erythroid cell numb.rs: vari able. inc......d to d=raK are dysplastic but are not neoplastic.

202

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

2 Erythroid na>pl..ia a. Acut. mydoid leuhmi.: erythroleuhmi. or erythrolruk.mia with erythroid prfflominan"" b. Prim:ory mydody'pla,{ic syndromn: mydodyspl:ostic syndrome or mydody'pJ.,· tic syndrome with erythroid prMominanu

X.

Sid.robJ""tic anem;" in dog,"

A. A di"l:nos;, of ,id.robJast;c anemia was basal on th. concu,,,,,nc p,ese""" of> IS % .id.roblast, in marrow :md of ring<'d ,iderobJ:.sts. B. Of th. =<" docs, six had nonrq;.nerati"" anemia.!, on. had. rege"er:l1i"" anemi., Ii"" had hypochromic .nemias, .nd two had microcytic .nemi.s. Dysplomic f.ature; in morrow included dysuythropoi ..is (asynchronous nud.... , matur>tion, binucl.ation, and nude.r f!'3l:mentuion or lobulation), asynchronous maturation in m.-ga\sis. and myelofibrosis. Xl.

Distemper indusions in dogs (Plate 4A .nd B) A. Erythrocyt. dist.mper inclusion. a", generally .... findings. They may occur in th~ ~.rly viremic stage and hefo .. clinical illness in dogs. They n.. y .ppear as pink or pale blu~ amorphom inclusions of varying shapes .nd . ius ...~ ",~ly obstrved in the United States, .nd a", usually more e",ily detected with the quick-dip suins (e.g., Diff_Quik) than the Wright naim. B. Rarely, simil.. cytoplasmic indusions a", round in blood nrutrophils .nd lymphocytes (= Plate 2D)

lA.BORATORY METHODS FOR ASSESSING IRON (F.) STATUS' Evaluation of an .nimal·s F. st.tus may he helpful in ronfirming an Fe-deficient or .n F._ o""rload nate, but ",rum [F. ] alone is .n unrdiabl~ reflection of body F. stores.

I.

Serum [F. ] A. Analytical concepts l. Bemuse n~.rly all F. in nonh~molyz.ed .. rum is bound to t",nsferrin, .. rum [F~] typimlly repr=nt' the amount of F~ bound to t",nsf~rrin. Fe in f.rritin may contribut. significantly to .. rum [F~] if the hyperfwitin.mia is marked. 2. Unit conv~rsion: J.4;ldL X 0.0]79] J.ImollJ.4; X 10 dUL = J.ImollL lSI unit, n~ ..est ] IlmollL)" 3. s..mple a. Serum i, p",ferred. b. Serum [F~] i, relati""ly stabl~ if the sample i, ..frige"'ted, fl"07.<:n, or k.pt at room temperatures for a fi:w hours. 4. Common principle of ,.rum Fe assays: F.... i, lihe"'t..,j from t",nsf~rrin in an acidic . nvironment .nd th~n i, reduced to F.... by ascorbic acid. F.... rraclS with a d~ or other compound to form a colored compl.., th>t i, det"",ed by photometric method,.

3/ ERYTHROCYTES Table 3. 15. Disorder. and condition. thai causc hyperl'cmmia F~

ov"load du~ 10 ",,~ss imake lalro!:"nic: excess Fe inja:tions or oral hemalinics Gene'lic defecl in regulotion of F~ absorplion Rd~ ... of Fe from tissu •• Hepatocyl< dam,,!:e Inc....
Of

b.moglobm.mi. (in vi'ro or

B. Hyp have: produced F~ loxicosis. {2} Fe inja:tions may be administerle.. breed,'" [n f'Nple, prim.ry h~mochroJrultosis occu .. becau.. intestinal abKlrplion of Fe is nOI . pproprialdy rcgulal+ COlI be transpon 400 IlgldL, but the [Fe] deereas
204

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

Table 3. 16. Di..,rdc" and condition. thai cau"" hypoferremia F~ ddici~ncy

• inc,,",=d F~ loss: chronic external blood [0 .. Dec",,=<:! Fe intah or intestinal absorption Shjft of Fe to sto~ sites • Acute inHammalion 'Chronic infl:omfilation Other or unknown m«honisms Dexametlt..50M inj~ions in cattl. 'Young .nim.!, {foal., killem, calves} Dog. with rong.:nilal ponosysumic shum. • A rel.tively common dioe .... 01 condition

2.

3,

4, 5,

6,

chronic gamointestinal h.morr~ (couK! leads to impo.ira! absorption of F. will contribute to .n F.-d.fici.nt ,tate, Gluroconicoids in attle mused a hypoferremia (from .bout 140 IlgIdL to 50 JlgldL) within 1 d .ft.. daamahasone (2 mg) was given intrav.nously.''" Portolyn.mic mums in dogs a, Dogs with congenital ponos)"temic shunts may have. microcytic anemi., :md ,om. may ha", ooncurrent hypof..",mia,''' In a~rimental studies, the .. rum [F.] did deer••.., in. group of 16 ,utgially induced portolynemic mums (from • me.n of 1291lg1dL to. mean of92 JlgldL), but the individual .. rum [F.] rem:linal WRl,'" b, The p.thogen.,i, of the hypof..",mia i, not enablishal, but data mppon th. concept of defective transpon of F. because of decreasal production of transferrin by hep.tocytes,'" Young animals a, Of the 2_ to 4_wk-old kitt.ns in a 'p«iftc_p:uhogtn_frtt colony, 70 % had hypoferremia (rd.tive to adult oon""ntrationsj th.t was associata! with microcytosis," This suggtm a !r.mi.m Fe-deficient stat. duting early growth, b, In 18 dairy alv.. « 3 d oldl, ",rum Fe collcent.. tiom w.re low.r in alv.. with Hct valu .. < 25 % than in tho.., with Hct valu .. :> 25 %, Only the most severdy

3/ ERYTHROCYTES

""

.n~mic coIf (Hcr = 9 %) had clinical sigll! of an~mia . Th~ co"", of tho appar~nt cong..nit>l hypof~rremia and F.-ddicient staU WlI.I not dot~rmin~d." c. Rolatin to .dult MCV values, fo.h han. po-ak microcytosis becw,""n 3 mo and 5 mo of age." Ho~r, th~ir strum F~ concrntratiom wer~ oqu:d to or gr ... t~r than concr ntratiom found in h~:dthy .dult ho", •." Stabl.d Dutch w;umblood fo:d. ( 1- 3 mo of age) that w~ro fa! freshly cut grass had low~r blood [Hgb], Hct, blood [F.], .nd ""rcrnt>~ transf~rrin saturation than ,imilar fo.h raisod on p"nur•. Th~ d.t> providod <"Vid~na. of Fo ddiciency in tho 'tabl.d fo.b.'" 7. Bta.Ust pl.. ma Fe is transport.d in tran&rrin, disord~f1 that come. 10.. of or docr ......! production of plasma prouins amId ams< hypof~rremia. R.n:d di ....., late prognancy, and hypothyroidism han bttn lin.d as hypoftrromic disordef1,'" but supportive data fur domonic mamnuls wor. not found.

[I.

TIBC .nd UIBC A. Analytico.l concepts l. Terms and uniu a. ITBC,. measure of plasma capacity ro corry Fe, is the maximum cona.ntration of F. that am be bound by plasma or .. rum protoins. B=ust mosr plasma Fo i, in mm(nrin {. complex of Fo and the prot~in apotransf~rrin}, .. rum [Fe] do""nds on .nd correlates with the .. rum [transferrin]. b. A tramftrrin mol~l. con contain two F.... iom, but, in h... lth, F.... ocrupies only .bout a third of.ll pluma or strum transf~rrin Fo_ binding ,iu •. UIBC, • measure of th~ tot:d nnu...! (opt~.ll Fe-bindifll: sites, .nd then tho bound_ F~ and f=-F. fr.ction. are "p"rat.d by ch~micol methods. Tho F." in th~ ,aturat.d transf~rrin molecules r...crs with a d~ to detormin. the bound [F.], which .. pmonts the TlBC. {2} Th. UlBC i. calrulmd: UIBC = nBC - .. rum [Fe]. c. In som~ clinical assays, plasma Hgb (hemoglobinc:mia) may co"", spoctral inter_ !n.ncr in :assay> that [nc:amre [Fe] {.... Laboratory Methods for Assessing Iron Status, socr. 1.B.4} and thus amId result in .rtonmut nBC and UlBC ,",ults. B. Incr••...! .. rum TlBC (Tabl~ 3.( 7) l. Fe ddici~ncy a. Pmpl~ with F. deficiency may Ita"" an incr"""! nBC b=me of the incr....d production of transf~rrin to corry available F~ ro crlls. b. Fe-ddicient dogs rypico.lly do not have increasod nBc.'

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

'0;

Table 3.17. Di..,rdc" and condition. thai cau"" increased TIBC inc,",asM apotr.mfcrrin production

F" deficiency: sp«i .. vari.obl. Other or unknown m.dunisms Young animals (fO. ls)

Table 3. 18. Di.."dcn and condition. thai au "" decreased TIBC D~.. ~

apotr.mfcrrin production • inHammalion Hepatic insufficiency lnn"asal transferrin lou (protein.lo,ing nephropathies, potentially other protein_Iming sUtc.)

• A ,.t1tiv
2 Young fool., esp«ially II':" I mo of "I:", h."" much gtrate, TIBC (;> 600 J.4::ldl) than namales or adult hom •. Colostrum j. transferrin rich, bUI rolo,era] intake d".,. not exploin the em;", inc",.... in TlBC during" foal's 61'SI month of lif•. C. D«r••.ffl5trum TlBC (Table 3.18) l. D~",..ro tr:msfcrrin production •. lnfLommalion: Apolransfcrrin is" ncg.tivc .cute_ph.... protein {i.e., its produc_ lion is da:r.,.Ks IL_I}, so inflam_ mation may lrad to hypotransf~rrin~mi .. b. H qJatic insuffici~ncy: B=ius< transf~rrin is a P1:lobulin producnl by h~pato_ cytes, Ii""r d~ that cau... hypoprot~in~mia may caus< hypotran&rrin~mia. H ypot .. mferrin~mia may pl.y a role in th~ microcyto.i. that d~""lops in some animal. with hepatic in.uffici~ncy. 2. Inc",ased t"u"ferrin loss a. ~""r~ glomerular lesions that produ~ a prot~in_lo.ing nephropathy may co"", hypotransf~rrin~mi .. Th~ rdative: molecular m ... of tr.mferrin is only slightly g",at~r than albumin',. b. Pot~ntiaJly, other protein_losiIIJ: st at.. {e.g., ,..,..,.. protnn_lming enteropathy and hemorrh3.f:<'} could .ho ca"", th~ loss of pl>sma transf~rrin :md thu. decrras< .. rum TlBC. III.

Percent transferrin satur:lt ion (% saturation) A. An alytical conarts I. PtrfrlU ""mfi,rin .atumtion is a calculated ~=ntagc: that .. timat .. the ~=nt"l:' of F.... binding sit.. on .potran&rrin mol«:ul .. that ar~ occupied by F~. 2. P~=nt tran&rrin saturation = (..rum [F~] X 100) + nBC B. B=iust th~ % tramf"c:rrin saturation is a calculated ~=nt"l:e, chang.. d.".nd on chang.. in the .. rum [Fe] and TIBC. I. Gr~.t~r % transf~rrin .aturation occurs wh~n .. rum [F~] is increased, TIBC is decrraonl, or both. 2 lower % tramf"c:rrin saturation occurs wh~n serum [Fe] is d=e. onl, TlBC is incrraonl, or both.

3/ ERYTHROCYTES

""

IV.

Stainable F. in macropha.ges of marrow. srl"'n. or liver A. In microscopic aamination, of form.lin_fix.d tissue or air_dri..:! cytologic p.. paration,. h.mo,id ..in i, ,,,,n .,. ydlow to brown granular or globular pigm.nt in maeroph"*,,. B. Wh.n att.mpting to quantity the amount of F. in 'to~ (esp«:ially for F. deficien')'}. the UM of.n F....,J"Cific nain (such ., Prussian blue) enables a more definitive ass=_ ment than does the US< of routine naim. HOWN... the p=" i, ,ubi"'tive and r
V.

s.rum Ferritin Concentr.ltions A. Analytical conarts I. Unit convmion: nglmL X I (XX) mUL X I ).Ig11000 ng = J.lglL {SI unit. n.. rm 10 ).Ig1L}" 2. Sample: s.rum i, p..f.. r.d. A [ferritin] i, .. pon..:! to b. ,ubI. for 7 d at 2- 8'C and for 6 mo >I - 20"C. Rc:1". t.d thawing and ..fINZing is not r
Table 3.19. Disorder. and condition. that causc hyperfcrritinemia Associat":! with increased total body Fe Iatrogenic ace" Fe injection, or or:'! hematini", G.::nctic d.fect in regulation of Fe absorption Iner.....:! apoferritin production • Inflammation Nu>pl .. ia: histiocytic ... rroma (malignant hiniocyto,i, ) Shift of ferritin from tissue to plasma Liver di ..... Hemoly.i. herci", in horses • A "1";,,,,ly oommon di..... or condition

208

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

2 Inn,,=<:! f. "it;n production a. In!L.mmat;on: B=me apof""itin ;. a positi..., acut.... ph... pror" [n pwpJ., th. F. saturation of f.,,;tin d= •..,e. with inflammation .... b. NropJ.,i.o: Hypen.rritin.mia has ~.n oonsid".d a mark.. for ~.ral human """plasm. and may k C;OUIffl by inft.mmation, ."""I.farM .rythrocyt. tumon, {h.moly,j,}. or inn.=<:! production by nroplastic ~ll,. HYI"',f.rritin.m;" h", oon associat.d with di"",miruotM hiniocytic ... room", (moligrt'lllt hiniocytosis) in dog .. '" It may:.!so occur in ,<""ti..., h.moph:agocytic syndrom • . 3. Shift of f"ritin from tissu. to pl.,rn. a. Lint di",as. such :as h.patiti, .nd n«ro";.: Tissu< f.rrit;n is ,de=d from damaged hqJatocytes and is measurw by fmit;" ""'1' that do nol .dectivdy me..,ure glycosyt.taj (or plrnna) f~rritin!" M""t dini",l OUS;O)"l pmn..bly m~asur~ gly<:osylatffl and nonglyc:01ylatffl fwitin. b. H~molytic di",a.=: Hyp<~rritin~mia may rda te to und~rlying inflammation or to inc",asffl transporT dema nds du~ to F~ r«rding from ly.;..d erythrocytes.

C. Hypofwitin~mia l. D«",asffl F~ sto~ a. ~rum or plasma fi:rritin concent.. tions d«rns. :as F~ stores d« reast. b. In """ple. fi:rritin concentr:ltions dec""", early in F. d.fici.n.!is. or d=scd serum [F~] is dec« tffl. Once Fe stores "'" dept"""" the d=sed plasm. ferritin roncentrations ",main .datively constant whil~ hematoJotic evid.nce of th. ddiciency (anemi. or microcytosis) d......Jop'. 2. Btcause .. rum f.rritin concentrations may be the net result of oppo.ing proce...s. an F.... d~fici~nt animal with inflammatory disns. may not be hypof.. ritin~mic. VI.

Reticulocyte Hgb content (CHr) and ",lume (MCVr) A. The ADYIA 120 h.matology analyzer dir«dy mnoures individual reticulocyte Hgb concentration and ",lume by light scatter. From these mu.ures. CHr (th. :IV.~ amount of Hgb in reticulocytes) and MCYr (an~ reticulocyte ",lume) :lte calcul>1ffl. B. Decreased CHr and MCVr were associatffl with typical findings of Fe deficiency in dogs." Th... valUe> may pron u..ful :as early detectors of F. d.fici~n
VII. Comp.ratin F. profile results A. I'.s.=sm.nt of th. F. "aCUs of an aninul is .nhanced if the laboratory .....y. at< grouped :as • profil •. B. As shown in Tabl. 3.20. the two major causes of hypof..",mia c;on be diff.t or disord.r.; chat can alter ",rum [F.]. TIBC. and F~ stores neffi to be con,id~red. BLOOD TYPING AND CROSSMATCHING J.

Blood typing A. Blood groUp' are imporTant for transfusion mfflicine and NJ. I. If th. transfu...,d ~rythrocyt...re recognized :as having foreign surface .ntigem. the .. are the two major con""lu.nces:

"',

3/ ERYTHROCYTES T able 3.20. Comrarativc Fe I:'rofile res ults Suin.bl~ F~ F~ d~fici~ncy

[nflamllUtion Ov~rload of F~ du~ to acess intah (diet or iairo!:"nicj [ncr~• ...d glucoconicoid, (ex<:qlt amle) Pathologic h~molysi, {in vivo} YouIli: animal, {comp"rw to IIU{U"'} Hep"tic inmfficiency Hep"tocyte nrc""i, Protein.looing

in

~rum [F~l

&rum TIBC

IIUrrow"

&rum

""

WRl-;

"

;

;

WRl-;

" ;

"

;

,

,

,

WRI'

WR!'

WRI- i

;

,l. (f""l, .nd kitt~ns)

i {foal,}

, , ,

"

"

",

,

"

, ,

[f~rritinl

; ;

-1-;' ;

,

n~rhror·thr • Murow umpJe. of bealthy "'''' do not baw .uinobLe ~ . • Phuru Hgb may c . .... «rO""""" v:o/.,., (oee tb. tat). < [Forritinl could be decr..",d bea .... of decr.....:! production or it could be incunITUge.

a. If th~r. are circulating .ntibodie. in the rC"Cipi~nt, th ...... ntibodi .. ",n atuch to th~ tran,fu...d cdt., which then mult. in a transfu.ion reaction and/or hemolysi, of the tran,fu...d cdls. b. If th~re i. not. circulating .ntibody in the re-cipient, the =ipi~nt'. immune system will be triggerw to produce on• . 2. Anti ... rythrocyte antibodies may be naturaUy occurring (i.e., pr... nt without p=iou. apomrc), or .cquiral if they devdop aft .. apomr. to foreign erythrocyte antigem. 3. Knowledj:e of blood groups is n=ied to undersund Nl. In thi. hemolytic , Ute of neonate., antibodi~, in the colostrum arc absorbed .nd then bind to the neonate·, erythrocyt...nd mediat~ th~ir d.. truction. B. Blood typing invol= methods to detrctthe erythrocyte blood.group factor.; on the surface of the erythrocyte>. Mon m~thod, in""l"" antibodi~. that a.., produced in laboratory animal, by injecting th~ blood·group antigen. For many y"ars, blood typing w., limited to • few spe-cialiud r...,. rch J.boratori .. that produced or developed blood. typing .."C"nt, (either polyclonal or monodonal). Blood typing in hones is still limited to , f,"", loboratories. C. Blood.typing cuds h."" been devdoped comm~rciaJJy for dog .nd c;ot blood. In these tem, a .mall drop of blood is added to a white card impregnated with an .ntibody (or other agglutinin). If the.., is , po,itive reaction, th~ erythrocytes agglutinate. I. For dogs, the blood·typing c;oro is used to determine wh~ther the dog is DFA 1.1 po,iti"" or ne-gative. The agglutinin i. an .ntibody.

210

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY 2. For cats, {h~ blood_typing cud is usn:! to determine whechu the at is ty~ A, B, or AB. Type A :mti~n is detttled with.n agglutinating antibody". typo B antigen is detected with a wh""t germ J..crin.

[I.

Crossmatrning A. Cro ... matrning proadur.. " .. usn:! to hdp d.urmin. wh",h.r an .!Iim:.!', blood can ~ ... fdy transfu",d into another .nimal. Cro.. matrning is similar to blood typing in that the [.bonto!}' assays involve amibody reactions to erythrocyte .ntig."", How ...", th~ ,,'" diff.rent in that the :mtibody is p ....,m in " =ipient', plasma (for major cross_ match) or in the donor'. plasma (for minor crossmatch). B. The Iwic >Sp<cu of the ......y or. as follow.: I. Blood is drawn from a pali'lII tru.t neul,,, blood {ran,fu,ion (the =:ipient) and from" donor. PJ:.S/IU or strum is .. pan ta! from th. erythrocytes, .nd the erythro_ cyt .. " .. w:ashed in saline ~ral time, to remo"" loosdy bound prot~ins. 2. M . jor crossmatch a. Th~ proadu", involns mixiJll: and incub..ting a dilut~ susp"nsion of p<Mntial donors ~rythrocytes with the =:ipient·, ",rum. Antibody bindiJll: is detecud by :ogglutination, hemolysis, or a Coomru' test. b. A romp.tibl~ major crmsmatch indiao.t.. that alloantibodies againu the donor. uythrocyt.. w~r. not deta:tffl. An incompatible major cro.<smatch indiao.tes that .lloantibodies against the donor', erythrocytes,.."", detectffl. c. Th... i, not . standard cro.<smatching procfflU.. in vecerina'1 mfflicine, so ..sult, may "''1. An incompatible crossmatch may occur when th~ :dlomtibody detected is not dinic:dly signifiao.nt. Con""rsdy, some signifimnt .lloantibodies .re not detectffl by :ogglutin. tion .nd rftJui", romplement ",.ctions or Coomru' testing for deta:tion. 3. Minor crossmatch (not done as frftJu.ntly., th~ major) a. Th. proadu", involv.. mixing and incub.ting a dilute susp"nsion of recipient·s erythrocyt.. with potential donor·, ",rum. b. An incompatible minor cros<match is us,",lly not. clinic:d concern kau"" it i, not ap«1ffl to alll. . . m. jor transft.. ion r.-action.

Ill.

Speci.. ""'"'tions A. N.tural antibodi .. ~.inst DEA 1.1 and DEA 1.2 h. "" not bn.n reportffl .• nd thus ..ja:tion of th~ first transft.. ion of blood of unknown t~ is not expecta!. Ho~.., if th~ donor dog is DEA 1.1 positin .nd the recipi~nt is DEA 1.1 nq;ati"" (.bout a 25 % chance), the =:ipient will d",dop xquirffl .nti_{DEA I. I} .ntibodie,. Th. DEA 3 system does hayt natural antibodi.. that am att:ock transfusffl erythrocytes: About 6 % of US dogs are DEA 3 positi"", and . bout 20 % of DEA 3_neg.tive dogs h. "" the natural antibody.'"" B. Cats ha"" natural isoantibodies against the .ntigen they are lacking: that is, ~ A cats han .nti_ B .ntibodies, ty!'" B caU h ave .nti_A antibodies, .nd ty!'" AB caU do not han iso.ntibodi~•. If blood t~. are not known or if a ao.t h .. had. prmow transfu_ .ion with untyp<,

3/ ERYTHROCYTES

21 1

l. Ho .... that lack an ~rythrocyt~ . mil:"n may h.n nalUral iso. mibodi .......

a. Horses lacking ~rythrocyte amil:"n A (EEA) may h.n .mi_Aa or anti_A<: amibodi ... b. Horses l. cking ~rythrocyte antil:"n C (EEC) will typically han. low tiw of .mi-C :mtibodi... c. hoantibodi.. to oth~r ..ydlfOCyt~ antig~m D. K. p. and U are rare to infrequem. 2. Ho .... =y ~ sensitized to erythrocyt~ .mig~m via blood tr:m,fusiom or during pregnancy. Th~ acquirw .mibodi.. that cou'" transfiuion reoctions or NI ar~ primarily ag. inst EEA, EEQ, and EEC. Th. antibodies may come an incompatibl. reaction in the =jor crolSmatch. D. Blood_t~ .ynems in cottl~ .'" extr~mdy compla •• nd thus it is . ... mially impossible to gin • compatibl~ transfiuion. METHODS FOR DETECTING ERYrHROCYTE SURFACE ANTIBODY OR COMPLEMENT I.

Coomb.· test (direct .miglobulin tm) A. PufJlOS': To detect ESAIg or romplem~nt on • pati~m·. ~rythrocyt ... usually to hdp diagnose immun~ h~molytic anemia B. Method for the direct Coombs' ten l. A p. tiem·. erythrocyt~. ar<: washw thrtt tim .. with .. lin~ to ",mon IIOnbound prot~ins.

2. Antiglobulin is addw (specieo-.pecific .mi_lgG. ami_ Ig.'vI •• ndlor :mticompl~m~m). S<:ra th.t contoin two or mor~ types of .miglobulin are coUw po/ywltmt anr;"ra. Antiglobulin! wiU bind to .ny IgG. IgM. or complem. m on the washw erythrocytes. 3. A source of frdt complem~m prot. ins is nttdw for a hemolytic r...ction in the equin~ Coombs' test. Th~ hemolytic r....ction is n«dw because equin~ ~rythrocytes m.y not agglurinat~ in th~ direct Coomw' t~n. 4. Positin r...ctions a. Agglurination indicates th. t • pati~m·. ~rythrocyt ....e coatw with hundrw. of inUIlUnoglobulin or C3 molecul.. and that the binding of the antiserum', immunoglobulin was mfficient to couse agglutination (Fig. 3. 15). {I} For agglutination to occur. ther~ must ~ the appropriate ratio of .mig~n and .mibodie•. To rwuce the po .. ibility of f. l.. _negatin r~.uln cous«! by the prozone r~spon"'. Coombs' tests .'" fttqu~mly completw with multiple dilutiom of antiglobulin .. ra. {2} If th~", is a prozon~ ....ction. th~ Coomb,' test might ~ negatin with a low dilution but po.itin at high .. dilutions: for n.mpl~. negatin .t • 1:2 antiserum dilution. weakly positive at a 1:4 dilution. and mongly positive at a 1:8 dilution. b. H~molY"i. sugg~sts that immune compla.. formw betWttn th~ .mi.. rum inUIlUnoglobulin and th~ ESAIg and that th~se immun~ rompln .. lw to complement activation and ~rythrocyt~ ly.is. c. Coombs' test. are not stondordiud and ar<: often unneceuary. given oth.. clinicol .nd labor.tory findings. Neg. tin r.. uln are common in cas .. that pr~ .. m. prog""". :md re;pond like immune hemolytic .nemias. Positive result. do occur in sample. from animals without dinicolly significont h~molY"i •.

212

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

""',

"'Ythrocyl&5

+

AntigloOO~n

_

Agoglutinalion

Fig. J.lS. Aggluti ... tion "':lenon of • l""i.iY< cani"" Coombs' test. W.m.d unm. ~brocytes .,e inrub.tNl with .. bbit onti·(dog jmmunoglobu~n). If th. dog', wash.d erythrocytes >t< rooted ...ith dog immunoglobulin, th. 1ntDerum wiU c""'"" .gglutin1tion of II.. dog', erythrocytes whm .1.. ooncenttotion of nbbit rnti-(dog immunoglooo~n) is :oppropri.... T..a m.y :oIso de"",_ erythrocyte-bound rompLem
n.

d~t",,{ion of ESAIg To det«, ESAlg or complement on a pati.m'. erythrocytes, usually to hdp

Flow cytommic

A.

Pu~:

diagnose immune hemolytic anem;", B. An:dytic principlu l. EDTA blood is ,h. prd'.rrffi ",mrJ. and should ~ kq>t cool during tnmpon. EIythrocytes are washal al I.... ,{ Ih,tt times with salin. or phosphate_buff.rM saliM prior to • dilute .rythrocyu su.p<nsion ~ing mad •. 2. W.,hgainst IgG, IgM. or rompl.m.nt. 3. If the patient's erythrocyu. arr coat«i with antibodies lor complement}, the fluore=in_lobd«i .ntibodie. win bind to them to make tho.., erythrocytes fluore=nt, <\, Flow cytomeuy identifie. thOst erythrocytes that are fluo=nt and thost that :ore not, A positi"" result has bttn identified by an incr....ffl p
3/ ERYTHROCYTES

213

Refe re nces I.

K..." Sf. Bondwao' Me. 5."""", G1.. Koury MI. 19"9}. n, ....

~f

in

.o... brl-idi,.tioa to ot»dy ,
VB' "'p"-"" in m"';o, kidn"...d livn. M~ R
2. SI<>ytbropoi,cio p...Jocti... , A puad.i ~ &.. ~IJ"~"''' rula ted ~ , ~ ",ion. Clin E..p PI... maool. Pby.iollJ096s-m. J. Deuyprt. EN. 1m. E.,tbropoi"i.. Iru I.e. GR. ""''"'''' I . Lokro. I. p... ok ..... F. Goo" Ip. ~, GM • .d •. """ .... /00, am"", H"""""oo- 10th odhloo. 169--192. PbiLoddpbi.. llppinrou William. &: Wdkint. t . H"""')" JW. 19"97. n, ,~, Pbpiolou. mrtaboI ...... ...d bOoch....ical WHine '1"''''''' fo. ao.lyoi. ofblood of coonmon dom"tk aoimal •. Y" Clio P.tboI. 12,25-32. 19. W,;.,., MG. 1937. M..Jifiatioon ...d ......... tion ofa mtdtidtanod blood "H rountinc; ' 1 - of blood ",.Iy.i. in Ytt";"'" ~''''''''''v. I Am Y" Med AttOC 191M II_·U S. 20. M"&,,,,'JW. Wa!~ I. I. O!.bo·M.boi W. Jon ..... NN. Eolt. MC. ZOO4. Pottabl, ~......,pobi """""". utd ...... """ocia! fo. "",.id, ""',«don of .........;. i" bovint -20(2) . I V" ]0<0"" M,d 19,}2S_}2./! . 19. H"""')" JW. 2001 . Ad.... .IV_;""'" H.."."""U BI..J..,../ Bo_ M...-- .ID.....,.;. An .......... Ph.iI..klpbi", WB

""""toO

Sawtd ....

214

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

B..od.. Sf. Ru. ... 1), G.inytl.rocyu val." in fooJ., Am J V.. Rr. 48, IH.!-lJ52. J2. W,ioo" MG. Kociba G/. 1983. Seq.....tial cbmp in ""Jum. Jj",a...';"n ...<1 miaocytooio .. oociated ..ith iron
J.O.

Gooli~JL.

s........

,"""«>
n. }t.

}S. }6.

l7. }8.

J9. .. 0.

oil. .. 2.

4J. 4• . .. S. .. 6.

.. 7. .. 8.

.. 9. 50. 51. 52.

Obi., J. Tajim. S. y.....'" O. lnab. M. H.d...... S, "hod, Y. 1m. H.....,p.biD typ< •• D«nora""n in aIv. • ..it!. powlocyt.ooi •. J Vrt Mod Sci SBii29-<>J4. T ''''..''' B. Hurold D. Rdna..Guon. M. Kaooh. J/. 1975. H""",,1ocJ of th, """natal alf. HI. F""I""'CY of «<>vnital iron d,bcl",<J' ••"mia. Con.dl Y" 6V;'H- H6. Wd.. DJ, Knot ....... A. P.p". Eq""', d~';'pbo.p~at< <>J2-642. Wd.. DJ, G«>e R. Sonith eM n, M1<... implia.tiono fo. =l,,-indoud pulmoouy ..........b.V in..cin& tbo.. -&kbnxl.. Am I v.. Roo S},]J8()....]J8S . B,..n." G. 1\.,.., M. 1972. P" .... , ...... of opindated ,od edI, ...d t!.d. ",J."'-hip .. tht dioootyt .. od.inotytt .......... m ...... ' A ",itia.l , ....... Blood 4(],J}}...} . ... Goo, RI. I...nd EM. Woi .. DI. 199J. Ecbi~ocy""i. i~ ...... " 5. "'.... (1990). I Am V~ Mod A..oc 202.976-»80 . llaJyJ.k SF. V... VI ... IF. Hm""" EH. F........ VJ. My.... CE.. 1985. Poikilocyt.ool. in doc • ...it!. cb"",ic d.o=",b~ do to%icooi •• Am IV .. R... 460'>05- '>08. Smith IE.. Mohand .. N. SboI.tt SB. 1982. Inttnctio~ of amphipad.ic d"". ,..;t!. ~. from vuio ••• ptciodtrooi. i~ • b..rt,. I Am V.. Mod hooc 1660 117()....117S . Waltoa RM. Bro.m DE.. H ...... DW • .\l,,..Jo. VP. H,,"~ JW. n..u MA. 1997. Modoani.".. of cd.i<><>mation in .... doc, Rq-t of "'" CO" • ...d , ...... of .... ~"".""". I AmAnim Hoop hooc 26.61~ . Wd.. DI. Motib A. ZOO}. Equi~, imm"",..,.,di.ted b ..... lytic antnt.i. uoociated ...it!. a."".;;.", /"fo"1"" i<>fectio~. Y" Cli~ P.thoI }2,22-26. ll<... M. 1977. BJ..JS-- &;,,"'~. Bt.~~,~. I~ .. m.tion ... a...;"opb .. M.\1. l.tt SE. 1994. Rrd ",ll """'pl.olo&ic altn.tiono i~ co"...it!. b,potic dioca". V.. ain P.tboJ 13,7- 12. O·Kr.k DA. Scb.dft. DI. 1992. HtmatoJocic t=icooi ....ocl"od .. ith nobici~ adminiatration in at&. I Y" In'"" Mod 6,276-282. Wd.. DI. Lolid. I. 1999. M,~"tic opod"""" ..ru. .id,robI .. tic diIftrnotiation in • doo&- Y.. ain Patbol

""&-

"I.""'"

,"""rocyt<.

21h S~J.

SJ. Hoff B, Lu..od. Con I S'. S5. S6. 57. 58.

59.

Comp Mod .9.} ...... 2. Scavd~ TD. Ho.~bucl.1, WE. Roth I.. Rrndano VT I •• d, lah._A. Ct", .. SA. F"oclt lW. Zi .. m« IF. 1986. P0"""P .... ic .b""" i" ",at" Stvnt. ""'" (1976-198'). I Am Y" Med A"", 189,JI7- J25. S.. ith IE.. Moot< K. A. .... M. Rind .. lmock, GA. lLd« A. 19I1}. H<=lituy ,lIipoocytooi • .,.;th prot<,...... d., to
3/ ERYTHROCYTES

215

60. Cl.apm .. B1., Glen U. 1?90. Inb"ited laod ..hi" "..i". J Small kim P TloSGAM. H"I'p" RP. 1991. Fami!i..l b,pioed Jj" ... in tJ.. dot; (D......., patoijobo..d). Y.. Q l},.J(l.4(J. 66. B...... DE.. W .. '" MG. Tlu.ll MA. G~, U. J.... CA. 1994. E..ytbOOCY" i..die< • ...d.clum, diotribv.tion in. dot; ..hb .....,.rocytooi •. Y.. P.tl.oI. Jb247- 2SO. 67. Boofaoti u. Com"'; S. P.ltoini.ti S. Bnt"",,10 W. 2000 . ~ . in 7 .... ted • ...dud od.n"""" •. Y.. Clin P.""'l }3,23 .....1J9. 68. Smith SE.. l.oodi JK. 19S7. Cobalt ...d .itamin Bu in ,wotinant oo""tio", A ........ J D';". Sci 40d2IS_ 1227. 69. S"IJ.., DJ. Elliot GS. ~ HW. Smit!. JE. 1992. Coop>ital d,.,«ytb.opokoi • ...d p""V ... i", .... ped. i" polkd H,..foed a/n,,, H"".",1odo<. biocb....iaL 1.0",....,..,.. <J"'><>1odo<. dectropbomic • ...d _ [ _ "'31 - 37. 70. HinchJiff< RF. Bdl."" GJ. LilIrym.. JS. 1992. U " of th, T odu.lron HI byp<><.l.""";' i" d,,,,,,;,,, ...... iou. m>«<>oi. induced by "",.. i", Kr EDTA oo"' ...... tio". Clio Uk H",.. atoI. 14.168_269. 7 1. G.",...... W. ,.." 0..1'1"" Y. Y,.,..jJd>,n RL 1991. K,_ '" K,-EDTA, n.. ",tiroa&uI.m ~f cI.oic< in .... tin. h "";... J Am Y" Med " ' - 218,1 S93-1 W7. 73. van Wyi JJ. Bartn JH. Ak=yd JH. MotuI.1y AG. 1913. n.........;. of 001'1"" ddKi....,. in d"p patby. A.cb Pathol lab Mod 124,(;16-61 ~. 76. H .... ill. G"o,[jA. Dwm RF. Mwpby JR. Whi" JG. Kdl"...,y... RW. 1973. Pkpica.l p",,,,,tioo of ,od «ll . .. ,d.,ed to d[",,, in .i",. IV. o.;,j.." drup po<>0--9379. Mtinkoti. JH . K..an AA. 2OOS. F,Iin, , Eid>' c .... (l98S- 1992). J Am Yrt Mod A.oc

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198. Gkru. 81. G],nn HG. Om""'" IT. [%S. Co_nit .. p"rpk,..k in th, do..->< cat (hi;, ,.-j, P,diminuy i~ on inhorit"." paturn. Am J V... R... 29d6H- 1657. 199. Wttb TK. 19SO. Po

cyt< ma .. . Am J Vrt R... 46,2175--2178. l()l. ,\1""", IN, M.t..Jf.,. EA. Zboun M. 1987. H-Prk< 1. Em, ... n CL. Hind. 11t VID«"'; FF, H........,..,j WR. 19118. H,«><>lytic .0"";0 in b.acI", Am J Vrt Rr. 49, 1020--1025. l()J. Pd blood ythro. J Am V .. ModA.oc 217,)o.!}-1491. 205. Too ...... Be, &an. CD, Kan,b, /J. Sd.alm OW. 1972. H,patic /'ail...., in tJ.. J.on.. "hl Y" 1'.", Ho4O--42. 206. p"",,,,,, ME. Kintu. pp, C.......p. PG, F"" PRo F,~"", OC, JOk<>OOD GF. Iledr. DY. 19l1J. Fdin< b1P"'tby" """li.ru Prrt...tmo",0'}' "'!u.rion of III "'''' •. J Am V.. Mod hooc 13}, [O}_ IIO. "lfJ7. Gla •• TM. H...ir; M. Ilawnc.rtn", C. R.u.d. C Eo 2003. ~_. ;"'n indo=! in d.oc> by ~~ •• dijf,,,,,, ~i&Io.alcituod, I,,·d •. y" Roo Comouuo 27,661-670. 208. R- RJ. AJ]", IR. 1985. H ..... toIo&i< '<''''''' .. =ocir and Ininin ~ y" CIin Nonb Am Eq..n.. P..n )'Ol61.....j76. 209. lyUrt.... G. ..! H. Job.no", K. 1977. T ,ai";~ , and ",..dr dunV "''Pin.''''T p«>pnb" ofbloc.J in bin,,,,ia in -dor;. """ to. NADH mbin. ..,,[_« d,fid."'J" in. doo&- I Am Yet ModAaooc 164,IOJO.-IOl,. 217. AW ... C Eo ~ II. Con~ LL 19I1l. M.t)"moV>bin. ..,,[_« d,fid."'J" and mrth~",ia in. doc&I Am Ani.. Hoop Aaooc 17,329-832218. Bak", OC. Gaunt SD. 19I1S. Niootin.",id.o-.admino din..dooti.k-mrth'mocl<>bin ...Iucta" activity in t. I y" 1m"" Mod 19,187_ 192. c....r.. AW. Blakr..ood BW. 198J. Acut< into.ia.tioo from • J.omatinic in • .....,•. I Am Yo< 227. RuJ.. LP. Nidd."", Mod Aaooc 132,616-61 8. 228. M.llao.,. TP. B......, CM . 19I1I!. [..,., t=icity in """",tal lOab. Equin, y" I 20.119-1 24 . 229. l<..i. HB. M.".", WA. 1975. I."""""" iroo
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230. H""", JK. Sonit!. BP, Ma.. J, Laa, VM. And"..,n Be. Goal"", TW, Pin<> MY. 1994. H,mochrom""';' in s.J,,, ""d,. J y" In"", I>W '" 10$-111. 231 . S",ith JE.. Ddl"'..... RM, Cipoiaoo JE. 1986. ~ cortkoo"rokLo iocte.... """'" iron co""",,"""'" in ro.""" bent. and po";". J Am Y.. Mod A..oc 18Ml%-ll9Il. 232. Gtafmty" D. Bondon M, Mand.on M. I.t.illain P. 1995. Tlot inflOKDCt <>fbilirubin. ~......,lpl. and tu.bidity on 20 ....t,tkal _ pa/'orn>od on .~",.... tk uoalyr. .. Rt •..Jt. <>f '" ;nttnty in .... pinli=l 100",•. J Am Yrt Mod A.."" 188,28$-237. 235. W",b BR. S...itb JE. Ddl",.,,,. RM. Smhlt J.\1. 1989. D",,,..od ..... '" ;ron and ,;nc «>netpt«n...tiom, I",pliatioo", fo, tit< """"''''''''''' of;"n ....... Eq..o.. y", J Suppl ,)4,IU-I90. 242. s.,.." RD. 1m A-.mtnt <>f i"", •• <>to. Oin lIOoch"" 290209-21 5. 24J. W«b BR. S...itb JE. Northrop JK. 1989. Rdatio ..... p of """'" f,..i';" and iron """"""trationo and"""", .. nl ;ron-bindin~ capacity to "".... "'" iron _to in """,. Am J Yrt Rr. SO,198-200. 240. '''' K.,,, J, Wold.";' A. W ... ",k.;' B, '"'" D.w"" C. 1997. n.. iron cont,o' of""'''' ftnitiru Pk,..ioIoVaJ import.",' and diau-ti.< val",. E", J Oin CIotm Oin lIOoch"" l5,SJ- 56. 245. N....Jand. CE. H""""" DM, Y..",ncdo. DY. 1990. Hyptrftnitin.",;. • .....;otcd...it!. mali" •••" hittiocytooi. in. """ J Am Y.. Mod A..oc 205,809--1l51 . 246. Andt .... GAo 2000. RaJ blood cdl ""';V'" and I*ood """po in tit< doc and ca'. In, Fdodm"" SF, Zlnld JG. Jain NC. ed •. &/"J"" V..........., H_1op, 5t!. edition. 767- 77 J . Pkiladdp~;' , l..ippinroW 14,190-1%. 250. K"d",kltnt G. Scltubtrtb HJ, Lt of "" .. mk """,. Y" J 169,30l- J.07. 251 . Bonka...ky HL 1991. Iron and d., li"". 1un J Med Sci 3Oh32-4l. 252. Z~.... la-J.Vdk> J. J. 1998. In."...Jiutioon . """ win~ EDTA-l""J"'ft"'Ihtpotocyt<. fot <=p",,-l hatmoc\obin-ltaprocJol>in compl",. In, J Bioc.I.,,,, Cdl BioI j009lJ-9ll . 253. Kino K. Min."""" K. W." ... bt J, T"""", H. 1987. Im",woobi.
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Chapter 4

PLATELETS Physiologic Processes ...................................................... 224 Analytical Principles and Methods ............................................ I. Complete Blood Count (eBC) ........................................ II. Thrombogram ..................................................... III. Methods .......................................................... Microscopic Features of Platelets ............................................. Thrombocytopenia ......................................................... Thrombocytosis ........................................................... Platelet Volume............................................................ Reticulated Platelets ........................................................ Tests for Immune-mediated Thrombocytopenia (lMT) ...........................

227 227 227 229 232 233 244 246 247 249

223

224

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

Table 4.1. Abbreviation. and ,?',nools in lhi. chapler [xl ATl'

ole DNA EDTA Fe lL-6 IMHA IMT

MrS MPV PDW

PSAl, QBC

RNA SI

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PHYSIOLOGIC PROCESSES I.

Platdcts .... mall cytopl.. mic fragrncnu of mq;a\<aryocytes (""!" = "luge," ""''lon = "nucleus," and CJ" = "<:tlJ") thaI :He dcrivffl from pluripotent hematopoietic nem cdh (..., Fig. 2.1 ). M :m unalian pl:udets ilia", mosr of rh~ following componenu, alrhough with .ptci.. variations: A. A phospholipid m~mbran~ containing glycoprot~i", imponant in edl _adl inuractiom and phospholipids imponant for coagulation B. An opl microtubular ring .nd .bundant actin .nd my",in, thar maintains disroid shap< and aUows for .hap< change: and pseudopod formarion wirh .ctivation D. A de"", tubular synem of endoplasmic reticulum chat sto= Ca>+ for pJ.rdet a.criv.rion and rhat is imponant in thromooxane '}'IIth..is E. ct-Granul.. th.r store numerous proteim involved in h~mOS{ .. is and ve....,l repair .nd wh~ contents a.. =reta! with .ppropri. rr stimuli F. ~n'" gr>nules containing Ca>+, Mg'"', adeno.ine diphosphate, ATP, .nd ",rotonin, all of which ... ",creta! with .ppropriate stimuli G. Glyroge:n stores .nd mirochondria for en~rgy

II.

Platder production: The proce;., of gc:neraring circulating pl atdm from hematopoietic stem cdls con b.: divida! into megmryopoi..i, and thromoopoie;i,. Ad"'luare mq:mryopoi..i.

4/ PLATELETS

225

is r~H~ctal by """'luau num~rs of meg. k.:IfYocytes in the bon~ marrow but does not C""rant« adequate thrombopoiesi,. A. MtfllMrytJpoi"i•• the prolif..ation and maturation of megak':IIYocytes. ocrun in hematopoi.tic ti"u~, mosdy bon~ marrow. R...ident mydoid proC"nitor ""H, respond to cytok.in~s, primarily Tpo, by und ..coing proliferation and maturation.' Limital baseline megakaryocyt~ production occurs in th. absen"" of T po.' B. Tlmlmbopok.is, th. formation of platdet, from meg.k':IIYocytes and th. ir ddivery to the circulation, is also primarily maliatal by T po,' but b,udin. platelet production am occur in th. abMn"" ofTpo.'Tpoi.alsoimport:antformaint.ining.dequateh.mato_ poi.tic nem cdl populatiolll. I. Thrombopoiesis occurs in the bon. nurrow and at other ,ites of hematopoiesis (• .g.. th • •pl«n). It also occurs in the lung., where megakaryocyt.. l~ after circulating fiom th~ bon~ marrow (Set Plate 4A [for all pl. tes , Set th~ rolor =cion of thi. book]).' 2. Platdm form from ut .... st:og. meg.bryocytes. They appear to ,hal dir~ctly into the blood by cytopwmic fragmentation or by the periodic ronstriction of megakaryo_ cytic cytoplasmic J>5C'udopodi. that at. nd into """"ular sinuses. C. Tpo i, producal prim. rily in hepatocyt .. , ",nal tubular epithdium, .nd momal ""H. of the bone marrow.'·' I. [n h~alth, Tpo is producal constandy and is dear.d by reaptor_mediated uptak.~ and destruction by platdets .nd megakaryocytes. ~· Therefor., plndet and meg. _ bryocyte ~s exen control o""r pl:asm. [Tpo), and the", i. C"n..ally:m inve"", rdationship ~t~.n [platdet) .nd blood .nd bone marrow [fpo). a. With decreased putdet JlUl.I, mor~ Tpo remains unbound and is :available to stimul ate megabryocytes. How...... , when megabryocyte hyperplasia .ccompa_ nies thrombocytopenia, more Tpo will become bound by megakaryocyt.. and the blood [Tpo) will ~ low.. than expected b...d soldyon the [platdet). b. k th~ [platelet) increast., more Tpo is bound .nd removed from th. circulation, so th~re it I... nimuution of megakaryocytes or other stem ""lis. 2. Tpo production inc",as.,; in ""nain pathologic states:' a. Inflammation caUle, increated IL-6, which indu""s hepatocytes to produa. more Tpo, which in turn may GlU,. thrombocytosi •. Becaust of inc",astd Tpo production, the plasma [fpo) is greater with inflammatory [hrombocytOlis th:m expected, given th. [pla[det). b. Marrow !!romal ""It. are inducal to produ"" mor~ Tpo in . t lun some throm_ bocytopenic nates. 3. Activa[ed platd." may rdease intact Tpo in vivo, thm increasing blood oon""ntra_ tions during putd.t ronmmpti"" sta[es.' D. Reticula[ed platdets: Just as the RNA content of circul. ting young erythrocyt.. (reticulocyt..) i. u..d [0 "'""ss erythropoiesis, circulaling reticulated putdm have betn measured to "'""" thrombopoi.lis in dog. and horst •. •.. Reticulated pbtdets have incru.ed cytopl:asmic RNA and h. "" betn shown to ~ I~" than 24 h old in dogs.'· lll.

Platdet kinetics A. Th. blood [platdet) is g~nerally establi,h~d by th~ rdati"" rat." of platdet production, consumption, and destruction, .nd by the ,hifting of platdets to . nd fiom the circul. _ tion (Fig. 4.1).

Bone marrow

@

0 Blood vessel

I

0 0

0 0

Endo!he i~IC8 '

,, ,

0 ° 0 0 ° 0: 0 0 0 0 0 " Platelet plug

"

•• •• • •

0

~-

@........""'•. o • PI~telet Fig. 4.1. Blood pJ.,.,kt ooncmtr:otions :oc...",blishod mostly by t'" ",loti... ..... of pl .. det prodoction, roruumption, :and dffiruction, and ~ t'" shifting of pl.",kts to :and from t'" circuhtion, In J>1thologic combiJl1tions of t ..... f:octon often oontribu", to . ooormn ph.. let roncVernl impon..,,,,, of this process is not known, Pl.",kts prod""od by'pknic "'autopo;etic ti.",. =y oontribu.. to t'" cirruhting pl.",let mas.! in hentb rnd ccy destroy pu ,.,kts tlut e>rry ... rue.: . ... oci. tesl rntibodie> or compkment, Agod or .t:.m.ged pu,.,kts moy be .imilody denro)"'d, o R.<jinribution: Splenic pu",let oeq .... tr:otion (~ibk) moy tesl""" clw: cirrnuting pb,.,let moss, ..,d spknic rontr:oction moy incre .... it, Pulmorury '"'I""'tr::ltion of pb,.,k" b .. been :woci. ted witb ~. bypoclw:rnu.. . oo endotoumi .. o M ... i"" transfusion witb blood products or oth.,. fluids m>y c"... . dilutiorud d
s"''''',

..,"'r

226

4/ PLATELETS

227

B. Platd"" production is .fI«t~d mostly by th~ degr.., of cytokin~ stimulation and th~ numb
Platelet function, {= Ch.pt~r 5 for d""ail, regarding the role! of pJ.tdm in hemostasis} A. The major function of platd"". i, to hdp repair """",I" damage and pr~ent hemor_ rhogt by participating in th~ formation ofhemo,utic plug•. Pla tdets are consumed in thi. ongoing process. B. Energy for pJ.tdet function> i. d
ANALYTICAL PRINCIPLES AN D METH ODS I.

Complete blood count {CBq: Quantitative and qualitati"" dau regarding blood platd"". are routindy included in mamm:olian CBC remh •. Major concepts !",naining to th~ CBC a" in Ch'pt
II.

Thrombogram: those portions of a CBC rdated to pl.td""s (thrombocyte!l A. Blood film ~:oluation l. Micro,copic ~:oluation of platdets on • suined blood film i. an imponant pm of the thrombogr.m, e!peci ally in bl=:ling pati~nu or when thrombocyto!",nia or ext""me thrombocyto";. i. present. 2. It ..rves thr.., mojor purpo=: a. It en.ble! deta:tion of platdet clumps, which may falsely da:""ase [platelet] v:olues. Some an:olyzers detect the prestnct of platdet dumps and rq>On their p".. nct, but others do not. b. It en able! enimotion of [platelet] for corroboration of automated ""Iue! or when .utomoted measures.re unavailable or unreliable. c. It ~n.bl .. evalu ation of platd"". for abnormal f~a tures.

228

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

B. Plud"" ooflctntmion I. Th~ p"'uln ananmlfion j. th. numb" of plald.ls ptr unit volume of blood (in clinical jargon, commonly r.ferrw to '" p/auk<
countwas individ"'" plndm 2. Breaus. MPV is an anrage, populations of la'l:" or ,m.ll platd...:, may bot pr=nt and de'l«t.bl. by micr05copy when the MPV is WRI. 11.1.0, luge pludm may bt =n microscopically but acludM from .nalysis by the instrument. 3. Unit: fl. {Ilm'}

D. PDW I. PDWi, an :wes511Y1It of platd", anisocytosis calculated from the dinribution of individual plmJe'I volum ... 2. Th,,,, ... stve",l different method, of calculating PDW. a. For ADVIA imtrumems. jt is the stand",d drn.tion of the distribution of plnd", volumes recordffi .. a ~":.ntagt of the MPV; that is, 100 X (mnwrd drnation of MPV)/MPV. b. For oth.. anal)'Z"rs, the cakulnion =y nclud. a ",nain proportion of the l..~t .nd mtallm platdets. 3. Unin: % E. Thrombocrit: t!Jrgmbo. ("plotdet") plus -<Tit (from krintin, meaning "to "'p:"ate"j; also called p"'«1n crit I. Thrombm:rit, Jik. h.matocrit, i, th. ?"",m"!:,, of blood volume filled by pJ.tdm (typically <: I %). It i. an as=smem of circuloting platdet mass. 2. UnJik. h.m. tocrit, thromOOcrit valu .. have not i>ttn routindy reported but Ita", become .vail.ble with mode", autom.tffi anal)'Z"rs. 3. Thrombocrit value. ~re calculoted from the MPV and [platdetj, so abnormalities or ",rors with . ith", of the", m=ur. m.nU affa::{ th. thromOOcrit value {Itt Eq. 4. Il.

10 /lJplnd", X 300,000 platdetslllL = 3,OOO,<XXl /lJIlL = O.003IlUIlL = 0.3 %

{4.l l

4. Comparffi to the [plotd.t]. thromOOcrit may bell'" represtm th. total pl. td", functional pot.mial .nd the thrombopoietic stimulllS." % or d""imal form {L/L or unitl.... j F. SN..al other plotd", values are generatffi by som. aruol)'Z"rs .nd =y be reponffi, although they ar. currently u..d mostly for invesrig.ti", purposes. l. MPC: mean platd", compon.m con",mration a. Th. MPC i, an .pproximation of the ave~ platdet density basffl on two"",, ,,gt. light ocaner of individual plotdm {ADVIAI b. It is u..d to assess plotel.t activ.tion, which lead, to granule s""retion .nd • d""reasal MPC. c. vatu .. are aff""tffi by .micoagulam, sample "!:'" sampl. storage tem~rature, and iatro~nic activ.tion, so they mllSt be interp .. tffi cmtiously. d. Unit: gldL 2. PCDW: mean platdet compon.m con",mration di
5. Unin:

4/ PLATELETS

229

3. V:dues for m~an platdet mn, lpg). variation in m.... n pJ.td.r m:lSS.luv pJ.tdeu. and pJ.td.r clumps may also k r.ported by some aruolyurs. III.

Methods A. Blood film ",,:duation of platdets I. Scan the f~athered edV and ,m.ar body for platdet dumps. If dumps are present. the following estimation of [platdet] will provide only a minimum value that may nurkedly und~renimote the true [pJ.tdet]. 2. One method of .stimating the concentration of individu:dized platdets requires first finding the appropriate counting window. wh.re leukocyt.. are Hatt~ned like &ied egg. to fip<>5' nucl.ar and cytoplasmic d.rail. a. Appropriate rounting window. cont.in fidds of .imilar blood thickness .nd therefore .imilar volumes of blood per fidd. Diff~renot:l in cell numkr Jl<'r fidd will reHeet difference. in cell numkr I"'r volume of blood (i .•.• cona.ntration). b. Smear thickness c:m k ju<4:ed by the degrtt to which cdls are flatt.ned out (fried rgg appear.na) rather than by the proximity of cells to on~ another. c. If fidds are chOstn to normalize the d~nsity of erythrocytes. thick.. ar.-as of the sm•• r (with mo .. blood volum~ Jl<'r fiddl will k ",,:duated in an~mic anim:ds .nd the [pJ.td.r] will k overestimated. 3. U.ing th~ 100x oil objeetiv~ (IOOOx m"l:nific;;otion). :.gain ....... for the presence of platdet dumps. If platdets :ar~ cluml"'d. report "dumped platdets" .nd recognize that smear .stimates .nd machin~ values for [platdet] in the ,ample may k faJ ..ly decreased. 4. If platd",. a.. w.U distributed. estimore the average numkr of platd",. Jl<'r I<XX))( fidd in at l....n 5 (prefer.bly 10) fidds. S. Conv~n the aver:og<' number of platdets I"'r field to an estimatal [platd",] by using th. following ronvwion factor: 1 platd.r' [(XXIx field = IS.OOO--20.txXl platd"'''IlL to For ex:arnpl•• if the mean numkr I"'r 1txXlx field wa! 4. th.n the ertimatal [pl>td",] would ranv from about 4 X 15.txXl platdets/Ill = 6O.txXl platdets/Ill to .bout 4 X 20.000 platd",.Jj.tl = 80.txXl platd",.J1lL a. Th •• rea represented by a 1000x microscopic fidd vari.. with the optics of microscol"". Th. diam.ter of the fidd of vi.w vari .. directly with th~ fidd numkr of th~ oculars •• nd fidd numkrs vary consider.bly. b. BecalJ.1le of this. the appropriate conversion factor varies comiderably. Th. 1:20.(xx) con""rsion may k mo .. appropriat~ for regular oculars. and the I: I S.(XX) con""rsion may k mo .. appropriat~ for wid~_fidd oculars. Either should provide • ....,ful minute. 6. Th. estimated [platdet] should th.n k comp:aral to species_sp«ific ref~ .. nce int~rv:ds for interpretation. To fidud~ thrombocytoJl<'nia. dog. {mon breffis}. cats. and cattle should Vn.rally have >t l.a!! 8--10 platdm Jl<'r lOOOx fidd. and ho .... should have at l~ast 5 pl>tdm I"'r lOOOx field. 7. Oth.. methods may k U.ffl to estimore [platdet]: a. Som. I""'pl. advocate .numeration of platd",. in 10 thick and thin .reas of the film and us<: of a 1:8000 con"""ion factor to e"imote [platdet] {the lower conversion factor reflects ...... m.nt of thicker areas with greater blood volume .nd more platdets Jl<'r fidd}. b. Oth." comp... th~ ratio of pl atdm to .ither erythrocytes or leukocytes in sever:d fidd •• and th.n u.. either th~ ~rythrocyte conantration or leukocyte

2JO

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY con~ntra{ion

to cakulat~ th~ estimatffl [platd",,] (~.g., for 5 platd.,u pt. lalkocyte and a Oeukocyu] of 20,<XXlIj.lL, the e;{im.ud [P1.ld.l] = 5 X

20,OOO/j.IL = IOO,txXl/j.IL). Th • .., methods requi .. quantiutiw erythrocyte or lalkocyte con~lIIra{ions. 8. Whil. estimating [plaId.,!], platdrts should be, ",""ssal for morphologic abnormali. {i •• (Itt the followiJli: KCtion).

B. Plude! OOllctntr>tion l. s..mple a. EDTA antioo>gulation of venous blood is routine for eBc. {= Analytical Principles and Methods, ,,",,I. I.D, in Chap,.r 2}, but cit""..! venous blood {I pM! citm. to 9 parts blood} can be, usN (e.g., when EDTA.indu=l plaut", dumping is .n.ptctal). Citme dilution requires {rull th. mea.urM [plotdet] be, oorr«tM (corr«1al plalde! OOllctlltrat;on = measural platd", collctntration xl.I ).

b. HqJarini=:l .ampJ.. should not b. n=!, kcaust plotd.! clumping frequemly ~".

u,ay

c. Pt.td.u can b. xtivatffl during blood colb:tion • • nd activ. tion cauoes dumping. Pl. td"u can dump b.cau,e th.y are hyperactive. b.cau", of slow or poor v"nipunctu .. tMniqu". or ba::;,,,,,, of dd"ynl or inadequate mixing of blood and "nticoagulmt. Plotdets of em and cattle are very pron" to dumping. d. Blood coll«tion tub.. containing citrat". theophylline. dipyridamole. and . d"no,in" (CTAD tubts) provid" platd~ inhibitory factors that hdp rfflu"", in vitro pt.tdet .ctivation." Th... ""'y b. p"niculorly u..,ful in cats. e. The.. are v.riou... ported ,tatemenU regarding the stability of platdet (X",,,,,n_ tration,.".>.! On average . con""mratiom . ppear rea,onably stabl" for 8 h.t room temperature . nd fOr 48 h .t 4 'c. Ho~er. con""ntr>lion. ""'y incr....., or d« ..... comiderably in individU

tion of p"nides that ar. within" definffl p"nide volume int""'..! (• .g.• 2- 30 fl.) Iu.ffl on th"ir d<"Ctrical impe<Jan"",. b. For mo.!l sampl... a properly .dju!lffl and calibratffl impedance cell count"r provid....l;..bl. platdet concentration. for mo,t dog•• hor.." •• nd catde. c. However. impe<Jan"'" cell coum .... cannot alway, accuratdy a = platdets: in ,om" samples. tho/ cannot diffe .. mi. te small erythrocytes from platdet, (• .g.• in iron defici.ncy). or lar!:" platdeu from erythrocytes (•. g.• caU and Cavali.. King Charles ,paniels). Cau have • &","ter vari.tion in platdet volume. with mo .. lor~ plotd.u than oth .. 'peeie,. so impedance m.thods are proble""'tic for d.termining [plotdet] accuratdy in cats." 3. Optical or lo",r flow cell cytomete" {Ott Ch' pt"r 2 and Fig. 25 for basic principl.. and .nalyze"l

4/ PLATELETS

231

a. Ass . .. m.m oflow ..,mgl. (volume) and high-.mgl. (r.fractin inda) light =tt" .nables !>Ht" diff... miation of plotdets &om erythrocyt.. than does th. use of dectrical impedance. b. Platdm a.. d.finn! a. partid.. with cenain light .. 5C;Otter charact"istics. and the number of th ... prnid.. in a given volume of fluid is the [platelet]. c. Ho~v". oth" partides. such", lipid droplets. con =tt" light similarly and be coumn! as plotdeu. 4. QBC (Ott Chopt" 2 and Fig. 2.6 for basic principl..) a. The QBC detect. the thidm... of the cell lay'" abovt the lymphocyte and monocyte lay" that has characteristic fluor.,cence of RNA or lipoprotein. b. Th. thickn . .. of this lay'" {. ... mially a thrombocrit} is convmn! to a [platelet] b...ffl on correlation nudi., with other a""lyze ... c. B«ause the value i. affectn! by platelet size, it may not alwaY' match plotdet concentrations based on other methods. d. Th. analyticol method is I... affrctn! by small platd.t dump' than are impffi .. ance methods!' 5. H.mocytometer {Ott Chopt.. 2 and Fig. 2.4 for b",ic principles} a. Pl atdets a", .num". tn! microscopically in d.fin.d grid regions .fter sample dilution and lpis of .rythrocyt... b. This manU

gnostic u..,. and may be p.. f"rn! wh.n many giant pl atdets are p.... nt. c. It d""s not .nable mrasuremem of oth" platelet paramet.... d. As with any method, pl.tdet dumping prevent> accurate mea.urement of [plotdet]. C. MPV I. MPV valu .. vary among analyze .. and methods." Electrical impedance methods may adudelarge platdets &om evaluation, and th. diluting fluids U.ffl to . ph.", platdets in opticalanalyzen may falsdy decr..... the v.lues.2O..l.o 2. MPV valu .. al50 vary with the amicoagulant, the sample storagt t. mperatu .. , and the sample storage time, though not necessarily in the same mmner with every analyzer. a. The.. variables usually havt rdatively minor effects {< 20 % diffr",n=}, but the effects should be consid"n! wh.n values a", imerp"'tn!, paniculorly for values ne .. the upper or lower ..ference limits. Refer.nce sampl.. may han been handln! differently from patient sampl.,. b. With impffiance meamrements, EDTA "'... shown to induce artifactualand time-dependent ina.,...,. in MPV that occurrrd within 5 min and could result in 3 h valu .. that ~re 150 % of basdine for room temperature sampl..."''' Artif.ctual increases also occur wh.n EDTA sampl.. are ..frigeratrd (at 4 0C)." c. MPV ina.a... ov" time in citr.trd sampl.. norrd at room temperature or 4 'C, but the inan.. is less chan with EDTA sampl.,.,,; d. When analyzed ovtr tim. on th. ADVIA, MPV valu.. inc",ased aft" an initial decrease, and the chonges we", slower at 4 ·C. MPV wa.! relotivdy scabl. when sampl.. w... anticoagulatn! by a mixture of EDTA and citrate, storn! at 4 'C, and malyud from I h through 3 h aft" blood collection." 3. Som. impedance analyzers cannot gen . .. te reliable MPV values in "",erdy throm .. bocytopenic patienu, 50 th. values may be unavailable when of interest.

232

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY 4. Pmiculau. such .. lipid dropl.u or cd! fragments may b. d.t=«I .. platdets, [""ding to faJ", MPV values with either im~n'" or J..., m~hods.

MICROSCOPIC FEATURES OF PUTELETS (~ Pl. to 7 .nd 8 ) I.

Gene",l

A. In blood films, pJatdeu are p",,,,m individuaUy or in mull to l.rg<' dumps (."" Plou 7J- L). B. Clumps usually =Ul1 from in vitro plaid., activation, but .ntj~Jant_induo.d agglutination of nonhuman platdeu h.., bttn reponal." Clump. are mon frequent in fdine :md bovine sampl... Thq may b. round throughout a blood film but ... btu found .t its feath,,«I~, .
Fralme. A. Shap< l. NOJUcrivated pJ.tdm are discoid and . ppt •• mostly round. oval, or dong" o in blood film. mad. from non_anticoogulatN blood {st. Plate 7F }.

2 Pl. t.],,, "-rome spheroid with EDTA .ntiroagulation (also with citrau, but to. I. "", degr«) and rna .. commollly ~ppe~r roulld. 3. Elollgat~ plndm. which probably "'p....,m ulldividffl mrgakaryocyt., I"'udopod segm~ms (propfatrlnsl . may bt more lIumerou, during stimulatffl thrombopoi."i, {Ott Plm 7E}. 4. Acrivatffl pl atdm may ru.", periphpist, thry may bt reportffl. 4. Bemus< MPV is.n a"'rage for.ll platdets, mbpopulations of large pl.tdm may bt pr=nt without all illcreaKd MPV. Also, large platdecs may 1I0t bt indudffl in MPV calculatioll' if thry.", too large to bt dett"CIffl as platdm. 5. 011 some blood films, mallY platdet. appear larger th~n normal but 1I0t larger than . rythrocyte!. Th.,,. platdets may not bt .. ponffl as large, but the MPV may bt incr ...","",. 6. An incr ..... in the numbtr of gi.nt platdets ruggem accd~ratffl thrombopoiesi, withill • f~ days of .<;;Imple collectioll, uSll:dly btc.UK of. de!tructi", or oollsump_ ti", thrombocytopenia. It may bt accompanied by an incr ...","", MPV {... Platdet Volume, sect. II.A}. Howev
4/ PLATELETS

213

a. With Wright staining. nonacrivata! pl>td~s h.ve cl .... r to p"1~ blu~ cytopl""ms containing uruoll pink to purpl~ gr.mul~s. The.. granul .. contain num"ous substanct. that con "" rd<"llK
.iu.

THROMBOCYTOPENIA I.

Gen~ral

roncqm

in which [platd~l i, 1..:. th an a valid low.. ..fe .. n", limit {platdet dumps must "" .I=nt}. Some h..Jthy gr'}'houndsJ<.-.!' and ~rh"P' Shiba [nm'" have low.. platdet con",ntntions than oth" dog•. B. Thrombocyto~nia rdl«ts a pathologic process and is • diagnO!tic probl.m. not a di..,...,. Its Jruljor s.ignifican", is th~ potentiation of bl=:ling when platd~ con",ntra_ tions a.. markedly decnased. Also. its p.... n'" may suggest specific disorders or di..,ascs. C. Clinical signs: P~ec:hia~ and ec:chym05<::l ar~ th~ hallJrulrks of .. ver~ thrombocyto~nia. Mucosal blttding {~pinaxis. h.Jrultochrua. or mden.}. hematuria, hypheJrul •• nd prolonged h~morrha~ aft .. accidental or intentional trauma (~.g .• venipunctu..) a.. common. D. Thrombocyto~nia am occur in Jrulny di~."-" but th,,~ ... limila! patho!:"nic m~chani,ms to "" considered (... Fig. 4.1 ).

A. Tlmm.'-:ytoprnu. is • pathologic

[I.

nat~

Dis,"""" and ronditions (T.bl. 4.2) A. PHudo_thromlmyropmu. is fat.. thrombocyto~nia that may occur when not .ll the platdets in • sample ar~ counted. It con be smp«ted wh~n autoJrulted analyzers indiane th~ pr=nct of platd~ dumps. It em al"" "" r«ogniud when micto.\lCOpic examination " 'veals platd~ clumps or yidd. an estimau of [platd~l that i, mbstan_ lially greolt" than the mU'lura! [platd~l. l. It often occur> b.c.u.., blood coU«tion caus.. platdet .ctivation and aggregation. Therefo ... platdets ... dumped and not counted individu<>lJy. 2. It allO occurs with autoJrulta! analyurs. panicul.rly impedanct analyurs. when = y I...g~ platdeu a.. present (e .g.• in em .nd Cavali" King Charl.. . panid.) but not d~a::ted b.c.u.. they n ett
Table 4.2. Di....scs and a>ndition.

mac aUK

thromboqropenia

Abnormal pla1e1et distribution (Kqueslrafion ): spleoomtgaly. uvere hypothermia Decreased platelet production (m),dosuppression of One 10 aD hemic cdl linn) Acquirtd ameg:ak.:uyocyt ic tMomoocyroJl"ni>. Drugs (Ioxian,,) ' Predictable: cbemoth~utic ag..ntS, c:strog..m in dogs (novnous, endo~nous), bracun fern poilioning (rumin:mts), rriromhtane m)'rotOlim Idiosyncr.l1ic: phenylbutazone. meclofenamic acid. 1rimerooprim-Julf.lnlelhonzole. griseofulvin Infecti"". (uswlly multif.ctorial: .- the t..:t) Irradiation (whole body or ":1ensive): prior to autologoul marrow transplanmion Marrow rq>I:oument: bone marrow nn>plasi>. (primary hemic or metwatic). myelofib .... i•• ostn>Jl"trosi. Megakaryocytic leukemia Myc:lonccrosis: infcaions, nn>pwia, tODcontJ Decreased platdet survival (accderatffl pI':l1del dtsrrucrion and ron$l1mption) Immunologic ' Primary (idiopathic) IMT S«or>dary IMT Drug induced: gol d s.alu. sulfonamide! Infraiou. (u5II:illy mul tifactorial: see the ten) Nn>natal all oimmune thromboq10penia Nn>plasia (usually multifaCtorial; 5« the 1..:1) P0;5nransfu,ion purpura Sysremic immunf-medialed disease: SLE, Evms' syndrome Nonimmunologic Blood 10», KIlle and sc-ve~ ant iroagulanl ~nticidC$ Platder iCliv:nion with aculer:utd consumption or utiliution LocaIi=! intravascular co:agulalion: hemangiosarrom:o, ~morrh~, thrombo.i. 'DlC: envenomation. hqMlic disease. infections. massive necrolis. pancreatitil. nroplasia. o~rh=ing, 2pticc:mi>. Drugs: plOOlmi"" sulfate En~nolNtion (wilhout DIQ ' V:Ha.Iliti slendoc:uditis: Rocky Mountain .paned w",r, c:mine ~r!",JVirus i..&ction, hemolytic umnic synd rome. dirofilariasis, angiOiltrongylosis. bacteri.l endocndilis. hemolytic uremic syndrome (rotanenu. and lenal gIom...war vasculopathy of greyhounds) Hemodilulion: massive infusion wiTh colloids. crrnalloids. pLatelCf-poor blood produCls Idiopathic m..ro:uti.m or multif:.aoria! (d.crnsed production and decreased . urvival) Idioparhic Ihromhocytopo:nia of Can tier King Charles spaniels "Infcaionl: A1WpWsm" platJl. An4plAs>1W pbttpt]Wphilum, &bnl4 amu, &bniA pb»"I, bovine viral diarrhea virus, c:mi ne di.um!"', viru .. coni"" parvovirus. CytAUXZOt11I fty.. EhrlifhiA spp.. equine infccliow anemia virus, feline leukemia virus. fdine immunodeficiency viru •• HiJNpt.u",., cAp,"IANml, LriJhmllniA spp., L~purpiM >pp., 7Mi!nw spp, Endoroumia "Nn>plasia: carcinomas, hmungin=ma and. olh~ .. rromas. lymphoma, l. ukemias Drugs Hypophosphatemia wociattd with hyperalimcnration Anaphy\axis " A rclui.-dy o:>mrnon diooue Of condition Nooe: Usa of >peciIic disotdo .. ot condilio .... . te no< complete bu< ale pmping i l l gian' platdra . hould br ""","
234

4/ PLATELETS

235

B. Abnormal platd", dipping of platdets in organs because of destruction by th~ MPS. Howev... in this textbook. irreversibl~ .. moval of platdm from circulation is regard..d :as d""r..... d platd", survival. not sequ~stration. 3. Abnormal platd", dimibution may rontribut~ to or {rarely} cau.. markal thrombocytoptn",. 4. Disord~rs a. Splenomegaly . ppears to am... plenic pooling of an ""chan~.bl~ platdet &action in some human conditions. though platd", survivals may abo be da:"",..d." Similar dfa:ts of splenomegaly h:IV~ bttn presum..d to OCCut in oth... ptcies. b. ~~re hypothermia (e.g., a rectal t~mptratur~ of 20°C) =y cause reversibl~ platd", r..distribution to Ii""r . nd .pl... n in dogs." c. Endotoumi. hal bttn 'SSOC"'tai exptrimenu.lly with transient pulmonary platd", pooling in dogs. but pJ.tdet conmmption .lso occurs ......, C. Da:re:ased platd", production l. Adequat~ platdet production requim; • healthy m~gahl}"O<:)'I~ popul. tion; th~re must be enough megak:uyocytes and they must be shalding platdm. Mq:.bryo_ cytes con be unhealthy becau.~ of g~ne ..lized bone =rrow disn ... or because of megakaryocytNptcific di ..ases th.t ~ith~r diminioh megakaryopoiesis or impair thromoopoi .. is. Unexplain..d bicytoptnia and pancytoptnia 'ugge'lt the J><>Ssibiliry of generaliud bon~ marrow di,~ase. 2. Idiopathic thrombocytoptn", in Cavali~r King CharI.. spaniel. i. likdy cau....! by da:r ......d platdet production rather than a comptns;:ot..d consumptive nau, but it is includ..d below {= Thromlxxytoptnia, >eet. II.F. I} und .. idiopathic conditions until bemr characteriud. 3. Acquir..d .m~gahl}"O<:)'Iic thrombocytoptni. is tardy repon..d in dog. and can ........ Some ca... =y be immune m..diat..d, :as in people. a. Bon~ marrow "'pi.. " and co.. lamples must be of excellent quality to ronclud~ rdiably th . t megakaryocyte> are ab .. nt. b. In people, it =y occur with . ntibody_m..diat..d or ~ll_m..di.t..d" destruction of megabryocytes or their precursors, or with antilxxly_m..di.t..d denruction of requir..d cytokin.,..'~" c. Antimeg.karyocyt~ .ntilxxli.. =y .lso impair thrombopoi~si,'''' without megabryocytic hypoplasia or .plasi•. Dog. inja:t..d with .. bbit .nti_[canine plaul",) antiserum devdoptd =rk..d thromlxxytoptnia with morphologic change; in megakaryocyt.. that mggest..d the possibility of imp.ired megakaryo_ cyte function without m~ryocytic hypopwi .... PW= from some people with IMT inhibits megabryopoiesit and thrombopoiesi. in cell cullUm;. 4. Drugs {toxicanu} a. Drugs with predictable and dose--deptndent mydosuppressive dfa:ts {""ampl..} {I} Antineopl:astic ch~mothe .. ptutic agents, including alkyl . ting "i:~nts, antim"'abolites, and antibiotics {~.g., doxorubicinl, frequently indue.. thrombocytoptni. 1- 2 wk .ft~r drug administration."'"

'"

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY {2} Esuo!:"", from aog~nous sources" or .ndog.nous sour~, (•. g., testicular s"noli cd] neoplasm," roor. Ova,i<>11 gr.mulo.la cdl "roplasms," and ra .. inw.t;tial cdl nwplasms" in dogs) can induce thrombocytoptlli<> or .plomic pancytop in ruminants," as may trichoth=n. mycoroxins." (4) Albc,nd:u.ol. has bttn implic;o{ffl in a dog." b. Drugs (roxicollu) with idiosyncmic {i. •.• sporadic and unprfflictabJ.j mydosup_ pressi"" dfects (n:lmplel) {I} Idimyncratic mydosupp'<:SJion jt 'Usp«tM much mor. oft.n than it is prov.n. Rda p.., of thrombocytoptnia with r'Ct..:!, potential offending drug, are withdrawn, but ,ub~uent impro""ment mayor may not be due to drug withdrawal {2} Phenylbut37.0ne and m.clofenamic acid are nonneroidal .nti_inflammatory drugs th>1 have bttn associ atal with bone marrow hypoplasia and bicytopeni. or p:mcytopen", in dogs :md prob.obly in hors .. {only phenylbuu7.0ne}, .,....... {3} Trim...:hoprim_mlfadiaz.ine and trim.rhoprim_sulfonamide ha"" bttn asso1 doses conunonly us.d, HOWN. ., potenti_ at":! sulfonamides pralict.bly inhibit ''"'Iuent",l st.", in fol ate pathwaY', thm ,uppressing DNA ,ynthesis, This may rontribut. to aplastic :memia and thrombocytopeni a in affect":! dogs, Thrombocytopenia c;ou...i by mydompp ...sion should be diffe"'nt",t..:! from immun._m":!iat":! platd...: denruction cau,..:! by trimethoprim_sulfa--
.ft.,

4/ PLATELETS may al,o k

COUIffl

by >q>ti""mia and/or

~ndotonmi.

:lSSOCiatffi with

=~

..

~nt~ritis.

d. Production failu .. IlliIy rontribut~ to thrombocytoptnip
238

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY (a) Destruction of on~'s own cdh it an .utoimmun~ ph~nom~non only if th~ body'- immune 'rn~m is di=tal :ogainn autoantigens. (b) All""ntibodi.. from colostrum or tr:muusion are dir=al toward alJoant;gens :md cou"" alJoimmune chrombocyto!"'ni .. {cj Antibodies to .diOrkd amigen, callS< ~nd"y IMT thai j, nejlh" autoimmune nor alJoimmun•. {5} IMT that i. part of .. more wid .. pr~ dist"", (e.g., SLE) ;. abo often calla! "ronda') IMT despite an autoimmune pathogenai •. b. Idiopathic lMT (prim:uy, often p... umro to ~ autoimmune) {I} Idiopathic IMT is wdl documented .nd rdativdy common only in dogs and pmpl. but 'ppta'" to occur in other spa;ies, '" wdl.-->< Immunoblot_ ting and immullop,«ipitation nudi •• h;ov. confirmed a role for pJatdet membnne amigen latgen in at least some cas •• of conine idiopathic lMT." Antibodi .. m.y .lso tUl:"t m~ryocyt~. Diagllosis is prim>rily by aelusioll. but inc",~ PSAlg or pmiti"" m~gakaryocyte immunolluores_ """"" results may support:m immun~ pathocen~sis {Ott th~ T~m for ImmulI~Mediaud Thrombocyto~lIia =cioll}. {2} Commoll J.bomory f~atures" are thrombocyto~ni. (usu.Uy < 50 X 10'I).lL alld oft~n < to X 1(01).lL); MPV iner ......!. d<"Crrased. or WRI; all~m", (in about SO % of cases); alld m<1::obryocytic hy~rpl",", (meg:obryocytic hypopl,,", call occur). c. Drug_illductd IMT {I} fuposur~ to ""ruin drugs may lead to iner ... sal PSAlg .nd therefore a=leratal pJ.td~ destructioll btcau.. of the production of drug-d"J"'lId.m or drug_illd'~lIdem amilxxiia (a) Drug_i"d~pmdm' a"tib"Ji~J do not require the drug for billding; they m.y billd to a pJ.tdet epito~ that i. cross_r ...cti"" with the drug or. mffilbolit•. {b} Drug..kpmdntt an,iiwdirt. which a.. mor~ oommon ill ptopl~ than in domestic mammab." can thtoretically bind to . ny of th~ fullowing; a drug or a metabolite adsor~d to th. platd~ surf."" •• plndet surfa"" amigtn apo...! by the pres.n"" of th. drug or a metabolite. or • combilled drug_platd~ nttll SUI]l<"Cted to caUl" IMT ill dogs. Gold salts {auranofill. gold ..ooium thiomalat•.• nd possibly .urothioglucost}" alld sulfoll. mid .. {with or withot1t trimethoprim}",'110 han bttn strongly

4/ PLATELETS

239

implicata! in dogs. Platd~t antibody ..say results hav~ bttn supponivt of drug_inducedIMT. {5} Methim37.0l~ and propylthiouracil hav~ bttn incrimirulla! in cats,'OI.IO' ~nd p when passivdy ~cquira! {=inly via colostrum} matern:01 alloantibodies to paurnal epitop<. on a neonat~'. platdets circulate in th~ neonat~·. blood and ma!ine platdet destruction. The maternal alloantibodie.... produced when th~ dam is sufficiently up<>5'd to feral platdets polRSSing paternally deriva! antigenic det .. min:mt. that are =x>gniud as fo ..ign. It h .. bttn describtd in hones, mule fo:o1s, .nd pig•. "2-"· {l} It must bot differ~ntia[a! from other ca""," of thrombocyt0P"nia in neo_ nates, esp«ially "]>lis. Confir=tion entails det=ion of inc......d PSAlg in the neonaU but not in the dam, :md demonstration of the pre""n"" of antibodies in the dam', blood that react with paternal and neonatal, but not =ternal, pl.tdet •. Valid . tal t<::st. may bot unavailable. {3} Thrombocytop rommonly in animals with neoplasia. The cau"" is often multifactorial but =y include immunologic mechani.ms. {l } Antibodi~., possibly in the form of immune mmpl""es, h. "" bttn indi=tly implicata! in ma!i . ting thrombocytop
,.

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY h~mic

and nonh~mic n~opl .. m,.j" lMT has bttn ow;oci.tal with Iym_ phoJrul in dogs and houes."""

g. P01llr:muwion purpur:l. human transfusion rn;ipi~nu. thi, condition occu" about I wk :aft~r transfusion. It is aslOCiatal with RV~" thrombocyto~nia and high titers of platdet_rraccive alJo;>ntibodies that m.diate th. dmruction of transfuS«!, '" well as the !"Ili,m'. own pl.tdm. {2} Rardy, posm.nsfi,,;on thrombocyt0J>"nia has oon reportal in dogs. but the

{I}

[n

patho~n • ..,'

have not oon establishal. III h. Systemic immuII<,-maliatal dis"" .. such :as SLE ... couW by gtnual B_ lymphocyte activation and the production of autoomibodi•• dir«:tM "l:.inttn impliau<"d in ""me cases by indir'""t assap for putdet_".ctiv. or m~I}-ocyt....".ctive antibodies in p.tient plasma." {d} Dog. with lMHA are oft.n prothrombotic. "" thrombocyto~ni. in !Om. of th.m may r.lI= platd,t consumption inst....d of. or in addition to. immunologic destruction."" 3. Nonimmunologic causes of d,"""ased putd.-.: mrvival a. Blood loss. acute and ....." {I} Wh.n blttding .ccompanies sn.~ thrombocytopeni • • hemorrh3l:' i. probably secondary to th. thrombocytopeni<> rather than ..v." thrombocy_ to~nia being caUsM by th. blttding. However. platdet loss and consumption at sit.. of h.morrh:.ge m.y calJ.1le or rontribute to thrombocyto~nia. {2} [n dog>. ex~rim.ntal acut•• sner. blood loss via phlebotomy causn:! mild to moderate thrombocyto~nia {up to 50 % rffluction in [platd.t]}.'.....,· {3} Thrombocytopenia is sometimes p" ... nt in bleeding dogs th.t have antico_ "l:"unt rodenticide toxico,is.'lO Pl.td.u a" probably conmm<"d at an a=l.rat<"d rate by use at sit.. of h.morrhage throughout the body. With extensive hemorrh3l:" putdet loss contributes to thrombocytopeni<> •• nd thrombocytopeni<> may become more sever. during routine tre.tm.nt with fluids. translil!ions. and viumin K,. b. Pl.tdet a.ctivation with a=l.rat<"d consumption or lJ.1le: Any dilOrd.. a""""i<>t<"d with increased platd.-.: activation may a.ca:l.rate platd.t consumption .nd l....d to thrombocytopeni<> if platd.-.: production doel not com~nsate. {I} localized {rontroll<"d} intrav:ascu.lar co"%,,l.tion {e.g.• with hemangiosar_ coma. thrombosis. or h.morrh3l:e} may be associat<"d with platd.-.: consump_ tion at the site(l) wh
"'"lI'

4/ PLATELETS

241

{al Activation of th~ cwgulation = d . l""d, to th. ~n~ntion of throm_ bin (f""tor lIa), a poUnt platdet .ctivator, {b} Thrombin activat.. pJ.td.ts, and .ctivated platdm rd~• .., platdet_ activating mb. tic di..,=, infN:tion', massi"" nrcro,i" panc""atiti" nroplasia, o""rh~.ting. or sopti~mi .. {3} Drug, and fOf.rin, con indu,,", ""'~" thrombocytopenia in h~parini=:l and nonh.pariniud dogs, appar~ndy through. di=t proaggrq;. tory dfrcr,'''' {b} Foreign m>trrial, ustd within th. VlI1Cular .yst~m {e,g" tubing and coth.urs} ."" t.. ted and chosen to minimiu platd~t activation, but accd~rated ronsumption may occur, {4} En""nomation (without DICj: Th~ num~rom type. of ""noms from num.rou. aninul sprcies diffi:r in th~ir .ffrcrs. V~nom from "'m~ m.h. nuy contain pl atd.t_activ.ting factors th at indu~ thrombocytopenia in th~ abMn~ of DIC,'" Oth .. v~noms indue. Dlc" JO {5} Vasct1liti, or ~ndocorditis: InHammation of blood """"I, or endocordium con ah~r ~ndothdial cdl, or l~.d to expolUr<: of sube:ndothdium, ""ch of which am lead to a prothromootic stat~ and platd~t adhe,ion, aggregation, .nd stCmion, Vasculiti, h., infretious, immune-mediated, .nd ch~micoJ musts, {a} Infretious cow;.,. of VlIKllliti, includ~ Rocky Mountain "",md f~.., conin~ h..pe'viru. infrcrion, dirofilariasi., angio,trongylo,is, infeaiom conino h.patitis, and "'Iuin~ viral art~riti., {b} Thrombocytopenia i, commonly assoeiated with b""t~rial ~ndocorditis and may ha"" a multifactori.l patho~n..i., {c} Endothdial cdl damage: .nd nrero,i, l~.d to thrombocytopenia without DIC in dogs with h~molytic ur~mic .yndrom~ {~,g" cutanrou, and .. nat glomerular vaoculopathy of grryhounds associated with ing..tion of Shiga toxin produced by &chmchid wit),""'" Wh.n .valuating gr~ound" it i. important to rrcogniu that thoir platd.t con""'ntration, in health at. oft.n low"," than rommonly r~ported conino r~f~ren,,", interval.. E. Hm.ooilution: M. ... iv~ dilution of blood with platd~t_poor fluid, (~,g., crystalloid" colloids, plasma, or padrd ~rythrocyt .. ) m.y couse mild to mod~m. drereases in blood [platdet],' .... ,. F, Idiop.thic or multif""torial mechani,ms L Idiopathic thrombocytopenia in Cavali~r King CharI.. spanid, (inh~ritrd giant platd~t disord~r in Cavali.. King CharI.. 'panidsl'JO-'" a. A rong.nital di,ord .. r~ported to ha"" .n autoKlnul r=iv~ inh~ritan,,", paturn,<J b. Th~ di",rd~r is common in th~ brttd. It is :wociated with large: platd~t, (mdcrothrombmyut), particularly wh~n platd.t concentrations ..~ < lOO,OOO/IlL It may be: r~f.rrrd to •• a mtltTtI.hrombtKy"'pmia.

242

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY {I} Th~ rdationship ~,""n {hrombocyto~nia and macrothrombocytosi, is und..,., b«:aust invtnigato .. have U>ffl va,ied ddinitiom of thrombocytopt_ ni. and nl
dogs. c. PJ.td.t rona.ntntion, of 50--100 X lo'lJ.lL at. common, and rh
d. Aff<'CIffl dogs h.ve no dini",] blttding problem . •. This brttd also has a high incid.n"" of mitral val"" di",as., but there i, no cle.r ,dation,hip bttwttn the conditions. f. Ikcognition of thi, condition i, imporunt to pr• .,..,nt unn=ary thuapy and to employ appropriate method, fOr a",unte plaid" oonctntration!. {I} Microscopic h.mocyto~{.r connn or mimau. from blood un..,." ...

u",fuJ. {2} Automat«i method •. particularly impalan~ method •• are unrdi<>ble when a mbstamial numb« of pl atdelS are larg"" .nd infections ...... ~,,' '" ,., {I} Etiologic agent, ITequemly associ>!ed with thrombocytoJl'nia include cenain ba.ct«ia (EhruchiJl .pp.• nd AMp"''''''' 'FP')' fu"l:i (e.g .• Hiirgplas",., 'pp.), viru ... {e.g., equine infectiou, .nemia viru,}, and protozoa {e.g., L,iih",.,nm 'pp., &Imi" 'Pp .• :md TMikri" .pp.}. {2} ThrombocytoJl'ni. occurs wmewhat less frequently, but not unexpectally, with oth« organism, {e.g.. Uprg'pfra .pp. and fdine immunodeficiency viru».''''''' b. Infectiou.s thrombocytoJl'nias may be coused by "..iou. oombinations of .uppr~ platdet production {e.g., direct infection, immune .uppression, or local effeets of inlUmmation within the bone marrow}, altered platdet distribu. tion. increased pl atdet consumption, .nd immune.mediated or nonimmune platelet destruction. c. Anaplasnw p"'fJI (formerly Ehrlichia p"'lJI) org:mi"ns." rich:ttsial bacteria that infect platdet, and couse conine infectiOn! cyclic thrombocytoJl'ni •. {I} Infection may be mbdinical or have clinical maniftnations.'" {2} r ...,itemw may be marked but are often mild, so org.nisms in blood films may be difficult to identifY definiti"dy. {3} Molecular diagnostic techniques ha"" been devdoped. '",''' 3. N~pl .. ms'" a. Many types of n~plasi<> a", .uoci<>ted with thrombocytoJl'nia. including corcinoma.... rcomas, lymphomas, .nd leukemias. In one 'lUdy, approximatdy 10 % of dog, with n~pla,ia _'" thrombocytoJl'nic. though sometimes becau.. of concurrem infectiom or th«apy. '"

4/ PLATELETS

may =ult from many m~cl,.ni,ms. alon~ or combinal. [n 'pa:ific cas". c><= a" mu..tly >p«ulatin. Potential m~nism. include the following: (I ) D,""",asaI production (a) Mydophthisis is a pr.sum«i contributor to thrombocyto~nia in dog. with multiple mydoma, acute lalhmia, or chronic lymphocytic leuhmia. Other cyto~nia, are ,omelime. pr=nt. (b) Mydodysplasia (c) Estrogen ,,",,mion by the n""plasm (d) Chemoth..apy (2) O,"""asaI platdet survival (a) OIC oa:urs in some canerr p"lienes .nd may contribute 10 throm_ bocyto~ni. ~n in dogs th at h."" /lUS1 erlln""plasia or hepatic m=na~.''''''' Howev.., most "",luaud dogs with n""plasia .nd det:r<",«i platd" survi""l (kinetic uudies) had hy~rfibrino~nemia, and fibrinogen half_live. we .. not d,""",ased. ''"'''' This dot. not suppon Ole. (b) Y"",ulitis or thrombo,is within a n""plasm (e.g., hemangiosarcom., a m..lignancy of endothdial erlls) may acederate pJ.tdet coIIJumption. (c) Saxond." y [MT may .lso oa:ur and has i>ttn incriminat«i in dogs with errtain types of n""pl .. ia {e.g., lymphoma} by u.. of indir= platdet and megmryocyte assays." {d} Oth.. contributors may include hemorrh . g<' =ndary 10 • n""plasm. Jq>sis =nd..ry 10 immunosuppression, .nd destruction (phagocytosi') by n""plastic macrophages {e.g., m..lignant histiocytic n""plasia}. {3} Abnorm:d platde-t dinribution: Splenomegaly or hq>>tomeg:dy inducffl by n""plastic infiltration {e.g., hemangiosarcom.} or ,econd..ry organ conges_ tion may be associat«i with plaldel Kquestration. 4. Drugs: Ai; :dr....dy nOl«i (Thrombocyto~nia, ...as. II.CA. D.2.c, IDd D.3.b), the thrombocyto~nia """",iat«i with drugs may be <""-u .. d by mydosuppression, "ccderated platde-t d~ruction {immune or nonimmun. }, or multiple m~IDi"ns. [n some cases, the pathog<'n..i. of drug_inducnl thrombocyto~nia is not clear.''' 5. Hypophosphaumia C>Usffl by hyptdet ...ctions in some sl"""i...''' b.

Thrombocyto~nia

243

244

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

THROMBOCYTOSIS I.

Gen~r:ol roncqm A. I1mm.lMcyrg,jJ is • [plaid",] grnt~r than a valid upJl<'r ""ft",oc;., limit. B. It =y ..",It from rffiimibution or inc~ production of platdets. lncreasffi production may bt asroatal with hemic neoplasia involving mq;. karyocytes, or it may occur ., a =onda,y ...acrion to ocher conditions. Familial thrombocyto= including thOst Ulustd by mut.tion, of the Tpo r~{or oo:ur in J'U'pJe but h:IVe not bttn reportal in animals. C. Appropri.te ref"en~ illt..""l, for [plald.t] may b. s.hiftal upward for individuals at high :dtitudes.''' D. Strum [K'] may krom. incr.=
[I.

Dis"""" and conditions (T.bl. 4.3) A. Hemic neopla,", (dona/thrombocytosis) l. Primary (.... mial) thrombocythemia: • ra .. chronic mydoproliftmi"" distasr that has bttn "'portffl in a few dogs and em {Stt Plat~ 7I}.'" ->''' a. pJ.tdet ronc;.,ntmions have bttn mark~dly inc==! (1<XXl-5000 X IO'Ij.lL).

b. Lorge. pleomorphic. or hypogranular pJ.tdets Im}' ~ p ...ent and .
Table 4.3.

m......,. and oondi!ions that cause thrombocytosis

Hemic n
Increased production 'Inflammation: infection. immune maliated. our!:"'}'. trauma Nonhemic n
Blood lms • A "1";,,,,ly common di..... or condition

4/ PLATELETS

245

e. Oat> :uelacking reg.rding the prrvalenc. of thrombotic or hemorrhagic tenden_ ci.. in .nimal, with clonal thrombocytosi •. 2. OIh" chronic mydoprolifemi"" di...,.,." polycythemia priJrulry erythrocyto.is. and chronic mydoid leuumi. 3. AmI< mtients with iron deficiency. Th. specific cause is not known. but blood coneentmions of measured thrombopoietic cytokinu (e.g .• Tpo and IL-6) ha"" not bttn incr• ...d in hUJruln pati.ms with iron d.fici.ncy and thrombocy_ tosis.'" Cross-reactivity of Epo with Tpo rea:pton i, also apparently not the cau ... ' " d. Vinca alkaloids: Vincristin•• nd vinblastine "imula,. thrombopoiesis that can l",d to thrombocytosis without increased MPV.'''.l '' ln patients with IMT. these drug, Jruly l",d to inc .......:! platdet concentrations by o[h" mechanism •• including inhibition of the M PS and th".fore decreased platdet destruction. e. Recovery from thrombocytopenia (rebound thrombocytosi'l : Thrombocytopenia may srimulal< enough thrombopoi..i, that produ<:tion exa:eds consumptionl denrucrion .nd blood roncentration, transiently overshoot the up~r ref"ence limit during rrcovery. Thi, can occur aft" withdrawal of mydosuppression. with recov",y from IMT. or aft" blood lo .. ."~''''''' · f. Pomplenectomy: Splenectomy callS<::! thrombocyt<>lis. sometimes marked. aMOCiated with increased thrombopoiesis .nd increased blood roncentrations ofTpo.' ''· '''Thrombocytosisistransientbutmaype"i;tforw..,h.Other

V"'.

".

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY ffi«h.nisms th at may contrjbut~ to (h~ thrombocytmis indud~ d«""asffl platdet

d.nrucrion or d«re.iffl soquestn t;on. In g. Blood lou, es~:olly chronic, ru.. 0011 :ossoci.tffl with thrombocytoli. in stV~ral 'p<'Cie;, but iron ddici.ncy, inflamm.tion, .. bound thrombocytmis, or Du>pJ..,i. may au"" or contribute to thrombocytoois in many of th... ca, ... Thrombocyto_ ,is omociatrd with blood 10.. moy =luire th. pr=nc;., of. spl..,,,; it occurr«i with repeat«i phld>otomy (chronic blood lou) in nonsplenectomized r.obbits that we .. supplementM with iron. but did not occur in iron.supplemented, .plen«_ {omiud nbbi( •. '" h. HypuconisoJemi.: Hypu,,".and ..d&ct ,.lotion. ,hip is not drat. Thrombocyto.is may rdate to underlying or concurrent

conditions. prffinisone administration to hnlthy dogs r~SIllted in no inCJu..,"· or • qu~tionabl~ incru",'" in pl.rd"" oonetntr>tiofll.

PU-TELET VOLUME I.

[ntupretation of MPV ... Iu.. is limited by inaccunci.. and inronsisunci.. of routin~ MPV nteasurem~nt and limited knowl~ of f.cton influencing platdet ,ize,""" e.~ially in dontestic s~i ... A. Valu .. should b., interpreted rdati"" to rd'",enet intervals t<'ner>ted from sampl.. handled in th~ same manner :as th~ patient samples. B. Anticoagulant, norage time, norage tem~r>ture, .nd analyzer all .ffect the r.. ult •.

[I.

Among individuals, popul.tions, .nd .~i .., platd"" volume is generally in""",dy related to th~ [plndel]''' . nd MPV values tend to be greater with thrombocytopenia .nd lower with thrombocytosis.'" A simil ar rel ationship was found in h..Jthy cats.'" A. [ncr~~""" MPV l. This usually suggests ~ccderated thrombopoie.o 3. Abnorrruolly large platdeu ..~ produced in amain congenital platdet disorden, so MPV may be inc",ased. Larg~ pl. td"". _'" r~ported in the first dogs de",ribffl with onerhound thrombop. thia,- and they are p....,nt in many Cavali.. King CharI.. spaniet. {,.,. Plat~ SC).'''.....lO'...,

4/ PLATELETS

247

4. MPV may inc,"""", with physiologic thrombocytosi" possibly kcau .. of mobiliza. tion of the spl.nic plml<'! pool, which i, thought to be ovmepr... nt":! by la~ platdets in some sp«i ..." 5. [nc,""astd MPV is ",sociat":! with hYJl<'nhyroxinemia in pn>ple .nd mi""." 6. Acute infection with the Kaw:.kami.Theilen main of fdine leuk.mia viru, induc:.d production of ma.crothrombocyt...JO, This w .....ociat":! with decrement' in [platdet] but no significant change in platd<'! mass. B. Decr.astd MPV l. Dog. with IMT may han danalffi MPV valu.. mo,"" ofien than dog> with thrombocyt0P'ni. for other r.-awns, but valu .. may also be inc"....:! or WRI .... [nc,"""",d numbers of platd.t microparticl.. are detect":! by flow cytommy in blood of dogs with [MT. 2. With..".., .. thrombocyt0P'ni. (< 5 X 10'Ij.lL), MPV may be affect"! by nonplatdet d.bris in the s;omple or instrument if ""cial pr<"CI.utions .re not taken. Thi. d.bris is i"'ignificant ""eple.'''' '' How...", MPV w .. WRI for ..nn of nine dogs with primary bone marrow di",,,,,,, and increastd in t"..o of nine. The Ian" two dog, did not han meg.karyocytic hypopl ..ia .... Ill.

An inc"astd PDW indian...n inc"...d population of large platd.IS, .n inc ......:! population of small platdet" or both.

[v.

Platdet volume is . lso subj.crin ly assessaI by microscopic in'pa:tion of blood films (Itt Microscopic F..tur .. of Platdet" ..cr. [I.B). Popul. tions of larce pl. td.u can be d.tect"! ...en when .utomat":! analyurs miss th.m. Int"p.. tation is limil.. to that for inc,""astd MPV.

RETICUUr.TED PUr.TELETS

I.

R.ticulat":! platd.u: young platdelS containing incr"""! RNA: analogous to .. ticulocyus A. As.=sment i, primarily. =ch tool at this time, but tming may be availabl. in spa:ializallaboratori... B. Reticulat":! platd.t P're
II.

Method A. Platd<'!s a.. typically incubat":! with thiarol. orang •. which bind. to platd.t RNA and gr.nule nuclaltid .. and emits .. f]uorua:n"" that is d. tecud by flow cytom. try. The P'ra:ntagt of platdet, with iner ...,..:! f]uorr=na: is d<'!"min..:!, and a micul. t":! [platdet] can be calculat..!.

'48

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY B. An inc"",,,,,, in f1uore=n"" may rd.t~ to an incr~ .. in plnd", "';u or to .n iner ...... in RNA ron""mration within th. plaldees, d.plion time, or fr ••h versus fixal).'=->07 V:uiations in m",hod. complicau im.rpm.r;on of .. ponal findings. C. With Klme m",ho,j" reticula!,"", pbtd.! p"""mages iner ....", with ",mrJ. fixation .nd with ""mr!' ,to!>&" for 18 h at eith.r 4'C or 21 ·C.'

III.

Imrrpretation A. [n ma>ry. increaM' in miculatal pbldets suggtsal thrombopoirsis and may therefor. help with the foUowing: l. Diff.rentiling wler;n thrombocytosis nom prim:ary thrombocythemia 3. Id.miJYing wh~1I • pati~nt i, r,"",v~rillg from bon~ marrow mpp",.,ion (~.g .• aft~r ch~moth".py)

B.

Th~ f~

5{udi.. rq><>rt..! to

dot~ g.n~rally

.upport the afo .. m~ntion..! thmry.

l. [nc..asal r~ticulat..! pl>ld" ptrctnt>g" han bttn pr~ .. nt. wh~n evaluat..!. in

mon dog, .nd hor... with thrombocytoptni:u em=' by deer",,=' platdet .urvival.··"' .... 2. Erythropoietin .dmillinration to dog, wa, "-""'Ciat~d with incr~"'..! numbrrs of reticulat"! platdets .nd platd" hypls th.t abnormal reticulatw platd" r.. ults may han ",me sptcificity for platd" diwrd.",. However. samples with incr",,=' reticulat"! platelets w.'" 1I0t report"! in this nudy to confirm that .n incre... in reticulat"! platd", could br d"ectai C. Reticulat"! pl atd" v:du .. h.ve usually b.~11 .. port"! alld int"pm..! '" pt=nt>g~s rath" thall .t concentrations. I. Analogout to int~rpr~tatioll of immatur~ ~rythrocytes {miculocyt~,}. on~ must con,id~r the possibility that reticulat"! platd", I"'=m>g~ illo"", may br r~lati"" and not truly indicative of incr.. =' platd" production. 2. How"",,. ,,"~II with ages may br iner",,=' in ~~ .. thrombocytoptnw without inc",.,.. in reticulat~d platd", concentrations beau.. ther~ may .imply br too f~ platdets for ..... n a high reticulat"! platelet pt=nt>g~ to yidd an illc.....w micul at.-d [platdet].'"'a. Reticulat"! platdet roncentration. m.y al50 br ,uppr~=d by th~ mark.-dly reduced platelet Ii£. SpallS in.."... thrombocytoptnw. Th~ rdativdy few platd",. must br collsum.-d at an .ccd~rat.-d rat~ to maintain normal vascular intq:rity. "~ll

4/ PLATELETS

249

b. Also. th~ pathologic proct:S! consuming or d.. troying pl.cdeu rruoy target young and old platdeu alih. D. Th. clinical utility of «ticul.ta! platdet ...... m~nt in dommic rruommals is uncl~ar and :!W.its fimh~r nudy in a vari~ty of disord.rs. TESTS FOR IMMUNE_MEDIATED THROMBOCYTOPENIA (IMn I.

A ... riel)' of speci..tiz
[I.

Sample conditions (•. g., .nticoagul.nt, norag. tim~, and storag<' t~mp
III.

Platdet assays A. Direct platdet assays th at test for . ntibodi .. on th~ mrf. "", of . patient', platdets {pSAlg} are r«:omm~nda!. l. Mon current """Y' are How cytometric; PSAlg is detecta! by lluore=nce_t:oggnl, ~..... p«ific, .nti_immunoglobulin antibodi ..... ·I.~I.,.I . . .Ill.1I . ...' " 2. Positive r.. ults gtneratw &om prop. B. Indirect assays t.. ting for antibodi.. in a pati.nt·s pl..m. or .. rum that can bind to "normal" pl.tdets from h~..tthy .nimals h."" bttn u.s
[v.

Direct megakaryocyte inununolluorescence assaY' have bttn u...:! to detect the p".. n"", of megakaryocyte-associata! immunoglobulins on . patient', megakaryocytes, usually on ,me'ars of. bone rruorrow aspint•. ""''' A. Testing require, good bone marrow aspirat .. with ampl~ megakaryocytes, so .anlpl.. are not .lwaY" adequate. B. [f .ntibodi~. directa! against platdet, al"" bind ilia=! .pitopes on megakaryocyt.., results may"" positive.'''' C. Fal.. _pooiti"" results may occur if megakaryocyt.. are dormgffi and cytopl..mic immunoglobulin uth" than surf""", immunoglobulin is detect.d. D. Th. cumulative reporta! diagnostic .. mitivity for clinical diagnoses of canin~ IMT it about 50 %, which is too low to r«:ommend."

25.

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

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=-

00'

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255

4/ PLATELETS

139. Sinrh MK. Umb WA. 2005. [diopatl.i< tkrombocyoopm.i. in c.,.. .... Kin, Cb .. ko ","';.1.. Au. y.. ) 83mlO--703. 1.(0. eo..... SM. Ilartp JW. Gompf RE. H.y .. JR. MOJ"'" ill. Snik el pIa"lrt acrintinc facto. in dovdopm,n' el tkrombocytopoaiptaoo-polym' .... d"in .".,ti.on. J M<>I Diap> 7,J08-316. lSI. Cloiobolm·o..i. A. 2000. ModoanI",u eltkrom~ in do, • ...it!.~. Comp<..d Cootin E.dU< p,",. Y..

cc.

imm.""""'·

Lao,.",..

"""r.

""_ti.on

22,lOO6-10I8.

IS2. G..n..k", CI!.. B.. ioodo....oi. in 16 dor;>. J Y.. In"", Med h75-M. 154. O·O"....dJ MR. Slidrt .. SJ. W,j,d", PL S-t. R. 1981. PI .. d .. ond fibo.j""V" kin el ............"cl.. ed pl.,,!.. oo......nptioo ........ pIa"lrt "''Y'''' inbibi"" •. Blood 59,1252-1258. 156. Bloo", JC. L....i. HI!.. Sdk.. TS, Dddat A. Mo'P'" m. 1987. lb, b"..""P'tholou el afonicid· ond ropoirtin t.v.lo in J"'W" Boli ............... .;,;tin, d" And ... [m) Biom'tro",I43,~j...90.

161. Ba .. MC. Schul"" AI'.. 1998. E-utial thoombopp<. PE. M...ddJ CP. T ...d JM. J.in NC. T.blin F. Zlnld JG. 1989. PmbabI, ,,,,,,,;oJ thtombo
'"

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

166. C';D GR. K.....bmi TG, 22611--64J.

Jain

NC. 19l!S. Radi ....n_ind..ced

~aotic

1..1..",.. in •

.J.o&.

Y.. P.d.oI.

167. Hamilron TA. Mon;..., WB. o.NOI. DB. 199!. Cytooin< ... ~, d..motl.,,,py'" KUt, ~ ],..krrni. in , cat. J Am Y" Med Aaooc 199,J59- }61. 168. P..k HM. Doot<. AR. T.ohbo. .... RE. u. YM, L,.,.. YS, u. SJ, Kim HJ, Sue JH. 2006. CIinlcaI, hi ..........<>Iociad ...<1 immwoobistod'micallindinp in a co .. of ~ .. tic kuhmi. in • J.o&. J V.. Di.~ l",..,ot 18.187_ 291.

169. Fidd ME. 1930.1\, .If"" of <mOtion ~n tJ.. blood pI_kt «>Un<. Am J rkpiol9Jo:MS--M8. 170. a..mb..1ain KG. T ~nc; M. r""iopon DG. 1m. ~" of .... ad.",,~bI. .plmic plat.kt. .dc...d ,iroolati.on d.dnt: ~ioduc«I tkrombocytool •. Am J H ..",ooI. }4,)61_ 168. 171. F"odman M. Ah".k. N. iWl"'Wn S. 1977. p",«>« of. D"mpknic pl""kt pool. Blood 50,41 9--425. 172. ldo< OD platdrt ,iz<...d n.....ba in poni", V .. Roc IOI.4ll8. 173. D •....,nAA. Ocnon D. 1%9. E=d.. -indoced "'~.;.. kn H......roI. 4;!,241--206. 17'. Sdunid, KG. RaUB.,"", JW. 1984. ~ d.anc" in th, in.i,.. dimibution of "'[".J.bd\ed p1atd.... Saad J I-/a"" • .,l 31,159--166175. M...p.... MS. ""'abO"" AI<. Kup NS, Tint: V. o..b1in BI<. S . P<>d~ J. o.blin SA 1998. H.moraIIy ""di.t«I thrombocyto." in m';", Jo."" au"";pn.,;.." throm~. B, J H",motol II" 126-]J}. 177. H""""", AS, 199!. TIu..mt.o.y...,;. in and 00'" A ",.... pecriv< ...Jr. Comp H ...... .,I In, 1,181_ 186. 178. S.llon OC. lnin< JF, Palma K. Millilin E. Grind"" C. Co...""" P. I '!97. lhromboq",," in 24 t.o.." (1989l?SIt). I V" [n'"m Mod 1)'24-29. 179. Gut! G, ['I""" M . Fimnd C1. W~"C GY, Fed",j,ci MG. Ba..d<. NH. R..bin SC I?SI}. H~ [[....6 J,,·d. in oocitic floOd "",..!a", ..ith ~ thrombo.yoooi. in paci ... " ";d. <&lion an«>. II. J H ...... .,J S},4jJ-41!. ISO, H _ DF. Db.JiwaI R'i. Si ... n DD. Ki«"'ll BE. 1m. Pannoopl .. tic i. indoced'l""mic thrombo..nboli.m in. at. J Am Anim H""I' """" J5,48.}--486. lSI. lnin< SP. 1m. TIu..m....,.,.. •. [0' L« GR. ",-"", I. Luk....J. Wi""""" MM. cd.. Wm .... b.' am"", H..-.!.o. IOd. odi ..... , 1643-1660. Ph.iL.ddpbi", U"""""" Willi ..... 8< Wilkin •. 182. Abn H, Guv M . 197J. ['I."Ie, .iu in ... rombocytopc..; .. and d.romt.o.y...,;. of vat;"'", ~ • • Blood 4),587- 598. IS9. [cbikaw. N, Ki""" K. Sh.imodai .. S, bhida F, [", T, Kajib.... S, Tab ... T. Ka,~ T, Kiyoa.... K. I ?SIB. Ck~. iD "rum "'rombopoirtin kvcla aft<> .plcnertomy. Acta H ....... ol 100, lJ7- 14!. 190. Jain N C. 1986. Qu.litati", ",d qu • ..m.,;.." tti L DjakLrtti M . 1981. lb, ,1Fert ol parti.J oplcnectom, ... pi_ie, pn>ical ,1Fert. of 100>,.,,,,.. .dminj .... . ..... of anti_infI... m....,. doo ... of 1"'.....;..,'" in """,. Am J Vrt Rr. 5},IOJl-IO}7. 195. Co."'" L 19I19. n , "Iati.ooabip ....... '" m'~ ploidy and pl.",J" ..olwn,. Bloc.! Cella 15,81_ 107. 196. lninJ. B-manJD. 198J. n.. in""'''..!a............. n platelet volume and pI.teI" nom .... , Abnonn.~tic . in ....... toIoci<
"'to ....

cc.

do,.

_bu."

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Chapter 5

HEMO STASIS Hemostasis ............................................................... 261 Platelets fThrombocytes). ................................................... 262

I. Ph~iologic Processes ............................................. II. Platelet Concentration ............................................. III. Platelet Function Tests ............................................. von Willebrand Factor (vWF) ................................................ I. Physiologic Processes ............................................. von Willebrand Disease (vWD) ...................................... II. III. Analytical Concepts ............................................... IV. Decreased von Willebrand Factor to Antigen (vWF : Ag) Ratio............ V. Interpretive Considerations ......................................... VI. Genetic Tests .................................................... Coagulation ............................................................... I. Physiologic Processes ............................................. II. Analytical Concepts ............................................... III. Whole Blood Clotting Times ........................................ A. Lee-White Method ........ . ..................................... B. Activated Coagulation (Clotting) Time (ACT). ....................... C. Synbiotics SCA2000 Veterinary Coagulation Analyzer ................ IV. Activated Partial Thromboplastin Time (PTT, APTT, or aPIT) ............ V. Prothrombin Time (PT) ............................................ VI. Thrombin Time m). .............................................. VII. Fibrinogen ....................................................... VIII. Other Specific Coagulation Factor Activities (II, VII, VIII, IX, X, XI , XII , PK, and HMWK) .................................................. IX. Proteins Induced by Vitamin K Antagonism or Absence (PIVKA) .......... X. Russell's Viper Venom Time (RWTI ................................. XI. Endogenous Anticoagulants ........................................ A. Antithrombin (AT) .............................................. B. Protein C ..................................................... C. Protein Z...................................................... XII. Coagulation Factor Antibodies (Inhibitors) ............................ XIII. Anti-(phospholipid-protein) Antibodies ............................... Fibrinolysis ............................................................... I. Physiologic Processes ............................................. II. Fibrin or Fibrinogen Degradation Products (FDPs). ..................... III. Fibrin Fragment D-Dimer ........................................... IV. Other Assays..................................................... Blood Vessels (Endothelial Cells) ............................................. Major Bleeding Disorders: Findings and Pathogeneses .......................... I. Diagnosis ........................................................ II. Pathogenesis..................................................... nlru!llbu~i~ ...............................................................

262 262 263 265 265 265 266 267 268 268 268 268 274 279 279 279 280 280 282 284 285

286 287 288 288 288 291 291 292 292 293 293 293 299 300 300 300 300 304 310

25'

Table 5 ,1. Abbreviation. and symbol. in this chapter III ACf ADP APC AT All' BMBT

0.> DDAvP ole DNA EDTA ELISA

It,·

FOP,

(I '" an.lyt~) Activ:llt
conc~mr:uion

+ fibrin~n

fratm~nl$

GPlb

Ha HMWK INR lSI

I-XIII L\1WH MPS M, PAl P[VKA

PK PT

PTT PZI RM SF RVVf 51 T AFI TAT TF TFPI I_PA

IT IT_ u-PA

.WD .WF vWF:Ag vWF:CBA WRJ

260

Glyroprorein Ib Hematocrit High mol~cular -'ght kinim>gen International normaliznl ratio International Sm,itivity I nda Inxti~ coagubtion faclors one through thi"~n; terminal "a'
ooor hal Men aaivatt
phagocyte 'Y"tem molrru..L.r rna.<:< Pluminogen activator inhibitor Proteins induced b)' vitamin K antagonism. :ibstnct, or deficM.OC)' Probllikrein or prdullikrei n (synonyms) Prothrombin ti~ Activatt
5/ HEMOSTASIS

26'

HE..\lOSTAS[S HmwifasU it th •• mst of bl • .ding or th. illl.rruption of blood flow through ~ v.... J. Th. t.. m is ..lto us.d mor. g. n....JJy to ref.. to th. illlricat• • nd bal. nc.d physiologic proc,,,... th .. t m.inuin blood in. &.ely flowing " .. t. but .. Uow th. rapid form ..tion of localizal solid plugs to .....J injured v...ds. Normal h.mo" .. sis d'p'nds on th. complex illl.ractions of its mojor romponelllS: pl ..,dets. OO
I.

(Fig. 5.1). [I.

Abnormal h.mo,tasit cau= h.morrhag. or throm!"",is. Laboratory testing of the indi_ vidual compon.nts of th. h. mosutic syst.m m..y b. Uiffl to discover. explain, monitor, or prognostica,. th ... p>lhologic ,Ules.

Plat.....ts

" ,,'

-,

PI1mary

"

tIemos\Qtic

!

,

"

Blood """",,Is :

~.!.-

pkIg

- ', '

,

,

... i ._1-../

•• S
.~-

tIemos\Qtic

\, , ,,

,

, Coagulallon

,

"-

Fig. Sol. H.""",totis in h..lth. Normal he""",wis is mainuj....d by tt.. numscuhr injury. The foUo...u.g ....,n'" oorur with blood......J

d:un>s" • V :ISOron>triction redu"",,, blood los~ :and actin",d ..,dotb.lial ",,[Js ""press both produombotic functions to limit b~ng md :ontithrombotic functiom to ~mit dotting. • Pt.",lett adhere to apo...d mb.ndotb.lium, 'I',.. d to patch the <\d'e
TAR • When ooagulation path""Y' ar • • ctiv:oted, fibrinolysis is also. FibrinolY'is helps control the en.nt of oo..,.,l .. ion by bre:dcing down fibrin, too. contributing to th. f",m .. ion of "" "Ppropri... >econdary he""",,,,ric plug ""d promoting ... ..,tual ,emov:tl of th. plug to maintain normal blood 80... Abnonnaliti .. of ""y romponent of thit '}"I<m an up .. t tt.. bat""", md leod to .ith.r hemorrbage or thrombosis.

262

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

Pu"TELETS (THROMBOCITES) I.

Phpiologic proa=. A. Stt Ch .pt~r 4 for plaulff struClur~, production, killetics, :md nonh.mostatic functions. B. H.mosutic function! I. Plude" are required for fotJrultion of priJrulry hemostuic plug. to repair smaU v:uwl." d.f«u. Th'7 amplity minute Slimuli into nplosin production of fibrin to form more .«ur. S<'COnd" y h.mostuic plug•. 2. Pl. td", hemosutic functions em k divided into fin major c;otrgories: a. Adh .. ion: Platdeu adhere to and 'p,rad over (p.tch) ~rturbffl.ndochdium or npo,ed sukndothdium, mOSlly vi. vWF ch.t bind. to plald.1 GPlb and coll"i:"l1 in th. ves.d w:oll. b. Aggregation: When . db.rene;., or pbtd.t 'gonj"" (e.g., ADP, ooll3J:en, plald.t_ activ.ting f.ctor, or thrombin) activate r«:q>ton for platdet membnne glycopro_ tein Clo.~" plotdm ~r. Pla,d.ts also rd..... platdet agonim that .ctiv.te oth~r pl.rdet •. • . Clot (coagulum) retraction: Platdets within ~ thrombm f.cilime v.uund dOJUre .nd vessd p:!tency by a comracta~ process inmlving .ctivaud platd.". inter_ platdet bridging via the fibrinogen r=ptor am,p" .nd platdet ""tin and myosin.' 3. Mediaton rd~~..d from platdets during th~ h~mo"atic process rontribuu to local tissue repair.

II.

Platdet roncentration {ofien called pbtd" count in dinical j:argonj A. s.. Ch~pt~r 4 for analytical concepts and disord~ .. ~uoc"'ted with thrombocyto~nia and thrombocytosis. B. Thrombocytopeni. may cau •• mucocutaneou. h~morrhagc: when ..,,,,,re. H~morrhag. from thrombocytopen", .lone i. not expected with. [platdet] abo"" about 30 X 1001J.ll and may not occur with a [platdet] < 5 X 10'1J.ll. I. Thrombocrit m.y be: a better reHection of platd" h. mostatic potential than is [platdet], bc:cau.. larg. pbtdm =y have gr•• ter h.mostatic potential. 2. H~morrh"l:~ may occur " g... t~r [platd~t] if there .te roncurrent platdet fum:_ tional deficits or increased vascu.l .. trauma. C. Thrombocytosis has bc:c:n """"'"'ted with thrombosis or h.morrh"l:' in poopl~. Similar r~ports .te rate in dom<::S{ic mammals. I. Thrombosis is not expected with r....cti"" thrombocytosi,.

5/ HEMOSTASIS 2. Thrombo,is . uociat.d with hum.n clon.J thrombocyth~mic disordw; m.y rd .t~ to .Jt~r.d function of platd~ts. ~ndothdi.l cdls. or l~ukocytes. It is not pr.dictabl~ baKd .
Platd~t

function tests A. BMBT is a nandardittd in vivo test of primary h~m"'tasis ustd in dog. and cats. Mucos.J m~mb .. nes or .kin sites' han bttn u•.d in l"g~ .nimob. Th~ ,kin blttdi"l: tim~ is l~", t<~atabl~.· and cuticl~ blttding tim~s .....,'" =ondary h~mo,wi, in addition to primory h~mo,t;ui,. I. An.Jyticol con""pts a. Units ..~ not nandardiU"d. Results ..e sometimes rqmrtal in =onds {e.g., 202 s}, minut~s .nd =onds (e.g., 3 min 22 s), or minute. (e.g., 3, 3.4, or 3.37 min). Im~rob.strv~r and illlraob.strv~r imprecision' mggests it would be. most .ppropriate to r~port to the n... rest minute. The BMBT ofhe..tthy dog, has bttn rq>On.d to range from I to 5 min, but it usu.Jly i. Ie.. th:m 4 min.'"' b. Procalur~: The upper lip is roU.d out and usu.Jly secured with . gau,"" strip .round th~ maxilla. Timing bq;ins when a sundardiU"d cut {or duplicote cuts} is mad~ in the muro,.J surfa"" of the upper lip by ming • 'pring_load.d devi"" {e.g., Surgicun devi"".}. Th~ cut is ,m.Jl ~nough {S mm X I mm} for primary hemostasis .Jone to r..olve the blttding", coagulation .nd I:"n~ .. tion of fibrin at< not n 4-5 min in dogs) with modeme to mark.d defects of prim. ry hemo,wi •. a. Thrombocyto~nia (Table 4.2): M .. kal thrombocytopenia is. rontraindicotion to BMBT beau.. it i • .Jready mown th . t BMBT will be. prolongal. Th~ dq;IN of thrombocytopenia required to prolong the BMBT i. not drar, but it i. commonly sUtal that BMBT prolongations moy occur with pla tdet oon""ntra_ tions of < 70-100 X 10'Ij.lL This decision threshold v..ies with oth~r factors, such as me"" platdet volum~, vWF: Ag, . nd Hct. b. Thrombop. thia (pl. tdet dysfunction ) (T.ble S.2) {I} T~rms used to describe. a disord~r of abnormal platdet function include mrombtJpathu., thrombopa'hy, thrombocytupa,hy. and th"'mbocywpamkJ. The precist definition and u~ of ~.ch term vary. n,romb~pathu. is used h..~in as a g~neral t~rm to indicote any disorder of platdH function. {2} Thrombop,uhia should be. ruspect.d if BMBT is prolonged, but platdet oon""ntr>tions, Hct, .nd vWF: Ag values ar~ WRI. {.} Her.d.irary thrombol""hias an:: unrommon. Additional reading is avaiJabk' {b} Ac:quiral thrombopathia occurs ooncurrently with di .... ses or oonditions that are usually diagnosed by oth~r findings. Th~ thrombopathia may be. mbclinic.J or it m"y comribute obviously to morbidity. c. vWD: BMBT con be. used as. KINning ust for vWD in brtt
264

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

Table 5.2. Di..,a..,. and conditions thai ca u.c thrombopathias H~rffliury:

CaL> di'cylglycuo] gU. Spin thrombop.thiaJ, Ch&liak HiC",hi syndrome (Persian em; Hereford, Brangus, .nd J.pan ... black cmle). cyclic hcmatopoiesi, {grry collie}. deDIc_gram". stol'3J:' pool dis..,. .. of American cochr spanid" Glanzmann', thrombasthenia (g''''! Py",n=, o{{crhounds, hom.), idiopathi,

thrombopathi. in a Thoroughbred, plardet procwgulam defect ofG..rmm mq.h.rd" Ihromb>. {ollcrhound1---Origiruol "",",nil Acquir«i 'Drug aposu .. : anesth<:{ics {b"bitum.. j, anti. inft.mmatories (nollSuroidal.ntj. inlUmmatory drugsl, m.mbran~ac{in drug. {local anmhctics, p... drc"holosporin.), antiplatdets (ticlopidine), dietary faaor.; {gulic, ethanol, ""fT.in.} Envenomation (cenain venom.) 'Inc""ased FDPs {increased fibrinoly.is, DIC) Hepatic dis ...... H)'P"rglobulinemia (multiple mydoma, dulichiolis) Immun.... mediated thrombocytop 'Renal f. ilu"" (utemi.) Synth..cic rolJoid, (dextran, h..castaKh) • A r.tllively common dis.... or condition Noto: Thromoopathi. (d.c",-" pl.",J.t function) .boukl be consi"'md wb." (I) p
d Anemia: This may prolong BMBT, Prop<>S
agonist! -'· e. Vascu.l .. disease: BMBT rardy may be prolonged with certain ""ocular dis ........ f. Antiplatdet drugs: Aspirin inn.. ..d fordimb cutanwus blttding tim.. in hone,' and also increased conin. BMBT valu .., although the 1m.. w... lIill WRI. Other . ntipl.tdet drugs oft.n do not prolong the BMBT."-lJ g. Afibrinogenemia: BMBT m.y be prolonged in patients with afibrinogen.mi., presumably beau.. of the lad: of fibrinogen for interplatdet bridging.' .... '· 3. Defects r.urined to ..rondary hemonasis .nd the formation of fibrin (•. g., hemo_ philia, but not . fribinogenemi<» should not prolong BMBT unl.... a larger v...d is cut." 4. Blttding time h ... not been u..ful as • oc=ning t.. t for predicting the occurrence of acessivc: surgical blttding in human p.tienu." B. Clot (co.gulum) .. traction is medi<>ted by platd..c., 50 some thrombopathias (•. g., Glanzmann's thromoo.lIheni<>) (Table 5.2) or muked thrombocytop
'65

5/ HEMOSTASIS 6<J.-90 min of clotting but i,

mor~

rdiably

d"'~rminal

by stand"diud tests of clot

r~tr:lction:"

l. Blood {O.S ml} i, co]J= al into cold salin~ {4.S ml} and maintainal at 4 'C until b.:ing IrOmfc:rral to a gl..., tub.: and mixal with thrombin {IO UlmL final}.

2. Th~ tub.:, ..~ th~n pla.c:ffi in a 37 ' C w>t
of th~ clot and th~ volum~ of ,urrounding ..,rum. 3. T~sting should not b.: don~ if th~ patient has thrombocytoJ>"nia or has r=ntly =c:ival aspirin or oth~r cyclooxygc:n= inhibitors, b.:cau.. poor clot mr:lction i, aJ>"Ctai 4. Clot r~tr:lction is not impairal in vWD. C. Oth~r 'J>"Cialiud USIS ... u..d to assess platdet respon= in vitro.' The.. [<m are av:nubl~ in labs with 'J>"Cialiud aJ>"rtist and aJuipm~nt induding flow cytom~t~f1, ~rrgom~tef1, and th~ PFA· ]()() analyur {Dad~ Ikhring, Miami, FL}.ln addition to d"',""ting and chara.ct~riI.ing d,""r~ platd", function, thu e tests em also d"''"''t hyJ>"r:lctin p!'tdm as may occur with inf=ions {~.g., fc:lin~ inf=ioUJ J>"ritonitis or heartworm di",ase} , malignanci~" th~ nephrotic syndrom~, .nd oth.. dioord~f1.''''''' A sll'lJ>"Ctffi sJ>"Cific thrombopathia m.y b.: d~monstr:lbl. by sJ>"Cific [tion and «<mion nudies or by mol
I.

Physiologic processr. A. vWF i, a large: muhim ..ic plasma glycoprot~in {M, " SOO.OOO- 20.000,OOO} important in platdet adhesion and aggrlion: vWF contributes to platd",. platdet bridging via GPlb and th~ p!'td.t integrin a ",JI,. 3. Th~ utg"st multi,""" of vWF at< the mon functional.'"' B. Mon vWF is producal by endothdial a:ll,'~" and ster.tal constitutivdy or noral and «"=ncage of total circulating vWF in most 'p«ies {~.g., em and J>"Opl~}, but conin. platdets rontain v. ry littl . ....,., C. S,""r",al vWF forms noncoval.nt compln.. with cwgulation factor VIII and seN.. as a nabilizing and prot'""ti"" carrier mol«:tJ.J~ for factor VII!.'"'

II.

von WilJebrand di",= (vWD) A. vWD, which i. a disord~r of primary hemonasi, cousa! by. d~fici~n but i, r:lr~ in cattl~," cots," and horst • .'l-" B. vWD is usually an inh~rital disord~r, but acquirffi vWD occurs rardy in peopl • ."''' Acquirffi vWD has not bttn d ...ly do<:um~ntal in v"'erinary sJ>"Ci~,. l. On. of tho di ...... :lS.'lOCiatal with human acquiral vWD is hypothyroidi,m, and tre atment of th~ .. people for hypothyroidism has resolval th~ concur..nt vWF d.fici.nci .....

'"

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

2 An association bttw«n hypothyroidism .nd vWD h .. aha bttn sug!;"!!aI in dog., particularly in Dobtrrnan pinsch~rs." How"""" stud;.. han producM oonHicting ,emits, .nd Ih. ooncurr.n~ of common di ..ases does not pro"" a Clu.. _and ..df,",,1 r~h{jonship.l> Th. finding of vWF: Ag v:du .. that w"". WRl for hypothyroid doc' I>Onffi in ho ......"·J.! 3. Tyl'" 3: Thi •• ..,,,~ fom,. which inml= an ab .. n~ of.ll vWF muhim .... occun particularly in Chesapeake Roy retri..,~n. Dutch kooik~rs, Scottish t.rri~rs, and Shetland sheqxlogs ......, D. Clinical and laboratory signs of vWD l. Mild 10 ~.re muemal h~morrh.ge (.piscui., gastrointestinal h~morrh"i:" or prolongffi estral blttding). culaneous bruising. and prolongffi hemorrhage from nonsurgical or mrgical trauma (•. g., "il docking. d.wclaw removal in puppi.., or tooth extr:oction); hemanhrosis and h~matom •• in hor..,s 2. Absence of petechia~ may hdp diff~rentiate vWD from platd~ disorders. 3. Prolongffi BMBT without thrombocytopenia and without prolofll:.d coagulation times 4. PTT may bt mildly prolongffi because decr......d factor VIII co"i:ulnion activity {which =y bt d~notffi FVIII : C} occurs .. rondary to rffiuctions in circulating vWF. a auri" molecul. for factor VIII. How..,... in contrast 10 human patients wilh vWD. FVIII : C in dogs with vWD i. uullily;> 30 % of the activity in ",f".n~ pl",,=, so PIT is usuaUy WRl, ev.n in dogs with tyl'" 3 vWD .nd th ..~fore no vWF.'.I.'U.,," PTT may bt prolongffi in horses with vWD." III.

Analytical concepn A. Sample l. Pl""m. from blood drawn into sodium citrate or EDTA; citrate tubes should bt fillffi 10 give a 1 : 9 volume ratio of citrate to blood. Th~ vWF: Ag = y bt =rkally decreased in sampl.. with don or in vitro h.molpis. bUI lipemia has no significant ~ffect"7 2. Th~ vWF : Ag is rq>Onffily st.ble for al lrast 8 h in canin. plasma or whole blood storffi al room temperature." How..,... values .'" Jignificantly increased at 24 h aft .. collection when whole blood sampl.. are storffi at room temperatu .. , and values .'" inc",.,..,j at 48 h (but not 24 h) aft" coUection when pl •• ma i. storffi at room t~mperature." Th... iner...... s did not occur wh~n sampl... w... re&ig~ratffi. It is gtn~raUy recomm~ndffi dut pw= bt collectffi promptly .fi~r blood collection, frozen, .nd mipped onrnight with ice. B. Unin: % . UldL, or UlmL rdatin to 100 %, 100 UldL, or 100 VlmL, r.. pectivdy, of vWF: Ag in poolffi plasma from healthy individuals of the same species. Units may bt written to indicate the species of the pati~nt and ref... nce sampl.. (e.g .• CUldL for

5/ HEMOSTASIS

e.

[v.

,.,

conine {q s;omples}. Vuiations in th. comp",ition of pooled pl:umo from the ",ferenc;., aninuls can affect resuhs. vWF: Ag >.<Say........." l. ELISA: vWF i, Ulu:dly measu=i by a quantiutive: EL[SA with 'peci""pn:ific antibodies to vWF (i .•.• vWF: Ag). V:du .. < 50 % u. usu:dly comide=i d",,,,.... d. ELISA testing has lugdy replaced drctroimmunoassay methods involving dectro. phoresis of pl:umo vWF in agaro .. gds containing antibodies to vWF. 2. Muhimeric .n:dysis {immunodectrophoreJis} a. vWF multime .. a", "p"rated by agarose drctrophoresis so that the rdativ• • mounts of diffi:rent sized multim ... can be determined. b. Differentiat.. typ<: I vWD {high molecular w.ight multime .. present} from type 2 vWD {high molecular weight multim ... absent} 3. Functional >.<Say, a. Botrocetin cofactor assay: Platdets agglutinate in the presence ofbotro<:e{in and vWF. The rat. of this vWF-d.pc:ndent plotdet agglutination corrdates wdl with the amount of plosrruo vWF: Ag except with type 2 vWD. [n typc: 2 vWD. botr<>«{in cofactor activity moy be markedly reduced while the .mount of plalma vWF: Ag moy be WRI or mildly decreased. This is becouse the mo", funnional large: multime", are d.fici.nt. b. vWF collagen.binding activity (vWF: CM) moy be m.... ured in canin. plasmo by an ELISA that det"'ts only the vWF that can bind to collagen. Th. "''''y therefore measur .. the quantity of functional vWF .nd preferentially detrct. the more functional loq:. multimers. The ratio of vWF: Ag to vWF: CBA moy be increased in type 2 vWD becom. of. dea.ase in large multim....nd therefore • g",.ter d",rea", in functional vWF than in vWF antigen.

Decr.a..d von Wdl.br.ond factor to antigen {vWF: Agj ratio (Table 5.3) A. D",reased plasma vWF : Ag is indicative: of th. vWD trait or a carrier nat. for it. dq>ending on the dq:re. of the dea..... Clinical.igns of impai=i hemostasis may not be pr... nt. Th. following are common guiddin.. for ELISA vWF: Ag resuh. in dogs:" l. Dog. with vWF: Ag v:du .. < 50 % .re considered carri... of the vWD trait. Thry ue at risk for clinical di, .. '" and u. likely to tr.onsmit the trait to offspring. Th. rid: of clinical vWD i, greater .t low", vWF: Ag values. 2. Dogs with borderline vWF: Ag v:du .. of 50--69 % are at little or no ri,k for clinical di",.... but may be curie.. that can transmit the trait to offipring. Rc:pc:ated teniDi: may clorilY if affected (v:due < 50 %) or not (value> 69 %).

Table 5.). Disea .... and conditions that cause deaea ... d vWF, As vWD: vWF : Ag ll'luaUy < 35 % ' Typ<: [: all vWF multimers present but proportionatdy d.ficient Typc: II: deficiency of vWF multim.rs with high M, Typc: III: ab",nce of.ll vWF multime .. , vWF : Ag .. 0 % 'vWD corri" (rardy symptomatic): vWF: Ag us,",lly 30--70 % • A lel1tively common di..... 01 conditi"" Note: Hemolysi.! 01 dotted umpJe.. lJUy "'.... m:uhd fili< d.creases in vWF: Ag nlUH. vWF: Ag uni,,: % of <=lIt fOJ" plasma poo&.d from t...ltby
'68

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY 3. Dog. with ty~ 3 vWD h."" . .... mially no vWF: Ag.

4. Mon doc'

cru.t bl~ ba::.u"" of vWD ru.v. vWF: Ag values < 35 % .

B. DoC' with at I•• n 70 % plasma vWF: Ag.", oonsid,,«1 frtt from Ih. vWD trail. Thry ,,< nol at risk for dinical dj"", .. and Ita"" a vtry low ri
Im.rpretivr comiderations A. Stt the forogoing KCtion {Analytical oon~pts, sa::t.

m, A} for df=, ofimproptr

"'mpJ. p,o.:;.,Ming. B. Intraindividual biological variation: Th. amount of plasma vWF : Ag has bttn shown to vary ooll!id.rably from day to "'"y when hnhhy or di",asaj (vWD) dogs ...

.ampl«i ..,ially." C. Exe,ci..,: Amounu of lh. p[",ma vWF: Ag in dog. fll"Y bot incrrasffi after ""'1 nt.nuous ."ore;..,.'''' Th. vWF: Ag in ho"". inc..asnl as mum :as 100 % within minm .. aft., SI",nuou, aerei ..... D. Pregnancy: Subst:mtial inc,",=. occurl'ffi in bitch.. at parturition. with less" incr~=. in th~ last trimest~r of pregn:mcy and for th~ fim 1- 2 wk aft~r parturition." E. DDAVP ( 1-d~amino-8_D_arginin~ v:lSOpressin}:" l. DDAVP incr,"" ... th. pl:uma vWF: Ag in dogs with ty~ I vWD or in dogs without vWD by rd~a,inc vWF from stores. probably in . ndothdi. l edt..'" [mproval h.mosusi. aft" DDAVP =y not b" du~ to p,",f~rential rd ..... of larger multim. " :as in palpl • . [n on~ study. th~ vWF: CBA incr~=d proponionally to th. vWF: Ag aft~r DDAVP administration to dogs. without . disproponionat~ incr.... in large: multimers." 2. DDAVP am b" usa! dfective:ly to incr~= the amount of vWF in blood donor animals wh~n .dmini"".d 30-90 min prior to blood collection." F. vWF: Ag values may:dso b" incr..,ffl by . pin.phrin . ... ~ndotoxin." azotemia." liver di ......'" .nd oth~r illne:s.=. VI.

G~nHic

tem h.ve: bn:n deve:lopffi th. t am dHa::t .som~ vWF gtn~ mutations in ~r:d brttd. of dogs :md may b" u..ful in detecting carri.rs in .pecific breal .... "' ..

COAGUlATION I.

Phy.ioloCic process . ..·~ A. Coagul.tion is not .ynonymoll'l with hemost:uis. It is only on~ compon~nt of h~mosta_ .i. and .lthough intimatdy associata! with ~ndothdi:d cdls. platd~ts. th~ fibrinolytic .yn~m •• nd oth" blood ce:ns. it is distinct ftom these rompon.nts .nd c:m b" rvalu.ta! indeparatdy in vitro but that have: considerabl. cross talk in vivo {Fig. S.2}.

5/ HEMOSTASIS

,.,

l. TF {~minsic} pathway

a. This pathw:l)' is initiatffl by TF npre;d on v.scuJ.r smooth muocl~ cdl.. activatffl monocyt ... or other extr:ava>cul" ""lis. Endothdial ""lis do not normally np .... TF. and it is not ""rrain wh.th.r th..,. do wh. n nimulatffl. b. It .ctivat.. f:actor X .t the stan of the rommon pathway. c. It is inhibitffl by TFPI. d. It is imponant in initiating thrombin g.n.r:ation. 2. Surf."".inducal (intrinsic) pathway a. This pathw.y is initiatffl wh. n the ronract coagulation facton HMWK. PK. and f:actor XI[ ronract n.gnivdy clutgffl surf.= (•. g.• coU.gtn). b. It .ctivat.. f:actor X .t the stan of the rommon path"..ay. c. It is inhibitffl by AT. prouin C. prot.in S. and protein Z. d. It is .lso .ctivatffl ~nd"y to TF and common pathw.y activation. {I} TF·YII •• ctivates factor IX. {2} Thrombin activat .. factors XI .nd VI[1. •. It is imponant in propagating and .mplilYing thrombin gtn.ration initi<>tffl by the TF pathway. 3. Common pathw.y {common continuation of surf."".inducal and TF pathways} a. This pathw:l)' is initiatffl by activ.tion off:actor X to f.ctor Xa by .ith.. the surfa.ce-inducal path"..ay or the TF pathway. b. It l.-ads to th. formation of thrombin from prothrombin by the prothrombin... compln. c. Thrombin acts on fibrinogtn to g. n.rat. fibrin monom... and th.n multim.rs. which ... cross-linkffl via factor XIII •. d Th. pathway is inhibitffl by AT. prot. in C. prou in S. and prot.in Z . • . Thrombin activation of factor V amplifi.. th. common pathway. E. Coagul:ation factors (Tabl. SA ) l. Enzymatic roagulation f.ctors circul ate •• imctiv. pr""nzym.. (zymog.n.) until th..,. are activatffl. a. Th..,.:ar. producal primarily in h.patocyt ... Th~ production offactors II. VII. IX. and X is vitamin K d.~nd.nt (Fig. S.3). Th. "K" in viramin K i. d.rived from th. G..man KDagulatiDm Vitamin (i .•.• "coagulation vitamin"}." b. Factor IX i. sa link ffl . [u !:I'n, is on the X chromosom •. "'... c. Half.liv.. of pr""nzym.. in h.alth: In ~pl •• half.li"... t:lng. from. few hou", (f.ctor VII) to "",ral da,.,. (f:acto", II and XIII). with most t:lIIl:ing .bout 1- 2 d." Half.lives of coagul ation factors in domestic .nimals a.. presumffl to b. simil ... Th. half.lif. of factor VII was < 5 h in f.ctor VlI-tor and cofactor for factors VII .nd VI[ • • • nd the major activator of coagul ation in vivo. It Gm ndiat. ctll signaling and

TF",,/" VII

l

TF-VIID

V, VIII, XI

fibrin XII/a

Fig. ';.2. Co.gul1tion coscod<. Tho coagubtion <»e:>oi< begins ..ith .ctiv:otion of th. TF (extrinsic) or swfx<_inductt.w.y. in vivo . • TF p1tbw:.y: It".... originally tbought to R<)Uir< utuvurular . "i",n;on and ..,.. th.refo", ... mod extri~n... This p. th.....y is initi ...d by TF relu>«l from, or .. p<>S"c0v:0"'" monocyt .. , aucropbages, rnd p<>SSibly . ndoth.Jial ""lk Th_ ""lis ern b. oro",,"'" by rndo"";n rnd =Clint.. f>ctor X (common p>tbw:ly) rnd f'xtor IX (.un."".indue«! pathw.y) in tho p'C\ll", injury. In vitro. k""lin. ,i~c .. C<~"'. di. toauc<:e-indu""d p. th...-.y i.s thought to propaga'" coagul. tion via tbrombin feedback on f.tcton Xl. V and VIII. Swf..,. :ocIintion p1"J'"" minor roL. in :ocIinting coagubrion in vivo: pati.nu with .ingl< contact factor ddicienci .. (•. g .• f'xtor XII. HMWK, or PK) do b.Y< clini",l bl.ed.ing dioordl! n.

,,,,,,J,,,

""tor

'0

no'

270

211

5/ HEMOSTASIS

.p~.rs to pmicip;>te in =eral nonh~monatic proc..... ' (~.g., 'DJ:iog~nesi, .nd meuswis). b. Fibrinugm i, a positin acut~_ph,..., prot~in pnxlucrd by h~p;>tocyt ... It hOIS a half_lif. of 2- 3 d in h.-ahhy dogs"" and is conmmal during co~ .tion :as it i, conv-nal to fibrin by thrombin. c. Factors Y and VIII markally accd~rat. coagulation by f.cilitating surf""" a({achm~nt and locala.tion of co~.tion factors. (I) Factor Y may bt producrd in hep;>tocytes, meg:obryocyus, lymphocytes, and v:ascular ,mooth mu..d~ crlJ.. 71 It h.. a half_lift of about 0.5- 1.5 d and is consumal by Arc during co~lation. (2) Factor V][[ may bt producrd in muhipl~ cdl typt" but h~patocyt .. apprar to bt most imporunt. (a) It i, sex linkal. It. g.ne i, on the X rnromooome." (b) It circulatrs in a nonroval~nt complex with vWF but i, distinct from

,WF. (c) Th. half_lif~ of human factor Villi, .bout 0.5 d, but it is Ie .. in the abs.ncr of vWF, its carrier prot.in. (d) It i, consumal by APC during coagulation. (.) It i, a "",itive acute-ph ... protein, iner...ing in pi"""" with eRrci.. and inflammation."''' (3) Factors V .nd VIII ..., ronsumal during clotting and at. not prestnt in .. rum. d. EDTA, oxal.t~, and citrate function:as in vitro anticoagulanu by binding fCa""' and pr...",nting it from interacting with coagulation proteins. In vim, ther. i, alwaY" .nough fCa""' for roagulation "".n with hypocalcrmia. F. PhY"iologic inhibitors of coagulation hdp pr""~nt acessi"" co~lation. Deficiencies of these anticoagulanu are associ.tal with thrombotmbolic di ...... Som~ of th~ inhibiton con bt m~alUrai

Fig. So2. ~ti~~"

• Common path..-.y: This is th. COJIUDOn rontinu .. ion of the TF and mrfioco-indu=! path......}". beginning with ><1iv .. ion of net"'" x. F:octo. X. rompl .... ..-ith net"'" V. (><1iv ..«1 mostly by thrombin) and fCa" on. phospholipid surne< to form the :octi¥< pl'fI,hytmlbiNlJ' """'plex• ...mcb mults in the . nzymatic ",,,,",,"';on of prothrombin (fxtor II) to thrombin (fxtor II.). Thrombin then ><1iv;.... put<1or XIII., in tb. p.... ne< of fCa". c"","lin'" fibrin and .. infor"", the ..-oonw'Y hemot",ic plug. • Prot';n pro-rof.octon (facton V and VIll): Proteolytic cle.vage le.ds to cof.octor ><1ivation :and :o<:<JJl"' through fxtor Xl •. • Prot';n C: APe in><1int .. f:oc.o.. V. and VIll ., .nd promot .. fibrinoly>i.t vi. t_PA. • TAFI: TAFI. formed vi. thrombin and thrombomodulin inhibits fibrinoly>is by cle.ving pwminog
1 't

d

~l~" ~ > ~ lt~! l1t~

lil~11 ~Jt~ ~ ><

t

t

n},

H;d

~~l~ ~~ I Jl~ - ~ i~;~~x=~~~ /:S~~~ • • • ~oil

1

~a

~

]1~~e

.

~li~i ,,~

'1!

!

~~~~~J~J~~~~~I~r!~~

~ t>t~ · J ~ l~ HHflJJlf l~

JwI 111JlwW!f:Jl

l JJ

, , j,lljjjj

~

j.1{

! l

H l 11 1"

J

i i i j1 - t j; t" -j! 1 II l t , i _ lj 1: I f j ~ 1 ljIH!! I; •• . j

W

Jtl

- g _·

..~. "'~ _= '"

b

a

0;

f

_"'

~ ]~1

.. j

1~ ~

'I ' !'"z~ 1::

_",!ii ..,

c;:.S;:S;:1:Sx>1>1>!

]

:dl:

5/ HEMOSTASIS

213

Liver

,._., #

..E.' ":',m;. K

I,~_m

vitamin K epoxKl&

___ --

Corboxyl~!ad

FM:!ors II, VII, IX, X Blood Fig. Sol, Syntb""is of vit:unin K--deptM md also produe<1ion of bilo .ads. • In hepatocyte<, vitamin K b.co""" roduc«l to in .ctiY< fo,m ('MUC«l vitamin K). Redue
I. AT (ilion for ATlIlj i, th~ mojor inhibitor of coagulation
aNivity.) a. It is a prot~in (M, " 58.<XXl) producal prirn>rily by h'p"tocyt.s. b. It bind., inactivates. ~nd ",moves most coagul ation .nzymes from ch. circulation, most imponandy thrombin (f~ctor lIa). factor lx.., and factor x... c. AT... nzym~ oomplo:.. are rapidly d ... red vi<> rrceptors on h'p"tocy<es.'"

274

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

d. AT activity is m >rkffily enh. nad by heparin (exogtnous, or endogenous from mast ",lis) and hqJar.m mlmle on endothdial <,<,Us (Fig. SA). 2 F'rotrin Cis. viumin K--lor present on mon endothdial cd] membran ••. c. APC then inacciv.t.. f. cton Va and VIII. by proteolytic cl •• V3i:' in the pr=n'"

of f:lelor V" and m.mbran~bound protein S {the S nand. for ·~.ttl.:

wh".

th. protein ~ first d.scrib«i}!' d. Prorrin 5 is • vitamin K--eior productd by endothdial cdh. hq>. tocytes, .nd megakaryocyt ... • . Additional anticwgulation occur> by this pathway b.au"", thrombin bound to thrombomodulin cannot d •• "" fibrinogen. and th. romplex is int>tocytes. It also circulates in pJ.tdm and in pl.!ffiO, mostly bound to lipopro"ins. In the pr=nce of ft:.", TFPI inhibit. TF_Vlh by forming •• ta bl. quater_ nary complex: TF_VIl ..... f. ctor Xa- TFPI." Thi. inhibits furth .. g.n ... tion of f:><:to," DCa .nd X. via TF_VII .. 4. Oth.. circulating inhibitors of co~ulation indud. PZI, hepo.rin cofactor II, oc,_macroglobulin, and ct, _prot.in .... II.

Analytical concepn A. .........y optima..tion for on •• p«ies (e.g., humans ) does not n=ssarilyoptimize for oth.. 'p«ies.7>-", V.., ..inary assay. for routine co'4:"lation testing are not .tandardized, and many are not optimized for the speci.. b.:ing tested. This compromises their ",nsitivity in d.tecting impaired coagulation. B. Proper .. mpling .nd "'mple handling are critical for .CCI1rat. results from most co~ulation """Y"C. Sampl. collection I. For most test" u.... citrat. v.cuum tub.: or plastic .yringe rontaining the right

amount of citra" for the volum. of blood to b.: drawn. Drawing into. 'yringe w ithout anticwgul:mt .nd th.n tran.f.. ring the sample to a citra" tub.: is not rN:Omm.ndrd, b.ausc: co'4:"lation may b.:gin duting v.nipunctu .. and prog .... far . nough to .It.. h.mostasis test results prior to mixing with the citrat •. 2. Sampling through nonhq>arinized cath.ten may b.: don., if necessary, .fter flu.hing the cathmr with 5 mL ",lin. and diKarding.t I.... t the first 5 mL of blood removed {.t lea,t.il: times the catheter d.ad .p""'}....... 3. Sampling through hq>.riniud cath..,." mould b.: avoidrd, though a .imilar flush_ and_discord approach producrd results for /Mil/thy dog. that did not differ .ignifi_ cantly from direct venipuncture result .... 4. Care .hould b.: tak.n to minimize a.ctiv.tion of platd..,. and th. ro~ation and fibrinolytic 'Y'''ms.

Liver

Kidney

_

proIhrombin

•

IITombin

fl2l"

~ IITombin-AT c;:onogeX

n

""fracliooal.cl Mparin & heparan IUN.~

5.".

Fig. Antithrombin path",)". AT .. tbr rrujor pilysiologic :mtiooagubnt in blood. It .. ptic.Uy. AT activity i, mo.rloodly ~nItanood in rhr preompt.us (TAn d.ioo<>ci.", from rhr hrparin 0< hoop....., ouI ..", and _ d .....1 From circula.i"" by htpotocyt<>. 1M hop"';n and hop ... n MIIh .. K' .. eml,.. ... and ••• l.aibb40 (4) for fonning mo .. oomplaa. An,ic:oacula. tiM by AT ""d hop ..... limi ... . "aII ..... dotting, bu. AT and hrp'.in do not inh.ibi. aoagul.ation """l""* bound to 6brin or pla,.1ns. lberd"Oft, loeaIi...-d and a>ntrolled coagula.ion an pr<><:eed ..b..., ....oed. UilWH moJ.,rnt.. also ~ o:>nfunnationol dtangn in AT th ... nalW i, to bind to and inbibi. f""to< Xx, bu. ".,. .brombin. • o..c.eued pLum. AT activity and _ntrotion O«W" via ...... ru"'f";"" "'''''0 intraYuculu coagubrion .. inctaOed 0' af... injecOon of."ogmow hepa.rin. Deanoed COllcaluaOono may aIaoOCCW" from dea.-.t It.epWc production at from ""CUI;"'" lou d ... to poot<xu. """",d..y to ."" prodoct;"" of inBaaun.-y W1.

275

216

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

a. Tnum.r;c v.n;punctur. «poses blood to TF, thm initi.ting oo3l:uJ.tion that can produ~ dOllrdumples or em", pJ3td~ .ctivation :md clumping. b. Fredy Bowing blood should bt coUectal by "clean" nnipuncrure on the first

.Iumpt. Probing with the nNdt., ..mpli"g through" hematom., or airing and 'Nm
r<::stffl. c. If coUec{;on is difficult, a n~ ""in mould bt .decta!. d. An ex=;"" v.cuum =1 caUst turbul.nct and pbtd.t activ.tion. •. Th. fim f~ drops or , complete rub. of blood may bt discardnl to dec"" ... TF in th. em sampl., but this is not n~ssary with "cl.an nick.." 5. Th. blood .nd :mtico"l:ulmt should bt mind immediately and thoroughly, but

gtmly. D. Sample I. Mon co"i:"l.tion tests: c;trat.d pl:asm. 2. Point-of-co"" instruments: citratal whole blood or citntffl plasma 3. ACT: whol~ blood in 'Pfi'itomaaous nrth 4. u-....Whit~ dotting tim~: non-.mtiooagulatffl whole blood tellffl immffliatdy 5. Citratffl samples a. Siliconiud glass tubes ha"" I>«n u.=I routindy for hemosusi. tming. but the use of plastic tubes is b.:roming common. Although statisticaUy Jignificont differences in ten =ults havt bttn document"! in comp:uisons of plastic and gl... tubes with human .. mpl ... diff~"[I(:C" havt oon .mall and rudy of clinical .ignifinnc:e." b. PlasJrul or whole blood should b.: antiro;;ogulatffl with trisodium citrate at a 1:9 (anticoagulant to blood) mio by using 3.2 % or 3.8 % cimte tuoo {check with the laboratory for • p.. fer~n""}. c. Th~ citrau oon""ntration .ignificontly affect, th~ ..suit. of oo:.gulation tem on hUJruln .. mples {3.2 % is usually recommendffl) ... ..,·· and it con have .ignificont ~ffects on some results with conine .. mples." The m3J:nitude of the ~ffect i, not known for most oonditions or most sPfi'ies. but a standard citrat~ con""ntration with refe .. n"" intervals generatffl by using that citrate con""ntration. instrument. and method is recommend ffl . d. Similarly. it i. important to Jrulintain the 1:9 citrate to blood volume ratio b.:cause overcitratffl5amples (too little blood) may havt r..!uc:..d ooaguJ.tion activity (prolonged times) and undercitrated samples (too much blood) Jruly b.: hypercoagulable (r..!uc:..d times). Problems as.!OCi<>t"! with mboptimal filling of hUJruln .. mples appear to b.: greater with 3.8 % citrate tubes than with 3.2 % citrate tubes." e. Evtn with th~ correct blood volume. Hct may affect the appropriate .mount of citrate to me!' {I} With anemi<>. plalma Jruly b.: und.rcitrat~d because ther. is mo .. plasma with the sam~ amount of citrate. This may result in falsely shonen..! coagulation times. but the dq:r .. of the .ffect was of quenion.bl. dininl significan"" when PT and PTT results for unadju<;{ffl sample, from anemic hum.n patienu were comparffl to PT and PIT results &om pairffl .. mples adjust"! to 3.8 % citrate.'" {2} With erythrocytosis. plasm. may b.: overcitratffl because ther~ is less plasma with th~ same amount of citrate. This may prolong roagulation times

5/ HEMOSTASIS

217 ba:.use th~ exa:s.s citm. binds too much of th~ C~l<- add..:! to th~

'=y

sY'um. {3} Drcr=d cicr.u to blood mios ~r~ ra::omm~nda! wh~n Het :> 55 % in ra'pl . ...... Incrns.d ratios m"}' ~ indic;ot..:! for Hct < 25 % , but th~ nttd has not bttn drarly shown." {4} Th~ following fotmul. {Eq. S.I} may ~ usa! to calrulat. th. citr>t. mlum. rrquir":! fot ~nticoagulating a volum~ of blood with a ginn Hct:>55 %: C = 0.002 X {IOO - Hct} X V C: cicr.t. mlum. (mL) Hct: blood h~matocrit (%) V: coll= ..:! blood mlum. {mL}

(5 .!.)

E. Sampl~ processing and subility" l. Tim. and t~m~ratur. ~for. p""", .. ing" a. Excq>t for whole blood clotting =Y', studies of human blood indict. that whole blood .amples may ~ rdJig~rat..:! or hpt at room t~m~ratur. for up to 8-12 h (PIT a.!S>y for nonhqmini=:l pati~nu, IT a~, and coagulation f.ctor ~ruolY'.. ) or up to 24 h {PT =y} ~for. processing.""" b. Ho~r, a good g<:n~ral =omm~nd ation is to "",ntrifuge and r~mov~ plasma within I h (room t~m~r;oture storage) and test within 4 h of "'mpl~ collrction, b«:ausr thi, tim~ &.IIY is n=ary for ctnain t .. u and ctnain sampl......., 2. Cc:ntrifug.tion .nd plasJrul h.rvest a. Aft~r ronfirming th~ abs.nct of dots in the sampl., blood should ~ ""'ntrifug..:! for I ()""I 5 min at high g_forct (e.g., I 5 min .t 1500x g), and the platd.t_poor pl.. JruI should ~ r~mov..:! by plastic pip<m. b. PlasJrul that c nnot ~ te'ta! within 4 h should ~ froRn. c. Excessi"" platelets (10- 200 X 10'JIlL) remaining in th~ plasma b«:ausr of inadrquat~ ctntrifugation forces will not int~rfere with PT .nd PIT .ssaY' for routin~ diagnostic work but will int~rf~", with h~parin monitoring {vi a PIT ..uy} .nd test. for oth~r inhibitors of coagulation ...... 3. PlasJrul 'to~ time .nd temp
'78

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

d.

R..frig~ratffl

{I}

plasma stongt

pw.m. from Mil/thy dog. had Jess stability at 4·C than at room t~m~r:I.

CUre. Values for f.ctor. VIII and IX w..e daoreastd by 48 h (th<7 appta'..!

to k dropping by 24 h), and PIT Wll prolongrd by 72 h {it .ppta."! to dropping by 48 h}." {2} [n a similar study of human plasma, PT was S{abJ~ for only 24 h at 6 'C, and factor VIII activity WlU de<:r~asing by 8 h. Samples from h'pariniyd pati~ms w~re stable for 8 h, although .ctivities of factors V and VIII were dropping by 8 h."

~

•. St.bility of value. in .... mple< from sick or hepariniud dogs h.., not bttn rq>Onffl .nd may differ from st>bility in samples from hrahhy dog.... Ef&cts of hqJarin may k decr•• ....:1 with of pJatdet factor 4 from platdets duriIll: no~." f. When mailing ,amples for testing. froun plasma should k pachd in jet and mailal to arrive f'DUII within 24 h. ( I) S;,mple. should be, testa! promptly after thawing, :md thawing mould be, rapid to minimiu cryopr«ipitate formation .nd thetefore 10.. of hemostatic facton, {2} Concumntly collecting, pr=ing, and mailing. sample from. healthy patient of the same ,!",cies am be, used as eviden,,", that sample handling did not indu,,", abnorn",l /'<::Iules, <\, Hemolysis: Sample. hemoly=:! by in vitro facton .hould not be, tesud beau", oo"l:"l.rion .nd platelets may have bttn activata! by the .. me f.ctors responsible for hemolysis, F, Instrumenu: A v.riety of automata! coagulation analyzen, including point-of-core instruments, are rq.lacing manual .nd fibrometer methods of measuring coagulation times for human :md veurinal)' .. mples, Specific methods and .. mple requirements val)' with the analyzer, End_point detection of. fibrin dot varies with the type of analyzer, L Electromechanical methods a, ElectriGlI conductance: [ncre..«i electrical oonductanct is detecta! betw«n a sutionary electrode and a moving electrode as fibrin forms (fibrometen), b, Electrom"l:netic viJCO.ity: Analyze" detect decreased movement of. small metal ball suspend«i in the lanlple as it osciUates in an electromagnetic fidd, 2, PhotooptiGlI methods detect changes in light transmission or light Jamr as fibrin forms, 3, Optical methods may function better with a low [fibrinogen] but may generate inaccurate results when optical interferents are pre"'nt (e,g" lipemia, Ox)-globin, hemolysis, or icteru.), <\, Synbiotics SCA2CXXl Veterinal)' Co"l:ulation Analyzer: The dotting end point is ba=! on optical detection of decreased ntovement of. small volume of blood as it is pumped bad: and forth within. ch:mnel in the test cartridge:, G, General analytical.pproarn: In vitro analysi. enabl .. independent testing of different pam of the coagul.rion web (.intplifi«i to a ''Y" ) so that defects may be, localized within the surfact_induced (intrinsic), TF (utrimic), or common path"..a)" (Fig, 5,5), Testing generally bq;ins with common "'r«ning um, with more specialized tests u.s«! when indicat«i, H, Severe hypothermia IIU)' contribute to hemorrh:.ge, in pan beause oo"l:ulation is a tempenture ",nsitive enzymatic pathw.y, However, oo"l:"lation testing i. routinely

,.1.=

5/ HEMOSTASIS

219

"' ,, "

",

VIII

PTT "

,,

x V

ACT '

" I

, ;:TT

Fibrin .. Fig. s.s. &h.JJUtic "'p",.. nution of th. co.gubtion cascode .. ...,.]u.t. Fibrin fo,m"ion;' th. ond point fo,udt "' ... PTr rnd ACT .-nlu"o =..,.,I"ion f.oClon in th. mrfac<·indue
_..

done at or near normal body tem~ .. ture, so routine assays may not idomi!y hypothormia.induced CO
Whole blood clotting timo. A, lff.Whit< method'''''·' L Thi, i, • ,randardiud, but ..rdy u...:!, in,onsiti"" mrthod of scretning for d.f,""cs in tho ,urface-induced :md common pathwoop that u,"" non.anticwgulataJ wholo blood immaJ;"tdy .fter coU,"",ion, Activation of cwgulation is init;"tal by tho glow tub., 2, Protocol. r«lui .. multiple analyses, st:mdardization of blood ""lumo, cle.n glass u., tub.. of a nandard ,ill:, :md a wooter b.,h (25-37 'C), ~oral modification, of the method h."" been u.s..d. ,0< V.r;"tions in protocol, induding ""nipuncture t (Itt Coagulation, =t, IV), B, Activatal CO
200

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY a. Surf."",.inducal activation: Blood (2 ruL) is drawn by .traumatic illl:u1u nnipunClur~ imo a p ....... >rntM (37"C) ACT vacuum tu~ ronuining ,ilacn>us (di.tollUceous) ~.nh, which acrivau. the mrf=_inducffl {intrinsic) pathway. b. The blood is mix..! by five in""",;o"" and the ru~ is inrub. tal (37°C) for 60 •. Th. IU~ is th.n ch~kal {visually while tipping it} ....'1 5 or 10 s for th. first

ddinite

~id.n""

of a clOi.

c. The tim. from contact of blood with s.ilaca>m ..,mh to the initial ddinite signs of a clot is the ACT. d. Units: ,,",,,lid, (nrarm 5 or to., d~nding on the f=iu.ney of inspffiion) 4. Interpreti"" colISid .."'tiom a. lnt.rpr..ution is similar to that for PIT (Stt Coagulotion, =1. IV), but th .. 1m has Jess diagnonic stns.itivity .nd may requi .. :> 90--95 % deficiency of a single fxtor for a proJong«i result. b. ~""r.. thrombocyto~nia « 10 X 10' pludm/J.lL) is commonly stat"'" to prolong ACT, b=ou.. of dee""as.,d phospholipid availobility. HoW<"Vrr, ACT v:dues m.. y bt WRI with ..",~re thrombocytop
Activ.tal panial thromboplonin time {PTT, APTI, or aPTT)'" A. A scr..,ning coagulation t.. t of th~ surf."".inducal and common p. thways B. Principle and method l. Surf. "".inducal activation: Citrat"'" test plasma is incubat"'" {37 'Cl with an a""ss of proro,,!:ulont phospholipid. (partial thromboplastin) .nd .. surf."" .ctivator (~.g., kaolin, silicate;, or dlagic acid) to activate th~ rontact f.cton. Th~ chd . tion of ft:r+ by citrate in th~ sample limiu activation btyond f.ctor XIa. a. The p.nial thromboplastin ""agtnt consists of procoogulant phospholipids dr-void ofTF, .nd th~ ..fore th~ ",,"Cent i. unable to activat~ th~ TF pathway (f. ctor

VII). b. In th~ orij:inal P1T assay, th~ glass tubt {rather than add"'" partimlat..} v..as the surfaa: .ctivator.''' To diffrrentiat~ tests with .nd without addal particulates, th~ current tm is r~f~rr"'" to as the IlN;/Kl,d p"rtill{ thromiNIpfilstin tim~ (APTI or .PTT). Howo-v<:r, PTT is uonl :u th~ abbreviation in this text for simplicity. Th. origin:d t~st without .dd"'" particulates is no longer usffl, '" thrr~ should k no confusion.

5/ HEMOSTASIS

281

Tabl. 5.5. Disca .... or co ndition. that may caus. prolonged PTI Ddici~nci ..

of functional.urfaa-induced or common pathway CO~ul'Hion facton Acquired (usually muhipl~ d~fect.) Decr ... sffi production 'H~patic d~ (necrosis. cirrhosis. pono.yn~mic .hunt) Vitamin K .nugonism or ab..,n~ ·Decr... sffi vitamin K recycling in hepatocytes: antic=gulant rod~nticid~ ingcnion. coumadin overdose. moldy sw..,t clov~r ing..tion {Mdiluru, .pp.}. ph~noprocoumon toxicity Dec=d vitamin K absorption: biliary obstruction. infihmive bowel di"'.... chronic oral .ntimicrobial•• exocrin~ pancr... tic insuflici~ncy Incr ... sffi co~.rion factor inactivation .nd ronsumption ·Di...,miruoted intraVllKUlar coagulation or coagulopathy Localizal consumptive coagulation or cwgulopathy Dilution of roagul.rion faCtor.s: .cut~. massive blood loss u ... ted with massive trarufu.ion of plasm •• poor fluids; e.g.• packed RBC. and crystalloid or colloid fluid •• including Oxyglobin "",Iurion H~redimy {wually .ingl~ defat. but may k multipl~} Intrinsic pathway: PK, XII. Xl. IX. Vlll Common pathway: X. V. II. I 'Inhibition of surfaa-induced or common pathway co~ ation factor.s: heparin. FDPs • • nti.(phospholipid.prot~in) antibodi~,. antibodies to coagulation fxtors • A rcl ..i""ly oommon disc ... or condition

Not<: ACT may :Wo be prolonged in most of th... conditions. but ACT prolongation cequir .. V' .... ddicits in factor :octi';tics.

a defined incul>ation tillY •• measured amount of pr~rmed (37 'C) calcium is .dded to counteract the efrec" of ciuate and ~nable the =d~ of coagulation to proettd to th~ formation of fibrin monomer•• which polymer;", to form .n insolubl~ fibrin clot that is dcrect~d optically or datromcch.nically. dep"ndiJ\i: on th~ irutrument. TIu tim, from "ddif;on Df,,,/c;um chlu,.;k fI} ,lu, d#«tiDn is fb, PIT. 3. Units: seconds {usually r~port~d to nearest 0.1 •• though rq>nning to the n~.r~st half.=nd or second may k more appropriat~ for som~ methods} 2.

Aft~r

chlorid~

C.

Int~rpr.tive coruid~ratiom

J. Valu .. g.... t~r than a valid upper refcr.n~ limit .bould k co",id~red prolonged.

V.lu .. arc ",metim.. interpreted by romparing them to v.lu .. from a concurrently t.. ted healthy .nimal of th~ ... me >pecies. but thi, i. not rcrommended. Neither i.< requiring a 25 % incr~= o,""r th~ URL to conclude that th~ PTf is .bnorm.1. 2. uboratory•• pccific .nd .pcci.... pccific refcr'n~ intervals mun k used kc.u", /"<Sui" vary substantially with variatioru in .pecic>. analyzers. r"'i:,ms. and protocol.. 3. As ~uine data indicat~. values in healthy n=borns {< 24 h old} may k great~r than .. fcr~nce interval. est.blished for .dult.. """" 4. Th, test is rdatively inscmitivc. bur. for optimized • .,ays. rcmlu ,hould k prolonged when there is .bout • 70 % decr..... in the a.ctivity of a .ingle

282

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY co"l:"luioll f:OCior (30 % of ap«t~d activity). [n on~ srudy of various PT reagents

and m.tho,h. th. f.ctor VII activity n=:l«i to kttp PT WRI v.n,"", from 16 % to 39 %.'" 5. Mild" rffluctions in individ,",J f.ctor .ctivities may k d.uctal when multipl. f.ctors are .if«taj. 6. PTT ..sayod at 37'C may ovtrestimat. in vivo coagul>tion in muk.dly hypoth". mic !"Ili.lI1S 1>«:..= enzyme activiti", "" temptntur. dep, hemolysis, Oxyglobin ",lucion, and icterus interf.re with PTf ......Y'dul d.t~ dot formation optically. 8. Ex~ssjvt cilnt. or improp (Itt Table 5.5) l. Pure defici"ncies of "ilh" f""cor XII or PK are typically not :associ.ta! with clinical h~morrh"l':e. but thq.", associata! with prolongal PlT remlu in most assay .ymms." A prolongal PTT cau..d by PK ddici.ncy Jruly be correcta! by increasing the incubation tim. of plasm. with th~ particulate surface .ctivator or by using eJJ"l:ic acid ror surfa"e .ctivation.·" 2. Carriers of h.mophil", cannot be detecta! by PTT because thq haw only a mod~r.ot~ decr=e in factor activity (.pproxiJrultdy 50 % of nornul). 3. Unexplainro prolongations in PlT. without other ""id.nce ofimpairro h.mosta!is. haw been "'portro in cats.'" E. Shortenro PlT i, not reliable ror detecting hy~=gulability. though inc",ase. in fibrinogen. factor V. or factor VIII with inBammation may mon.n th. PTT.'" F. PlT ha, been used to monitor unfr.octionated heparin th.rapy. but targets of prolongation u.srd for ~pl~ (~.g .• 15- 2.5 times basdin.) do not ap~ar to ",Beet appropriat~ :mtico"l:ulation in animals. T arget prolongatiofll vary with the PTT assay:md r~nt'. in part because !"Inial thromboplastin ",.gems have diff.rem ..,nsitiviti.. to heparin."' ·.. • PlT woos Ie .. prolongal in dog, th:m in pwple for a giwn plasJrul heparin activity. p".. ibly becau,. of gr....ter factor V and VIII activity.'lOlMWH h", minim.! .ffect on PTf because of reduced anti-f.ctor lla activity.'"

V.

ern.

Prothrombin tim~ also calla! one_nag<' prothrombin time (OSPl) A. A scrttning coagulation test of the TF and common !"Ithw>ys B. Principle and method l. Test plasJIU .nd a C."_thromboplastin reag<'nt {comaining phospholipid:md excess TF} .'" 5q>amely p~rmed (37 "C). 2. A m~asured .moum of th~ prewarma! C,'+_thromboplastin "'.gtnt i, forcibly added to a measured amount of the pr~rmed plasma so that TF will activ.te f.ctor VII in the TF pathw>y. Activ.tion should proceed along the common pathw>y so '" to form fibrin monom.... which polym~riu to form an insoluble fibrin dot that em be deteetro optirnlly or electromech:mirnlly. de~nding on th~ in
5/ HEMOSTASIS

283

Table 5.6. Disca.... o r co ndition. rhar may cause prolonged PT Ddici~lIci~.

of functional ~ularion facton in rh~ TF or commoll parhw;oy •Acquired (mu:dly multiple def<'Ct.); I« T abl~ 5.5 H~redirary (usu:dly .ingl~ d~f«:t) TF pathw;oy: VII Commollpathw;oy: X, V, 11,1 • [nhibitioll of coaguJ ation factors ill the TF or common pathway: FD P., OUl.ti.{pho.pholipid. protein) amibodie., antibodi~s to co3j;ul.rioll facton, som",ime. h~p",in • A ,.1";",,ly common dio.... or condition

C.

D.

E. F. G.

5. Reagtm. alld pl"'ma Jruly b. diluted to prolong PT and illcr..... the an:dytical sc:nsitiviry for d"'<'Cting .bnormaliries. Fibrinogell may b. add..! to dilut.d samples to ~nsur~ .d. rin h«ausc: of the presc:nc< of hq>arin ina.ctiv. tors ill m""r thromboplanin r<"al:ems, but it m.y b. prolongnl with c< Shortrn.d PT is not rdiable for detecting hypc:rroagul.biliry but may occur wirh increased [fibrillogen] or f. ctor V.'" PT i, usc:d to moniror w.rfarin therapy (rargc:r PT .. 1.5- 2.0 rimes balelillc). INR i. a unid... ratio {Eq. 5.2} used widdy ill hUJruln m.dicine to hdp nalldardizc: the r~porrillg of PT v. lues to hdp correcr for diffe .. na:. in rhromboplastin r,,,!:,,nts .mong laboratories."" [n Eq. 5.2, refe"'na: PT is the mean of a valid ",fe",na: im.rvaI for the speci.. in question, nor a random control .. mple value. The lSI {lmemational Sensiriv· iry Index} is a lIumb.r determined and provid.d by rhe rhromboplastill reagtm Jrulnufactu .. r for rach lor of re.~nt by ming • particular PT method. The lSI, which reH<'Ct. rhe rdarive .sc:nsirivity of the rrag<:m to factor deficiellcies, is determin.d by calibrarion ag.insr all imemarion:d ref~rena: preparatioll. Thromboplastim with higher lSI ""lues .re I... sensitive than a", thromboplastins wirh lower lSI v:du ...

INR = ( Patiem PT Referena: PT

)W

l. The IN R Wll.! devdopcd .nd u,.d for moniroring or:d .miooagulant rh~rapy

{w.rfarin} ill people.

{5.2.}

284

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY 2 It m ay ~nabJ~ rno .. m~.ningful romp •• ison, of PT results among labon tori... but does not dimirulte .ignifie>nt interloboratory diff... nq,.'" 3. A ,.liable ref... nce m..an for th. sp«i.. in question is raJui=' for v..!id "'" of the [NR a. If. locol human hospital i, UIM for ro~.{ion (est, of veterinary !"'tienu, INR "sldts may bt reponffi by using . human n"" me.n. Sum values " e not

,.f...

v:olid for nonhuJrulII ""mpl... b. Sub,rituting a control sample value for the ,d'",.nce ruran is not '=ptobJ •. 4. Exampl.. using ch.1 NR a. Diff... m pa,ient> and diff"ent thromboplastin 'liem I. [SI = 1.5: PT = 12.0.; n"" mean = 8.0 • {Eq. 5.}:!} {2} P.tient 2. [SI = 23: PT = 12.0.; n"" mean = 8.0, (Eq. 5.3b) {3} Patients I and 2 had the same PT (12.0.) when patient 2 was teJlM with. I... ..,nsitive thromboplastin rli.nt I. [Sf = 1.5: PT = 12.0" r~fe .. no:: m~.n = 8.0 , {Eq. 5.}:!} {2} P.tient I. IS[ = 2.3: PT = 10.3" r~fe .. no:: m~.n = 8.0, (Eq. 5.3<:) (3) Wh~n patient I was tesud with th~ less ..",itin r
,.f... ,.f...

([")" ,.0

Patientifll NR = _ .-

= 1.8

([")" = 2.5 .= 1.8 (_103)" ' .0

{5.J a.}

Patient if2 [NR = _ .'.0

{5.Jb.}

Pati~ntifl[ N R =

{5.Jc.}

VI.

Thrombin tim. (TI). allO caUM thrombin dotting tim~ (TCT) A. This • ....,"'.. the thrombin.induced oonv~",ion of fibrinog.n to . n iruoluble fibrin dot without being .ffectM by thrombin gtn.mion. It diffi:r~ntiates hypofibrinogen~mia. dydi.brinogtn~mia •• nd eff«t. of heparin. FOP •• and paraprot~ill1 from oth.. co<= of prolongtd PT and PTT. B. Principl. and m.thod l. Thrombin i. addM to pr~"..armed test plas/IU (J7 .C). Thrombin deaves fibrinogtn to form monom.rs. which polym~rizc: into an insolubl~ fibrin dot that am be d.tectM optically or d«tromech.nico]Jy. depending on the in
5/ HEMOSTASIS

285

2. Lob •• p«ific refNen"" interval. should be used for irmrpmation beau .. of m0 D. Prolongffi TI'''' l. Hypofibrinogenem", or .fibrinogenemia: Th ..e is an inadequate .mount of fibrinogen to fOrm a normal fibrin clot.,6",...m a. inc....ed consumption from locolized oo>gulation or DlC b. Congenirn or he ..dit:ory deficiency (rare) c. POllibly beame of hepatic failure and deerea=' fibrinogen production 2. Dy.fibrinogenemia: Polymerization of fibrin it imp.ired beau.. of abnormal fibrinogen molecule •. ' ..... '" a. Hereditaty or .cquired production of abnormal fibrinogen (rare) b. Acquired fOrm th.c may occur with a variety of liver disease. 3. Heparinized patient or ,ampl~ Unfractioflated hep.rin interfer.. with thrombin a.ctivity via AT. so TI can be u=' to monitor unfraction.red hep.rini7..tion. llo TI prolongation by heparin can be normalized with heparin neutraii7.;Otion. TI i. affected relatively little by LMWH.'" 4. inc",ased [FDPs]: This interf.... with fibrin polymeri7..tion .nd thrombin', action on fibrinogen. '" Fibrinolytic th ..apy can be monitored with the TI. S. P.raproteinemia: Abnormal immunoglobulins from multiple mydomas may interfe.. with fibrin polymeri7..tion.'''' 6. Sy.remic . myloidosi. (reported in peopl.): Plasma from .ffected patients may contain .n inhibitor of fibrin polymerization. lJ. 7. Hyperfibrinogtnemia: Rarely. in people. hyperfibrinogtnem", hat prolongffi the TI. but the cau.. is not clear.'''''''' 8. TI is unaffected by decrea=' a.ctivity of any coagulation factor (including factor XIII) other than fibrinogen. E. Shortened TI: This IIU)' occur with inerea=' or activated clotting factors. or with soluble fibrin in the .. mple. but it is not a reliable measure of hypercoaguJ.bility. VII. Fibrinogen A. [Fibrinogen] measured .. fibrinogen a.ctivity'17.'2I"JO l. Calculated from TIa-. •• modification ofth. TI a. Pl .. ma is diluted .0 th at fibrinogen is rate limiting. b. Greater concentration, of thrombin a.. u=' to override much of the inhibition cau=' by heparin and FDP •. c. TIa-. val"", of sampl.. with. known [fibrinogen) and the",fO", activity are u=' to construct a re~ .. nce (standard) curve from which a te" plasma [fibrinogen] can be enimated. 2. TIa- is in""".,ly proportional to [fibrinogen] when high con""ntratiom of inhibitors and d,..fibrinogtnemia ..e abs.nt. 3. TIa- is mrasured in ....::onds. but valu .. fOr [fibrinogen] are rrad from the ",f..ence (standard) cu"", .nd reported in units of mgldL or J.lmollL 4. Unit convmion: mgldL X 0.0294 = j.tmolfL (SI unit) 5. Heparin will not int..fere at clinically th ... p
FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

'0;

6. FDP. h.vr a minim.! df"", on ITa... in human .. mpJ~, unt ... pr...nt at high COD"'ntr>t;on, concu,,,,,ndy with hypofibrinog..n.mi
B. [Fibrino!:",,] may k ffi=urM by d.laotion of fibrino!:"" antig." l. Anti_fibrinog.1I antibodi•• ar. usa! to d.t«t fibrinog<'D amic<'fl by immunoas .. y.

e.

2. Prolonged IT .nd ITa... with d"" ....al fibrinogen antig.n indicau hypofibrinog.n.mia. 3. Prolonged IT .nd ITa- without d"",nsN fibrinog.n .mig.n suppon the pr=n~ of. dysfibrinog<'n.mia (wo). [Fibrinogen] by h.at p=ipitation j. not prrci.. or .ccuraU '"OUgh taltniqu. for us< in hemostasi, uscing.

D. Interpret;"" coruid.ralions l. PWm> [fibrinogen] .. H=, the baun", bttween production :md consumption, not production or consumption alone. 2. Aced.ratM consumption of fibrinogen duriJll: hyp...:o~labl. sUI.. may k m.,hd by inc=>td production :lSSOCi<>t.d with inHamJrultion or pr ~uine data indicate. value; in he;o/thy newborn, {<24 hold} Im}' be lo~r than ",fe",nc;., imerv:d, establi.hed fur adult •. ""''' VIII. Other .p«ific <=gIllation factor :octivitie. (II. VII, VIII, IX, X , XI , XII, PK, and HMWK) A. Othu .p«ific roagulation factor = p Jruly be u,.d in .p«ialiud veterirutry laborato_ rie, if ",r""ning tem and clinical findings suggest factor deficiencies. The assay. Jruly be U.M to characterize :ocquir.d deficiencies but usually are done to identity her.ditary deficiencie; (Table 5.?}.''''''''''''

Table 5.7. H eredilary or con~ilal ooall;Ulation factor deficiencies rel!oncd in animal. &p«Ced TeSI Resull. Pathwar Faclor

Condilion

SE;ie;

PTI

IT

IT

FaClor VII defi,iency

Do,.

WRI

;

WRI

; ; ; ; ;

WRI WRI WRI WRI WRI

WRI WRI WRI WRI WRI

;

;

Tissue factor

VlI Surfac.,..ioouccd

PK XII XI

DO Villa Common X II Combined II, VlI,IX, X

PK deficiency H:agtJruln Inil FaClor XI deficiency H emophilia B' Hemophilia A"

FaClor X deficiency Hypoprolhrombinemia Hypo_ or afibrinog<'nemia Vilamin K-
DoC" horse. DoC" cats DoC" cats, cnde DoC" cats DoC" cats, cnde, horse.

Do,. DoC' Dog., goats

; ;

;

WRI WRI

;

;

em {Devon raj,

;

;

WRI

pmsiblr dOI2

'The.. conditions h.Y<:on X_~nked '=SSM tummission rnd nrely occw in r.m.Jes. No ..: Hemorduge i.s not :woci. te
5/ HEMOSTASIS

"'7

B. F.ctor ddicienci .. a.. indial.!..:! if. I: 1 dilution of test pl:lSm. with normal plasma oorrne:u prolong..:! PT or PTT (provides the missing f.ctor or factors). wh ......, failute to ruff<et ~ulation time, support' the prose:n"", of.n inhibitor (on: =t •. XJI and XIII). C. Clotting . Udys for ,~ific factors ...... the .bility of the p>ti.nt pla,1IU to rorrect the PT or PTT of .~ific faaor_d.ficient pla,ma,. ,., l. PIT is used for . rin or lMWH in a .. mpl • .'''ln thi, chromog.nic ...ay. iner....d hq>arin . ctivity i. :usociat":! with ina.....:! inhibition of f.ctor Xa..,d th.refore d.er..=' forllUtion of a chromogtn from a substm. for faaor Xa. IX.

Proteins induc.d by vitamin K .ntagoni,m or ab",n"", (PIVKA) A. PNKA are the inoomple-tdy g.mm.-carboxylat":! vitamin K--d.peooent coagulation factors (e.g.• d....y-carboxy prothrombin. oft.n refrrr":! to as PIVKA_ll because prothrombin i, coagul. tion faaor II) that . re produc.d by hq>. toqt.. during periods of viumin K antagonism •• bsc:n<:<:. or deficiency (= Major Bl=ling Disord.rs. =to Il.B ).'!! B. In those: conditions. pl:lS1IU con""'ntrations of normally carboxylat":! coagul.tion facton are d<ere-as.d •• nd PIVKA .'" """"..:! . nd circulat. in the blood at incr .... '..:! ron"",n_ trations. Th. pot.ntial of PIYKA to beoome activ..a1 to functional roagubtion enzym.. i. "",..dy limit..:!. C. HUllUn assays l. Clinical immunoassay> are used in human m..:!icin. to ,~ificaUy and dir=ly me• • ure pl:lS1IU [PIVKA], but they are not u..d in vet ..inary malicine. They can detect .bnormaliti.. in the vitamin K- dependent factors that PIVKA_",n,itive ro"l:'-'larion t.,ts cannot.'" 2. Charge diff... ne<:. ~tWttn PIVKA . nd their carooxylat":! forms also .""ble detection by high_perforllUn"'" liquid chrom.rography.'''

'88

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

3. inn,,=<:! plasma [PIVKA] may occur with vitamin K ddic;eIlCY, vitamin K .nt:ago_ ni,nt, or as a result of production by malign<>n1 hepatocyte:! in human poci, nu with hep.tocellular co,cinonus. 147 D. Th. Thrombotest PT, • modificolion of Ih. PT a=y cru.1 js PNKA .. mil;"", har bttn r.f.,rM to :as a PIVKA test, but i{ is not .ptcific for P1YKA and does not dir«:tly m ..am.. [P1YKAj.'''' '''

l. It is a PT WI that d",,",,cs da:.......d .ctiviti.. of cwguJ.tion f""cors II, VII, and X

from .ny em..,. Fa.ccor V .nd fibrinogtn

:or. not """,=
kc.UK

thry ... provided

in Ih. assay. 2. [f PNKA ar. p....[1( in Ih. sampl., Ih. Thromooles{ PT will ~ furth., proJongffi ba::;,...., PIVKA 'P!""" to act :as COntl"'liliv. sub,trates th at dday Ih. gtllualion of thrombin. P[YKA do not .pptar to im"fu. with function:'! faclors in vivo.'''

3. Cal1Se'l of a proJongffi Thrombotm PT a. Vic.min K antagonj,m or ddici.ncy: Th. Thrombot~st PT may han gt
{I} In a subset of th ..~ cats t~.ted with vitamin K" ThromboteJI PT valu .. we" WRI 3-5 dafter tr
Russell'. viper ... nom time (RVVT): Th. t.. t is not widely offerffi. Di=t activation of foctor X by venom of the Russel]". viper lead. to a dot. Prolonged dotting tim .. indicate an .bnormality of the common pathway (factor X, V, II, or I).

Xl.

Endogc:nou. anticoagulant. A. Antithrombin {An, also known as .ntithrombin III (AnH), it assayed to provide information .bout • patient"> anticoagulant statu. I. Plasma AT is usually m
5/ HEMOSTASIS

289

Table 5.8. Disca.... o r co ndition. that Cil U"" decreased antithrombin

(An activity

D,""rra'M production H~rMiury (not .. ponM in .nimal,) Acquir~d

Hq.atic dista'lt Inflammation (polSibly, in Hyper.. trog~ni,m

Klm~ 'pa:i~,)

to. 'Prouin_losing

n~phropathy

Prot~in_losing ~m~ropathy ~,,~ h~morrhag~

Consumption: incr~• .ed h~patic clnr.na:: of AT ..,nzym~ compl"".. Loc.liud coagulation (~,g" hemorrhagic rr.UIlU) or thrombo,i, 'DIC

' SqJsi, 'HTarin oodmininr.cion • A rel. tively common dioe ... or condition Note: Noon .... m>y b.",. 10..., AT v:ol.,., tb.,. .dult>, 3, Units a, Units

ar~ %

.ctivity compared to ~ith" • 'pa:i~'-'P'""ific or human plasma pool to have: ] 00 % activity, 8«;oUs<: of 'I"""i.. diff~"'n= in AT activity, .nd r~portM v:due, VlIry with th~ 'P'""ie! used for th~ .. f,,~na::

COn"d~rM

.. f,,~na:: pooL""'" b, AT a150 has bttn rq>OnM in UlmL or UldL, whu~ I UlmL or ]00 UldL, "'I"""tive:ly, i, arbit"'rily assignM a, th~ me;on value of th~ ref~r~n"" ""'pl~ pool. 4, Subility: AT .ppe.n to ~ st.ble in citmM pluma for 6 010 at - 70'C and for 6 wk in whol~ blood storM at 4 'C"" 5, Int~rpr~tive: con,id~ .. tiom a, Foal. and human neonar .. have bttn mown to have plasm. AT activiti~, that ar~ substantially lower than aduh v:due!,llo,,,· This must ~ consid"M wh~n im"prcring AT value! in young pati~nts, e'l"""iaily in [h~ ab .. nce of "1:"_ matchM r~f~ .. nce im~rv:d .. b, AT activity llUy ~ ove:rcnillUtM when mcarurM by !lOme thrombin chromo_ genic ....ys bccau.. heparin cofactor II .ctivity llUy ~ dffcctM in .ddition to AT activity.'""'''" 6. D,""",asal AT activity''','"''· ..• (Fig. 5.4 and Tabl~ 5.8) a. Dccr""M AT activity may ~ eith~r. cause or an ~ffcct ofhypercoaguJ.bl~ (prothrombotic) nat~'. Oth~r clinicl .nd labo""o'1 finding, should ~ U'M to help dcrermin~ irs pathogene,is. b. Dccr""M production {I} InhuitM d~fici~ncies hav~ not bttn reporrM in animal, but occur in pcopl~ as type I {d,""",asal amigtn .nd activity, a qu amitative: disorduJ and type II {d.cr.....,J activity but norm.1 amoun" of .mig~n, • qualitative: di,ord,,).''' {2} live:r di ..... (including ponosY"'.mic ,hun,,}'" may cu .. or contribute to AT d~fici~ncy in """,,,,I ways. including dccr..... d AT production relatM to d'""r .....d hepatocellular IlUlS.

""

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY {3} InHamm. t;on may d,""""as. AT production in >om •• ~j ... AT w:lS shown to be a nbbi{,'" .nd cots.'<>-,.,· ..• (4) Esuo!:"", may rontribuu to AT ddici.ncy by causing a mild to mod.rat.

d,"",,,,,,, in AT '1mhesi,'''''''' c. AT loss

{I} Lik. albumin, AT may be [OS{ from Ih. body in aninuls with prouin_losing n.phropathy and with prot.in_Io.ing hrotic .yndrom. p.ti.ms, mouth om., f.ctors ... involv~ ' .. {3} ~r. h.morrhag. may contribut. to AT Joss. d. AT collmmption: inc,rasN h.p. tic d.""mre of AT ...,nzym. complexes {I} Loc.li=:i c=gulation or di""minata! conmmpti"" co"l:'-'lation ,um (Itt Thrombo,is, Sffi. II) may cou.., d«r~ased plas/IU AT activiti.. when hq.atic clearan"", of AT_enzyme complex.. exettds AT production.''''·''''·'''-''' {2} SqJsi. has bttn """",ina! with a prothrombotic condition and danaied AT activity.",·m {3} Hq.arin the"py (unfnctionata! and LMWH) aa:d~nt .. th~ u .. and, th,,~for~, hq.atic d~"n'" of AT.I ...ding to d«",asa/ plasma activity.'n.'n Secou,. AT i. ~uira! for h~parin', fulJ antiCO to hq.arin and may be hq.arin r..isunt.'''' 7. lnc",asa/ AT activity a. This is of unknown diagnostic utility .nd not considered a probl~m. b. Exogenom conisol oodmini
8. Thrombin_antithrombin complexes (TAn a. These form wh~never thrombin i. generated in the presrn", of AT. b. Th~ [TAl) is measured to ...... for activation of CO
d. Decreased hq.. tic clearan", of TAT could theoretically contribut~ to incr ...sed pla,/IU [TAT]. e. Inc",ased [TAT] may occur without appr«iable d«r ...... in AT activity. f. Equin~ .nd conine plasm. TAT ha"" been measured with. human EUSA, primarily for research purpo... ."~",.."·

5/ HEMOSTASIS

29'

g. UnitS:).IgfL or nglmL h. TAT may r~adjJy form in vitro if collation ttdllliqu .. art poor .nd coagulation OO:Ur5.'"

B.

Prot~n

C l. This is infraju~ndy assayed in nt~rinary medicin~ but provid~s information about a pati~m's amicoagulam and fibrinolytic UaNs. Low plasma oon""mrations or activities predisp<>S< individuals to thromoo.i, ba:au.., of decreased inactiv.tion of f.cton Va and VIII •• nd ba:aUM of d~cr~ased fibrinolysis. 2. Anti!:"n con""mration can b. m~ .. ural immunologically (by ELISA.. radioimmuno_ ..... y. or Laurdl rock" dectroimmunoauay}."" or activity can b. m~asured by clot_ based or amidolytic-based functional .....y•. ". a. Anti!:"nic a1Stssm~1It does not detat functional d~ficiencies. including tho .. inducnl by vitamin K anugoni,m or d~fici~ncy (prot.in C is vitamin K dt~ndtm).

b. Rr",lu of functional .....,.. v.ry with the mtthod. and ¥ci .. _specific modificatiom =y b. rajuired. ' .. HO Amidolytic .....,.. do not ...... all functional aspect. of the mol~cul~ and do IIOt direcdy m~asur~ its capability of inactivating factors Va and Vill a. How...... the .......,.. ar~ less struitin than dot_based ..... ys to coagul.tion inhibitors ",ch .. h~parin. Some human pati~ms have had .bnormal dot_based reswts but normal :mtigen and amidolytic test ..",hs. 3. Results .. t report~d :l.! % activity or amigtn relative to ... f~r~n"" plasma pool comidered to haVt ]00 % activity or ami!:"n. 4. Decr~ased proltin C activity a. Decr ... s..d production {I} Her..ditary ddicitnci .. (t~ I. :mtigtnic; or ty~ II. functional): A functional deficiency was id~mifi..d in a ooh.'" {2} Vitamin K antagonism or deficiency (prot~in C is vitamin K dt~ndtnt) {3} Decr=
{4} linr di ..... with decre.sed functional hq>.tic m ... b. Decr ... s..d survival: oonsumptin magulation and its pr..di'p",ing causes {S« Thrombosis. >«t. II}:'" srp.is'" c. Inhibition: Ami_{phospholipid_protein} antibodi .. =y inhibit th~ function of protein S. protein C. or factor V.'" ,''',''' d. AI; ajuine data indicate. protein C activity (not :mtigen) vatu .. in hralthy nronat .. « 24 hold) =y b. greater than rd"t .. na: interval. establiili..d for .dults."o," , C. Protein Z l. l'rotrin Z is a vitamin K-ttn :u.sociat..d with .bnormal h~most:ui •• mually thromboc:mbolic di..,a..,. in ~ple.'" 3. Protein Z is simil.. to albumin in molecu.l .. rnass. and plasma con""mration, have corrd.t..d positively with AT and nq:.tively with proteinuria in prople with

292

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY n~phro{ic 'rndrom~. Urin:.ry loss of prouin Z may contribute to th. prothrombotic state «ry patienu.

XII. Co"l:"lnion f""ior .mibodi.. (inhibitors) A. Antibodi .. to coagulation facto," "'" commonly ",fWM to .. inhibiflm. Th= inhibi_ tors must k diff.",nt;"tM from endogenous physiologic anticwgulants, a ogenow antiooagubnts, and .nti_(phmpholipid_protein) antilxx/ies. B. Conditions associaud with coaguJ.{;on factor antibodies I. Antibodi .. moy form sponuneously as !"In of an autoimmune disorder. 2. Antibodi .. =y wllh from ~atal tr.mfusion. to hemophili<>cs who denlop antibodies to th. ddiciem factor. e. CoaguJ.tion factor antibodies may p='i,post !"Ili.ncs to h. morrhag<'. D. Approach to d<:{«:ting coagulation f.ctor antibodies I. Cwgulation factor ~ntibodi .. prolong which""~r coagulation tim .. a", de~ndent on th~ ta~ f:octor, usually PTT, so unaploinal prolongations ofPTT, PT, or both should prompt oomid~ration of rd.v:ant ddici~ncies and inhibitors. 2 Wh~n PTT it prolongffi but PT is not, unfractionat.d hqmin .hould k oomid~r.d a potential cau",. !«:sampling, analy.;' with ~ hep:orin iruoctivator, or .val uation of IT {prolong.d with h~p:orin} could k con';d~r.d. 3. [f heparin cannot k incriminat.d, th~ coagulation times should k d~ .. min.d by u'" of mixing studies that as=< th~ df,""ts of diluting the sample I: 1 with platd,t_ fr"" normal plasma. Co"1:"lotion times mould k asseJMd immaliatdy and aft~r a 1- 2 h incub..tion at 37'C kca"", 5Om~ ~ntibodi., at< tim. andlor t~m~ .. tu'" de~ndent.

a. Imm.diate oo"'""tion of the oo~uJ.tion time, with no prolongation upon funh~r incubation, suppom a factor ddici~ncy {including tho .. caus.d by vitamin K antagonism} rath~r dun a coagulation factor antibody. F~ctor allOn.d in vet~rill
5/ HEMOSTASIS C. Allhough inhibilor> of in vitro co>i:ul. lion, clinical IIUnifmalions .'" Ihrombosis or Ihrom~mbolism, not h.morrh>i:. ... D. A .. ria of spa:i:aliud t. m m.. y b. ri:ulnion tima wh.n mixed with p.ti. nt plasma'" 3. Neutralization of the inhibitor by increaKd phospholipid in the t..t syst.m 4. Exdusion of other co.gulopathies FIBRINOLYSIS I.

Physiologic process.. A. Fibrino/ytis is the .nzymatic degradation of fibrin. It rounteracts co"i:"lation .nd helps r.. tore normal ns,.::l archit'""ture and p>t.ncy .fler h.morrhage has been controlled with. oecondary h.mosutic plug. B. The fibrinolytic .yst.m, like the coagulation system, i, a complex network of zymog<'ns, mzym .., activator>, and inhibitor> (= Tabl. 5.9 ). C. Fibrinolysi, is initi.t"! simuh.neoudy with coagulation and is normally loc:diud to the h.mosutic plug. l. Wh.n coagulation occur>, plasminogen {inaai"" zymogtn} binds to fibrin. 2. t_PA rel....d from slimulat"! .ndothelial cells bind, to the fibrin_plasminogtn complex:md proteolytically d •• "". plasminog<'n to form pl.mtin. u_PA from circulating cell, or damaged ti ....... may play. similar role. 3. Plasmin .nzYllUtically degrad.. fibrin, fibrinog<'n, factor.; V•• nd VIlla, vWF, HMWK, and other prothrombotic factor.;. 4. Plasmin r.::l.ased into circulation i, rapidly inhibit..!, primarily by plasmin inhibitor {a,_antiplasmin}, .nd dear..! by th.li""r. D. Th•• ctivation of plasmin i, promoted by APC and inhibit..! by PAl. which APC inactivates. E. Plasmin degradation of fibrin and fibrinog<'n produ= FOP, (Fig. 5.6), which con b. m.asural to assess fibrinolysis. F. FOP, .ppear to be d .....! from circul.tion by the li""r (hepatocyte< .nd macroph. gu) and kidneys {via cocabolism or excretion}."'''''' Nonhepatic pam of the MPS IIUy:also b. involved.-""

II.

Fibrin or fibrinogtn degradation product, {FOPs} {fibrin frat:m.nu + fibrinogtn frat: m• ms } A. Purpose: Th. m.asu",m.m of FOP, i, primarily used to del'""t inc=std fibrinolyti, associat"! wilh excessive cwgulation. It :also may b. u...:! to delect inc",ased fibrinogenolysi •. B. Sample: .. rum or plasma l. Serum .....ys: Blood is coll'""t..! into spa:i:al tubes containing thrombin or Born»: nnom {"'ptila..,}, .ilh.. of which con b. u...:! to comume fibrinogen via coagulation. Soybean trypsin inhibitor or aprotinin are present to inhibit plasmin and th.",fure in vitro FOP formation. 2. Plasm • • ss.ys: Blood is roll,""t..! as for ""r..,ning coagulation tests (... Coagulation, sect. II); citral..! pla,1IU is used.

,,,rox

. ... ............ ....... .... "

...,-x .. ........ ' '" , 0 .... ['.

•

".

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

Table 5.9. Fibrinolytic factors and their major functions Function

«,. M:KT<>gIobulin

Binds, inhibits, and

Fibrin Fibrin f!'3i:mc nt D-dim.r

Major protein in a stabl. thrombus; source of FOP, FDP that contain D.dimrr moietl .. ~nuat.d by

cltllS

pl ... min from circul>1ion

factor XIlb- m«\iated cross-linkiJli: of fibrin; indudes high .nd low mob:uJ •• w.ight products

Fibrin f!'3l:mcnts (FDP,)

+ fibrinog<'D fragment.

Fibrino!:"D {factor I}

Pl ... min P[",min inhibitor (PI, oc,_antipwmin)

Pl ... mino~n

FngllY[I{, of fibrinogen or fibrin producnl from enzymatic d
Pl ... mino~n activator inhibitor I (PAl_I) Pla!mino~n activ.tor. Ii ....... t~ (l_PA)

Inhibitor of fibrinolysis pnxluc:ffl by ~ndothdial ""U,; inacciv.t.. t_PA and u_PA Conv~m pl .. minogtn to pla'min; impon:mt in fibrinolysi, within the v:I>culature Pl ... mino~n .ctivator, urokin ... typt Converes pla,minogtn to pl:wnin; imponam in tissue (o.PA) remodeling Thrombin-act;v.t>bl. fibrinolysis Zymogtn activated by thrombin and thromoomodulin; TAFla dnve. pl..,minog<'n_ inhibitor (TAFI) binding .ite. from fibrin, thus inhibiting fibrinolysis • T he nom."",]1t"", follow. recommend. tions rrude jointly by the Scientific .nd Stond.rdiution CommittN of the In""wtio",J Society on TItrombosis..,d Ha<most .. is ..,d the CommittN (Commission) on Q"..,ti,;.. and Units (in Clinic.J Chemistry) of the lnt.,.utiona] F.d...tion of Oinical Chemistry (lFCq and the In,,,,wtion.J Union of Pur<..,d App~.d Chemistry (lUPAq.17J

c.

3. A> with other test. of hemostasi., cue mull be taken to pr .... m or minimize co"l:"l.tion during roJJ«tion .nd pfO<".t:S!.ing. Wh.n pla.ma is the test sample, clotted .ampl.. should be discarded. 4. Stability: It is often r«ommended to t.. t pl.""" within 24 h wh~n it i. no=' at 4 'C and within 20 d wh.n it i. frozen at ~ 20 'c. Analytirn concepts J. Lua agglutination immunoassaY' a.. used.'" Oilutions of t. n .. rum or pla,ma ",e incub.t. d with lata beads cQ>ted with antilxxli.. to human FOP •. Agglutination of th. beads indicot~. inc.. ..,.,j [FOPs]. 2. Antibody sp«ificity a. The sprofic .. ""tivity of :way antibodi .. with different FOP ffal:m.m. from v<:cerinary '1'«;" i. IIOt known, but erou_reactivity of some polydonal .. ag.nt. with conine FOPs h.., oon ,hown."" Clinirn correlation. with dis ......lso suppon that th ... is at lean partial cross-reactivity in dog•.

5/ HEMOSTASIS

29'

b. Two FOP ......ys. on~ .. rum . nd on~ plasm •• did not appear to hav~ clinical utility in id~ntifying fibrinolysis in hot=; with colic ~"'" th~re w~ .. positin ... ults in h~ ..lthy hones and few incre~ values in hot=; 1l1Spectffl of having DlC."'; c. Th~ pl .. m . ......y uses a monoclonal .ntibody to FOP, that does not r~ct with fibrinog~n, thl1.'l allowing th~ u.. of plasm .. (which conr.ins fibrinogtn) .. th~ test s;;ompk d. Th~ .. rum assay u..s. polydonal .. ntibody to FOP, th .. t also binds fibrinogtn, thus n=itating th. in vitro .. moval of fibrin~n by m~ns of r.ptil .... ot thrombin. 3. Unit: usually j.tgIml (m ..y .. Iso k r~portal in nglml or j.lgll) 4. Ikportffl .. miq""ntit ..tivdy as th~ following: ... uss than. low d«i,ion threshold (•. g., < 5 j.lglml with plasm.. teslS and <10 j.lglml with .. rum Ust,), bdow which values ar~ consid~rffl norm. l) b. Inc....ffl {5-20 J.Ig1ml with pwrruo tests and 10-40 j.lglml with .. rum tests} c. Inc....ffl mor~ (;. 20 j.tgIml with plasm .. un. .. nd ;. 40 j.lglml with .. rum tom) 5. Int~rp .. tin consid~r:lliom a. Prown.: With • high [FOP,], ~Iutin ..tion rruoy occur ..t tho great.. dilution but not >t th~ lesstr dilution. b. F.. I.. r.sults {I} s.rum ......y results may k fal ..ly d.c,..• ...:! by FOP incorporation into th~ clot .. it forms.'" {2} s.rum ......ys m..y k f. lsdy incre~ by inoompl~...moval of fibrinog.n from th.... mpl. du~ to tho pr... n", of dysfibrinogtn.mia {rard or h'p",in {if thrombin tath.. th .. n .. ptil .... is u...:! to .ctivat~ clotting).200 {3} G..n.ration of FOPs during blood coUrction can cau.. f. l.. incr ...... with pl ... m.. or .. rum """ys. {4} Results m..y b. fal .. if .ntibodi.. do not bind to targ~ FOP, or if they bind to oth.r prot~ins. c. As tfflsigns .re oft~n .. b.. nt wh~n infl .. mrruotion is 'J.SOCiatffl with .. n inc.......d [FOPs]. d. H~morrhage: Bloody fluid roll.cral from body caviti~, aft .. h~morrhage oontaim high ooncrntrations of FOPs, and .. n inCl"~.K
'98

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

Table 5. 10. Di""a..es or conditions thai cau.e inerene.! concentrations of FOP,' inc,",asM fibrinolysis Localiud intravascular e<»guialion 'Di ... min<>tffl intravascular ~Jation 'SqJsi. and oth~r inflammatory condition. [nt~rnal h~morrh"l:~

lnc,",asM fihrinog<'Dolysi. Ennnom.lion. Administration of pl:.smino&<,D activ. to", Ex~ssj"" rd~.", of t_ PA (hypot~mj"" mock, surgical trauma, h ....utroh) D'"".. ..,«I FDP d .... ran'" Li"", di",=

Iknal

f.ilur~

• A ,.t1tiv<[y common di..... or condition ' FOP, includ. D-dimen. but ina....d fibrinog<nolysi.s :d""" will not inc.r .... [D..dimerl.

Howev"" an incroasN s.rum [FDPs] was not dct""{M in dogs when hcmorrh,,!:, into tissue W:l'l «p
a. unain cn",,"omalions''' b. Administration of plasmin~n activators {~.g., t_PA or m~ptokin ....}m c. Ex=sive ~ndothdial rd~..e of t-PA, for which causes ar~ not cl=ly ddin.d but may include hypot.nsive shock, surgical trauma, and heatstroke''' d. It may .ccompany fibrinolysis ousociat.d with thrombotic dis ..... .''' but some con.O' b. Oecr....d ... nal function truly contribute to increased [FOP.] in some pati~nts with renal failure.'" c. Theoretically, d«reased MPS activity could contribut. to incr....d [FOPs]. E. Effect. of incre...d [FOPs] on other hemonatic functions and tests I. Prolongs valu .. for tests with clot .nd points {PT. PIT, IT, ACT, and Ltt_Whit~ clotting time} 2. Impairs pl.tdet function 3. M«hanisms of .nti~ation and .ntipl.tdet .ffecrs a. FOP. compete with fibrinogen for the active sit. of thrombin .nd thu. truly inhibit th. conversion of fibrinogen to fibrin.>.!'·'''' b. FOPs compete with fibrinogen for platdet_binding ,ites .nd thu! truly inhibit plotdet aggregation.""' c. FOP. associat. with fibrin monom.rs and m.. y disrupt normal polymerization.'1J.m 4. An incr ... sed concentration of FOP. promotes .n antithrombotic and proh.mor_ rhagic nat •.

29'

5/ HEMOSTASIS Ill.

Fibrin f"'l:m~nt D_dim~r A. Thalry: It an ~ UoN to asse .. inc",_d fibrinolysis auoc"'ta! with OO.<Say•. As with th~ FDP assay. wh~n s..um ,ampte. are usn!. in vitro D-dim~r forJrultion must ~ pr~nnta! and D-dim~r fragments may ~ lost into the dot. 2. Subility: It is recomm~ndnl to test plasma within 24 h wh~n stora! at 4'C :md within 20 d when frozen .r - 20 'c.m C. Analytical con<:qlts l. Although. canin~ >.<Say was drvdopnl.'" it i. no longer :lVailabl~. and routin~ ou.says usn! to dH.<Says a.. specific for fibrin formation. but antibodi.. in som~ kits bind human D_dim~r domains without pl"'min_inducal d~gradation.'" {4} Some antibodies dH<>.!itin r<:sulls in animals suspa;ta! of having DlC b..,a! on im~rf= CJit~ria. {2} A point-of-a", immunochroJrultographic .....y with. monodorW antibody for conine D-dim.. (not cur",ndy available) romparnl well to EUSA .nd latex .gglutination >.<Says with anti_{huJruln D-dim..} antibodi~s.'''

300

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY 3. Unit: J.Ig1mL, nglmL. or J.Ig1L, tq>Ort<'d variably as D-dim~r units (D-DU) or as fibrinogen "'IuiVllI~nt unin (FEU); I nglmL D_DU = 2 nglmL FEU. D. [nn~~=' pJ>s/ruI [D-dim~rJ : [nt~rpr~t>tjon is simi!." to th.t for incrrasnl [FDP,], excqJI due v:du... hould

r~H
only inc,rasnl croll.Jink«i fibrin fornl
fibrillol)"i. {not fibrinogenolysis} or d«",,=d cl~.nn~ of fibrin degradation product. (not fibrinogen d for increas.. in the [FDP,]. a variety of conditions em", increa.= in [D-dim~r] without onn DIC or thrombotic distaK; for aample. with inHammation. with h~morrhage . and .ft~r surg..ry.' " a. T .. ting ...ial .. mpl~ dilutions of up to 1;8 .nd using incrrasn! d I<XX> ng/ntL)."" IV.

Oth.. assa,.. may be usn! to mrasure fibrinolytic components (e.g .• plasmino~n. plasmin inhibitor. t.PA. and PAI.I ) (0« Table 5.91 or fibrinolytic activity (euglobulin lpi. time test). but they are not widely usn! in clinical .. ttings. Thromboelastography and ...on:mce thrombography are more global in vitro tesU of hemo,uoi, that assess the functional contributions of platd~u. roagulation. and fibrinoly.is to th~ hem05u tic p""",ss. Funher reading about the.. specialized techniques is .vailable."'>"'>"

BLOOD VESSELS {EN DOTHEliAL CELLS} Although blood ""ssds (esp.cially th~ endothdial cell.) are ""ry imporunt in maintaining normal hemost",is via prothrombotic .nd :mtithrombotic properti..."· their assessment in the clinical po.thology laboratory i. limited primarily to ti!SUe biopsy and hinologic evalu.tion (e.g.. for vasculitis or thrombosis). V.!Cul.. l..ions .re umally obvioll'l wh~n hemorrhage is the result of mrgicol or accid~ntal v:..culu trauma. Di..",.. involving """ll vessels •• uch •• RMSF and equine purpura hemorrh>gico. may be associated with petechiae and ecchymoses cousrd by rombin ations of direct vaocular damage. thrombocytopenia •• nd thrombop. thia. Hemostasis tening may hdp exclude other di",rders but will not provide. sp<eific diagnosis. MAJOR BLEEDING DISORDERS; FIN DI NGS AN D PATHOGENESES I.

Diagnmis; The diagnosis ofblttding disorde", requir.. knowledge of the general ty~ of bINding di50rd~rs. conoideration of clinical findings •• ccuraU interpreution ofhemo,wi, t.. t r.,ulu •• nd recognition of h~most"'is test p.tterns.

5/ HEMOSTASIS

301

A. TyJ><'s ofbl=ling disordm I. Blood VeM diKlrd~rs ar~ typically identifi.d by nonh~most;;otic I<sts (~.g .• gross ."amin.tion. imaging. s..ology, .nd bio!"'y), but h~monatic test, ..~ u ..ful for ."cluding prim:uy blood di""rd~ .. and may provid~ diagn0.'ltic clues b=.u.. of =nd.ty ch.ng~s in blood corutitu~nts (e.g., thrombocytoJ><'ni. with ,""sculiti,). a. L,,~ vessels: surgical or nonsurgical trauma, inv:uion, aneurysm" and . nomali.. b. Small ......ds: vasculitides (inf«tious. immun~ m.di. t.d, and chemical) and vasculopathi.. (rare) 2. Blood disorders: typicolly identifial by h~monatic tests a. Impair.d prim. ty hemostasi" thrombocytoJ><'ni. , thromoopathi. , and vWD b. Impair.d sa::ond.ty hemosusi" heralita'}' or acquiral d~f«ts in coagulation c. Ex=si"" fibrinoly.is: DIC and som~ en""nomations B. Clinical findings a.., e... ntial for ~,t;;oblishing • final diagno.i., but the bl=ling patte"" br.,.,d, ~, and gend~r may ~ of panicular valu~ in directing diagnosti, plan,. I. Blttding pm~", a. Pet~",e and ~cchym""'. mould prompt consid..ation of sev... thrombocytope-nia or thromoopathia, though concUl",nt defects in =onda'}' hem0.'ltasi, may .lso ~ pr...,nt. Vascular di ....... m.y cousc: similar hemorrhages. b. Suocurana>u, heIlliltomas .nd hemorrhage into lxxIy coviti.. suggest d~f«ts in =nd.'}' hemost>.<'t.ch",e and ecchymos.. are ab .. nt. Ho~r, h~matom .. may form with platdet defects. c. Hemorrh~ through muco",l surf. ces (•. g., ~pistaxi" gamointminal hemor. rhage, or prolong.d estral ble.ding) IlliIy occur with thrombocytoJ><'nia, thrombo. pathi., vWD, or defects in coagulation. Prolong.d hemorrhage sa::ondary to venipuncture or surgicol or nonsurgical trauIllil is aloo not specific. 2. Bre<:d and ~ a. Br=l pr.di,positions for inh~rit.d diseases may hdp in sd«tion of .ppropriat~ diagnostic tests."" b. Hemorrhage in. young .nimal should prompt consid~ration of.n inh.,rit.d defect of platdets (Tabl., 5.2), ..WD, or coagulation bctor deficiency (Tabl~ 5.71 if another cause i. not .pparent. Mol«ular g~netic testing may ~ . vailabl. for som~ characterized mut;;otions in som~ .ffected .nd corri.. animal, (".g., vWD).'" 3. G.:nd~r a. H~mophili. A (f""tor VIII deficiency) .nd h.mophilia B (f. ctor IX deficiency) .re sex.linkal (X chromosome), rectlsivdy inh..it.d di,orders. {I} Male. are .ith.. :offecr.d or not. Th.y.re not corriers. {2} F.males may ~ f..., of the d~fect, carriers (heterozygous), or. rardy, .ff«r.d {homozygous}. {3} About SO % of the off.pring of carri~r females mat.d to unmect.d males will inh~rit a defective X chromosom., so SO % of IlliIle offipring will ~ mect.d and 50 % of f~mal. offspring will ~ corri.... {4} All fc:m. le offspring of a clear femal~ . nd an m«t.d male ar~ .. ymptomatic carne ... {S} F.mal~ offspring from a corri~r female .nd an .ffect.d male con ~ mect.d. b. Th~refore, the .. di""rde .. almost alwaY' occur in m. le.. Hemorrhage in female. is unlikdy to ~ coused by one of th= di,ordm.

302

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

Table 5. 11. Possible cause. of abnormal res ult
i Tim. ,l. Activity"

AT

of hemostasis

Possible caus..

Test or .nalyu

ACT

l es t.'l

[).,f«t in surfaa-inducM and/or common pathway. Renal or intestinal loss, consumptive oo:oguJ.tion,

hq>. rin .dministration, or dec.... M production i Tim~

ThrombocytoptDi. vWF ddiciency,

,l. ht~nt ,l. Activity

anem", Thrombocytoptnia, thromoopathi. H".dilary or acquirffi foctor ddici.ncies

D-dime", FOPs Fibrinogen'

i Conctntr:llion i Conctntr:llion ,l. Conctntration

(h YJ>OCO,,!:ulabl. .tat.) Co:ogulnion .nd fibrinolysis, d«reasffi d.aranu [ocreaK.tic production, increasN

Prot~in

i Conctntration ,l. Activity'

BM BT Clot retraction Co"l:"l. tion factors

C

PT PIT RVVT IT

i i i i

vWF:Ag

,l. Conctntration

Time Time Tim~ Tim~

i Conctntration

consumption (may ~ hyp<><:WglllabJ.) Inflammation, hypovolemia (d.hydration) Deer• • 1ffl hq>.tic production, .bnormal production (vitamin K .ntagonism or ab..,nct), [OSl, consumption (Jrulr b. hypsoprcssin

• Chromogenic ... Of' >1Se1< :octivity. but th.re :ocr immunologic .. "Y' th1t....,., ronomm.tion. ProtITrJ.trd .. itb fibrinognornW production of prot
C. Hemostasis tem: A .U/lUrulry of available ,,,,,,,,,,,ing and 'ra'ialized hemosta.lis tests is presenud in Tabl. 5. I I to aide in ..d«tion and interpreution of'ra'ific test,. D. M . jor patterns of hemost..,i, ten remIts: Hemo,t;" i. in bINding patients i, ben evalua ted by multiple tests (hemonasi, profil..). Examples of the major pattern. of common hemo,t;;otic ten result, in bleeding p.tients a.. li,ted in T .ble 5.12. I. Pattern I: Prolonged BM BT without thrombocytopenia or ,evc:r~ .n~mia suggem vWD or a thrombop.thia. vWF .nalysi, or platelet function studies rruoy be indianed. Specifically d~fining thromoopathi.., may be difficult. PTf mayoccuion_ ally be prolonged with canin. vWD if factor VIII is concurrently deficient enough. and it rruoy be prolonged in horses with vWD. 2 Pmern 2: Prolonged BM BT associa ted with moderate to seve.. thrombocytopenia can be aplained by th~ thrombocytopeni • • though concurrem def«ts in platelet

5/ HEMOSTASIS

J03

T able 5. 12. 1nrcrfrctation of the major fafrcrns of oon""on bcmonasi. test rcsulu Panern

PT

PTP

FDP"

Fibrinog.n'

Platdct.

BMBT

Inter~rctation'

,

WRI WRI t WRI t

WRI WRI WRI WRI WRI

WRI WRI WRI WRI WRI

WRI

,

WRI WRI WRI t t

•

t t WRI WRI WRI

6 7

t t

t t

WRI

WRI

WRI- t t

TIlfombop"thia, vWI)' Thrombocytopenia Intrifl!ic p.thw"}' d.fecr F:>etor VII deficiency' Common p.thw"}' d.fect Of multiple defCCI. Drs- or ofibrinogro.mia Fulminant DlC'

3 4

t

••

WRI WRI WRI

•

' ACT would mirror PlT multo in most dis. .... , but h .. Je.s .m.gno'!{ic ..."irivity :and tt..nfor< m ay b. WRl . • PJaun. [FOP, I, including [D..dimSSeS!r"en' of h.emoot ..i" fibrinogon <x>nam,mions "'" um.Jly detrr.b ted .. itb fibrinogen cona"" ..,i""" unLess prolongrd by bigh cona", ... ';o ... of hepuin or higb ronDOn,mio", of FOP" • s... tb. _, (M' jo, BJe.ding Diso,de.., >ret, to) for ..pandrd inl<'pmiv. com"""tI. 'P1T may b. prolonged in ho.... :and oocasion.uy in d"ll" .. itb vWD if factor VIII is concu,,,,ntly defici.n', 'This pattrrfl m:oy ilio occw .. i,b early ,ug.. of b.patic di,.... or viumin K :mugonism or :obs.nc<, I This p .... m appli'" only to tl>. ~"', fulminm, fonn of DIe.

function or vWF connOi be ",eluded. BMBT testing it nOt indicoted in p"tienu with mod.rat. to seve", thrombocytopenia becaus. prolongations of BMBT arc ",peeted and thus will nOt add new information, 3, Patt.rn 3: Isolated prolong.tion of th. PTT indicotes a .urfaa-induced pathw"}' defCCl, though inscJ>!itivity of th. PT =y may m.. k other abnormaliti." a, Acquirw PIT prolongations: This 1m}' be caus.d by h'p"tic disc ...., h.parin contamin.tion of the sample (',g,. inappropriate collection from. hep"riniud cath.ter). or heparin th ... py. and rardy is cau.w by vitamin K .ntagonism, 1Ur• • urfa.c<:.induced pathway coagulation factor or .nti.(phospholipid.prot.inl .ntibodies should be consider.d. b, Inheritw or cong.nital PIT prolo~ations (Tabl. 5,7) ( I) H.mophilia A and B COUlW by defici.ncies of faCtOr VIII and faCtOr IX. rr:spectivdy. a", th. most common inherited caus.. and may be associatw with .....'" hemorrhage:, Th.y ... at"'mdy .... in f.males b.causc th.y ... X.linkw r=si"" train, (2) D.fici.nci.. in factor XII and PK arc nOt .. ro ated with h.morrhage: wh.n . ither occurs alon., (3) D.fici.ncy of f.ctor XI is associatw with mild h.morrhage:, umally in rr:spon .. to trauma. H.morrhage: =y be ddayw, (4) D.fici.ncies may be multiple (' ,g.. factor XII plus faCtOr IX d.fici.ncy), 4, Patt.rn 4: lsolatw prolongation of PT indicates a TF pathway (faCtOr VII) d.fccr, a, Acquirw PT prolofll:.tions: Hepatic or h'p"tobili.ry di ....., and vitomin K .ntagonism or defici.ncy should be coJ>!id.red, F.ctor VI I h .. th. shon.n half. life of th. vitamin K-J"",nd.nt faCto ... and thrtt of the five factors in th. combined TF .nd common pathway. a.. vitomin K d'pend.nt, Th...fo",. th.

304

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

PT may

~

:of&cc«i by vitamin K antagonism or abstn~ ~fo", th~ PIT,

~,~ialJy wh~n

a PIVKA·.. n';tin PT method is usa!. b. Inhuitffl or ooIli:enital PT prolongations (Tabl. 5.7): F.ctor VII ddiciencies are rare .nd :associatw with mild h.morr~, uma/ly in "'ponsr to surgical or nomurgical tnum ..

5. Pm.rn 5: Prolong or dysfibrinogtnemi.>. which would b. =gniud by a low [fibrinog<'n] ). a. Acquir«i dj""rd",: Combin
gen] and d", ......... in [FDPs] h."" bttn rq><>.t<>rtrd in Devon rex cots and p,mibly found in a Labrador retri"""r}.'''''''' 6. Pm. rn 6: Prolongrd PT and PIT with hypofibrinog.n.mia and possibly prolongrd BMBT a. Acquirrd disorders: Hep>tic di"'''''' i, a consid"ation for thi, pattern {= the discussion of patt. rn 5}. Consumptive coagulation {•.g.• Die} could also b. conoid"rd. but thrombocyt0p"nia and inc".=' [FOP,] andlor [O_dim"] ." usuaUy expecrrd with Ole. b. Inh.ritrd or congenital disord.rs: A markrd decr..... in [fibrinog.n] along with prolongrd PT and PIT should prompt consid.ration of rare inh"itrd dJ"lfibrinog.nemia or afibrinog.n.mia. " Seve", hemorrhagic tend.ncies a" ex~trd. and BMBT may b. prolongrd. 7. Pmern 7: Prolongrd PT and PIT with incr•...d [FOPs]. hypofibrinogenemia. and thrombocyt0p"nia are typical of fulminant ole. what• ...,r the couse. Such a patient would app"ar very ill. likdy with .viden"" of multipl. organ durulg.. Findings with hepatic f.ilu" could b. similar. II.

Pathog.n.,is: The pathog.n.... of h.morrhagic d.f.cts in primary h.mon",i, rdat. to ,~ific .bnornuliti.. or d.fici.ncies in platd.ts. vWF. or the v. .... l w:.ll. Inherited disorders of strondary hemostasis are caus.d by various gt:netic .h"ations that r.. ult in d.er•• ...:!. ab .. nt. or .bnormal hemo,utic f.ctors. Acquirrd blttding disorders oft.n have. mo,"" compla pathog.n=s. The pathog.n.... of h.monatic .bnormaliti.. are di5Cussed for the foUowing .dected a.cquirrd blttding disorde ..: A. Hepatic di ....... '" l. liver di ...... con couse defn:" in the production and dearance of procoagulants. anticoagulants. profibrinolytics•• nd antifibrinolytics. The net result i, oft.n clini_ cally ,il.nt despite abnormalities in hemo,utic laboratory tests.

5/ HEMOSTASIS

305

2. Blttding is unconunon but =y occur ifhq>. tic f.ilur~ is 5tV~'" or asoociatffl with DI C. Oth~r J.bor.uory .-vid.n"" of hq>>tic f~ilur~ would ~ npa:1ffl in • pati~nt blttding b«:au.. of th~ h~patic failur~. 3. Pot~ntial abnormal h~mostatic test results .nd th~ir cau... a. Prolongffl PT. PIT, ACT, or IT {I} O,""",-d h~patic production of co.gulation factors b«:allSt of d,""reasffl functional hq>atic m..... (including pono.ynemic .hunt,"') {2} Abnornul production of vitamin K- {2} [nc",-d FOP production {fibrinolysi,} b«:au.. of a<"COmp:mying intrava,· rular coagul ation, which i, som~times di""minataj c. Thrombocyto~nia {I} [ncre.,.d .plenic =!uestration c;oustd by portal hy~rt~mion, .pl~nomeg..ty, and po..ibly d,""",-d thromoopoietin production {2} Con,umption ..-condary to accomp.nying intravasrular coagulation d. Prolongffl BMBT {I} Thrombocyto~nia {2} Acquirffl thrombopathi a • . Deer .... 'ffl [fibrinogtn] {I} D,""",-d hepatic production {2} Consumption ..-condary to accomp.nying intravasrular COCeS.'leS is not alterffl •• much as th~ function of th~ isolatffl process.. that a", testffl. Simil.rly, bl~ffling ..-cond.." to bio!"'y procedur.. in dog. and caU doc:. not .p~" to be pr~dictabl~ &om PT :md PTT result" ~lthough, in cats, th~ .. was an association between major complications .nd PTT prolong>tion of at I...." 1.5x the llYan of the r~fe .. n"" interval. >OJ Blttding complic;otion, in dog, and caU are mo", !Irongly associatffl with thrombocytop
FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

'0;

{2} SW'""t do"",, or .w~ vtmal grass comaining biohydroxyooumarin, a m~uboli{~

producffl from th~ actions of ""ruin moJd.?"""

{3} Th~rapeu{jc ooumodins: From an ovtrdo ... ba::;,...., of displ.""ment from plasma prouins by additional protein_bi"diIll: drugs {• .g., ph.nylbu(;;l7.0nej , or by ingmion of m~jc;o{ion im. nd.d for human p.ri.nu (includes w.rf..in and ph.noprocoumonj"· (4) Ex""", sulf.quinoxaline, a coccidiost>t with v;umi" K :mtagonistic

.ff«ts"'->" b. On"" absor~d. m= compounds inhibit the enzymatic ~cyding of oxidiud vitomin K back to its reducal .nd functional form in h'patocyte,_ [....ding to production of cwgulation factors with d«:om. prolong«i (within 14 h)"" .nd hemorrhage may occur. 2. Yiumin K ddiciency (hypoviuminosis K) is rudy ddicient enough to COUM hemorrhage. a. Yiumin K i. absorbed in th. int..,in. aft.r i~tion or production by intestinal bacteria. b. Causes of clinically significont vit:mlin K deficiency have not bttn clarified in most species. but th. following mould be considered: (I ) Prolon~ anorn", or ingestion of an abnormal diet moy muse or contribute to vitamin K d.fici.ncy. In rontrast. normal diets contain ",a ss vitamin

mu,

K. ",~" (2) Gut st.rilization by antimicrobi;d, Im}' couse or contributr to vitamin K deficiency.'''''' {3} Malabwrption of vitamin K (a fat.solubl. vitamin)'"

{a} Intrahepatic or extrahepatic cholmasis (decrrased f.t digestion and absorption. :md ther.fore decreased vitamin K .bwrption) (b) Intestiruol mal absorption dis ....." (e.g .• infiltrativr bowd diM• ..,) (c) Exocrine pancreatic insuffici.ncy {drcr ......! fat digc:nion and .bsorp. tion. and th.... fo ... drcr ......! viumin K absorption} c. Dimini,h.d. but nOi absent. vit.min K moy lead to • subclinical mixture of normal cwgulation factors .nd PIVKA. Vitamin K supplementation wa. 'lSOCiatrd with a cor=tion of prolon~ PIVKA·..",itiv. PT values in cots with intestinal or h.patic dis .....".''' 3. Cause:! of abnormal h.mostatic test reswts a. Prolongc:d PT. PlT. or ACT (I ) Decre.,.d amounU of functioruol vitamin K-Jrpend.nt cwgulation f""tors participating in the surfa",. induced (factor IX). TF (factor VII). and common {f.ctors X and II} pathv..ays {2} Although PT is npected to be: prolongrd be:fore PlT. th.re may be: prolongation of PTf .lon•• PT alone. or both valu ... drpending on the optimization of the .....Y' and. perhap•• the .pecies involved.'" b. Thrombocytopenia {I} If p .....,nt. thrombocytopc:n", prob.bly .... ult. mOJily from coIIJumption .t multiple sitrs of hemorrhage. {2} Wh.n moderate or markrd. BMBT prolong.tion would be: rxpectrd • .. pecially if the patient is anemic.

Liver

,; "

.

Blood

Poorly carba
Fig. S,7. F.If..c", of viumin K :mugonism or deficimcy on hepatocyt~ production of vit..nin K~nt roaguhtion f:octo," {II, VII, IX, and X}, Ci,dd M"'..b", in the figw~ denor. th. thr ... following proc.=es th .. ,<sult in producrion of defK:tiY< f:octo," II, VII, IX, md X: I. Anugonism: IngestEd rntico"ll"unt rodenticid .. or other rourrurins:rn: . bsorbed in tb. inresti ... , In h.patocyt.., th." inhibit til. ~nzyrrutic r~ction of viumin K .poxide b.dt to r..!u=l vitamin K, whiclt i" tb. fo,m of vitamin K "'quir..! for thr viumin K-dependent corbozyJ... enzymr to carbo,,),b .. thr co"ll"l.tion proteins, Foilw. to lKJ'C1r vit:unin K Ie. ds to draourd 1JIlounts of ",ducrd viumin K md incrrosed 1JIlounts of vit:unin K rpoxide, Foeton II, VII, IX, ...d X ore "ill produced, It~, Tbry ~n"" tb. blood md cirrul ... in • perm.,..ntly hypofunction:d 0' nonfunrtion:d .Ute, Thesr poorly carboxybted or nonc:orbozybted co. gubtion haotJ :orr known .. PIVKA (prot';m inducrd by vitamin K . nugonism) rnd in proplr an b. "",.. wed ..itb lp
''''

308

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY c. F:OCior .n:dysis is cypirnlly not 0=1}', but ddici~nci~, in viumin K--J."..nd~m f:OCiors would bt flOIM. d. Oth~r .bnormalities hov. not ktn widdy ducribal. but da;'...... d [FDP,], induding D_dim.., :md inc,rastd [fibrinog<'n] may r~",h from deer""...! coagul.tion .nd co~u.ncly da;""asM fibrinolysis. ProJong«llT "porlN in ra'pl. corrdati:ulam blood componenu a.. denroYffl or removed from circulation (consumffl) and FOP, ~'" ~nemffl. 3. It may ~ri .. from DlC. in which cas< bl...ding is common. a. DIe is a .yndrome of consumpti"" coogulation occurri"l: throughout the v:lSClllatu'" (dis .. minatffl .nd imravaICul..). b. It is ~lwaY' sa::ondory to • condition that caU,.s unb..tanced or acessi ... coaguhtion. {I} Causes indude ,ev"e tissue n«rosi •• angiostrongylo,is. h.... cstrok.. di ... mi. natffl nroplasia. endotonmia. "P,i,. pancr..titi •• hepatic di",=. or some envenomations. {2} Co.ogulation is often initii:ulant factors do.. not kttp up with consumption. a"",ssive coagul ation am le~d to d.f«tive cwgulation (i .•.. consumptive coagulopathy). 4. Consumptive cwgulopathy may ..i,. from localiud consumptive co>i:ulation. in which GIS< bl..ding is ""e.'" a. Ex=sive roagul ation in • localiud a",~ or org:m GIn produc< labomory ,ign' of consumptive coagul ation and. rardy. dinical bl...ding. Cor=tion of the localiud problem may ~ curative. b. H uman aample! indud. ""nic aneurysm and large h'lIlIDgionus. With the .. conditions. di"",miruotffl intraVll.!Cular 3fcorrw may ~ examples oflocaliud consumptive coagul.tion in dogs. How",... the chonges h. ... UJu..tly bttn im.. pmffl as Ole. S. Clinical signs vary from subclinical to .. rious hemorrh>i:e. ,hod:. or .igns of multipl. org.n failur., all1sffl by thromoo.mbolism or hemorrhagt. Sign, of the und..lying dise;o .. may al", ~ pr=m. 6. Consumptiv. cwgulopathy can "" rrcogniud when the typical laboratory and dinical signs .crompany ~ condition known to trigg'" the proa:s.s. Typical finding. are thrombocyto!",ni
5/ HEMOSTASIS

309

drcr .... snl AT activity, or {{: Fibrinog~n is oonrumM a, it i, t..",formM imo fibrin during actlsive cwgulation, B=<= fibrinog~n is a politive arut<'-pit= prot~in, incrnsnl h~patic production llUy occur in inflamm>tory sUt~, and mask th~ hypofibrinog~n~mi. typiGlI of consumptive coagulation, b, Thrombocyto~nia: Platd~ts .'" consumM as th~y patch ap<>Rtion by hq>atocytes. Thrombin allO binds thrombomodulin on ~ndothdia! ctlJ m~mbr:mes, and the oompla is im~rna!izal and d~adM' [f the "'t~ of enzyme inactivation and removal r~main' ~at~r than th~ "'t~ of production :md rd ...... of n<'W proc:nzym.., plasma activiry of th~ .. coagulation factors drcr .... sc:s, {3} FOPs &om fibrinolysi, int~rf~ .. with fibrin formation, d O=.... 'M AT activity: Mt~r AT binds to .ctiv>tM coagulation ~nzymes, th~ complexes .r~ removed nom th~ circul.cion by hq>atocytes, If the rate of .. mova! "lIUins gr ....ur than the ",u of production . nd rd...... of n~ AT, pl asm. AT . ctivity d= ......" e, Incr • ."M [FOPs] .nd [O_dim~r] : With activation of coagulation pathways, cro.. _ linkM fibrin form" with concur",m . ctivation of fibrinolysis :md plasmin_ mMi:nM deg",dation of fibrin and possibly fibrinogtn, Thi, l.ad, to th~ incrnsnl forllUtion of FOP, .nd O-dim~rs, f, ProlongMIT:md ITa-..: Thi, can be G1U'M by hypofibrinogtnemia and incrnsnl [FOPs], g. Prolonged BMBT: Thi, can be G1usnl by thrombocytop
310

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

weight rolloid, (dalran., oxypolygt'Jatin, and h"" ..curn) may have addition'! df«u on herno,us;" such .. cau,ing gr.ater d«remem, in Ih. amount of plasm. fibrinogen and vWF:Ag. Th•• ffea, dq>-m Without .d"'luate functional fibrinogen, fibrin clots cannot form, thus prolonging PT, PIT, ACT, and IT. B=<= fibrinogen j. the mojor im.rpl.tdet bri<4:. during >ggreg. {ion, BMBT may""

prolonged. F. Inhibition of filllction:d ~Jation f. ctors"" l. Hq..rinizalion a. HqJ.rin .dminim.rion or rdra .. from "roplaslic mast

~ll,

may prolong coagu.i.lion {im.. and predispost p:uients to hemorrh3l:" (,.., Fig. 5.4 for th. m~anjsm of ""lion). b. Unfraccionalal h"lmin prolongs PTf .nd ACT v:du .. but typically not PT v:du ... PT assay> !:"n,,:dly ronuin a h~parin inactiv;;ltor. c. LMWH d""r ...... f.ctor Xa activity .nd mayor may not prolong PIT. d. AT activity d""",.sts aft" h~parin .dmininntion. 2 FDPs: Th")" contribut~ to h~morrh:ogt. prolong coagul.tion tim .. (PT. PIT, ACT, and 11), and d""r ..... pludet function. 3. Antibodi.. to ~lation f;;octors:"" a. As with human h~mophiliac p"ti~nt, r~iving muhipl~ tramfiuions, dogs transfu...d for h~mophilia A and h~mophilia B han .Iso prodUcffl antibodi.. to the f.ctor th")"I;;ock.""'" b. Antibodi.. may:dso form as .n autoimmun~ phenomenon, and th")" may have reactivity to co"l:"lation factors other than factors VIII and IX. c. Antibodi.. am prolong ~lation tim .. of the aff<eted pathw.y or p.thways bec.u.. of antibody_mediated inhibition of th~ urge! protein. d. Normal pooled pla,ma doo; not corr<et the prolonged coagulation time, wh~n mixed with p"ti.nt plasnu; prolonged ~lation tim .. r~H""t an incr.....d propensity to blttd.

THROMBOSIS I.

I1mlmwlu i, the formation of thrombi within the vaocular 'Y't~m. Thrombi ar~ in vivo "clot," but adher~nt to th~ vossd or h... rt wall and romposed of varying amounts of fibrin and blood ~Ils. Thrombi that form und~r high_How conditions (an"i;d) contain more platden. Thrombi that form under low_How conditions (venous) contain more .rythrocyt~. and f~er platd.n.

II.

Thrombi form wh~n the normal bal:m~ betwe~n prothrombotic f.cton and .ntithrom_ botic f.cton mift, to favor thrombosi. (i.e. , thrombophili.ol. Th~ specific reasons for this mift are not rompl.tdy und~rstood for many condition •. A. Th~ shift may occur with dilOrd~" involving one or more of the foUowing mM.ni,ms: l. Endothdi.ol cdl activ.tion or damage: 2. Platdet activation 3. Activation of coagulation 4. Blood st;ui.

5/ HEMOSTASIS

311

5. Inhibition of fibrinoly.is 6. O~fici~ncies or abnornulities in anticoaguhnt proteins (e.g .• AT. protein C, protein S, factor V, and po .. ibly protein Z or PZI) or abnormalities in coagulant proteins (e.g., prothrombin)"" B. Acquiral condition. associated with thrombosi. include th~ following: l. Vascu.litis, endocarditis, intr:lvascu.lar COItheuriLotion, .nd injection of chemical irritant. 2. Conguti", hean failure, fdine COIroiomyopathy, and v.scu!:'r defect. 3. Surgical or nonsurgicol trauma 4. Malignant n""pl",ia 5. Sepsi., endotoxemia, and «J.uin~ colic and colitis 6. Immun.... mediated hemolytic anemia in dog. 7. Hypothyroidism and hYP"radrenorortici!JII 8. AT deficiency from protein_losing nephropathy, ~nteropathy, blood 10M, or liver failu", 9. Prouin C deficiency 10. Anti_ (phmpholipid_prouin) antibodies {.ntiphmpholipid :mtibodie.}" II I.

Thrombi may COIUse clinical signs by obstructing blood flow and cousing ischemi. , by forming emboli (fragmenu) that travd to dinant sites .nd COUK ioch~mia, or by consuming platelet • • nd oth~r hemonatic factor.;. which may l~ad to h~morrh .ge. Thrombi may occur in • singl~ ar ... b«aUst of local disturbances (e.g., ch~micol irritant. injaoted into the jugular "'in) or at multiple sites b«a.... of more systemic abnormalities ('.g., DlC).

IV.

Clinical .igns ar. variabl~ :md d.~nd on the und~rlying dise= and location and ..,,,,,rity of the lesion{s). 0Y'pnea may occur with pulmonary thromboembolism, hematuria with renal infarction, edema of the head with cranial vena cov. thrombosis, .nd distal . ortic thrombosis may co .... rear_limb wealme .. and p"in, dao",ased femoral pulst pressu", :md quality, and cool extremities.-.m

V.

Potential hemostatic . bnornuliti.. A. Inc..ased [O_dimer] is ev:id~nce of cross_linked fibrin formation and degradation and th~refo .. intravascu.lar coagulation. B. Inc..ased [FOPs] can b. ev:id~nce of fibrin degradation, but it could b. the remit of fibrinogenolysi. without intraVllSCular m . gulation. C. Oao..ased AT .ctivity COIn b. evidem:" of conmmption coused by intravascular coagula_ tion. but it may """,It from AT loss or decr• ...d production and may contribut. to intraVllSCular magulation. o. Inc... ied [TAn i. evidence of increaied thrombin \:"neration .nd th~",fore intravascu_ lar roagulation. E. Oaoreased prot~in C activity con b. .vidence of consumption coused by intravascul ar coagulation, but it may al", r~sult from other COUstS, .uch '" vitamin K . ntagonism or h~ralitary defici.ncy, in which COIst it may contribut~ to intravascul. r coagul ation. F. Thrombocytopeni. COIn b. evidence of conmmption coused by intravascular coagul. _ tion, but ther~ are m:my other cous ... G. Oao..ased [fibrin~n] can b. evidence of conmmption COIused by intravascular coagul. tion, but incr• ...d production ou.soci. ted with inflammation may m.ok short_ ened fibrinogen survival.

312

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

Refe re nces DKIouy-P~vnt J. T oci F, Satn N. r ..qurt /,\1, Un.. A. F...,..."'« JM. 1996. Pk,..iop.t!.o!ociad oipfia."", of <1talytk pbo.pl.olipido in d" V"' ..1ion of thrombi." s....in lboomb H"""" 2h IS7_ 1&l. 2. Solum NO. 1m. P"""'-l"lan, "'I'"" ion iD pIat
l.

YaK BioI

1~2S41 _28.j6 .

J. Oodoit S, R- JP. 2001. Fibrin dot '
C ... y" J JOo.J«--J4S. 7. Saro I. """,",00" GA. r ...., BW. 2000. An. into""-....... ..,J i",,~ mod, ofbuocal m~ bI«
V...........,

P«>'"

...va.!

II .

11. I}. 10. IS.

16. 17. 18.

19.

20. 21 . 21.

2}. 2• .

2S. 26.

.j1<>idal ""ti.in8......,."''1 dru, admj";,tration in tI.o dor;. NZ Y« J """In-lSI. Gu~ E. Ui..n.. EF. 1965. Plat,k< io ~,.;tal a6brin.>~0"";" n",mb Di,th H"""",,~D..."j. and • ci.culatinc; antibody ap;"', Iib.~ in. biclo.>n f,j" d.o&- Y" Clin Pathol Jo(,14S-ISS. Broob M . Catalf&mo J. 199}' B"",al m " " ,. . blrodin& ci"" ;" p""loovd in ,...;"., mod,~ of ptimaty b""""taci.c «d pl_]", «activity in b,,,,.....,,....i..fecttx] d.op. Am J Yrt Ro. 5O,IS#-IW. G ..... RA. Ruuo fA. G.«D' RT. Kabel. AL I S18 S. Hrpoalb.omin,m;'_,d,t«l pl"d« ~1P"'"""thl.,. iD ..... do", .... tb n
r....."""

RDd"".

""'o-

te«thl.,.

,.,,«

oc.

5/ HEMOSTASIS

313

27. P...... Mr. T • .....n... MA. lo ......n GS. 199!. Von Willobond facto,;n I,..." of .....bod ania, pla,d.... Am 1 v.. R... 51,119_ 125. 28. B.noon RE. loionoon GS. Dod.do WJ. 1981 . Ilindin,; of Jow..moIocub.-""'&b' =in< f.c"", VIII "",,,,lan, from "'" Wdkb....d pla.",. '" ani", farto. Vlll.....,h,od ""tivn- B, J H""""toI 49,5-1 I_HO. 19. S..Jli..an l'S, G..bbo ST. 0Id00..y TWJ. And, .... E\l . Wbit, JG. c. .. Ib."", JI.. Dodd PA. McDooald 11'. 1994. Bl ..... " fooJ. Vet Roo 157,}Z2-J24. }}. ~ RA. B....... MB, Bain IT. Bpc. TD. 2001 . CIinicaI~ , Von Wilkb....d dioc." in. tI.oro..rhh "".<m,o' ...itb tbyr=in<. Clin AppI T .... "'b H"""" 7,11}-11 5. }7. A'V''' S. l.otI."'p CD I•• McDooald 11'. 1990. PI ..m. yon W..ucbuod f"""" «>nCCUtntion...d tbyroid funccl"" in dop. J Am Vet Mod Ao.oc 1960921-924. }8. P.ncic", DI.. Jo~ .... n GS. 1994. Pi ...... yon W..uobund factoc ~n concentration in dop with kn-hpoidi .... J Am Vet Mod A..oc ZOM 550-1 5H. }9. SodJ .. JE. 1"0. th, S.boo ...",i_ "" "'" WdId....d Foe"" of doc Scimlilic ...d Standudi.zation Commi"", of doc lot ..... tionol .'iocicty on llorombooi • ...d Hoc ....... i. 1994. A ... iood dauificotion of ",n W,Ikb..nd <>type in Dob..", ... pinodo. .... Am J Vet Re. 6Z,.J6 ....}69 . .. I. B....... M. Ilaymond S. Ca,oI ....... J. 1996. s..... .•0C-929 . .. 2. SIappcndd. RJ. Bcij" EGM. "'" L.a.wcn M. 199B. T1f'C 1II """ W..u..bcando l"'l ond SI.. Jo",," DW. B"",h MB. Dodd WJ. 1990. 0iniaJ .nd w",...,..,. r.,.",,,,, of • ""'"' ""'" of YOn Wdkb....d .Ii"... in Slortiand doc.pd.op 1Am Vet Mod A.oc 197, IJ42-IH6 . .. 5. Vcnta PJ. II I. Y~'(;wLn V. Bm ... GJ. Sd.aIl WD. 2000. M",.tioo a~ .n.,; """ W..ud.cand" . d;""" in SrottiaI. J Vrtlntcm Med IHO-I9. .. 6. S.,...,. T . P...·, BW. M..."H PD. 1995. Factoc VIII oc,;,.ity in ani", '""" Wilkb.and dioc." . Vet (]in Patbol 24,81 _90 . .. 7. Mo ... J. M'1"" KM. M,inkotb IH. B..... d]A. 1996. T , mpo<"01 vuiatioo and foe _ aifoctin, m' .... 'm' ot of ani", '""" Wilkb.and (,"0'. Am J V.. Re. 57,1211S-129J . .. s. M....dJ PD. P"'l' BW. 1991 . Stabili.,. of conine fac"" VIII acthl'J"" .<><1 """ W..uobond facto. ~n «>ne Inva' 9,JI4-}17. 52. C..II.., MB. Gi ~ V . C ,,01 ....... JL Z00 5. EHect of "'-nopcc.in "" yon w..u..bc...d fac"", oruicl""", in Dobc.man Pinocb ... ...i
,,,ci.co•.

314

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

55. T .." ... cin, MA. HaboAW. Jolonoon GS. 19S6. Foe ... VIII rompla in ~ p"an • .n.,. ... b.....iouJ tr Wdkb....d' • .Ii...... Am J Y" R.. 59,111_ 118. 58. J o k _ IB. 1m. 0. ....0,,""';0 ...bu>«. th, bindin& of p ...... "'0 Wilkb...,d facto. to =Ilauo in pi .. ",.. fro", ""nn.1 do", and doc. ,.;th trati.on. in ,,YOtny MI, T ..non';"" MA. Johmon GS. Adamo HR. 19017. E..pn;m,ntal
W'",

61 . 8",...d]A, Moyen KM . Voo W' alt<md in azot<mic d.op"';:'" proIoovd bl«din,; tim" J Ub Oin Mod IU5s.-t;2. 62. Badyl.k SF. Dodd. WJ. Van VIcrt JF. 19l!J. PI ..... > ooarJo."'~ fact,,, .boo.malitia i~ do, • ..itb ........II, occw,;", k,l"'tlc tt W,1Iiamo !< Wollin •. 60. ,.... 000, BA. V,_SA. SI_..dd RI. 2004. DNA 'minc;fo. typ< II[ YO<> Wdkb....d dit<...,;" o..tcIt Kooikrt do"," I V" [at<m. Mcd IS,2S1--2S8 . 65. G"""""r; CoS. Ortlm .. CI.. 1m. Blood ""'l"J.ci.on""[ 6brin.>lpi •. [~ , u., GR. Fo. ...... I. I.uhtu I . Wlnttobo M'\l. od •. W"""h-' 0;.,;..1H, .... H)d, oditio~ . 684-760. Ph.il.ddplti", l..ippinro
""'o-

2Ik~6_..j2 .

70. O ·DoondJ MR. Slid.. ... SI. W.;.J,a PL. S~ R. 19I! I. Plaid" and fibo.i"", ,,, kinrtia in coo;", """"' •. c....c.. R... OI,lJ79-I.J.Il}. 71 . RDd"". GM. 1985. VucuIat ....... tIt muock from tit< cite ...... " , R..k of"";" • • b,l"'to
"".I,....

B.."'"

76. Gtillin IH. 2001. Control of ~.tioo "actio.... ]'" Eo Udrtm... MAo CoIkt BS. Kippo TJ. s"li,"' .... v . ..... W;u;..., H"""'''D . 6dt ottitioo. IH5--1449. N ... Yo,," MeG .... ·HiD. 77. S..... L H.X. Dalil>Od. B. 1997. Syo,~ rofacto. function offKto. V and p«>t
padt_,.

5~,2J6-2t1 .

SI . Liociandro GR. Btoob M. Catalfanto II.. 2000. Coo, .. , facto. cy in > Gtnn ... olo.ottb.itcd p<>int< ... clioiaJ..i.d""", of~. I V" [a' .. ~ Mod 14J011_lI0. S1. Mio
itlto.,

5/ HEMOSTASIS

315

DOC.....,

BJ. M,,.. DI, Dimond fP. Kearn.. W. H""", MK Ill. 19'96. D~ yol."". r.. dinic.Jly uo • ~ Y .. pui..., ,,{ p... ..,.j pI .. tic blood coIIoccioa r.. routino • ..J .peci.1izod """,Iati.on _P' A «>mp,dtto.i,.. ttuody. A.d. P.thol U, Med IJ0039--.t4. 87. Adrod DM, "'ouio OC. Madu RA. 19'97. Elfoc'''{ J.~ YO J."," ....Ii.", dtta", «>""""ati.on "" ... tin< «>apd.tioo ~ Am I Clio P.tI.oI 107, IOS_ IIO. 88. yon Pap< KW, AIaad f . Bok .... I. 2000. Pt.",I .. function .....p .. whl. PFA_IIX1 io p'ci.o", mcdia.ted..itb >«ty! •.Ji
,..bo.

"Thro...b

~

98,29S- 299.

89. Stclooi. T . B.oo ... MB, E..b HN. 2000. EHect,,{ ci....., n«ntuti.on. Am I Clin Padtol 109,S9$-S99. 91 . O'B';.o SR. Sdk" TS, M. RM, MuI" AA. 2006. Elfoc' .... routino ...d .paciaI ~tion "''';'''; vat.-,,{ d....., .~ ...;....m..., io p'ci,,, .. ..ith b~ h<",H<>«<> A. T """"" f . T ""'Iuillo V. lub.t G. SoIano-G.Jkco L 2006. Snbility ,,{ . m...! aoi ... pi ..... 1'0 .......... n ... "'.tint;. V.. aio P.tho] JS,20t-207. 9S. Adrod D. K...... D, Ma. .... RA. 199Il . TIt. dfoc' ,,{ tim< ..,.j ""'P"""" "";'bk. OD ,....,;"., """,Iati.on "' .... Blood ~ Ftb ......p .. M6J-0i70. 96. It- LV. 0k0r0d..J. AO, P........ IR. EIeJt<>
".tio,

316

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

Ito. Bartoo MH, Mo .... DD. C ...... N. ColI ..... C, r. .... KW. 1m. H ......... tic indic<. in buld" fooJ. fro", bird. to OD' month of~. J Y .. Di.~ ]"""" 7<J30.--J8j. Ill . Bartoo MH, Mo.n. DD. Norton N, Pro ... KW. 1m. H,,,,,,,tati.< and fib.inolytic indic<. in _tal bb ...itlo. """om,d ""ti.=nia. J Vrt l""m Mod Ih2<>-}S. Ill. Miod.kr R. DioJrid.. M, NoIt< I. 200.1. SnuitiYi.,. of djff..,,,,, p«>tI..ombin cim, ....,.. to facto. VII «"""" fu... W 1l,71- 7S. 117. K...... M, S"'p"" Y. y .......ki N. Kit"..... ] . Hun.da Y. Horti I. 2OO}. Mod.ani .. """ obort...; .. , PT and Al'TT in ""'" and "''', Effect of u..~o on YT and APTf. J T osi.roI s.; 2.'j,jJ9--44J. lIB. Mi..hlr R. ZOO}. Hqoouin io .itro ..... iti.ity of th, .ainted parti.J thrombopl .. tio rim, in =in< pIa_oo """'" of • low "",huIao .... ;po, koparin in i.tdth, do"," Am J V .. Rro 6hS9l-...S9S. 122. Hall DE. 1970. s....itivity of diil'=nt thrombopl .. uo ... , "'" '" f.c"" VII ~ in th, blood ofbt.r!, dop. Lab Ani", ·HS- S9. 12l. Miodo. s.w.., V..... ~·-,. H""",,!.rJ. S... odttion. 10.JO.-I0}6. PhiJ."";',,. 2nd <>do ...... 170}-1717. N .... Yool, Chwdillillvi_nt. lll. Wdli.",. JE. H""""" RR. H"",u" J. McDo~ J. 19111 . ClwK'"iz.oion of tbt inh.ibioion of lib.in • ......bIy b, Iib.~ f_,ot D. BOod, .. J 197.661--66.'!. Il4. O ·Kant MJ. Wiodom GB. Dtai ZR. Aodobold GPR. 1994 . w.ibitioa of6btin ""'........ polymtri ....... by ""do",. i"' ......p.,bulin. J Clin PathoI47,z66-268. l}S. G ..,inc,"" DA. G"",.\1A. Danid. TM. Kyit RA. s.,..;, EJW. 1?91 . Inbibi"", of th, ... "'mbift tim, in .,...... " .myIoidooi., A «><> «>apdation ab"""",aIi.,.. Blood 77,2637- 2640.

5/ HEMOSTASIS

317

136. Cu. ME J•• G.bdd DA. 19S6. H~,;""V"'mia .. a cao","( "",loop obrombin dottin& ci .... Sooth Mod J 79,S(;}-S7(I. 137. F.kkr WA. McDo ... ~ J. 1983. T!..ombin dottin, tim • ...d fibo.j""V" «>0«0'"'''''' io pati= .. ,,,,,od ..ith CF. !lo,;n EG. Jl.o..i., EJW. CarroU JJ. Joi" JH. uo ...... JG. Marl" RA. T ,;pktt DA. 19'90. ~ fot da. d."" .. ;"";"n,,( fibo.jnovn ;" pia ....,,, App......! ~lio •. N.tk.,,J fo. Clinical L.bo",<>-1019. P\ilo CF. s....i< EjW, CutoU JJ, D., HJ, Joi" JH . Ln..b.o JG, Mati., RA. T';pI,tt DA. 1m. Drt«IIuI.. arcinomaa. C an«< 74,IH}-I5-i1. 148. c.....r SA. W.."" K. Co.brtt J. R.od.oJpk]F. &b HN. 2000. I'rotoino itwokod by';""';' K abo=c. • .,.J cIottin, ti .... ;" dioiaUy ill ca ... J Yo< 10«<.. Mod 14,192--l97. 1.. 9. H, ..I= HC. Ydthmp JJ. H,,,,,,,A. t...dip EA. 1963. N• .....,,,( protbrombi .. bOooyntb .... ~b"""" io vitamin K-< ""taconi.t' ("PIYKA0). Iru Kitk RW. od. C"""", V......;-,. v","'1':1 [)(, S-o An;.-J P-';". SI}-SI S. 1'hiladdpNa, WB So""""' •. 1St. [l""",.ki EA. Drobatt Kj, HV' D, Sootti M. Giv< U. 2001. T!..ombot .. (PNKA) , .. ...-Ita i .. 2S doc. ,.;th acqui.."J...d J.....Jjtary roopoIopathi ... J Em .., Cdt Care 907}-7fJ. ISS. l'u.t..J. N . Btaoo U, 1\0...,. R. lutt H. 19'97. AotithNmbin_1ll "Ii,;ty in pia"". ofb",ldoy...d .ic1 catt!o. Y" Roc 140,17_ 18. IS6, Miod.h R. No/t, I]A. 2000. H""",n.;', lnoroductioo. <>Y«Yinv, I..,,,,,,ty tedniqu ... 10, Fddman BF. ZinlI JG. Stb edition. SI9-S25. 1'biIa91.

Co.nmj,,,,,

,,""Ioy.

""'o.

318

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

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16 • . !'Inca L\.,!Iodin G , c..cui ..m M. CoIhon D . 19'9S. H .........tk balanc< inlIammat..,.....cion; ...id. oped.! to antithrombin HI. R.>m J PJ.y.ioI J2m- 76. 16S. Wdl... EG, Bo..d«aW< MK. T yk. JW. 19'9J. Plat.,] .. , aotitl.rombin, ...d Iib.iDOIytk octirtti" in ta...ino-en,; ..... in at> ..it!. bcut di.".... Am J Y" R... SS0614Il. 167. Kobayad.i N . T ,krda Y. 1m. Studio. of -.dioI, prop...,.." rortiool, thrombophJobici •• ...d typ/><>id .... dn. on .,... ... " .. and cataboli .... of aotitl..ombin ]][ in tho ~ Thromb H."""" 370111_ 122. 168. a.",AC. Kraj. BJ. 1m. UK oi=ombin"", ti"""pI....in.vn oain_ 1'0. """tic du<>mbol";. in . k~ ,';n....k do&- J Am V" M«I A.oc 212;SJ9--S43. 169. lola JH. R..ru.=i G, Mann"""; P.\l. 1994. Abno.mal bkedin, and ",...,.,.boo .. in "0.1
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nl- 9lS. Philaddpbia. JS lippincott. 170. W,kh RD. Watkin. JP. T.yIoo TS. c..I..n ND. Cart.. GK. 19'92. Di-minated in...vaonoLa.~.tioa . ooociated ..ith ""Ii< in 2J 1.0.",. (19M-1m). J Vrt [nt<m Med 6,29--J5. 171 . do~. A.\1. F=man LM. SIu... SP. B.....b MB. Razan.li fA. Ru.h.JE.. 2OOJ. H<mOOt.tk <~. i~ CQ.Urtnc """ •. J Yrt Int<m Med 17,6701...-679. 172. H,Ild., ..... LJ. Slapp o~ b,p&d~ "''''PJ'. """.do ~ 1076-1079. 175. Jaroby RC, o..;~ ~. fr. O,,~ T. Gouolin R. Fold", ... EC. 2001. lIiodoomic.l baoi. fo. ... , k~t..tt« O«D in Cuhi", ~, . A.do. 5.'1: IJ6,IOO}-IOO6. 176. a..pital AD. H,..ddd. 5R. Uoyd L. Pi.p<. D. 2001 . n.., ,if«<. of <> .mitl.",m · bin HI l...d. in """ •. Am 5"" 67,25}-1S5. In Ni,..,., RW. PfoIlmdoofBA. Stwk A. l=>pinc RJ. Sd.""P MC. Hack CEo Po"'". M. 1~5 . n.., ~ of i.....tin. brt. ·"tradioL d<=mrtb.""" ",d dop<>id ho"""", on tho oeartio.. of ~.." ...d ......... pnKOin. by Hl'<" MJ. Pu ... KW. M<>l'<" MJ. Pu ... KW. 1996. UK of ""J"",.linluxl imm""" . "",,~, ...., to m' .... ' thromb;.,....."cithrombin HI rompl<=< in bo." • ..itb ooli<. Am J Yo< R.. 57 ... 56-462. 185. M ....J' .... H. W.to'; T. Mi",. T . SoLi M. TaLl.aohi T.!Co" H. Y.....ya Y. A....., K. Edam.", K. Saem"'''''", i~ do ~ • ..ido mali""", """".n. Yo< R.< 156,8}9-840. 186. Bartol. PC. Sd.o... [ M. '""" B..!,,,...... AA. 2001 . Rcducti.on of ~ """'" d", to in vi.... actinoion of ooapo.b.tio~ . Clin lab 4M4~51 . 187. Wdko EG. Pu", KW. DwoanA. Men .. MJ. 1990.~nk ....J' /"0, p..,tdn C drtonnination in bo. .... Am J Y.. R..o j h 107j....1079. 188. Wdko EG. Pu ... KW. M..... IN. 1991. U", of~....tJ' dn·d.ped....yo /"0, P"""" C...d ~ i~ bo.", • ..itb of ooIk. Am J Yo< R.. 52,Joij....}51. 189. Jok_ IS, M.rti.. CA. 2000. Compo"';'" of th, kwaan p."""in C actinto,. 1'",,_ <><> ... , ""ti,..ted porti.[ thrombopl .. h ..J;,;....,. H ..... S·O' P\,'l..kIrh'" I i rr ......... WiII .. _ ... W,II"~

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191 . Ed .... LM. Mon" DD. P..... KW. ~ MR. 19"93. H~bI.. . tat< • ..oci..,od..ru.. ~ ofpo<>tmb Ro. 55,297- 307. 19J. E-n.,n N1.. Sofa O. Smintov MD. E..ooo CT. 2000. Anripbotpl.olipid ...cibod.i.. ...d tt.< p""oio C ""tt.""y. J A~toimm"" 15a21_u 5. 194. py-", cv. E.t<>uf MP. 1m. n, ioohtioo of • 0"" .....&.; ... "'0,;.;..., po<>t0';0' pt-n.. Ili.ocJ..... s.... Tuno5a5j....2S6. 195. V... Wrtt P. Oddford P. H"p" P. ZOO4. Munrioo ....ilhio tit. p.. t SV. 198J. n.., dcu"." of b......., lib.iooc'" f_,m . X ...d Y in mi,,,, A ......-. mediated by tit. ft'C"'m' D .«olitm of bwaan 6btinovo fta&m=' D in "" ... a1 ... bjacm .nod "" ....... with ~"'. cinho ... Thromb H."""", 4Ht6-I"9. 202. R.j~ ... S. I'i"", SV. 1986. Clo....rn-ri .. tioo of .. u..... p«ptot". b lib.in(~) "'uad.tioo product •. Blood 67,1224_ 1228. 203. AJdune"<>D"'~ "C1 41 ,159-165. 210. R.imondi P. ~ O. t K';"'ro.iodoced 6btinovo prot",Ipi •. I Clio In",,, 87,1()81--109(I.

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5/ HEMOSTASIS

321

244. Bartol 1M. Tbompoon 11 . Min";" SM. 0;....,. TI. 2000. H,,,,,,,,,k~ di....... m< 21Z,SS7....sS9. 246. lutu G. Ron.hild W. Ehvt" I. l.tppd., I . Ku."d"....m K. 2OOJ. Cut <>niam in ... ' <10&0 n=eut ~ I AmAnim H_Auoc 15.691-f>97. 2So. Woodr BI. M"",ky MI. lUy AC. G,.,n RA. 1992. Co.pIopathic dl;""1'" ,,,,,J 0;""'" p,.",.;..•• }.d odition . 102}-I06J. Pbiladdpl.ia., IB Lippi"""". ]A. Rwk IE. 2002. Ma.iv< tno.fwion in d.op. IS ""'" ( 1997_2001 ). I Am 258. lutkm.in LI.. R..uaoki EA. Yrt Mod Aoo<>< 220, 1664-1669. 259. I""" PA. T om..ic M. Gm...,. PA. 19"97. Ot.cotic. k,modilu.tional • • ..t l.tmonotic _ <>f iootonk .lint and bydooxytd.yl •• <eb ...t.tion. in~....nuJ "","to. Am I Y" R... 5S,SolI - SoiS. 260. ~ KT. H .. liliu SC. Fddm.n BF. 1991. H .........tic d,foe" uoociated...it!. "'"" infwion .at". ofdo. T,.......fV-m.., 1""",.1 M"""..... Du..,.., of th. D., ...J c..t. 5th odition. IS29-IS41. Pkiladdpl.ia., WB 261. Gt.o....ki MM. M" " ..·M ..... PF. Eob HN. Bart Sc. 2OOJ. Elfoe .. of oxypoln:d.tin and dtno.n 70 "" b,,,,,,",,tic ,....;ab!t. in <10.: •. Y" Anat .... Anal, .JO,2()2-210. 26l. ffactoo IX "ci,;ty in. labtadot f ~ .. b ...... ito in b,,,,,,, ,..;th "",i" Fjy., "'"'" (198.!-I9"9S). Eopoin, Yrt I J},IOS- I09. Ill. No .... CR. Grilf"ti......d woi" in .... clinical w.o...,.,.. ock..cn. Y. I'ropatic • • ..t ....... in thrombooi. and b......... oi •• P... , Appl Ck"" 69,IOH- I079.

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Chapter 6

BONE MARROW AND LYMPH NODE Bono Marrow: Major Concepts and Terms ..................................... Bono Marrow Classif ications................................................. I. Bone Marrow Hyperplasia ....................... ........ ........... II. Bone Marrow Hypoplasia ....................... ........ ........... III. Bone Marrow Lymphocytosis ....................................... IV. Bone Marrow Mastocytosis ...................... •••••••• ........... V. Myelofi brosis..................................................... VI. Myeliti s ...................................... ........ ........... VII. Bone Marrow Necrosis ......................... •••••••• ........... VIII. Myelophthisis .................................................... IX. Bone Marrow Hemic Cell Neoplasia ............... ........ ........... Interpreting Results of Bone Marrow Examinations .......... ........ ........... Lymph Node: Major Concepts and Terms ...................................... Lymph Node Classifications ................................................. I. Hyperplastic Lymph Node .......................................... II. Reactive Lymph Node ............................................. III. Lymphadenitis.................................................... IV. Lymphoid Neoplasia (Lymphoma and Lymphosarcoma) ................ V. Nonlymphoid Neoplasia (Typically Metastatic) ......................... VI. Other Findings ...................................................

324 334 334 338 340 340 341 341 341 342 342 357 358 363 363 363 363 363 365 365

323

324

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

T able 6. 1. Abbreviation. and ,?',nools in lhi. chapler

ALSG AML

eBC CD CMMoL D NA

E"" F, FcLV G:E G_CSF GM_CSF M:E

M6_Er MDS MDS·EB MDS_Er MDS·RC MPD MPO

NK TCRo.~

TCR')':'i WHO

Animal uuhm;" Study Group Acuu mydoid lalhmi<> Complete blood count

Clune. of diff..entialion Chronic mydomonocytic I.uk-mi. Ckoxyribonucleic acid Erythropoietin [ron

Fe/iM leukemia viru, Gr:mulocytic to erythroid Gr:mulocyt. coionr-stimulating f.ctor

Gr:muJocyulmacroph"l:' coiony- Slimui.ting faclor Myeloid to ..ylhroid Amu crythrolcuhmia with erythroid r=iominance Mydodyspwtic .yndromc Mydodysplmic .yndrome--ac... blasts Mydodysplmic .yndrom.......,rythroid predominan"" Mydodysplanic .yndrome--rcfractory cyto!",nia Mydoprolifcmivt di",= MydoJ><'roxid= N .tural kill" « ~_Ant;gen_bjnding h.tcrodirncr ofth. T_lymphocytc r=plor IT -aU '=plor} ~Anticen-bindinc het
BONE MARROW: MAJOR CONCEPTS AND TERMS I.

Compmition of bone marrow {medullo oss ....} A. Bon, mIlm>w i, th. ti..,u•• ndo...d by conical and cm cellou, bon• •• nd it consists manly of h.matopoietic cell, •• dipose tism •• :md ,upponiv. ti.,ue. When h.matopoi_ etic cell, predominate. as in young :mimals. the marrow .ppurs red. When adipose ti"ue pralominat... a, in the diaph}'!"'li rq;ion' of long bon.. in h.althy .dult,. the marrow appears yellow beaus<: of its high fat content. Th• .,;tent of red marrow {""tive h.matopoi.,i,j diminish.. as namat .. grow to adult ,ize. but red marrow remain, within bon.. of the axial (ilium. ribs. n.rn"' .... and veneb ... ) and proximal appc:n_ diculor (hum..i and f.murs) ,kdeton. y.llow mar row con convtrt to red marrow during pathologic nates that ,timui.te hematopoiesi,. B. Nutrients are ddivtrM by ve"",]' branching from on. or more nutri.nt (medullary) art.ries that .nt.. through the cortical bone. New h.mic cdl, ent.. blood through th. wall, of medullory ,inu,es. C. Hematopoietic cell, develop in • supportive .,;trava,rular microenvironment controlled by. myriad oflocal and ,)"temic cytoki n...

[I.

HrmalrJ/H'inir ulli.re precursors to h.mic cell, round in the blood or ti.,ue. Microscopi_ cally recogna.ble h.matopoietic cell, indud. ctll. in the proliferation pool •• nd the maturation pool. of the megakaryocyt•. erythrocyt • • and l.ukocyt. lin.ag...

325

6/ BONE MARROW AND LYMPH NODE Table 6.2 . Noo ..,nclalure of the erythroid ""ries Rubricre synem' Rubriblm Prorubricyte a,>ophilic rubricyte Polychromatophilic rubricyte Normochromic rubricyte Mcurubricyte

Erythroblasl system'

Normoblast ,ystem'

P~rythroblan

Pronormoblan Ba>ophilic normoblast Ba>ophilic normoblast Polychromalophilic normoblan

Basophilic procrythrobJ:.st Basophilic erythroblast Polychromatophilic erythroblast Early onhochromatic erythroblast Lote orthochromnic erythroblast

Early onhochromalic normoblm Lote orthochroJrultic normoblast

, Tho wu.J nomencu",,,, in ..... rinuy modici"" • Nomencl. nu. u..d primarily in hum. n modici""

A. Megakar}'ocyl.lincage I. M'Cd.myorylrS account for < I % of heJrullopoiClic cells. butlhey.re vcry large polyploid cells. each wilh the capability of producing many plndCl •. 2. Thrombopoielin and olher cytokines {s .. Ch apter 4} S{imut.I' Ihe formalion of mature megakar}'ocyles from megmryoblam Ihrough mdomifl),is: the nuclem divides repeatedly wilhout cytokinesis to form cells of prog.... ivdy greater ploidy {mon about 16N}. a. MrCakll'}obld"" which a.. the first microscopically rccogniI.able cells of Ihe series, are similar in ,iu 10 rubriblam, mydoblasls, and monoblasls, and havc single nudei and dcq>ly basophilic cytoplasms lhat may form bleb •. b. Promr£llMrytXJlrS develop from megakaryoblasls and ha"" what appear 10 be two or four nuclei and scanl amounU of basophilic cytoplasm Ih'l m.y form bleb, (pt.le 9A [for all plales, J« th. color ..aion of Ihi, book]). c. Megakaryocytes vary in ,iu and ploidy. A> Ihey dcvdop from promegakaryocytes, Ihey b.:comelarger, have: more nude.r lobulalion., and have: more .bundant, paler cytoplasms Ihal break up imo :mud•• te platdels. Malure forms predomi_ nat. (Plat. 9B). B. Erythrocytelineagc: (Table 6.2) I. [n VCIerinary medicin., Ih. nuclealed erythroid precursors .re usually named using. ,...bri_ prefiL The cell, wilhin Ihe .. ries.re Ih. rubriblasr, prorubricyte, basophilic rubricyte, polychromalophilic rubricyte, normochromic rubricyte, and meurubri_ cyte, allhough Ihe.. is a continuum of dcvdopmem making cell subcl""ificalion difficult at limes. MCiarubricytes gi"" ri .. 10 rCIiculocyt...nd JrullUre erythrocytes. 2. A> cdl. proliferate .nd JrullUre (... F~. 3.1 ) from Ihe firsirecogniUlble ,tage, Ihe rubriblast. they decr..... in ,iu and cytoplasmic basophilia while incr .... ,ing hemoglobin conUnt and associaled eosinophilic ,uining {plate 9C}. 3. [n human medicin., :md somelim.. in VCIerinary medicin., Ih. nuclealed erythroid precursors arc named by u.
'"

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY C. Nonlymphoid leukocyt~ {mydoidllin~age< l. Nonlymphoid leukocyt~ p,a:ursors are oft.n ooJ]""tivdy J.beI.d mydoid cdl .. HowrY.f, there ~'" diff... m m.~ning' of ",}"oid and the prefix ",}do_. Thus, a word ~nning with ",ydo_ rno!! b. int.rpm«i in collta!. a. My!_ or my"". mranings: {I} Combining form denoting the rdatiomhip to bon. marrow

{2} Ikfwing to the nonlymphoid hemic cdls of th. mq:. bryocyu, lrukocyte, and erythroid ""ies {as in mydoprolif.rat;", distaK} {3} Refwing to granulocytes, as in mydomonocytic l.ukemia (4) Oft.n t=d in .~ific r.f... nct to spinal cord b. My/Did meanings: {I} Penaining 10 or d.,ivn! from bone Dlarrow {2} Pmaining to .11 nonlymphoid l.ukocyte pr«:ursors, as in th. mydoid to erythroid ratio {3} Pmaining 10 nonlymphoid hemic cd], of the meg.k.:IrYocyt., leukocyte, and erythroid .. ries, as ill .cut~ mydoid l~uhm", {which includ.. pur~ ~rythrol~uk~mia}

{4} H.villl: th~ .p~rancr of th~ myelocyte {5} Pmailling to spillal cord c. MyliKytr m~ning" {I} Int~rmaliat~ pra::ursor crJJ of th~ gr:mulocyt~ ",ri.. {2} Any cell of th~ grey man~r of th~ lI~rvOUS lyst~m d. Myliris m~illg" {I} InHamJrultion of bone marrow {2} InHammation of spinal cord ~ . My/ogmoul m~:millgs:

{I} ProducN in bon~ marrow {2} Pmailling to nOlllymphoid h~mic cells of th~ mq;.bryocyt~. lrukocyt~. and . rythroid ",ries. as ill acut~ myc:logc:nous (mydoid) leuhm", 2. Nonlymphoid leukocytes: monocytes. granulocytes (nrutrophils. '01inophils •• nd barophils), alld mast crJJs {= Chapter 2 and Fig. 2. I}. a. Nrutrophils pralomillat •. Th. Jrultu .. neutrophils in the boll. Jrulrrow a.. coll=ively r~f.md to as th. nfumlphil "rmlgr JH»I. b. Granulocytes are d~rival from mydoblam and promyelocytes, acquiring Ip«ific secondary granules .t th~ myc:locyt. lIag~ {llrutrophil myelocyt. , eosinophil myelocyt~, :md barophil mydocyt~} (plat~ 9D). c. Monocytes d~dop from monoblam and promonocytes, populations that are inconspicuom in bon. Jrulrrow from h~.hhy animals. Macroph"*,, d~rivffi from monocytes, are pres.nt in low numbers. d. Mast a:Jl precursors are 1I0t d~t=abl~ ill ch~ bone marrow, but low lIumbe:rs of .. sid~nt mast crJJs drrival from hemic "~m a:lls may be present. D. lymphocyte lineage I. lymphocytes .nd plasma cells are pre:s<:nr in relatively low numbe:rs. 2. All lymphoid crJJ. are drrival &om a common bon. marrow precursor a:U {Fig. 2.1). 3. Cd], destinal to be: B_lymphocytes matur~ in th. boll. m. rrow or in 5Om~ oth~r tissues, wh ..= cells destinal to be: T_lymphocyt.. migrate to ch~ thymus alld m. tu .. th.re.

6/ BONE MARROW AND LYMPH NODE E.

327

D~lIdritic

ctll, I. D~lIdritic cdl, "'~ 'peci..tizal for uptah. tnmpon, proassing, alld pr .... ming antigen. to T_lymphocytei. Th~ .. ar~ , ...,,:01 subtypes, indudillg LmguhallS cdls, int~r.;titial d~ndritic ctlls, :md plasmacytoid d~lIdritic cdl .. ' 2. All d~lIdritic ctll, ar~ bon~ marrow d~rived, but th~ir origins .. ~ not compl~tdy und~mood alld th~ir p=r.;or.; ..~ not r«:aglliud micr05COpically. 3. D~lIdritic cdl, "'~ bdi....d to originat~ from both mydoid ~nd lymphoid precuuo ...l.' F. Bon~ marrow nudrat.d ctll diff~r~ntial munt ill hrahh I. Th~ expect.d ntio of 1I01llymphoid leukocytes to ~rythroid precursor.; (mydoid to ~rythroid ntio) v. ries with th~ species and th~ illdividual. Av~~ ntios in h..ahhy dogs, cau, hor..,s .• nd c;ml~ ~r~ reportal to k 1.25, 1.63, 0.93, .nd 0.71. respectivdy.' 2. [n huhh, "",Us of th~ n~utrophil and ~rythroid ...,ri.. pr.domin.t~, .nd th~ majority of ctUS in each linrag~ ..~ th~ mo .. m. tu", cdls.' a. Mydoblasts alld promydocyt~s acrount for < 4 % of th~ lIud~~t.d ctlls. b. Sq;m~nt.d and b.nd n~utrophils ~r~ th~ pr.dominant leukocytes. c. c.Jls of th~ =inophil ..,ri~. um:olly .ccount for < 5 % of th~ lIud~. t.d ctlls, wh~rras ctlls of th~ basophil...,ri .. a.. ra ... d. Rubriblasts .nd prorubricyt.. acrount for < 4 % of th~ nud~~tal edb. ~. M~t:lrubricytes and lm·_st~ rubricytes ..., th~ pralominant nud~t.d ~rythro_ cyt... Polychrom. tophilic ~rythrocyt~s ..~ pr=nt. f. Lymphocytes usu..tly .crount for < 5 % of th~ lIud... t.d ctlls ncq>t in cats. Up to about 20 % lymphocytes hovr bttn "'pon.d in h.. ahhy cats. g. c.Jls of th. monocyt~ .. ries mnstitut~ < 2 % of lIud ... t.d ctlls.

Ill.

[lldications for bolle m. rrow u .. minations A. Wh~II"""r th~ following a", unuploin.d I. Dec",=
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FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

3.

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lysosomal IY.

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Mffhods A. Complete description. of. bon. marrow biop'Y {colla:tion, fixation, staining. and aamination of bone marrow from a living :mimal} are ~yond the scopt of thi. tnt. Procedur.. of a bone marrow biopsy are descrikd in S<'V"al sour"""w, B. Bone marrow oolJ'""tion and processifll: l. Typically. bon. marrow umpJes are colJa:t.d from ,ites that are «preted to Ita"" actin hematopoietic ti,sue. a. In dog. and cau, ouch {issue j • .,.~{ed in the iliac crest or in the mMuJJary Clvili.. of proximal femu," or hume,i, b. In horses, such ti.,u< is most .ccc:ssibJ. in the 51emob"",. [n cattle, medullary caviti., of proximal ribs cm k ..mpJed. 2. Th. two major m~hod, of ooll=ing bon. marrow sampl.. are .,pir.otion and core

coll=ion. a. Aspiration: A R"""mhal or illinoi, biopsy

IIrffil~ is illsble forms r'"'luir. steriliutioll .lId ,h ar~lIillg. Disposable forms are .""jJabl~. b. Om roffinK- A J.mshidi biopsy IId~ is illstnffi imo the cortical boll. adj.cem to boll. morrow, the .tylet is ",movffl, the lI.ffil. it adv,"ced I illch or mo", imo th~ Ixm~ marrow with • forceful twistillg motioll, the IIde is roytffi 360' severn time!, alld thell the IIrffil. is withdrawlI. Aft.. the IIde is withdrawn, the .cylet is usffi to push the co .. Iampl. out the top of the IIrffil. (1I0t the tip .lId that is ""rrower thall m"" of. Jam,hidi IIde', bo",); this promote • .. tri..,.l of a cylilldrical co .. of tissue. 3. Proce... illg boll. marrow aspirat~ samples a. To rffiuce clot form.rioll .lId autolytic challges, it is critical that such samples be proccsstd {sample dimibutffi .lId fixed omo gla ... did..} withill secollds of collectioll. A v.riety of .mear, "squash", roll, .lId imprim methods may be used to produce ,lid.. that ha"" mOllolay.", of imacr cells md foci of bolle m arrow panide! that may be ,yillffi alld examilled. {I} Aft .. ..,pintioll, drops may be placed 011 a Jeri .. of glass ,lid.. alld quickly sp",.d by eith.. a wffi~ blood ,m",r techllique or a hori7.0mal "squa,h" t..mllique. The Ian.. bett.. 'pread, cells that a", presem withill panicles. However, drops may be large, r.. uttillg ill ,mean that a.. too 10llg or too thick. Th...fo .. , olle mould comiderliftillg P"'" of •• ch drop up with the elld of. 'p",ad.. .tid•• lId th.1I u,illg the .pr~.d .. ,lide to m.ke squash p"'P.ratiolls with thi •• mall .. amoum of sample. Repeatillg this proceu COli yield m allY good smea ... {2} Oth.. techlliqu .. COli be Usffl to deer.,... hemodilutioll of the filial UII.,.'" alld maximize ceUularity md marrow particle dellSity. {a} Aspiratffifluid COli be expressed at the top of illdillffi dides so that the blood flows dowllward, l.avillg mo", panicl...lId less blood at the applicatioll poim. The ,hort.dgt of a .pread.. slide COli thell be used to

6/ BONE MARROW AND LYMPH NODE

329

lift p:mid~_rich m>t ..ial "p'atrdly from the top of th~ slid~ and mah horizontal squash prq>.ration •. (b) Ah"'IlItivdy. coU.crrd fiIlIt~riaJ am ~ plac.d in • watch gt... or P~ri illill with amicwgulam. :HId th~ panid~ am ~ e';(rKtrd and spmod onto s1id~ without th~ co"",rn of doning. Antico:ogulants m"}' indu"" anif:ocu. b. Aspiration may yidd a "dry tap" containing e=ntially no material. in which = additional :ospir>tion mempts or oor~ biol"Y .. mples a.. ~uirrd. "Dry tal"" do not indicote cru.t the nurrow i. hypocdlulor; th"Y may oo:ur with bon~ marrow hyp",,,,,llulariry in a variety of disord~ ... c. For roU!in~ microscopic naminarion •• th~ air-drird sampl.. a.. Slaintd with a stain thai is u.s.d for blood film. {a ROJrulnowsky Slain such as • Wright Slain or Diff_Quik}. A Prussian blu~ Slain Jruly ~ applird to ....... th~ .mount of ,tainable F~ in a sample. d. Proce ... ing requir~m~m. for cytoch~mical or immunocytologic procalures should ~ obtainrd from th~ rdevoom laboratory. 4. Proa'lSing cor~ biopsy samples a. Afi~r making imprims by touching or rolling th~ ror~ on a gw.. dide. the core ,ampl~ i. placed in a fixative. B5 or unk .... fixatin is pref~rabl~. but routin~ buff",td form.lin is adequate. (Note: k""p the air-drird sampl.. far a"oay from th~ finti"" kcau .. formalin fumes am sev~rdy alter th~ ,taining propr~"'ntati"" sample typically comain. ,mall pieces of tissu~ known as bon~ marrow fracm~nu. bone marrow uniu. bone JrulfJOW grains. bon~ marrow partid... or bon~ marrow .piml... Th~ bon~ marrow fragments that contain adipose tissu~ will glist~n. Bon~ marrow fragrn~nu should not ~ confu..d with dottrd blood or platdet dumps. b. Th~ appo:aranct of bon~ marrow co.. sampl.. will vary from hYp'rcdlular rrd tissu~ to hypocellular fatry tissu~ to cortical bon~ or blood dots. 2. Microsropic examination: A complete microJCOpic namination of bon~ Jrulrrow sampl.. invol"". the cruract",i7.;,tion of the bone Jrulrrow tio.ru~ archit.crure. diff~ .. mialion and ~num~ration of ""ll, •• nd """luation of individual cdl S1ructu... Such """minations ar~ ~st complerrd on qualiry nainrd hi'cial Slain, can ~ tim ... ronsuming. Frequ~ndy. just :as much dinically useful information con ~ learnrd from a subjectin .....,ssm~nt of cdl population, by an np
3JO

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY {I} Th~ hematopoietic cdJul.rity of bone marrow f!'3l:ments rdlffi:' the hematopoiffic cdlulnjty of the bone marrow, and the rno", fragmenu thore are to ""amine, the W{{U is the assessment. HowN«, hemampo;"';c ""UllJ.rity of bon. marrow is best a,sustd in a co", sample, espa;ially when th". is bone marrow hypocdJul"ity .nd il j, l1nd~r wh",h.r fragrnenu of fat .'" nom the bone marrow or from utramMulJ.ry adipou tissue. {2} Cdll1J.ricy of the p",,:oration i. not the same as the heJrultopoi",;c cdluJaricy of th. bone marrow. If bone marrow fngm.nts. on .vtr:agt, .'" < 25 % adipou tissue, th. bon. ffi>rJOW is hy~rcdlul>r (pl". 9E). [fbone marrow fragrn.nu, on avt~, ... ;> 75 % .di~ ti .."., the bone marrow i. hypocdlul:or (Plot. 9F). [fbone m:orrow fragments.", not pr=nt and the prqmation is ""I}' cdll1lar, the bon. marrow i. likdy hYP"rcdlular and unlikdy hypocdluJ:.r. If bon~ marrow frngm.nu a.. not pr ...~m .nd th~ prq>.ration is not vuy cellular. th~ bon~ Jrulrrow may k hypocdlular or th. ""-mpl. m"y k poorly rq> ....,nt;;uiv. of bon~ marrow tissu •. b. H~Jrultopoi~ic popul.tions {s,,", Figs. 2.1 .nd 6.1} {I} Numkr and struCtlm of megakaryocyus {a} Th~ apa;t~d numkr of Jrultu .. mq;akaryocytes in a .. mpl~ vari~. among sp«i.. and i, highly d~nd~nt on th. quality of the .. mpl~. ~ally th~ num!J.,r of bon~ marrow frngm.nts in aspirates . [n h~.hh, [h~r~ is usually a[ l~..,t on~ mq::okaryocyt~ ~r marrow fragm~nt. [f more mq::okaryocytes a.. found than a.. ap=ed, then mq;aka,yocytic hy~rpl",ia is p.."'nt. [f f. wer are p.."'nt, then m~gakaryo oons<cutiv~ gr.nulocytic (or mydoid) and nucl....ted ~rythroid pr,""u""rs and then dividing th~ numb.:r of granulocytic {or myeloid} cdt. by th. flumb.:r of ~rythroid "",lis. For exampl~, if out of 500 counted cdls th~ .. w~ .. 300 "",J]s of th~ gr.nulocyte ",ries and 200 nude;;oted .rythrocytes, th~ G: E ratio would k 300/200 = 1.5. {b} Most authors .... m to ""'" G: E and M: E as .ynonynu. How.""r, • M: E ratio may indud~ monocyt~ p..cursors or mq:.karyocytes in the myeloid c;;oltgory, and a G: E ratio mayexclud. thest "",lis even though it may b.: difficult or impossible to d~finitivdy diff~ .. nti.te mono blasts and promonocytes from gr.nulocyt~ precursors. Th~ diffe .. n"" betwttn thest ratios would ",,,ally k insignificant btcau.sc: the", art u"illly fi:w r«:ocnizable "",lis of th. mono
(c) Oth.. "",lis (e.g., lymphocytes, Jrulcrophage;, mast "",Us, and stromal "",lis) .'" not included in th~ G:E or M:E ratio,.

Eryhoid pool

EooIo .......

@

" ," tJ -""'''" @

_COlO

l,.·oAoocyIa •

""'......... (() G:E = 41 :63= 0.65 M:E" 43:63" 0.68

.ha.

Fig. 6.]. Sdo..na.ic "'p'jOt pooh .... ..,11. of .1:0. ncy"" mly t.. ooosido.Rd p .... of the m~lo.id pool 01" may t.. ~ullcd .. I oepont • ..,U ]i~ and no. indo>dod in ,ho M:E ratio. MIlt cdls..., ... mit'l101ly difI"of.n,i ....d 0..... ulh 10m..- ,ban bemaoopoieOc cello and Ire DOl includood. in the M: E ra.io. Lymphocyte and plum. oo:lh bdong the n~loid lympboid pool 8, band n..."ropIUl; CFU-E. colony.rorming unit-<:I)'"Ih.w; C FU.G, colony.fotming uni<-v>nulocyt.; Mb. mydobl ..., Me. myftocyf~, Mmc. ......... J'<"loo:ytJo; M., "",.:uubricyto!: Pit. progranuloqu; Pt. prorubti. cytr; Rh, rubribt..., Ik, rubticyt.; ..,.[ S, "l"",nt..d ~trophil.

'0

331

332

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

(d) A G: E or M: E ratio is mimatal in hi,tologic m"ples, but it is mbjectiVl" and w.aI manly on ch~ mor~ Jrultur~ granulocytic and ~rythroid cdk Immature ~Us :ore difficult to diff.mllia'e ddinitivdy into the appropriate cdlline •. {oj The G: E or M: E ratio (p1.u 9D) m.y be, increasal bn::aUst of gnnuJocytic or mydoid hYP"rpla,i., uythroid hypoplasi., or both. If the morrow heJrultopoietic cellularity is increastd. granulocytic or myeloid hyp"'pJasi in bone marrow ofhe:dthy .nimah are small. but occasional larger form. and pla.ma edls an ~ found Hem>topoietic stem cdl, have: th~ appc:aran", of s.mall lymphocyt~. but are apc:cted to ~ present in v~ry low num~rs. c. Other edb: M""t edl •• macrophage •• fibroblasts. oneocwu. osteobl""ts. and other stromal ",ll"l< usually presc:nt in very low num~rs. Their nunt~r> may ~ subj«tivdyategoriud "" normal or inerea,«i. Incro:ased numbns em indiau or suggest cenain p"thologic processel.

6/ BONE MARROW AND LYMPH NODE

3D

OF, (I) In aspirat~ ~nd oor~ samples, th~ F~ pigm~nt in h~mo,id~rin will "'ng. from ydlow to ydlow_gr..,n to )'tHow-brown (pl.t. 9E), A mor. complet~ """""m~nt of F~ .tor:ag. C>II bt d.t~rmin..! if th~ umples """ !lain..! with an F. stain such as Prussian blu~, {2} Th~ .mount of F. pigrn~nt in th~ ..mpl~ i, estimat"! and r~port~d by using a variety of t~rms: (a) Abstnt or d= ...,al (b) Ad«Juate, within normallimics, or mod~rat~ (cj Incr~a !etl or abundant (3) H...lthyem lock detocubl~ marrow F~ 'tores, but F~ moy bt pr=nt bta"", of h~molytic disorde .. , .ft~r a transfu,ion, .nd in m)'tloprolif~"ui"" disorde ..,'l,lJ Stores may bt absent in h~ahhy young animals of .ny 'pecie., 3, Comparison of hinologic and cytologic enminationt a, Hi,tologic enmin. tion of rore ""mpl~, (I) Adv,mtag •• (a) Tissue architoctu", (how cdl, ..~ arr.nged), nocrosi" infihrati"" pattern" focollesiono, and mydofibro,is em bt better :asse<sal, (b) Bon~ marrow "",llulority con bt btller ass.un!, (c) Megakar}'ocyt~ numbtr con bt bttt~r:use:sset!, (d) Abnormojities of bon~ and "",,,,Is con bt better as=sal, (e) Further tissue .ettions con bt cut for 'pecial ,uins, (2) Di""dvant:ag.. (a) It i. mor~ np<nsi"", with gre.ter turnaround tim., (b) Cdl diff~r.ntiation i, mo", difficult, especially if =tions a", not cut thin enough (wg~t .. 3jlm), (c) Phagocytooi, ofh~matopoietic cdl, i, difficult to detoct, (d) M~ny dyspla'tic f~.tur~' .'" difficult to detett, (3) Potential problems with 00'" "'-mples include .hort """pIes of un""ruin repr=nution, sampl.. con,i,ting mostly of oonical .nd ,ubcortical bone, sample damag~ rdatal to prior "'pimion, cru,h .nifact from traumotic collettion, improp
334

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY (c) [nt~rpr<:Ytjon' are limit,"", if marrow pa,tiel..... not obtain,"",. (d) It may ~ difficult to determine wheth" a s;ompJ. is repr=ntatin (i.e., to cliff.mlliate a poor 5ampJ. from hypocdluJ.r bone marrow). {3} Potential problem, with cytologic prel"'fation, includ. colJ'""tion of ntm· ffi«iull.ry 5ampJ.., "dry taps," ex<:es.!ive hemodilution, don«is;omples, chick sm..ars, lyw:l cdl., and poor staining. c. Combining n>minations of co.. and :aspir>te marrow 5ampJ.. provides the most complete information. (I) Cytologic ~:dl1ation of aspirate, is almo>! alw:.y. indiauffl. {2} Histologic aamination j. "",ful when marrow ",mit«tu,. is !nJuir«i to ",.ch a di:ogno.i., myelofibrosis is susJl«tal (conn«tive Ii ... "" afolia!.. poorly), or hypoplaSlic sUI.. are 'Il'l~{nl (•.g., when pancyto!"'lIi<> js pr=nt) and th.refor. an ..,pinto our yidd f<'W cd], and a pam", that may not ~ distinct from poor .. mpling. {3} Wh~n both .amples :ar. coll<etrd, car. should ~ taken to avoid th~ first coll<etion procrdurr &om diminishing the quality of th~ KCOnd sampl • . A corr cm k artifactuaUy alurrd when coll<etrd aft~r an aspiraer in the same artl, and an aspiral< may ~ morr h~modilutrd if collin many proteolytic and lipolytic rITzym .. that .nabl. the digestion of edt.. b. &.mpl.. should ~ coll<etrd as soon as possible after an anima]"s death so th~ they will ~ adequate for .... aluation (guiddine: <: 'I, h for cytologic as..,..,ment). c. Sampl.. should ~ coll«trd from ao.nc:eUou. bon. in th~ proximal humerus or femur, or from the axial skdeton (e.g. , ilium, rib, or sternum). Although it is =i~r to roll«t .. mples from the diaphysis of long bon .., thest regions are not reliably rqJ .... ntatin of he"",topoietic activity. 5. Flow cytomeuy has bttn u...:! to ...... bone marrow aspirates and provide partial diff.rentials" but is not routinely used for clinical cases and cannot r~place micro_ scopic .... aluation.

BONE MARROW ClASSIFICATIONS I.

hyptrp!AJia i, a term that indicates an incr ......d

num~r of nonneoplanic cells in bon. "",now b<eausr of a stimulus that causes on. or morr edl lines to proliferate. Th. stimulm mually =ults from .n inc",asnl ntt,>irtir. Epo stimulation of pr~cursor cells ltl'" to a prolif~ration of erythroid cdls, erythroid hyperplasia, and in"",asrd rei="" of reticulocytes, nucle_ atrd ~rythnxytes, or erythrocytes into blood a. It is usually =:onda!)' to hemolytic (Tabl. 3.lO) or blood loss disord~" {Ke Fig. 3.8}. Th. m«l,.ni."" that ltld to ~rythroid hyperplasia begin immrdiately aft~r th. on",t of an~mia, but the remhan! erythroid hyperpla.ia may not ~ pr=nt until a f~ days lat~r. Th~ dq:rtt of erythroid hyperplasia should correspond to the duration and lenrity of the an~mi ..

Eo",

",.,m>w

h~matopoietic

6/ BONE MARROW AND LYMPH NODE

335

Table 6.3, Disordcn and conditions Ihu cau.e erythroid, granulocyti c, or n,,'Sakacyocytic hypcrpla.ia in marrow Erythroid hyptrpl",ia Eff«live erythropoiesis 'Sn::ond"y to hemolytic or blood loss disordm Sn::ondary appropriate erythrocytolic disord ... Righl_lo_l.fl munts, congcniul or ~cquired Chronic pulmonary di=.c Hyptrlhyroidi,m Sn::ondary inappropriate .rythrocytolic disord... Iknal ncopl",ms, C)"lIS, or dis ......,. Olher ncopl",ms {hepatomal Ineff«tive erythropoi.. is 'Immune-mediated nonreg.n.",tive an.mia 'Nutritional: F., copper, folat., or vitamin B" deficiency Cyclic h.matopoiesis of grcy oolli.. and F.LV_inf«ted cats Granulocytic hyperpl"'ia Eff«tive granulopoicsi. Inflammatory 'Infections: bacteri:d, fungal, viral, protozo:d 'Immune_mediated h.molytic ~n.mia 'N«rosis: hemoly.is, h.morrlugt, infarclS, burns, ncop!...ia, sterile inflammation Sterile foreign body Othe.. or unknown mechanisms p..",ncoplanic n.... trophilia Neutrophilia ofl.... kocyt. adhesion d.fici.my G-CSF adminimation Estrogtn toxicosi. {e:arly} Cyclic h.matopoi ..i, of grey collies and in FeLV_infccted am In.ff«tive g",nulopoiesis Immune-mediated neutroptnia Diphenylhydantoin and phenylbutazone toxicosis (suspected in animals) Chronic idiopathic nrntroptnia (G_CSF d.fici.ncy) Megakaryocytic hyptrplasia 'Recovery &om thrombocytoptnia: withdrav..al of myelosuppression or in ... poJl5C: to a disorder tru.t cau.scs d«reased platdet mrviv:d Inflammation: inf«tion, immun._mediated, surgery, t",um • • A rclllively oommon dis< ... or condition Not<: lnfolmllion :about eosinophilic. b1SOphi~c. mononude"'1'lugocytic,lympboid, most ""n, >nd g
b. !keD"""ry "PP"pri"" {ryllmKytonc ,w"rdnt: r... ist.m hypoxia or incr.~..d tissue demand for 0, promot.. Epo rd.ase, which cau.scs .rythroid hyperpl"'ia and .rythrocyto';s (..., Chapter 3 for disord ... ). c. !keD"""ry inapp"priatr nythroryrgtk disDrdnr: Inappropriate Epo production in th. ab",n"" of hypoxia em..,s .rythroid hyperpl"'i a and .rythrocytosis {s~ Chapter 3 for disorders}.

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY 2 bujJmiur "JtItropoi~'ir. Erythroid hyp<rpl:lSia occur> concurr~ntly with a non~n_ «"i"" or poorly reg~n~r.lti"" anemir:m"" of maturation arrest with ~rythroid ,,~ prior to th~ targtt "",U "ag<' oong num.rou •. " Macrophages containing mid_ to lat~_stagt nucll~ 9G). Wh.n eorli~r 'U~ ""II, ar~ targtllffl an:ock, th~re truly b. pure rffl ""H aplasia {Itt Bon~ Mmow Cla""ificotion •• =t. II.B}. b. N utritional d~fici~nci.. {I} Fe-d~fici~ncy an~mia: In th~ Fe-ddici.nt stat<. an~mia d....dops despiu erythroid hyp<rplni •. Erythropoiesis i, dd'ecti"" lat ...stage ~rythroid prtdy into the blood. Reticulocytosi, may b. pr<:Stnt. but th~ reI~... of r<:tirulocyte, is not ~nough to trlttt th~ nttd. As the ddici~ncy prog ....... the rubricytes may a~ar small~r and have I... h~moglobin in th~ir cytoplasms. (2) Ra .. GIS. . of cop~r. folal<. and col>:damin defici~ncies:""· Col»lamin ddiciency, which m.y occur sa::ondary to .bnortrull inustinal robalamin re<:<>rtffl in giant ..rnnau:z",rs. b.agI.. , Bord~r rolli ... and All'ltralian m~ph«d,.17 Dy'pi>scic chang~' may b. pr~ .. nt. B. GrllnuUxyric hyprrpfil'ill: Unle .. nata! oth~rwi .., gr;onulocytic hyp<rplasia (also coHed myloid hJpnpiasill) typiGlUy i, char:ocurizal by an incrl~ta! stotagt pool and. I~ft mift. Orh" forms of granulocytic hy~rpl",ia ..~ id~ntififfl :lS a.'inophilic granulocytic hy~rplasia and basophilic gr:mulocytic hy~rplasia (r;o,e). Btcau..... rly "'4:<:s of nrutrophil and monocyt~ prgniud wh~n th~re i, neutrophilic granulocytic hy~rpla,i
I. EjJmiur gmnuu,poiftir, Continual nimulation of neutrophil pr«ursor> by G_CSF,

GM_CSF, or ""rtain interleukin, I~s to granulocytic hyp<rpl:lSia, in"'~asffl rd..... of neutrophil, to blood, and typically a neutrophilia (with or without a loft mift) (Stt Chopt.. 2). a. Inflammatory: Th~ granulocytic hyp<rpla.
6/ BONE MARROW AND LYMPH NODE a.

Immun~_m«iiat«i

naltrop when m.gaka,yopoiesis is p<:r>i
C.

D.

E.

F.

G.

337

338

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

Table 6.4. Dioorocn and conditions that cause hypoplastic .Ia te. in marrow G~ncr;oliud

marrow hypoplasia '[nf=ion in bone marrow: bactcri"!, fung"!, viral, protozoal Toxicosis: braden fern, ~phalosporins, chcmOlhuap
'F.LV_indu=! erythroid hypopl,.. ia Endocrin.: hypothyroidiun, h~""nocortjci,m. hYl""'ndrogcnism Drug indu=': chlor:lffiphcnicol Sd=i"" gnnulocytic hypoplasia Infectious: p"rvoviru. (dogs, cats), F.l V, T"""p/as""" Ehrlichid (chronic) Toxic: br>chn f~rn, ch~moth~rap"utic drugs, chloramph~nicol (cau), rstrog~n, gri=fulvin, ph~nylbuY7.0n~, diph~nylhydantoin [mmun.... maliatrd nruirop"nia Sd=i"" m~ryocytic hypopl:ui. Toxic: br>chn f~rn poisoning (ruminant.), ch~moth.rapeutic age:nu, rstrog~ns in dogs (aogrnous, ~ndog<:nou.) , grisn>fulvin, mffiof~namic :><:id, ph~nylbuY7.0n~, It imHhopr im _sulfam.thoXll1.OI. [mmun~_maliatrd: am~gakaryocyt ic thrombocytopc:ni • • A rel. tively common
ar< concurr
&nt mArrow hypop/asu. is • nOIl!p«ific term that indicat.. a da:reased number ofh.mato_ poiHic cc:ll. in th. bon. marrow, H ypoplastic disorders may be caused by .b.. nce of lIimulating age:nts, pre:so:ncc: of inhibitors, dirtet damage: to th. morrow, or dir= dam"l:' to individu al cell lin.. (Tablr 6.4), A, Grn~raliud marrow hypoplasia I, Ap/d,tir "!lrmia ("p/asfir /"'nr:I"'pmu.) is the p athologic syt. in which ""'~ .. gtn~raliud marrow hypoplasia cau .., nonr~n~rative .n.mia, n~utropc:nia, .nd thrombocytopc:nia , 2 G.:n~raliz.d marrow hypopl ..i. i. most rdiably identifird with. good cor~ bon~ m arrow biol"Y .. mplr that consists of wh at should be active: marrow 'p.ces, T.ngtntial cores somHimes yidd mostly subconical marrow that moy not be r"P .... nytive: of the h.matopoietic lIat~, 3, M .ny disord ... or rondition. cau.. gtn~raliud bon~ marrow hypoplasia , and other dilOrd ... ha"" been :u.sociated with it,"17 but id~ntilYing .nd proving the cause: of

6/ BONE MARROW AND LYMPH NODE

339

the dilOrd .. is &.fo" the animal b.ame clinicolly ill. B. M/UtiW "Jthroid h}/'Op"',iIl is • p.thologic staU in which the numl>.r of erythroid pr«ursors is d«"asffl but the gnnulocytic :md m.g. karyocytic cdl lin.. a.. not. I. Am rrd all IIp''',iIl is a p.thologic state in which the.., is .d«tin .rythroid aplasia or ........ hypoplasia. It co ..... d",dopment of normocytic. normochromic. non,,_ g. ,,",,n concurrently with immune sph..ocytic hemolytic .n.mia. The "'fa of .if«t.d dogs con suppress .rythropoi.. is. Bone Jrulrrowa.minatiolll r",.al markalerythroid hypoplasia. The.. han b..,n Jrulny "ported """s in dogs" '" and ra" reports in cots." b. McDnM') purr rd ull "p"";., has ocrurr.d """,nd"", to t",.t'""nt with ~mbi_ nant human Epo in dogs,'"'" ho ..... J1 JJ .nd cots. Th. sda::tin erythroid hypopwia th.t occurs in some F.LV_inf«tM cots:md possibly in p:uvovirus_ inf=.d or parmviru'_VlICCinatM dogs i, also ron,id"M • form of =nda'1 pu" r.d cdl aplasia.It 2. Immune_mMiat.d d.struction of nudeatM erythroid cdls: When . nemia it co"""" by destruction of .. rty erythroid pr<cursors {e.g.• prorubricyt..J. an erythropoietic stimulus .. pands th. rubriblast companment. hut erythroid hypoplasia will be pr=nt b.a"", cdl d.struction pr""nu the accumulation of Jrulny taur stag< erythroid cdls. The ",ri.. will I>. left_.hift.d and han th. 'Pp"'t:Ifl"" of a m. tura_ tion .rrest b.ginning at the target.d st,,!:e. 3. F.LV_indu=i erythroid hypopla.tia a. Th. Kawakami_Theilen strain of FeLV (A, B•• nd C subgrouJ>5 pr... nt) cou..s s",e.. erythroid hypoplasia in neonatal kittens but not in w... nling or adult J

cots. • b. W ... nling cots and old.. cots infa::t.d with :moth.. strain of F.LV (A .nd C subgroup' p".. nt) also con d",dop erythroid hypoplasia and nonregenerative .n.mi .. " 4. Anemia of inHamJrulto'1 d~ (... Chapt.. 3): Decr ...sn! erythropoi..is is pan of the pathog. =x>gttiud becou.. it i. mild or b.au .. roncurrent gr.nulocytic hyp<rplasia may mal.:. it difficult to d~ .. min. wheth .. th.", is an absolute or a ",latin d«r..... in erythroid precursors. However. the presen"" of nonregc:neratin .nemia in th. ah",n"", of erythroid hYJ>'rplasia supports suppre=d erythropoi..is ..."n wh.n erythroid hypoplasia i. not .pparent. 5. Sda::ti"" erythroid hypopla'ia cousn! by endocrine disord.r.;: The nonteg. ""'1 mild :md not ra:ognizM in routine bone marrow aamin.tions. The pr=n"'" of nonrq;en.rative anemia in the ab .. na: of .rythroid hyp<rplasia suppons suppr=d erythropoiesis. 6. Drugs: Chloramphenicol con indua: a lralllient erythroid hypoplasia in dog,>' :md cou." Th... may I>. roncurrent mydoid and mq;.karyocytic hypoplasia in

m". C. M/UtiW grtlnukKytir hypop/mill ("tr"nulocytmj,) is • pathologic state in which lh. numl>.r of granulocyt. (neutrophil ) precursor.; is decreasffl. but [h. erythroid .nd

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY m~'yocytic

cdl lines . "" not. Th~,~ a,~ not ~nough =inophil and luwphil pr«urson in health fo, • d«= in th ..e lines {o co"", gJ>nulocytic hypoplasia. I. s.-ver:ol inf«tion> and drug. han bttn r~po"al to co"", ",l«tive gr:mulocytic hypopl .. i. (fabl~ 6.4). How",""". ,in"" many of th~m han :also bttn associatal with hypopl .. i. of oth~, cdl lines. th~,e m.y ~ th~ app'.ran"" of sd"",i"" granulocytic hypopla.i •• but d:nnage to othor cdllin~. might not ~ det«tal in the .:nne marrow sample. In parvovirus inf«tions. the granulocytic hypopl",", could ~ amsM by damage to ""lJ. with mitotic potent",i. depletion of naltrophil pooh ba::."", of ex",,,i,,,, tilSl..l~ d~mand. or endotoxin_inducM d:nnage [0 marrow ",U,."'''' ln conin~ monocytic ~hdichiosis (Ehrlichia cani-I) . hypopl ..ia (typicolly gtn~ralizM) may ~ .. en in chronic inf«tions. wh.""as hYP'rplasia i. "",n in acur. inft.ctions. 2. Animal. with immun<,-maliatal nrutrop'nias con han ~ithor ",l"",ive gr.nulocytic hypopl .. i•• crompanial by. ld't .hifi {... rly_s~ to midstage dmruction} 0' by indf"",i"" granulopoi..is with granulocytic hYP"pla.i. {latNt"l:~ destruction}. D. &Iu tiw ",~kJ.ryocytic hypopfmid is a pathologic stat< in which the num~' of meg._ karyocytes is d«r~.sM. but the erythroid .nd gr.nulocytic cdlline. are not. I. Sevtral inf«tion. and drug> han bttn r~po"al to co"", ",l«tive megaka,yocytic hypopla,", (fabl~ 6.4). 2 Rare ca... of app.""nt immun<'- mali>lal .m~'yocytic thrombocytopen", ha"" bttn .. ponal in dog>." Indfn:ti"" thrombopoiesis 01 megakaryocytic hypopl .. ", m.y contribute to thrombocytopenia sn:ondary to immune_maliatal damage to meg.karyocytes. Ill.

Bom",.,mnu ljmp/u"yltJli. i, a neoplanic (0« Bon. Marrow Cl .... ification •• ..cr. IX.C) or nonneoplastic .ccumulation of lymphocytes in th~ bon~ mallow. Nonneoplanic lymphocy_ tosi. m.y occur from infiltr.tion 01 loc:d hyp'rplasi •. In ti ..m • ..crion. of bone m.rrow. th~ lymphoid ",lis m.yoccur in nodule •• in gorminal ""nte", 01 as • diffu .. popul.tion. A. Because bone marrow i•• lymphoid tissue. an inlUmmatory di ..... may co"", lymphoid hYP"pla and plasm. ""lis may be prominent. B. Small lymphocyt.. may ~ up to about 20 % of the nucl~atal cdl. in bone marrow !amples from healthy con. but the mean valu~ is .bout 5- 10 % ." In cont",n. < 5 % (um:ally < I %) are exp«:tal in dogs. canle. and ho ..... C. A v.ri~ry of disord." that involve stimulation of the immun~ syst~m m.y cou.. bone marrow lymphocytosis. Exampl.. include canin~ ~hdichiosis.' syst~mic lupus eryth.matosu ••"" and C,i=fulvin [oxirosi •. "" Wh~n bone marrow lymphocytosis in cots was ddinal as > 16 % lymphocyt.. with> 50 % h~matopoietic cdlulariry. it was associatal most commonly with immune_maliatal .n~mia .nd pur~ ral cdl aplasia." Lymphocytes we,. mostly B_lymphocytes and wor~ oft~n pr.sent in aggreg.tes ... opposed to diffusdy distributal T_lymphocyt~. in = of fdine chronic lymphocytic leukemia. D. Wh~n thor~ is granulocytic .nd ~rythroid hypoplasia. th~ lymphocyte population m. y appear mo .. promin~nt but ~ ,datively incr~ased "'the, th.n ab.olutdy inc, ...sal.

IV.

Bom",.,mnu """rocyr~liJ i•• neoplastic (s.., Bone Marrow Classificotions. sect. IX.B.S ) or nonneoplastic .ccumulation of mast cdls in the bone marrow. Nonneoplastic .ccumul._

6/ BONE MARROW AND LYMPH NODE

341

tion, may b. calbl mOlSl cdl hyp<rpJ.sia, but mast ~lIs may accumulat~ by homing of rruut cdl pro~nitor aIls rather than by inc",a,..,d production of mast cdls through cdl divisions. A. Undi!fe",miat.d mast cdl pr«:ufllOrs originat~ in bon~ marrow but a", not =ogniud as a "'Parau ~II popuJ.tion in routin~ bon~ marrowaamination •. Ckcasional mast ~lls a", found in th~ bon~ marrow of h~.hhy dogs, cats. and hor.o; and probably r~pr ..ent a .. sident population. B. Bone marrow mastocytosis has bttn """,n.d in dogs with aplastic anemia ...• regenera_ tin anemia. Fe..ddiciency an~mia. hypoplasia s.-rondary to a &rtoli cdl tumor, and lymphoma." C. Bone marrow mastocytosis prob.bly t<prestnts a rractive sUte assoaat.d with an inflammatory process. Concurrent plasmacyt05is is common.

v.

Myelofibrosis A. Ml'u,fibroli. is a pathologic sUte of incrras.d fibrou, connective tissue and collagen in th~ bon~ marrow (PJ.te 9H). B. In dogs. myelofibrosis is oft~n accompanial by a moderate to ~e .. non"'t"nerativr or poorly regenerative anemia without other cytopgt, or it may b. =ndary to a clonal disorder of hemic cdls {e.g., MDSl. in which m.di ators from the hemic alh stimulate fibrocytic hyperplasia."

VI.

Myelitis A. Ml'liti•• in the contat of hemic disord~rs, is bone marrow inft.mmalion. Som~ J>'Ople u.. the word ultrQmy~litis to di!fer~miate bone marrow myditi, from spinal cord myditis. whereas oth~rs use the word to indic;;ote roncut",nt bone inflammation and bone marrow inflammation. B. There at< """,raJ agents that can au .. myditis; the inflammatory popul ations vary with th~ agent. I. Fungal myditi, {histoplaml05is .nd bIOlSlom}'COsis} 2. Protozoal myditis (leishmaniasi.) (plat~ 91) 3. Bact~rial myditis. usually au...! by bact~"'mia or a penetrating wound

VII. Bone marrow n=mis A. Criteria for establi,hing the pr... n~ of bone marrow n«rosi, vary, a, does the atent and rdativr significma of n«rosi, when it i, repon.d. The term bone marrow n«rosi. may b. "",ictal to bon~ marrow samples in which n«ro,i, i, th~ primary and pr.dominant .bnormality. or it may b. appli.d wh~n n«rosi, is not the major patho_ logic process. B. Bone marrow n«rmi, must b. difftrentiat.d from coll«tion and processing artifam. both cytologically and histologically. I. Cytologically, suining and pres."v.tion .nifacts c;;on mimic cdl d~th. as can cell trauma and lysis during 'm~.ring. 2. Crush artifact and plasma pooh have bttn interpret.d as ev:iden"" of n=mis in sa:lions of bone marrow rores.

342

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

C.

Bon~ marrow n~CfO!j, h.. oon .qmrtal in association with many dj""rd~n, includiJli: toxicants, !>xu,",i "",is, monocytic .h,lichiosi•. emin. p.rvovirm inf=ion, fdine

panleukopenia, systemic lupus erythematom., lymphoma, di"",miruotM intr:""urulor coagulation. and hypoxia." How ..... , the atelll of th. 11«""';' has not alway. oon

,.portal, causr_and.df«t rdationship,.", gt'nerally unprown, .nd the clinical signifi_ c;on~

of th. finding is ofUn unknown.

D. Bon. marrow neero";, h.., oon ron,id".d idiopathic when unousoci. tM with .n underlying disr.!K or with drug aposure," though .!SOCi. n.utrop}. IX.

Bone marrow h.mic ",J] neoplasi<> A. Gen~ral concept! and t~rms I. Neoplasia of bon~ marrow hemic cdl. i, not •• common as many oth.. forms of neoplasia in dom~stic mammal •. Th~ gen~ral diagnosis ofh~mic cell neopla'ia is not difficult wh~n th~re ..~ num~rous poorly diff~ .. nti<>tffl ceUs in • blood or bon~ marrow sampl • • but ..ublishi"l: th~ cdl of origin can b" difficult. It can .lso b" difficult to diff~ .. ntiat~ n~oplasia of wdl-diff~ .. nti<>tffl h~mic cells from inHamma_ tory conditiofil. Specific diagn05<::'l may b" reachffl by. combination of exclusionary tem. immunoph~notyping and polSibly cytoch~mical staining of blood or bone marrow by means of p.nd, of .ntibodi.. and stains. bon. marrow ""amination•• nd mol«ular C"netic t<Sting {Fig. 6.2}. 2. Characterization and classification of hemic neoplasm, in domestic mammal,""'''' {Stt Tabl~ 6.5} has g~ne .. Jly followffl devdopmenu in undmtanding and classification of .
Tab lC' 6.5, aauifications of hem k cell nw pluia involvi ng blood o r marrow C lassification

Cdl typt'

Lymphoprolikrativc: naJplasia 'Acut~ lymphocytic It'Ukrmia Chronic lymphocytic lC'ukemia 'Lymphoma Plasma edl mydoma Mydoid neopla,ia Acu{C' mydoid leukemias' Acut~ m~lobLulic with minimal diffC'll'ntiation (MO)~ Acute mycloblanic without maturation (M 1) Acute m~lobl;Ulic with IlUturalion (M2) Acut~ promy~locyt ic It'Ukrmia (M3)' ' Acute myclomonocytic lC'ukemia (M4) Acute monocytic leukemia (M5)~ Acute erythrolt'Ukemia (M6)

LymphocytC' B., T., or NK.lYl1lphoeyte B·, T., or NK_lym phOCYle B·, T·, or NK_IYlJl ph oeytC' B. lymphoeyte Nonlymphoid hemic cdl Nonlymphoid hC'mic cdl Granulocyte Gr.mutocyte Granu[ocytC' Granulocyte Granulocyte and monocyte Monocyte EI'),hrOCyte, no ntymphoid lC'ukocytc , nxgabryoeytC' EC)1hrocyte, nonlymphoid leukocyte, nxgabryocytc'

Acut~ e'Y'-hrol~uk~mia

with erythroid predominance

(MG-Er), M~aryoblastic leukemia (M7)

Acute It'Ukrmia of ambiguoui lineage C hronic mydoproliferativc disordus Chronic myeloid It'Ukemia Eosinophilic leukemia Basophilic leukemia Primary erythrocytosis Polycythemia ",ra Thrombocyth C'mia M yclodysplasticlmyeloproliferativc: di.=.sc Chronic myelomonocytic leukemiaf Myelodysplastic syndromcl Mast cell naJplalii:f 'Mecutatic mast edl neopl:ums (cutaneous or viSU'ral) Mast cdl It'UkC'mia , A relo'ivdy oommon di
M~ar}'oqte

Unknown, miJ:eQ, or mult iple (II« thC' tat) Nonlymphoid hemic cdl N eutrophil Eosinophil Basophil EI},hrocytC' EI},hrocyte, ncutrophil, and megakaryocyte Mcpkal}'OC)1e Nonlymphoid hemic cdt Neutrophil and monocyte leukocyte, erythroid cdl, andlor megakaryocyte Mast cdl Mast u Jl Mast cell

lcu~ia Study Group of ~ Am«ican Sociecy for Vettrimuy Clinical P~thology." These Icu~i .. fall into the WHO category of_tr ~i" tn.~Ut "of othtTwiw ~1f1JfpriUJ.'" "TIx -M " .bbr",,;~cion syRnn i. uxd in the FrendH\mcrican. Bririoh d ..... fi ...cion .00 >Va> used by !he ALSG but i. no{ UHd in the wrn:nt WHO nommd.tun:. n.e ALSG clauilication ominN aam: myeloblastic leuknnia ""ith minimotl diffi:rentiation, a diagnosis requiring immunophmotyping.oo ultnstructun.l MlL1tion that ~ no{ availoble to !he study group. ' Human am ... pmmy.locytic Irubmia i. typically :wociatN with defined mumciolU:tnd" !herofon ca~i!Cd as AML wirh m-W'Tnft ~ obomlll/jti~J ... ,her than as an othe"";<e uncatcgori...t IrubmiL "!be ALSG did not ""paute M 5 into acute monobl""tic lrubmi:o (M50) and :acute monocytic kuh:rn.ia ( M 5b) kcauoe of difficulti... in difhrenti:U1ng rnDIlOblu .. &om pl'OlIlonocytes, but othen ~ m:>de .br

distinction in animals .. is donr 10 human dusdica.ion schetnes..

'!be current bWJl.1Il WHO system indudes a puu rrythroid kuUrni:o.. 'CMMoL U cIau~ here :lr_ :and a MDS. • ~ thr t=t for pr<>p<>.d MDS d:u';6cations.. 'llKa"", m ... <",U" origin • .., from a mydoid .tem "",U (..,., CIup.er 2), mast cdt nropluia;' ooosi~ myeloid neopl:ui.. J43

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FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

'"

InJlum;a j, from the G,,",,k It,,h, ("whit.") + halma ("blood") = "white blood " .nd ref.", to an inCJ~ bullY coat lay". It j, the prestn<:. of nwpJastic hemic ~ll, in rich .. blood or bone morrow ~u .. of a neopl>S{ic proliferation origi""'ing in the bone marrow or, sometimes, th. spl..,n. Not all hemic na>pl"i. originating in the bone marrow is co,,,id.r<'d J....hmi.; for aampJe, ffiydodysplasti, syndrome. pla,rna cd[ myeloma, :md some chronic mydoproliferative di.ulO; such as =mi:d thrombocythemia are not Jeuhmi.s. 5. An," kul«mid j. a r"lotivd), mot. rapid proliferation of less difftrentiatw hemic "",Us. It typically consim of many bl • ., ""lis, .nd ,h. illness is brief (unl ... tre>lro). The microscopic erit,,;. ud for considering cdl, to k blasts . '" neith" clur nor uniform, but CD34 expression ,uppom cl.ssi/icotion ... an acut~ leukemi .. 6. A ""'" all i. an imJrulture cdl with replic;;otivt pot~lItial. Bwu typically havt smooth or findy nippled chromatin .nd lack ft.tum of well-differellti
Th~ t~rm

6/ BONE MARROW AND LYMPH NODE

347

add ..>Mkaryobl ... u, mono· blasts ± promonocyt... and .typical promydocyt.. ) must . crount for;> 20 % of .ll nucle;md ~lls in the bone marrow (ncluding plasma cdls, lymphocyt.., macrophage<, and mast ~lls). (I) Th~ blast pe=ntag. dreision threshold used to ~ 30 %, . nd that i, what was .dopta! by the ALSG for animo1s. However, hUJruln conditions with 2()"'29 % blasts {previously MDS r:lthu than A,\tLj appear to bdla"" clinically lih thost with 30 % or mo", blasts (AML) (Fig. 6.2). (2) [n ~rythroid leuhmi.. , wh.n ;> SO % of the nucleat«i cdls in the m>rrow ar~ .rythroid, th.re may ~ too ftw bl ... ts or blast equivalents to m..,t erituia for AML Diffuent crit.ria ar. uK
""U,

~lls.

b. Fi"" g.nual categories of A,\tL ha"" betn de""ib.::d in human m«iicin .... Thq .re provid«i hu~ .... b.. is for und.rstanding categori.. that Jruly ~ defina! in dom.nic mammo1s. (I) AMI with rmmmt grlWk "blWntUlli'i,r. Thi. classifiction requi.., id.ntifi. cation of specific .. rogniud clonal g.netic .bnormo1iti.. (trandocations or inversion.) that d.fine th. nroplastic population. {2} AMI with multillm"gr dppldJi8: Th .... leuk~mi .... ithu .ri .. following MDS or MDSIMPD, or they han dysplasia in . t l~a!I SO % of th. ~lls in two or mo .. mydoid lineages.

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY AML .nd MDS: Th..., clon. 1 prolif~ration ari", months to yra .. aft~r therapies including :olkylating "i:"nts, radi>lion. and topoisom .. _ .... inhibito ... (4) AML n~t omrTWin catrtf'riud: This category is ustd for AMll that do not fit into one of the oth~r cat~ri ... including when g~nr lymphoid. This category includ~s bilinral leuhmias consisting of two distinct blast populations. combin«i mydoid and lymphoid I.... hmi .... undiff..~miat«i acut< leuhmias lacking lymphoid or mydoid mark ..". and biph~notypic .cut., l~uk~mi ... with lymphoid and mydoid morh ... 2. MJi/odyfp/a,tir rynd,."m" (MOS.) a.. a group of clonal disord ... in which th~", i, typically in~ff.ain h~matopoi..is (marrow prolif.ration of. cdl lin~ concumnt with cyto~nia of the urn., cdlline) and microscopic u 10 st.,m from neoplas_ tic transformation of multipotenti:ol h.,matopoi 50 % of aU marrow nuclrat«i cell.. MDS_Er is defin«i by blasts (mydoblasts. m.gahryo_ bl ... ts. monoblasts ± promonocytes. and atypicl promydocyt..) b.ing < 20 % (30 %) of non~rythroid cdls. and rubribJ",ts plus blasts (.. jU!! defin.d) b.ing < 20 % (30 %) of.ll nucl.,at«i ""lis (Fig. 6.2). b. The dysplastic changes that may b. Ob.. rvN in MDS indude the foliowing:'~:U (I) Dy""JilopoiniJ {Plat. 9K): giant cdl .iI.<. abundan"" of .rurophilic granules. hypogranular cytop!asnu. polyploidy or hy~ ... gm.nt«i n.... trophil nucl~i. or Pdgc:r_Hul't ""II fratu ... (hyposegmenution) (2) Dysnythropoir'is (pIal< 91.): multiple nud.i. nucl ... r fragm~ntation. meg.loblanic appearance. excessi"" cytopla.m rdati"" to the nucleus. abnorm:ol dimibution of siderowm... macrocytosis. or unequ:ol nuclear division {3}

Th~rapy_rdat«i

6/ BONE MARROW AND LYMPH NODE

349

{3} Dymugllkilryo/",i(f;' {Plm 9M} and dy"hrombqpoi~Jis: nonlokd large: nudeus, muhipl~ small nud~i, dwarf mrg:obryocyt .. (micro. mrg. karyocyu.), mrg. pludets, or abnormal cytoplasmic gr.onulority or vacuolation of platdets c. Microocopic det'""tion of dysplomic change. i. subj=iv~ , and the degr..., of ronoordana:: .mong dinical pathologists for recogniI.ing dysh~m.topoieis in vet~rinary mMicine is unknown. It is the autho,,· nperiena:: tru.t th~re is a gr .... t deal of variation in the stringency of individtill dinicol pathologist', criter", for labeling change. as dysplastic as opposed to within the normal .pectrum of variation. d. Clo.. ificotions (I) A w.ic veterinary MDS dassific;nion proposed byth~ ALSG divided MDS into three groups: MDS, MDS.Er, and CMMoL M This symm ~ modifi~d by Raskin by dividing MDS into two groups (MDS.RC and MDS.EB), thu.s g~ner.oting four dassifimtions of MDS in mommals: MDS.RC. MDS.EB, MDS.Er. and CMMoL" Three cotegories ofMDS are descrikd in the HiJtou,giwl CfIlssifiwtion of Hmrtltopoink Tumm of [)gmmir Animlllr." idiopathic mydofibrosislmydoid metaplasia (cotegorized as a chronic myc:loprolifer.oti"" dise• ..,), CMMoL, and r~fr.octory an~m", with na::u blasts (RAEB). Oth~r c;;otegories Ita"" oon proposed ...... ~· CMMoL h., most recently been cotrgorized ., a mydody.pl.,tid mydoproliferati"" neoplas", in people (Ott the following Ret. 3 and T.ble 6.5), not a MDS. (2) Th~ WHO dassific;;otiollJ of human MDS ..e provided h~r~ becallSt there is no d .... r ron",nsm on the dassificotion cotrgorie. or criteria for MDS in domestic animal •. {a} lUfirJrtory Iln(mill (RA): bone marrow with < 5 % bIas", < 15 % ringed .ideroblast, (nud.... ted erythrocyu. with. ring of stainabl~ Fe around the nude us), and erythrodyspl.,ia (b) lUfou"tory Iln(mia with ringd 'idnobfilm (RARS): similar to RA but with at le.,t 15 % ringed sideroblom (c) lUptory <]fOp",jll with muflilin(llg( dy
(d lUfou"tory Iln(mia with D:CrfJ bfilm J (RAEB./): cytopeni., with < 1000 monocytesllJL and < 5 % blasts in blood, one or more dysplastic a::JJ lines, and 5- 9 % bone morrow bl.,ts (/) lUptory Iln(mill with D:CrfJ bfilw 2 (RAEB.2): like RAEB·I but with < 19 % blasts in blood and 10-19 % bono marrow blasts (g) M)"u,dy1plastir 'Yndrom~. ulU"l=ifid (MDS.U): cytopeni., with granulo. cytic or m~ ..}"ocytic dysplas", .nd < 5 % blasts in bon~ marrow (h) MDS Ilssocilmd with isola"d ibl(5q): anom", with < 5 % blasts in blood and norm:ol to incr .... sed platd.t oona::ntr.otion; allO with normal to increased numbers of megakaryocyte. with hypolobulated nudei in bone marrow, < 5 % blasts, and isoloted dd{5q}

'50

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY •. Dome'tic ffi>Illffi.b and pn>pl. with a MDS may d.nlop MAL, and thus MDS

has bttn comid.=I a prd.uhmic disord.r. f. MDS must diso,d.",:

~

diff.mltiat"'" from nonn
{I} HUffliury poodle marrow dyscmj. (poodle macrocytmis) {2} Milibsorption of oob.J.min (viumin Bill in giant .chll."",n, B<ml.,

rolli .., Aum:oli.n ,hq.h.rd dog., and

~e.

{3} DY'.rythropoi• ..;. in English .pringtr spanids

(4) Congtniul d,...rythropoie,j, .nd progresJiv.

alo~ia

of pollM Her.ford

calves {5} Possibly idiopathic thrombocytop.<SOCiation with th~ following .cquir~d condition. in dog •• nd c;ots:'''''' {I} Toxic;onts: l....d toxicity, ch~mothenprutic drugs, onro~n, cc:phalosporin., chlor:lInph~nicol, ph~nob>rbital, and colchicine {2} Nutritional ddiciency: F~ ddici~ncy {3} Immun~mffiiaud di~ immune_mal"-tal hemolytic anemia and immun~_mal"-t.d thrombocyto~nia (dy.megakaryopoiesi.) {4} InHamm.tion (,ideroblastic .n~mias): s.pticc:mia, pyometra, pancr~atiti., hepatiti •. glomerulon~phriti •• and fdine inlb:tioll.S ~ritonitis {5} Neopl:.sia: lymphoma, multipl~ myeloma, and polycythemia vera {primary erythrocytosis by the crit"'"- of this tntbook} {6} Mydofibrosi., although dyshematopoiesi. associatal with mydofibr<>!is may provt to k n~oplascic in rome """,. 3. Myelody>plascidmydoproliferativt dist....,. (Table 6.5) include conditions that may havt mydodJ"Plastic and/or mydoproliferative fntures. a. Chronic my~fqm~nocytic Irulmn;Il fulfil], the crit.."- .nd occu'" in dogs. Th~ .. i. typically leukopenia .nd nonr"C"nerative .n~mi. with a hy~rcdlular bone marrow and .typical hyper..,gmented monocytoid cc:Us in blood, bon~ marrow, .nd often lymph nod... Fewer than 10 % of the nucl~atal cdl. are blasts. and the disorder hu a chronic cou",e. b. Oth~r condition. described in human pati~nt, havt not bttn recognized in .nimals: atypical chronic mydoid l.uk~mia. juvtnile myelomonocytic leuhmia, and uncl .... iliabl~ myelodJ"Plastid myeloproliferative di",a.-;e. 4. Chronic myloproliJirllnw dinllm: This group. which includes stveral ¥cific chronic clonal disorde .. of pluripotential stem cc:ll., i. charact~rizal by e~ive proliferation or .ccumul ation of differentiatal neoplastic cc:ll. that hayt few or no micro=pic or functional .bnorrruoliti.. (T.ble 6.5). Tools to detect donality .nd prove neoplasia are ~nerally unav.ilable for th= conditions in domestic .nimal •. Di.gnmi. i. olien b..ed on exclusion of oth~r conditions. but diff..ent"-tion from nonneoplastic conditions is difficult because th~ edt. a.. well diff~"'nt"-t.d {Fig. 6.2}. a. Chronic myrlogmow lruknnill (also known .. chronic granulocytic l.uk~mia or chronic mydoid lrnhmia) i. charxterized by. nentrophil"- .nd typically. leli shift. Chronic eo.inophilic leuhm"- {and po ... ibly hy~reosinophilic syndrome} .nd chronic basophilic leuhmia ar~ v.riants.

6/ BONE MARROW AND LYMPH NODE

'51

b. Polycytiumw """ . nd p,imJl'] "JfflrtKJfIIfi, must ~ diff~"'ntiata! from oth" causes of ~rythrocytosis (~ Ch.pt~r 3). c. M(gaka'Jocyu mydo,u or (umrial timmbiJcytiumw must ~ diff,,~nti.ta! from markal meg:obryocyt~ hy~rpla!;' .nd r~3Ctin thrombocytosis. d. Chronic idiopathk "'J''''fibm,is with (Ja,amrdullary h(mJlwpoi"u must ~ diff~ .. n_ ti>!a! from =ndary myelofibrosis. Proof th>l idiopathic mydofibrosi. i, a n~plastic ~ntity in domestic .nimals is cUf",ndy lacking. 5. Ma" uU dh(/U( (mdSftKJ""U): Mmocyumia Jruly ~ r (solid ti .. "" origin) and !'ulumias {primarily bone m. ffOW . nd blood origin .nd dimibution}. Lymphomas may .nt~r a leuhmic pha.. with bon~ marrow and blood involnment, but a pra!omin. nt tissue digoeytosis. The functions of the .. two cell tYJl"S .'" reAecta! by th.ir immunophenotypes.

3"

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY l. [n dog., htmupm,gorynr hiifi~ryrir larcuma i, a prolif".tion of 'pl~nic raJ pulp and bone IIUrrow ffi3oCrophag., du{ ap"", among och" marhrs, CDlld .nd, inoons;sumly, COle and COlIc." a. N""pJ.,{;C cdh in the bone marrow cdls may ap~.r rdatively wdJ diff... miatM or .typical. Nropl:lSIic ~ll, ... consistently pr=nt in th. spl,""", wh ... thry nuy haw more >typia. Orh" t;!S\le! may ~ in",,]val (e.g., Ii"", and Ju~). b. Most dogs hove a r~nerati"" anemia, thrombocyto~n",. hypoalbuminemia, .nd hypochoJesterol.m;", 2. Malignant hi.tiocytk saTCumd {"",/ifl"'nt histiucytl16u} of dendritic cdl origin is a malignant proliferation of interstitial dendritic edl, which npress, :nnong orh" m>rhrs, CD Ie .nd CD lie, but not CD lId." a. Plwmorphic populations of nroplanic ",ns are pr...nt in bon. marrow and 'pl..,n, lungs. Ii""" or lymph nod ... Phagocytosis may bt pr~ .. nt. b. Oth~r findings ar~ v>riabl~ and nonspaific, including anorexia, l.thargy, w~ak_ ness, and w~ight loss. 3. Th... nwignancir. must bt diff~ .. nt;"trd from {I} sy>l~mic histiocytosi., {2} nonna>plastic h~mophagocytic syndrom.. occurring in association with inf=ion. and oth~r malignanci.., (3) immun~_mffliatffl destruction of h~matopoi<'tic c~lI., and (4) ma.crophagic inflammation. a. Malignant histiocytic prolif~rations typically hav~ atyp;", though it moy bt mor~ .ppa.. nt in oth~r tissu .. {Pl. t. 90}. Th. numbtr of cdl. in th. bon. marrow is typically gr~at .. th:m in oth~r conditions, and ",,1I, a", mor~ likdy to bt in dun~ ... b. Phagocytic activity .ppta.. non ..l=i"" in h~mophagocytic histiocytic ..,como .nd h~mophagocytic .yndrom~ {authors' exptri.n",,} {Plat. 9O}, but i. typically .rI'""ti"" for a particular linrag<' and maturation s~ in marrow-di=tffl immun~_mffliatal an~mia {Plat~ 9G} and neutropenia. c. With inflammation, oth.. inflammotory cdls may bt pr... nt along with organ_ isms or n,""rotic ""lis. d. An~mpu ha"" ""'n mode ming flow cytom<'try to diff.r~ntiat~ malignant and btnign histiocytic bon. marrow prolif.ration •. " E. Methods of classifYing h~mic ""J] neoplas;" (Fig 6.2) l. Th~ major goal of cw..ifianion ",h~m~. i. to d<'t .. min~ .nd rommunicat~ th~ cdl of origin with hope! that it can bt u...! to rd;"bly providr prognostic information and th~rapeutic r«:omm~nd .tions. Two mojor probl~ms with current classification. sdt.m~s .'" (I) th. most .impl. and inrxptnsive m~thod, microJCOpic .pptarancr on • Wright_scain~d .. mpl~, has major limitations .nd can bt inaccurat., and {2} oth~r m~thods ar. not widdy availabl. :md ar~ exptnsin. 2. Microscopic .valuation ofWright_stainal blood films or bon. marrow prrp:rnuions a. Th~ primary valu. of th.,.. rvalu.tions is to d.t'""t th~ p .... n"" of na>pla.nic h.mic ""Us. This may bt obvious wh~n th ..~ is • largr population of .typical cdl., but it may bt difficult wh.n th ... ar~ frw.. cdl, or wh.n na>plastic cdl. ha"" ma tur~ f.atut .. oompatibl. with an inflammatory population. b. Finding ""lis th at ha"" nud.... r or cytoplasmic fratu ... that a.. uniqu~ or more common in ""rcain diff~rent;"trd ""II lin.. is th. first.nrp in char:lct~riI.ing th. neoplasia. Wh.n th. cdl. ar~ immatur~ or lad: diff..~ntiation. it can bt ""ry difficult to classify th~ ""It. rdiably by th.ir light microscopic apprar:m",,; that i., blast cdl. could br of one of srv~ral cdl types.

6/ BONE MARROW AND LYMPH NODE

353

Table 6.6. Ezpcctcd cytochemical . ,ainlng reaction. of norn,al or neoplasli c henuc cells in dog> and cats Cytoch~mical

Cdl

PO

Nallrophil

+

SBB +

CAE

lAP

+

+ (immatu.. )

Lymphocyt~ Monocyt~

Eolinophil B~lOphil

± + in dog

± + in dog

- in cal

- in cat

-' -'

NBE

- Ifocal + + (diffu..,j

±

+ ±

Megahr}'ocyt~

min.'

± ±

' PO. p.k 0' 'P"wic nnning lJUy oc
na>pl"ia is d ... ifin! mon accumdy by oth.. m.,hods aft.. first ~ing id.mifin! by vis,",l imp«lion. 3. Cytoch.mical naim"''' a. Thest ar~ hdpful fur diff~remialing the lin~ago'(') of l~uk~mic reUs. b. Th~ !>.sic premi.., ~hind ,h~ usc of cytoch.mical mim i. tha, l.ukemic ",lis may ronuin ~nzym .. or compounds tha, are coounon or unique to ",nain ",J[ lin ... For a . mpk nrutrophil. and monocyt .. ronuin Jl<'roxid=. wh ..~. , Iymphocyt ... ~rythnxyt~ pr«ursors. and mq;akaryocytes do nOI. How...... poorly differentiat"! mydoid pra:ursors may nOi comain .nough analyt~ for a positi¥< r....clion. or staining may ~ equivocaL c. Siaining may be mor~ promin~m in immature cells than in m. tu .. ",lis. For aample •• lkalin~ phosphal'" . ctivily is prcscm in canin~ mydoblam bUI not matu .. neutrophils. d. Th~ usc of cytochemical suin. for diff..~mialion of lruk~mias is limit"! to a few sp«ial hemalology laboratori.. and is of most v:due wh~n a pand of stains is used (T. ble 6.6) . nd wh.n as .......:1 in conjunclion wilh oth.. findings. e. Pmili"" lIaining of acUle leukemia ",Us for peroxidase. phospholipid. chlora",'a'~ .., ....... l.ukocyte alkalin~ phosphat .... or a_naphthyl butyrate .lIe""" (diffu..) suppom a nonlymphoid lincag<'. Suining with a_naphthyl butyrau .ste""" (diffu .. ) .nd chlor. retote enerase mppom • mydomonocytic popul ation. Cdls tha, apJl<'ar lymphoid in Wrighl_lIainn! marrow or blood ofl~n prove to ~ mydoidlmonocytic wh~n cytoch~mic>l stains are applin!. f. Some cytochemical.uins con ~ u5ed on approprialely fixn! pWlic...,mbeddn! ti"" ..... 4. Immu""phmofJPi"'~" i. Ih~ "'"' of .mibodi.. 10 d.uct cellular epitoJl<'s lhat help to classify ",J[ type'l. It is typically uKpWia. no' to ronfirm il. a. Th~ use of monoclon:d .mibodies has ~nabl~d the id.mificolion of sutf.", .mig~m on a variety of ",J[ type'l. If. group or dust .. of monoclonal antibodies r'"""l:nizo. th .... me amigo'n. th.t .migo'n is .... ignffi a CD num~r (Table 6.7). c.

Th~

..

it j

!

H.- ...

rt"l!~ ~

t·

i~{

rf '

~ p t~" 'L j1 1 ~ ! till j" II'

-j"

II :>:>:>:>

1. ,,

,"-

'ill e-

j'! ,jj

l j"

•

2

0

0

h, "t

,,f-1-I ., I ', ,.•

1" 0

0

B 0

B

.g

ml ,H"Of

~~t ~jln~ 11 ttl

:>

:>l..,

f Jl1ldhl~

11 :>

f ! ;

~

j ; _I.

,

••

jl

ii'ili

: !!lh 1If JhlHI

1,j8ij 1 1

~

-i

'@

!

:::

~

0

a

•,

, • l~ ) j~Ji ill ~1~

'i " 'r •I I" ,,1 I-

~~1

'0B ~ ~ ~

:i

t!~ . ~j

j

J

~ : 1

I!ll, I

g

'§

f

j

.g

l ~lll, t1

!

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I j,l

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! "i , -n - i ' < 1 ,J ~,,<,f ;';-1 , ;I"i , ~ a'!""i- t ,· -~ati~1 ~'i"'i 1j ull,Hll; 1"11 l .111.,,111 !llll :c '" <

~

~ f: uS

Ie0

!

~2;' :>l ..

f

1

1

1

It t i

1

l' ;

1

1

H tIm • 1 , '1 H ~ I I ';1 • 'il,l • t'ij " t t 1

,1 I pl. l ! 1

!, it

'1 i f

&,

!; J

1.J~

[

jl il} If min! } a , , , , l,' ~I, '1!t. 3l!, l nUl"'!'" JIl'

uuu

a~ g

l~~ , - ifl ~illfd t_",'§]~j t ·l

8

,

11 u

§8

..

".

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY b. Th~ CD .ntjg~'" ar~ best cha""'{~riud on human :md moo"" ""US, but many of th. same CD antigtns.", found on cdh of domesric mammols. c. Cdt. from blood, bon. marrow, or ocher tissue. may k immunophenotyptJ by How cytometry, immunocytochemical .uining of .ir-drial prepM. t;ons, or immuDohinoch.mj",] staining of fix""" or f.DUn hinologic ,,",,{ions. Some antibodi.s can ~ usa! on unfixal tjune but not on fix
con!ldlat;ons of .ntigen ap", .. ion t .... t rdl= the cdllin~ and .tage of maturation. Pamrns no mo .. likdy to rrffiict outrum. than individual :mtigtn .. ""tivity. Individual .ntigtn ,,-activity =1 b. misle.ding ~Ust most immuno_ ph.notyring .ntibodi.. react with ""ns of ml1hjpl~ Jin~~s, .nd neoplastic ~lls may h."" akrrant .ntiC"n a pression, e, Th~ following pand h .. bttn rrromm
f, lmmunophenotyping results should k int"p"t.,j in light of all historical, clinical, microscopic, cytoch~mical, cytogtn~tic, and mol~cular gen~tic findings that at< .vailable, g. Canine chronic lymphocytic l.... hmi .. : lmmunoph~notyping indicat.. th . t most ar~ T _lymphocyte nropl",ms, including /lUny granular lymphocyu leuhmiou, Tho/ do not apress CD34,!7'" {I} Nongranular T _lymphocyte lrnhmias at< typically prolif... tions ofTCRaP ~Us th.t lack. cominem pattern ofCD4 .nd CDS ap..,..ion, Granular ""U J.t,hmias are typically CDS+ and CDlld+ , .nd apra. TCRaP about {Wia: •• often .. TCR)6, {2} B_lymphocyte J.t,hmias aprest C02 I (withoIU T_lymphocyte marhlS) or CD79a .nd um:dlyCDlc, h, Canine acute l.... hmi .. , wh~h .. lymphoid or nonlymphoid, usuaUy ap..,.. CD34 ("'~pt for granular lymphocyt..), {I} Nongranul.. acute lymphocytic leukemia is usually of B_lymphocyte (CD79.-+-) origin, {2} Granular lymphocyte l.... hmi .. m.y consist of either T _lymphocyt.. {C03+, TCRap+, CDS«f\+, and CD lld+} or, prob.bly, NK_lymphocytes (C03- , CDlld+, .nd CDSa+ ), {3} MPO apr. "ion supports .n aClUe mydoid leuhmia oth.. than pu", erythroleukemia. MPO expression has not bttn d~a::t.d in some leukemias of megakaryocyte or monocyte lineage," " B_lymphocyt.. and T _lymphocytes oflymphoma.t are CD3+-, so thi, mathr is hdpful in diffe",ntiating pri/IUry acute lymphocyti, leukemw of >gr.nular lymphocytes (CD34+) from leukemic ph .... of lymphoma, j, Abe:rrant "'p""ion of CD IS and CD4S h .. bttn "",luatffl for recogniI.ing and classifYing .typical cdl populations. ..... 5, Electron microscopy may k u..ful in 5Om~ l.... k-mw to provide eviden~ of. particular cdlline: for aample, th~ ultmtruc{U", of l.... kemic cdl cytopl .. mic granules m.y be: distinctive for basophil, ro!inophil, or m .. t cdl granules, R.ardy, immunogold suining may k usn! to d~ect lin~.decti"" /lUrhl'S,

6/ BONE MARROW AND LYMPH NODE

357

6. Mola:ulu and cytogen~tic studi.. may b. ~mployal to dffa:t C"netic abnorm:ditiel that Jruly pralict b.h.vior and outoom~ of som~ n~opwms. Chromosomal abnor_ malitiel in c;onin~ In>k~mia cdh han bttn dffrctal by karyotyping but have off..al litd~ prognostic valu~ to dat •. ChromosoJrulI banding studiel :md mola:ular C"netic tmniqu .. a.. b.ifll: ~:duatal. Th..., includ~ fluo .. =nt in situ hybridi7.;,tion, oomp..atin g.,nomic hybridization, and micro ..",,...,. Currently, th~ most Usffl C"nffic t.ronique is th~ polyroe....., chain rraction {PCR} for ousessment of clonality:"61 a. With only rar., ua:ptions, na>plasia of lymphoid a:lh i. an unoontrollal prolif~ration or a.crumul.rion of one clon~ of lymphocyt ... b. Antigen_binding regiom of B_ .nd T_lymphocyt~ recq>ton a.. enrodal by th., CDR) region of th~ immunoglobulin and TCR gc:nel. Th~ CDR) region is produced by various ra;ombinations of ~~ralC"n~., gc:nerating num~rom genetic combin.tion •. Prime.. are Usffl to amplifY con",rval regions of th~", genes, .nd th~ products .re ",p:".tal by sa.:. If th~re i. a donal upansion of cdh with on~ p:mirul" CDR] region d"'a:t>bl~ with th~ test prim~"', .. po.ration wiU yidd a dominant band. c. Polym~rasc: chain .. action a=ys for B_ .nd T_lymphocyt~ antigen r.aptor g~ne rrarrangc:ments a .. u",ful to hdp diff...,ntiat~ lymphoid neopl •• i. &om inflam_ matory conditions. {I} The pr... na: of gc:n~ ..ar"'ngc:m~nts supports neoplasia. B_lymphocyt. and T -lymphocyte gen. rrarrangements usually occur in na>plasms of th. resl"""liv~ cell tY]lel. {2} Th•• I=na: of a deta:talC"ne ... rranC"m~nt docs not exdud. lymphoid neoplasia. {3} Clonal rearrang.m~nts of th. TCR gc:ne have bttn d"'a:tal in dogs with Ehrlimill nmu infa:tions, .nd lymphocyt. receptor g.n~ ... r",ng.llYnts som",im.. occur in nonlymphoid leuk.mias." {4} Th.......y ",mitivity varies with the twu •• nd proponion oflymphocyt.. that a.. neoplastic, but don:dity am b. d.tn:1al when as linl. as 0.1 - 10.0 % of s;ompl. D NA i. from na>plastic cdls. Th...fo .., it em b. US<:pla.
Int..pretation of th~ r.. ulu of bon. marrow ex:mlin.tiom is rasi .. if on. u"'. th~ bon~ mu row examination to an<w~r specific questions: A. Why d",. an animal han a nonr~n.rativ~ anemi.? B. Why d",. an .nimal han a persist.nt nrutro~nia? C. Why d",. an .nimal have a thrombocytopenia? D. [s th ... a na>plas{ic process in the bone Jrulrrow?

[I.

Complet. interp.. tation of most bon~ Jrulrrow s;omplel is only possible wh~n th... a.. CBC ..",In for the day the bone marrow WlI.! oolla:"'d.

3"

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY A. Hyp<rpJ:astic and hypopJ:.stic conditions can usually ~ r~ni=l jnd~pend~ndr of eBC findings, but th~ eBC findings hdp explain th. disorder. For ""ample, a hy!",'. cdlulu bone Jrulrrow with an incr•• G:E (M: E) ratio indicates th ... is mydoid hy""rpl .. i. {rardy DaJp[",ia} no matter what the eBC findings are. How~u. th. eBC

='

may indicate th". is indf«tivt neutropoiesi, {granulocytic hyperpl",", with ='" neutropenia} or .f&ctivt namopoi.,i, (gr:mulocytic hyp<rpla,ja with. marktd neutro_ phil",). The former cas< sugge>l' immun~m.diated neutropenia, wh.",as the lamr =

luppom an infLo=tory

p~.

B. When marrow ""Uu[:uity u ""i~ de"ly incml!al nor cl.",1y d=astd and ~r. js an abnormal G:E {M :E} r:llio, ~ eBC provides duo:as to th. cau", of th. abnormal ratio. I. If the G: E (M :E) mio is inc",=<:!, it =y ~ due to ''Yduoid hypopJ"'ia, gnDulo_ cytic (mydoid) hYP"rpbsia, or both, A nonr~n~ratin an~mia would support nythroid hypoplasia, wh~"" .. a n~utrophilia and hematocrit within th~ ""ftren~ int~rval would suppon granulocytic {mydoid} hYP"rpl .. ia, 2, [f th~ G: E {M :E} ratio is d«",,=<:!, it may be du~ to ~rythroid hYP"rplasia, granulocytic (mydoid) hypoplasia, or both, A rq;<:n~rativ~ anemia or erythrocytoli. would suppon erythroid hYP"rplasia, whe"" .. a l... kopc:nia would support granulo_ cytic (myc:loid) hypopl .. ia, C. Unl""". condition i, cl~.rly chronic and st;obl~, it is important that CBC =hs are COJJ«tM the ..m~ day as th~ bone marrow sample, L An animal may han a nonrq;<:n~rativc: anemia one day but a ""g<:n~rativ~ anemia the next day (or th~ r~vc:ru), 2, An animal may han. neutropc:nia on~ day but a normal to incr~ neutrophil concentration th~ n.,n day {or the r~nrs<:}, 3, An animal may han a thrombocytopc:nia one day but a normal to incr~...d platd.t con~ntration the n.,n day (or the =~rs<:), D, Examples of the corrdation ofCBC and bone marrow r~,ulu for fin dog. are shown in Fig, 6,3, Findings mu>! allO be interpreiM in light of other clinical findings. LYMPH NODE: MAJOR CONCEPTS AND TERMS

L

Terms and conditions A. Ll"'~pathy is. ~hologic state im,uving • lymph node, [n dinical disnseo, the lympMdmopathies cypiCIlly cause enlargc:d lymph nod .. (i.e" Iym~~), F"'Iumtly, but inaa:urndy, lymphadenopathy and enl:orgnllymph node: arc: considered .ynonym~ B, Th~ primary reason for a lymph node bio!"'y is to determine th. pathologic process that it ""using lymphadenomq;aly, P.thologic proct""" may be presc:nt in lymph nod .. that are not ~nlargnl, but not .. frajuently, Th. following are major dilOrde" or conditions that allIS<: ~nlarged lymph nodes: I, HYP"rplasia of lymphoid cdl, 2, [nHamm.tion inmlving lymph nod .. 3, N~pl .. ia of lymphoid ~Jl, 4, N~pl .. ia of nonlymphoid cd], (typically metast;otic n~pl .. ia)

[I.

Mffhods A, Compl.te descriptions of a lymph node bioJ>ly (coJJ«tion, fix.tion, st;oining. .nd examination oflymph node ti ......~ from a living animal) .. e beyond the Jropc: of this tat, Procalur.. of a lymph node bioJ>ly are d .. cribed in KVeral !Ourees,· ....,.

6/ BONE MARROW AND LYMPH NODE B. M.jor

f~atu=

35'

of a lymph node biopsy

l. £ampl~ coll«tion and p=.. ing

a. Typiudly.lymph nod~ 5ampl.. a.. coJl«ud from ~nla~ pS may bt u...d as. guid~ for coll~cting cytologic ,ampl.. from lymph nod .. or oth.. mp'rficial =s: (I) Slides should bt mad~ r...dy. (2) Sd= a 22 ga u~ nttdJ~ and a 5- 12 mL syring.. (3) Prepa", th~ skin ., for • v~nipunctur~. (4) lsolat~ and subiliu th~ lymph nod~ with on~ h.nd. (5) Inst" th~ nNdle into th~ skin .nd lymph nod~. (A syrin~ is not n=""ry at this tim~, but if using a nttdl~ attachffl to • syring~, mod ... t~ suction con bt .ppliffl aft .. th~ nttdJ~ is in th~ lymph nod~ to hdp hold ror.. of tissu~ in th~ nNdle. Fore.,!"l "'pimion should bt avoidffl, particularly whil~ l~"ving th~ n~fflle in on~ pI""", btcaUK this am COUK significant hmKxiilution.) (6) Cut. co.. of tissu~ with a sharp forward cutting motion followffl by an optional rotation of th~ nNdl~ to hdp fr,"" th~ co.. , withdraw th~ nttdl~ without leaving th~ nod~, .nd rep
,.,• ®®

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•

•

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."

U · ' .3

F".... )."

k

..... ........... _olClloC ........ _............... . . .

it

.... _ , -, ' .......... ..-n. ...... .. _

" ) , .... ,

Go ' , "' .... .., .......... _ . , ' .. . . . . . . . _ .... _ ...... _ , , ' _ ........... 01 ........... .. ... , ',.~ .... ..,... ... ......

n.,;]. o.a. .... , ,

, , , • , ......

---._'

' ....... «

""r;', L,m-o',

"" "'<

..... _ _ '

_""""' .... R

.... , '.,

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u ..... ,

( _ .......~

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......... ........ ,'" .., .... , ,, " d

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to

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'G, E . . . ~ ............. , it

. . . . . . . . . . . .. "_n.. "oO"';,.--. . . ' ''' ' , I ,.

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FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

b«:aUst Ih. gas can ..nrdy alt., th" suining pmpmi.. of th" cdh. Kttp th"m dtlJl, oo"",rd. and ~t ~mbj'lII t"mptratu ",. d. To pro=« ... mpJes for hinologic .valuation. thin sli= of aci..,d SOImpJ.. should be, pl. e«! in fixativ. for puaffin..,mbMdrd 5«tioning. Formalin is u~ mOSl oft"n, but BS fixation improv.. cdl deuil. 2 Cytologic aamination of lymph nod. "'pim.. and imprints a. This inmlves Ih. diff.r.miation and rn.ract"rization of nud.alal cd], and Ih. id"milicat;on of org.n;.."" or olh., non~Uulor ,lrueCUm (•. g., h.mosid.rin). {I } Th" typt. oflymphocyt.. """ d"wminro by th"ir nud..,., diamete;t oompl~ed on a quality stained sample with a quality microsrope by a person who is tr.in«i for such aaminations. 3. Methods involv«i in the histologic ex.mination of lymph node sections are beyond the SCOJl" of this textbook. Such exarninatiolll should be performed by =erinary pathologists. E. Cdh in lymph nod .. of healthy mammals l. lymph nodes &om healthy mammal. are not oommonly evaluated. How"",r. microscopim should have a cl ... r image of wh.t should be ..en in normal lymph nod .. 50 that abnormal cdl populations or other .ignificmt findings will be r=>gnized (Plate lOA). 2 The expect«i cdl populations in lymph nodes vary with the locotion of the lymph node. Mandibul ar and m..enteric lymph nodes in hffithy mammals typically h. "" greater Jl"=n~ of resident macroph3J:'" plasma cdh. neutrophil ••• nd lar&" lymphocytes than do other lymph nod ... 3. lymph nod .. from healthy animals oonsist of a h~ero!:<,neou, population of ... lls. SmaU to intermedi ate lymphocytes acrount for the vast majority of the crll •.• nd there a.. low Jl"=n~ of plasma crlls. large lymphocyt ... n.... trophib. eosino_ phil.. and macroph3J:<:S. 4. The size oflymphocytes should be ju~ whe .. crlls ... well sp..ad {diam~.rs are maximized}. They con be meamred with a micrometer or rompared to "crllular micrometers " to estimate sius: conine erythrocyte diam~er is about 7 ).lm: conine neutrophil diam~er it 12- 14 ).lm: and mature plasma crll nuclei have diam~ers similar to erythrocytes. a. One system da ... ifies Glnine lymphocyt.. by nuclear diam~er rdative to Glnine erythrocyte di.m~er. but there are gaps bet",,,,,n cotetorie"n {I} SmaU: 1- l.S times the diameter of erythrocytes (.. 7- 10 ).lm) {2} Intermediate: 2- 2.5 times the diam~er of erythrocytes (.. 14-18 ).l m) {3} Lorge::> thr"" times the di.meter of erythrocytes (:> 21 ).l m) {4} Erythrocytes from other species ... smaUer. so the cW.tificotion ""Ie must be modified .c:rordingly. b. Others have used different numeric di.m~ers for classifiGltion: n {I} SmaU: <: lO).lm {2} Intermediate: 10- 15 J.lm {3} Lorge::> 15 J.lm

6/ BONE MARROW AND LYMPH NODE 5.

363

urg~ lymphocyt .. with b:lSOphilic cytoplasms. findy stippl«i chromatin. and nuclalli :or~ oft~n .. f~rred to as rymplJQblm/t a. i, consist~nt with th~ t~rm. mydo_ blon. monoblast. and rubriblast. How~~r. oth~" re .. rv~ thi. t~rm for the reL.tivdy sm:dl lymphocyt~ without conspicuous nuclalli that prolifer:u.s in lymphoblastic l~uhmi"'lymphoma.

LYMPH NODE ClASSIFICATIONS I.

HYP'rplastic lymph nod~ A. L}mph notb bypnplmia is char:o("{~rizal by inc=std numbe,,, of B_lymphocytes. T _ lymphocytes. or both. Th~ proportions of diff"'~nt types of lymphocyt~s may apptar normal. in which cast hy~rplo5ia is suggtn«i by normal cdl populations in association with lymphad~nomq;aly. Th~ .. may be, inc...,.,es in l~ lymphocyt~. andlor plasma ""lis. in which cast th~ t~rms muti", or "am", bypnp"',ia a.. oft~n us«! in pIa"" of hy~rplo.ia, though th~ nodes ar~ ~nlarg«i bc,c,u.. of hy~rplasia. B. A vari~ of infa:tiou. and noninfectious di" ..... including bact~ri:d. viral. fung:d. and neoplastic disorons. C>CI l~ad to th~ stimulation and proli~ration of lymphocyte>. If there is g~""ralizal lymph nod~ hYP'rplasia. a synemic illn= should be, consid~m:I. If only one nod~ is hyperplastic, a di..... within th~ drain~ fidd of that node should be, colI!ider«i. C. Th~ .. mayor may not be, a conrurnnt inflammatory lymphocyto5i. in mammals with hy~rplo!lic lymph nod ...

II.

R~a("{in

Ill.

lymph node A. A node classifi«i as rrlUtiw typicaUy has incr.....d numbe,rs of plasma ""lls and/or largt lymphocytes (Plate lOB). The p"'''''nt~ oflarge lymphocyt.. i, exp«t«i to be, < 50 % in a rtlctin nod~ and is usually < 10 % . An incr~= in plasma ""lls indicates B_ lymphocyt~ stimulation. B. Th~ cau... of a r~a("{ive lymph node ar~ .,...ntially th~ same as th"", for lymph node hy~rplo.i .. Lymphad~niti.

A. L}mpm"Jmitis i. charactrri=:l by an incr.....d numbe,r of nonlymphoid inflammatory ""lis in a lymph nod~. On~ inflammatory cdl typt might dominate {~.g .• neutrophil.}. or thrr~ COlI be, a mixtu .. of inflammatory cdl. {e.g.• neutrophil •• macroph"J:~s. and eosinophils} (Plm IOC and OJ. B. Th~ cau.. of the inflammatory nat~ may be, within the lymph nod~ or. more com_ monly. in th~ node', drairtagl' fidd. For example. an allergic d~rmatitis may lead to an eosinophilic lymphadenitis. or a lymph node draining a necrotic h~morrhagic lesion may hav~ many macrophages conuining cdl debris and F~ pigm~nts. C. Th~ .. mayor may not be, a concurr~nt inflammatory leukocytosis. D. OrganiSJllS ruch as pyogtnic ba("{~ria. Myrobllm,ium sp. ( Plat~ toE). Hisrop"''''''' sp. (plate 10F). B/a,tomyus sp. {Pl ate 10GJ. Ltishmalfi/J sr. (plat~ 10H) . Proroka sp .• and Nrcrid",mia sr. may be, pr=nt. E. Lymphadenitis is often associat«i with reacti", {proplastic} changt •• and the term "acti." IymphadrnitiJ is sometimes usa! to reflect both changes. IV.

Lymphoid nalplasia (lymphoma and lymphosarcoma) A. Anatomic classification. histologic cl""ification. and st"J:ing of lymphomas are bqond th~ >COpt of this book.

364

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY B. Cytologically, lymphomo can b. di>gnoud wh~n thu~ it n~arly a .ingl. popul>1ion of atypical lymphocytes rather than the heterogtnam, mixture of typi",! cd] typ<' pr...nt in normal, reacti"", or in!L.mnllymph nod ... How.""" depending on the app<-ar.D"" of the ed[., lymphoma can b. .n ""'r or difficult diagnosi. cytologically. I. When cytologic p .. paratiofll consist of single populations of largt lymphocytes with prominent nud""ti, the diagnosis of lympho/IU is d"", (Plate tol .nd J). 2. Th. diagnosis is rno .. difficult when th. ~ll, .re ,mall to intermediate in siu or when subnantial numbtrs of nonn 50 % of

th. lymphoid cdls ~'" largt lymphocytes. C. Ovu th. past 30 yr, =ral classifieotioll systrms for lymphomas han bttll basrd 011 ti .. u. pmer"" cdl sizr., lIud",r shapes, lIuclrolar sius, . lId cytoplasmic granulatioll. M"'t cl ... ifieotiom wu. formulatrd for th. cl ... ifieotioll of hurrumlymphomas, :md th.ir ~pplieotioll to oth.r mammaliall lymphomas has bttn inoollsisunt. Id.ally, a cl ... ificotion sysum would ditr.rrntiau llropla,ms of diffuillg clinical behavior .nd progno.is that thry could r«:riv. the most appropriau ma~m.nt. l. Th. Rapp.port da .. ifieotion is ba..d primarily on sizes of th.lymphocyt .. alld pm.fIIS of cdl growth {llodular alld diffu ..}. 2. Th. Kid da .. ificotion i. basnl 011 lymphocyt •• ius and nudrar .nd cytoplasmic f.atur.s, .nd "'pan tes lymphomas into B_Iymphocyt. alld T _lymphocyt. llropl",ms. 3. Th. Lukes_Collins dousifimtioll is w.rd 011 th. sizes alld shapes of th. nud.i {•. g., small,lar~, deovrd, nondravrd, .nd convolutrdl alld 011 cdlular f.atures {small, immunoblastic, plasm.cytoid, .nd histiocytic}, :md sq.arat., lymphom'" imo B_ lymphocyt •• nd T_lymphocyt. nroplasms. 4. Th. Workillg Formulation syn.m us .. growth pm"", (follicular .nd diffu .. ), nud",r outline (dravrd, 1I0Ilcl•• ve
rum

6/ BONE MARROW AND LYMPH NODE

365

tic leuk-mi:.. lCD34+1 from lymphoma with bone mmow .nd blood involvement (C034-). HoWl"V". CD34+ B_lymphocyte lymphomas h.", oon reportrd." a. At a minimum. a B_lymphocyt~ mark~r and a T_lymphocyt~ mark.r should be, .....ssrd (often C079a and/or C02 I for B_lymphocytes .nd C03 for T_ lymphocytes). T_lymphocyte lymphoma, Ita"" a poo.. r prognosi,." b. Oir"'t cytologic .m~a .. may be, u,a! for immunocytochemistry. Alt~rnativdy. cdl. con be, oolla::ta! into fluid for rvalu.;;otion by a numbe,r of t.ronique" (I) immunocytocheminry on oonerntr.ta! prq>arations of the erH SlI,~nsiom, (2) immunohinoch~minry on erU blocks mad~ &om erH 'Il'l~nsions by ~ll~ting .nd fixing cdl., or (3) flow cytom<'try. Immunohinoch~minry m.y aho be, usal on &oun or fixa! tissue =tion,. c. Coap.... ion ofC03 .nd C021 or C079a has bttn "ponrd," and erll, Ita"" ltad eith.. TCR or immunoglobulin ..ceptor grne ",.rr:mgrments. Thi, abe,rrant npr=ion may be, a us.!"l diagnostic mark .. for malignancy. d. Lymphom.. may also be, asse..al for p53 tumor_JUppressor protein, which has oon s~nificontly incr..sal in high_grade lympho/IUS." 3. Polymera.. chain ",. ction an.lysi. for rrarrangem.nu of the immunoglobulin and TCR g~nes con aid in d<'ta::tion of lymphoma (..e immunoph~notyping of hemic neopla'ia in Bone Marrow Classificotions, =t. [X.E.4). It i, likdy th at cytog~n.tic and molrcular t.roniqu .. wiU be, of diagnostic and prognostic valu~. V.

Nonlymphoid neopla'ia (typically metastatic) A. Lymph node, con be, ~nlarged b.c. ..... of the growth of nonlymphoid neopl .. tic cdl, in the node. M<'taslatic cdl. con also be, found during biopsies of lymph nod .. that do not apprar enlar-gal. B. Many neopla,ms h."" th~ pot~mial to spr ...d to ..giona! lymph nodes. Tho.. s..n mo .. frrqu~ntly in the ~ripher:d lymph nodes include '
VI.

Oth" findings A. Ed~ma may be, suU.. ta! in aspirates by the 10><>5< arrangemem of aH, that ,uu~'t' di,~rsion of erll. by fluid. B. The presrner of nonlymphoid h~mic p=urso" (rubricytes, mq:abryocytes, :md granulocytes) indicat.. ntramrduHary hematopoi..i, or, rarely, nonlymphoid hemic nropl ..ia involving the lymph node. Lymph node hematopoi .. is may occur when bon. marrow damagr is nUnsi"" and nimuli promote prolif~ration of h.mic prrcursors in extrama!ullary sit... C. Th~ presrner of .rythrocyte, in the ,ample indicat.. h~morrhagt. [f only ~rythrocytes are found, it may be, difficult to diffe .. miate pathologic hemorrhagt from hemorrhagr cousal by sampling. Th~ presrner of ~rythrophag .. and 'iderophag.. ,uppom the oonclu,ion of pathologic hemorrhagr .ither within the lymph node or it, drain~ fidd. O. A diagnosi, of m<'ta'tatic mdanoma should be, oomid~ra! any time mdanin pigm~m i, found. However, m~lanin pigmem con be, found in macrophages (mdanophages) when the lymph nod.', drain~ fidd comain,. md.noma, necrosi" or inflammation of pigm~nta! tissu•. E. Various combinations of multiple abnormalities may be, pr=m.

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

'" Refe re nces

I. Znd.r M, Hk.onym~. T. 2006. T.,.....J. '0 .,.j,......u..,;,,( do, .. ....aip""" facto. ........ k of d.nddtk «H d,,-dol'''''''t. T....d. Imm.....! 27,)4<J.-14S. 2. W. L. Dakic A. 2004. o.",lopm"" of -~ pot
... H.,...,. JW. 2001. Ati.. 669-696. 10. J.in NC. 1986. Sch.J"" V..",'_., H.-.I.D ...... «Ihlo... 1'hiJ.oo. Y... ain No. ... Am Small kim P u~"Ii: c';"";, ~ by .... ,Dim.llt""'.,..;. mod, p<>up' A ..' .... p«tiv<..,.[, of IBI a... (1%9--1992). Comp H........,) Int J,125--IJ4. 26. W,;" DJ. 2006. Aplaati.<.......;. in """ ClinlropatboloOco.l f......., and ... "";"od ad." 1,\1, Macl..,.J IN. 1999. Com"'.; .... of bioIociad acti,;ty and ..frty of ,... ~ robin"", bwnan .tyth
.p"'"

"""o-

1.0""""

",,,,bin,..,,

60,6J6....6.j2.

6/ BONE MARROW AND LYMPH NODE

,.,

JI. StoL.I T. Randolpb J. MocLrod J. 1997. Pw, ",I ",ll'plaai • .ru. =<0"' ........ , hum .. «ytI>ropoittio ,..,,"""" in ....,.,.[ !.tack lIo..... ~ od",ini.traoion ~f '000"'........' hum .. <>ytiu<>poi,cio to tw<> J.~njk.. JL 0 ..... RG. 1980. n.. imm.""""'lou.f tl., ktiD< bk..nia .ino •. Yrt Imm.""llmDnm~pathoI M07- 16S . JS. lu.rtt O. G.I in tht patIoop>"i. ~f ,rythroid kypopla ~ L 10, J Cane... H ,28.J-2Il8. J6. W"",o AD. 1977. Cl.lorampbtniool ,«>tt W.lliamo II< Willin •. 40. Ucl......icr. JL 1'_ GS. M ~..IJ SD. M"""t'J'SC. 2004. Acquit«l ~tk duombocyrop«to';' io a do ~. Can y" I J7,]42-744. 4J. Rottman IB, Endido RY. Bttitod.......tt EB, D~ DE. 1991. Bont "'....... kypopla ~ . io a cat _ted ..hb ",... ful';o. J Am Y" M«I A.ooc 198,029- 4} I. 44. Walkt. D. Co..dJ RL. Clioktnbt. rd KD. 1''''''' B, Mtinkoth JH . 1997. Bont ................. cdJ byp"pl .. ia in <10", ...ith. apt..ic antmia. Y" Clio P.d.oI 26,106-111. 4S. "kM.... P. 1997. Caoio, .....<>< 2:17,26J-267. 48. McM .... 1'''1. 2OOS. a ...iJi.wi.on,,{ mrdoid 0 . .1' ....... ' A oompan';"" ........ Y" OJ,, !'athol. J4, 189--212. 49. Valli VE. lKObo RM, PuodiAl, Y.. o.~ W, MOOt< PF. 2002 . Hu..!.F" C'-;;'~u ./H.....,.,.Uti, T.......,. of v..-.;, An ......... W .... in~ DG At",«1 F.o.c.. Innino" .fP.tl.oIo". SO. Yudi",,,,, jW, Hania NL l!."""j,,~ RD. 2002. Th, W.dod H,,]tb ~ (WHO) d ... ifiati.on,,{tl., "'1'1oid nroplauo •. Blood lOO,1292--2J(12 . SI. Btu, JT. 2000. M,d"d,~ .. tk aynd ....... and m,d,,6bro.i •. 10, F,ldman BF, Zinld JG. lain NC. «I •. s,!.ob.o' V...........,. H, ...."""" Sth. «Iition, 68l-681l . Pbiladtlphia, Lippiooott W,lliamo &: Wtllin •. S2. Rookj" RE. 1996. M,dopoi"i. and "'1'1op.<>liftn,;.." dioord" •. y" ain North. Am. Small Anion PUC< l60 IOlJ--lOO2 . H. Wti .. DJ. 2005. R.oopitioo and d. .. iJi.wi.on,,{ "'.D'dopoic~. io tl., <10" A , ...i ..... I Y" Imnn M«I 19,1.j7- 1S4. S4. Wti .. DJ. 2006. &.tuaoion of d,.my.J"l'..i"ia j" ab, .4 ~ (l996--200S). J Am. Yrt M«I At."" 22M19.J-1197. SS. Wti .. DJ, AinJ B. 2001. CytoJock ...tu.cion.f I"'.....,....d .=nduy m,d"d,~ .. tk aynd ....... in tht do ~. Y" ain Patbol J.O<67- 7S . S6. Btu, JT. 2OOJ. M,,~ .. ia, Dilf"""tiatinc ""'pla.tic from """"""pla.tic ayndromt. of inp<>ieoi. in doc •. T=iooI Patbol JI (Suppl),#-48. S7. Y,m ... W, M ..." PI'. 1m. An. immwooph."",'Y!'ic ohody,,{ c..o... kukt ... ia. and I"',liminaty _ ....."" ,,{ dooality by poIymtra" doain <eo1 lmm"""""tlooI6H45-164. 58. "loot< PI', All'oIt .. YK, y""". W . 2006. C""j", ........p~ hi.ti.ocytic .arcom" A prolil'n.tiv. di"""",,{ CDlld+ maaopb, V" Y" PatlooI".M J2-64S. S9. AIfoltt. YK, M ... ", PF. 2002. Localiud ...d di......inated bi.tiocytk oarooma,,{ dt..drttk «H oticin in <10" . Y" P.tlooI J9074--1iJ. 60. Wti .. DJ. 2002. F.... cytomcttk tvaluation of ........pha~ dioord ... in a..iD< boD< ............ V" Clio P.tlooI JhJ6-(l . 61. Rookj" RE. Yaknciaoo A. 2000. Cyt<>d.....iaJ /'0, diapoooia of kokrmi •. ID' Fddnoan BF. Zinld IG. I.j" NC. «I •. ~' H_f.o, 5th. «Iicion. 7S5-76J, Pbiladtlpb;" llppinrott William. ,\( W,lkina,

v,,, .....,

,oot.

368

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

62. De.nGA. 2000. CD ~'" and immw.opb.,..,~ [D' Fdod",,,,, BF. Zinkl IG. Jain NC. ed •. ~; V-w...., H.."."""o. Sth edition. 689-695. l'hil.ddpl.i" Lippincott Willi ..... &: Wlli",. 63. y, ...... W. 2004. Ao.. <J'<>""uic ."""",ot of ....... topoirtic ""'pLui. in the ~ [0' S~ltb Aru. .... M,nin, of...., ~ CoII,V ofY""';D"J' r.d.oIo&i-t. (ACyp) and 39th Ana...! M"tia ~ of...., Aa...iaD Society 10. y,,,,.......,. Cli..ical Patholid "'aIi&n'D<>padooI 112,zH_252. 66. Comani S, G.1ain ME. Sp.~ v, Rlond"o F. G~ImiDO R. Sarto"jJi P. 2006. Flo.. "'" '''''' •. V.. r.d.oI4o,}2-41. 68. Bakn R. lumod", JH. 2000. lb, lymph..tk 'J"'<m. In, Co"'" "'tim 'fC'''''-D of th. o.z ""J Cst, 71- 90. St kuj" CV M~oby .

69. Mill. IN. 1989. Lpnph ...,.j, <10. Di.S"mK C:!'''''v.."J H....."""D ofth. o.~ _; Got. 2nd «Ii';"". 97- IOJ. 50 !n.... , cv M~oby . 71. R..kiD RE. M'Y'" DI. 2001. Atlu .fc.";,,. ""; F,{i", C"'!.D. Pbilad,Ipb'" WB 72. co....n RL. Do...,. KE. Moinkoth IH. 2OOJ. Lpnpb nod, C)""'I ........ " Th, R.EAL ...d WHO douifiatioon ~f Iympboid D«>pIa ...... An.. 0n00I II (Suppl 1),sJ-510. 74. Wilh..".. MI. Dole. K. Koo"man T. Sbwooan W. Cb.n R. G ...", L. B..b.. L. AV<%"f A. 2OOS. llno~ diffi"""i.· lion of """n, Iympb<>mail,..krmi. •• naf'<"uioo.. I Comp Pa""'l lJh107-2lJ.

Sa."""' •.

Chapter 7

PROTEINS General Concepts for Total Protein, Albumin, and Globulins .. . ... . ... .• .. . ... .. .. 370

Analytical Principlas for Total Protein, Albumin, and Globulins ......... . .......... 372 Hyperproleinemia .. .. .. . ... .. .. .. .. .. .. . ... .. .. . ... . ... .. .. .. .. . • .. . ... . ... 379 Hypoproteinemia .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. •. .. .. .. . •.. .. .. .. . . Hyperalbuminemia . .. .. • ........................... • ... • " .. .. .. • •. . • . .. .. .. Hypoalbuminemia . .. .. . ... .. .. . ...•... . . • • . ...•... . • .. . ...•... . • .. . ... .. .. Hyperglobulinemia .••..•...••..•...• _..•...•... • ...• • ..•...• _..• • ..•...•... Hypoglobuli nemia . .. .. .. .. .. .. .. ..• . .. .. . •.. ..• . .. . ... .. .. •. .. . •.. . ... .. .. Positive Acute-Phase Proteins.. .. . ... . ... .. . •.. ..•... .• .. . ...•... .• .. . ... .. .. I. General Concepts ..••..••..••..•.. • ••.. • ...• • ..•... • ...• • ..••..•... II. Fibrinogen .. .. . ... .. .. . ... .. .. . ... .. .. . ... . ... . ...•... . •.. . ... . ... III. Other Acute-Phase Proteins .. .. .. .. .. . ... . ... . ... . ...•... . •.. . ... . ... Immunoglobulins ..•...•...••..••..••..•...•...•...•...•... • ...• • ..•...•... Colloidal Osmotic Pressure (COP) (Oncotic Pressure) . ... . ... . ...•... . • .. . ... .. ..

385 390 391 392 392 392 392 393 396 398 405

369

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

Table 7. 1. Abbreviation. and ,?',nbols in lhi. chapler (TP:Fib). [xl ADH AL Alb

APP BCG BCP

C3 COP

CRP DlC ECF

EDTA Fc FPT

H,'

I",

19E IgG IgG(T} IgM M, Na,50, NH :

PLE PLN

PP:F pll'. RlD SAA

SI SIADH

SPE .TP", 11'. 11' WRl ZnSO.

Toul prol~in to fibrinogtn in plasm> x oon"'ntnlion (x = a""IYI<) Antidiumic hormon~ Amyloid lighl ch.in Albumin Acut~_pha .. prot~in Bromcre;ol gINn Bromcre;ol purpl~ Compl~m~m facror 3 Colloidal osmolic pressur~ C_r""cli"" prot~in Di""min'lcd intrav:oscul.. oo"i:"I'lion Exlra",lIul" fluid Ethyl~ncdi.minel<'lraa=ic acid CrysyJlizabl~ &agm~nt

Failure of passi""

I",nsf~r

H~moglobin

Immunoglobulin A Immunoglobulin E Immunoglobulin G Immunoglobulin G, SIlbty~ T (T i, for ICYnu, ) Immunoglobulin M Rdali"" molecular m.... Sodium sulfit~ Ammonium Protein_losing ~mcropalhy Protein_losing nq>hropathy Plasma prolein 10 fibrinogtn Plasma tOI>l prot~in by refracrom<'lty Radial immunodiffu.
GENE RAL CO NCEPTS FOR TOTAL PROTEIN, ALBUMIN, AND GLOBULI NS L

Phy.
7/ PROTEINS

371

prouins, triglyarid~, .nd cholest~rol, wh~r""s glycoprouiru contain prot~ins:md polysa.cch.rid.. (UI!:ar), B, Plamta contains albumin and globulins, including fibrinog~n .nd oth~r clotting factors, A major diff~"'n"'" k!w""n .. rum and plasma is that .. rum d""s not con!>in fibrinog<'n, C. Mon plasma prot~ins {.lbumin :md J:lobulins} a", .ynthesiud by hepatocytes. Th~ major ncq>tions .r~ th~ immunoglobulins that .'" produ=:l by B_lymphocyt.. and plasma cdls, Th~ plasm. half_lif~ of .lbumin vari .. among sp«i.. and g<'n~rally inc",as~. wich body size; r~porta! valu~. includ~ 8.2 d in dogs,' 2- 3 wk in c;ml~,j,' .nd an avtr"Co of 19.4 d (n = 5) in horsts,' Th". is • large variation in th~ half_life of th~ various prot~ins in th~ J:lobulin fraction, .nd th,,~ ar~ vtry f,"", publications "'l:"roing half_lives for ch~ pia",,,, globulins in domestic mamn",k Th~ .v":og. y_globulin half_ life in horses (n = 5) was 11,0 d in on~ study,' wher~as the half_life of [gG in foals was 26 d. ' D, [n addition to a wid~ variety of sp«ific functions, prot~ins .ho contribut~ to COP, which hdps maintoin intravasular fluid volum~, [I.

Prouin disord~" A, Protrin "yuras;Il is. condition wh~r~ th~ .. is:m .bnornul prot~in (abnormal nrucru",), B, Dpp,."trinmt;Il is th~ pr=na of normal prot~in at abnormal con""ntration or abnor_ nul prot~in (dpcflUi.) in blood, L Sdectivt or norudectivt dysprouin~mias a, Nomrin:th'f hyptrp,."trimmia: All prot~in con""ntrations ar~ inc.....M (pIlnhyptr_ p,."trinmtia), It results from hemoronantration, b, S,/utiw Irypt,p,."trinmtill: Tho [total prot~n] i. inc",asaI, and some prouin con""ntrations a", inc",asaI more th:m oth~ .., Typically, it r~sults from inflam_ mation or B_lymphocyt~ nmpl.,ia, c, Nomtin:tiw hyfX>p,.",dnnni4: Th~ [total prouin] is d~Cf~'=', and all prouin con""ntrations .'" d«r~asaI fpIlnirypoprottinimillj, [u ca<= i. a proportion.l lou of prot~ns or proportional d«",.... in .ynchesi" d. S,/utiw Irypoprotdnimill: Th~ [total prot~in] is d«r.....d, .nd som~ prouin con""ntrations .'" d«r~asaI mor. than others, Its cause is sd«tivt lou (typically small prot~ins .d«tivdy Ion) or .d«tivdy d~u~a=' synth ...is of on~ or mor~ prot~ins,

2, To det~rmin~ wh",h~r the drsprouin~mia is sd«tivt or nonsd«tivt, .. rum prot~in d«trophor•• i. may be: n«
a, If h)'Il'OOibuminemia .nd hypoJ:lobulin~mia are pr~ .. nt and dectrophoresis remits india" that all prouin fractioru are d«r..... d proportionatdy, then th~re is a norud«tivt hypoproteinemia, b, If h)'Il'OOibuminemia and hypoJ:lobulin~mia are pr=nt and dectrophoresis results indiau that protoin fraction. are not d«reasal proportion atdy, then there is • sd«tivt hypoprot~inemia, c, The .. m. concepts apply to hyperprot~in~mia ""aluatiofi[, C. Och.. than che hyperproteinemia of ddtydration, th~ most frequ~nt dysproteinemias are caused by alterations in prot~in conantratioru during inflammatory di",as .., There

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY are th..,., major groups of prot~illS who"" pl:asma colI"",mratiollS change ba:.Ust of inft.mmation. I. i'<1,itiw APl'i are th~ prot~ins tru.t ha", inn~~ plasma or ..,rum conctntr:u ion. ba:."", of all inflammatory P""""'" SM and CRP conctntratiolll may illc ........ ill < 1 d .... Conc~ntr>tions of oth~r APP. may be, illCreasN within 2 d aft~r th~ onset of inft.mmation.' Th~ir conctntratiollS incr"".. ba:.Ust of incre~ production by h<patocytes aft~r .timuluioll by cytokin~s such '" im~rlrukin 6 and illurleukill l. The major po1itiv~ APP. and som~ of their physiologic functioll1 follow. a. Fibrinogm. the pr,"",rsor of fibrin. is usn! to form =ndal)' h~m""tatic plugs at ,it.. of vascular injury. It is ab..,m ill ,~rum. b. Cmutiw prorrin promotes billding of compl~m~nt to Wcuria .nd inducts cytokin~ production. c. Sn-um amyloid A promotes r=uitm~m of inflammatol)' ctlls to inflammatol)' ,it~ . d. Haprogkbin bind. Hgb dime .. so tru.t iroll is 1I0t .vailable to organism •. ~. a,.Arid glycDp,."trin h", ..veral ami_inflammatory activities. f. CaulDplasmin i, an ~lIzyme th.t tr'lI1pom cop""r .nd h.. oxidas< .ctiviry. It..Jso hdp. con",rt ferrous iroll into f~rric iron ror transport in the pt.,m. in associa_ tion with transferrin. g. Fnritin ..,rves as a sto~ form of Fe {in Fe>' form}. Mo,t f~rritin it in ti .. u~s. but .mall amounU I""", ctlls and em" plasma. u 2. Nrgath't APl'i are tho .. proteill1 that ha....., d""reasN pt..ma or ",rum collctmratioll1 ba:."", of all inflammatory P""""'" Th~ir collctmratioll1 d""re. .. ba:.u.. of d""r .... sn! production by hepatocytes due to the .ctioll1 of cytokilles mch OIl int~rl.... kin 6 .nd interlrukin I. The major n~ti", APP•• nd some of th~ir phY'iologic fullction. follow. a. Alhumin i, th~ major contributor to pl :asma COP ... rv~s:as. ",urce of .millo .cids. and trallSpom many cationic .... b.'lIances (e.g .• C .... Mg'+, and drugs). b. Tranifrrrin i. the major transport prouill for iron. 3. Drlnyrd "'ponsr prDtrint ar~ prot~ill1 for which pl:asm. or .. rum colI""'ntratioll1 in"' ...... 1- 3 wk .fter onset of inflammation. The two major det.ynl responst proteins • .., immunoglobuliru .nd complemem. a. ImmunDglobuum • .., producro by B_lymphocyte, or pl:asma ctll, and ar. cl:assifiw by their h. avy chain, :as IgG. 19.\1. 19A. or IgE. Subc/:w;ification, also ai,t. b. Compkmrnt protrim (primarily C3). part of the inn. te immune 'Y'tem...cnmu_ I.,. in pla,ma in some inflammatory conditioll1. ANALYTICAL PRI NCIPLES FOR TOTAL PROTEIN . ALBUMIN . AND GLOBUUNS I.

[T oral proteill] A. Refractometry for measurillg [tot'! proteill] (pl"'ma or strum) I. Principl~ The d~grtt of light refraction in .n aqu~ou, ",IUlion i, proponional to the quantity of solid. in solution. ~u .. moll solid, in plOllma are proteiru. the degr~ of light ..,&action i, highly d",,"d~nt on protein colI"",mration. 2. The ..fractometer. totol protein 1C:ole i. c:olibratw with the OIlSIlmption tru.t chong.. in refractiv~ inda refl""t chall!;'" in prot~ill collctmration .!on~. A um""rature_ com""n!3.ted refractomet~r ;., recommended over • non-t~mp
7/ PROTEINS

373

a. It does 1I0t raJuir~ daily adjunm~nu b.... d 011 .mbi~nt t~mptratures. b. It will probably be, more :K:C\lrate. [f comptll>ated .nd noncomptllsated ref"",_ tom
[nterfer~lIces

a. BecalISt the .. fractive index of a solution dq>end. on the con"'ntntioll of solids in the sample, high colI"'ntration. of a variety of ..... bnallces (e.g., glucose, urea. Na+, .nd Cl) could increase th~ refractiv~ index and thus th~ toral protein reading. The total prot~in readillg is reponed to be, falsely increased by 0.6 gldL if th~ plasma gluco .. con"'ntration is .pproximatdy 700 mgldL (or 0.7 gldL) or the ur~. lIitrogtn con"'ntration i. approximatdy 300 InJ:ldL '0 b. Gross liptmia will increa.. the refractive index alld thus falsely increa.. the total prouin reading. c. Hemolysi , causing a plas= [Hgb] of 0.5 gldL did not int~rf~re with refracti"" index value> but mad~ reading of the dividing line ill the r~fractometer more diflicuh." d. Bilirubin coII"'ntntions at 0.4 mgldL did not interf~re with refnctiv~ index values." However, icteru. i. commollly listed as a cau.. of fal ..ly incre.~ values in clinical chemistry textbooks. Perhal" interferen", occurs at high~r colI"'ntrations. 4. Ullit conversion: gldl X 10 = giL (SI unit, n~..est I giL)" 5. Comment. a. The [TP..J is also referred to as the pIA,,,,,, "'tal ",lids roncmtrlltiQn b.caus<: th~ value i. affected by solutes other than protein. Howevc:r, most refractometer sao.les are calibrated for prot~in colI"'ntration, not total solids, alld thus other substance. (e.g., u.... or gluco..,) are interferents rath~r than th~ targtts of m~asurement.

Det~rmination of the [TP...J is part of a complet~ blood count (CBC) in m.ny veterinary laboratories b.ca"", it is a simple, quick, .nd inexptnsive method fur deta:tion of hyperprot~inemia and hypoprot~illemi •. c. Most rd"ractomet~rs ..~ calibrated for th~ normal prouins ill hu=n plasma. The calibratioll scale will vary amollg speci.. b.cau.. of the different composition of pl.,= prouins," but the difftren", is typically colllidered dillically insignificant. d. The [total prouin] ill serum may also be, estimated with a refractometer. The serum con"'ntration will be, lower than the pl:wna [total proteill] b.cau.. of the .b..,n", of fibrinogtn in .. rum. However. there are oth~r factors that calISt diff~ren= between plasma alld serum [total proteill] ev~n if mealUred by the same method." {I} H,O diffuses from erythrocytes duting dotting and thus lowers .. rum [total protein]. AI thi, ch.nge is rardy described, it =y ao.us. ollly minor ch an!:"". {2} Some antiCO
b.

374

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY fr~u.ntly mildly differem (" 0.3 gldLI but OC<'a5ionally diff.rent by .. much ... 2.0 gldl in <;;Imple:! that are not h.molyud, ict.ric, or lip"mic (authors' obstrvations). B. Biu,,,, reaction for mrasuring [total prot.in] {.. rum} l. Principl~ Copper binding to p"ptid. bonds c,rat.s .. violet rompl.,.; the numb.r of ptptid. bonds, :md th".for•• mount of color d u n!:", is proponion:d to [total protein]. Howenr, not all individual proteins .. ""I in th. sam. w:oy, .nd not all proteins are pur. polypeptide! that contain th. same amoulII of nitfOl:"lI by ~ight. Therd".. , [torn protein] determination. are not oompl.tdy accur:Ur . 2. [ntr,feren=: In .om. ass.ys, hrmolysis may cou"" .. positin int.n.r.n~ (r.g., Hgb at 400 rug/tiL will product a 12 % bias). Drxtran (polysacch..rid. usa! as a pl""m. """"nd,,) may abo cause a positive int.rf~ .. nct. SmaU pq>tid.. may react but contribut~ nry linl~ to total oolor chang~. NH.* may int~rf~ .. with th~ biuret ..:lCtion but not .r ron""'ntration, found in plasma or ..,rum." 3. Unit ronv~"ion: gldl X 10 = gil (SI unit, n~arest I gil)" 4. Comm~nt: Th~ biuret or modifiw biuret reaction is the moJi rommon spactropho_ tometric method of mea.mring ",rum [total prot~in] .

II.

Albumin conctntr>tion A. BCG dye_binding reaction {.. rum} l. Principl~ BCG pr~~r~nti :dly binds to :dbumin and produces a color complex. Th~ quantity of BCG_:dbumin complex i, proponional to the [:dbumin], though binding VOIri .. among spacies; for example, the binding ofBCG to bovin~ :dbumin is much strong~r m..n to canine and fdine albumin." 2. Int~rf."'nces a. Th. binding of BCG to globulins will result in a f:d.dy devatw [:dbumin]. Th. nonalbumin binding may l.ad to ,ignificmt enors wh~n the true .. rum [albumin] i. very low {< I gldl} compared to the con""'ntration of int~rf~ring globulin {• .g., a._macroglobulin}. b. In "'m~ :ways, Hgb at 0.4 gldl will cou.. a positin 24 % bias, wh ..~", triglyc_ .. id~. at 0.8 gldl will yidd a negati"" interferem,. of about 0.2 gldL c. Some BCG methods, but not oth~", are affected by th~ presenct of anticoagu_ lant •. Measuring [Albumin] in heparinizal plaUllii r"'mlted in gre.ter albumin v:dues compared to serum with a standard BCG assay {median differ~nct, 0.2 gldl; -0.6 gldl to 1.2 gldl}, bur lower values (median diff~ .. nct, - 0.1 gldl; -0.9 gldl to 0.1 gldL) with a modified .....y.'. About 50 % of th~ diff~r~n"'" in the .tandard:way wa. due to th~ p.... nct of fibrinogen. d. Unit conversion: gldL X 10 = gil (SI unit, nearest 1 giL)" 3. Comm.nt: BCG dye binding is the moJi common spectrophotometric method of me• • uring .. rum [albumin]. B. BCP dye_binding r.-action {.. rum}: BCP binding is t=d in ",me human medical laboratoti..., but BCP does not rdiably bind with :dl mammalian albumin molecules. BCP assays may gi"" falsely low {sometime> markedly low} ..suhs in som~ domestic .paci.. (e.g., dog.). C. HABA (2_ [4'_hydrm:yawben"'n~] _bem.oic acid) dye_binding r~.ctions (.. rum): unreliable in domenic mammal .. rum,17 bur the rrag<'nu a.. still available D. Protein dectropho ..,is con be used to determin~ [albumin], bur is used more for quantitating globulin fractions.

7/ PROTEINS

375

Ill.

Total [globulin] A. This is typically d.t.rminal by subtraction (.. rum). l. Principl~ AU pro"ins in .. rum oth" than ..!bumin a", globulins. 2. [Globulins] = [tot"! pro"in] - [..!buminJ. B. Pro"in d.etrophor .. is (.... the next ""tion) C. Unit oon""",ion: gldL X 10 = giL {51 unit. n.a,," I giL}" D. Conun.nu l. [Globulin] will be: only as accur>I< a. the m,,,,urM [torn pro"in] and [albumin]. 2. [Globulin] .. prno:nts the tot"! cona:nt.. tion of.ll .. rum prol 1000 pro"ins; •. g.. haptoglobin. t"",sf.. rin. ct,-m.croglobulin.lipopro_ t.ins. :md immunoglobulim}.

IV.

S<:rum pro"in d.etropho=is {SPE} for dff .. mining prot.in fractions A. Principl.. I. S<:rum prot.im >q>.rat. into 4~ major groups of on. or mo" bands baoM on th.ir ability to migr.t. through a:Uulo .. ~at. or agaro .. in .n d.etrical fidd. Th. dtes the proteins into 5- 9 protein b.nds. wh ..... u.ing agar".. "Jl'Irat.. the pro"ins into I 0-15 protein band •. A prot.in b.nd may rep"""nt one protein or ..-vc:ral proteim that have migrata! the sam. dinan",. c. Prot.in bands that represc:nt globulin prot.ins .'" grouped into d.etrophomic r 0.1 gldL i. need.d befo.. it con be detect.d by this method): {I} In most dog. cot. and horse .. ra. fi"" globulin regions can be ... n: Ct.,. (l,. P,. P,. and y. {2} In most cotde ..... only thm: globulin rq:ions ... ...,n: (l, P. and y. B. Calculating roncentratiom of the protein fr>ctions I. The protein cona:ntr>tion of .n d.etrophomic group is the product of the [torn protein] (preferably from a biurff r...ction) .nd the pera:nuge of total prot.in occupia! by. region. When. stainal a:Uulo .. ~ate .trip is samn.d with a d.nsitom.ter. stain.d proteins cau.. l<:s.t light to be tr.mmitta! through the strip to a d...etor. The d.e",...d transmituna: is recorda! .. a deflection on a d.nsitom .... sc:m or tracing. Aft.. the cellulose acetate !!rip is .cmna!. the resulting curve represents the rdative qu antiti.. of pro"ins {Fig. 7.1}. The .". under the curve rq>r=nts the total quantity of suin.d protein.

316

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

,

Ab

!

N.

'"

fiLl'

I

"

I I I I

IgM IgA

•

Celuose aaolate strip (shaded areas mpr"....,t stained prot...... ) Fig. 7. 1. &h.m .. ic ,.pnsenution of SPE r.,ul", (<,<Jlul ....."''''''' strip ond dornitomotnode, ond globulin mel;""" .. p:u:ot< into bands or meo"ns {•. g., (Jl ;:lobu~ns, a,globuli .... Jl,-gLobu~n~ I\:.-globu~m .•nd y-gLobu~ ... ). After r lKtropbo=is, th. mil' is.uin.d .ntb . protein ".in (• .g., Ponc.. u 5). Bmds tlut con";" th. mon protein ".in the duken, When scan ... d with. donsitom< ,.J.tP.<. . t>ining int<",ities of th. corr''I'''nding bood.. 0"" p..k may rq>cesent the ",jning of "n< protein {•. g., albumin} OJ m.y "'P''''''' th. sum of multip!' prot.Uu (•.g., the a,--globulin region
rnd PA. pre.Jbumin.

2. Old~r d.nsitom.urs are calibratw so that oompl~t. transmin:m"" through ch~ a=at~ mip doo; not deflttt the needle {uro respon .. j . In .ddition, densitomeu", may ~ calibratal so that ch~ most blockage of light cau..d by the darkest prouin b:md d~flects th~ n=:ll. n.... rly [00 % (maximum respon ..). The dark~st bmd {or the maximal relpon",} is normally the albumin b:md but can ~ found in the globulin fractions. N.....~r 'ystems u.. flat_bed scanne", to obtain digital dau chat ar~ transformw into rdatin pe=nt3i:el {e.g., Hde"" QuickSc;,n 2000}.

7/ PROTEINS

377

of th~ total ar .... und" th~ curve for ~arn region is caleulat.d to per",ntag<' of [tot"! prot~in] rq>r=ntal by ~arn dearophor~tic region. Then. th~ p<=nt,,*, a", multipli.d by th~ [total prot~in] to deurmine the approximate protein concentrations in urn dearophor~tic region. For exampl~. if the [torn protein] i. 6.0 gldL and d~ctrophoresis mult. indicate th>t 50 % of th~ stainal prot~in it in th~ :dbumin band. then th~ [:dbumin] is ca.kulat.d to ~ 3.0 gldL 4. Th~ con"'ntrations of th~ prot~in fra.ctions d~t"min.d by dearophor ...is will vary ba::.use of KV~ral factors: variable affiniti .. of nains for ",nain protein •• accuracy of the m~a!Ural [tot"! prouin]. a.ccuracy of densitometry. and th~ tion in domestic mamm:d .. When using "i:.rose gd dectro_ pho... is for canin...ra. the prouin migrations w~'" .imil.. to those list~d in Tabl. 7.2 e~pt C3 w;n a ~,_globulin and transferrin was . IJrglobulin." For som~ .. ra (~.g .• bovine) . only th,.., or fout globulin fractiom wiU ~ d~ect.d. D. Conun.nu l. SPE has limited diagnonic val"". It may ~ hdpful in diffe",ntiating th~ cau... of hyperproteinemia. characterizing hypoprotein~mias into ..leaive or nomdeaive cat~gori ... ler..,ning for a monoclonal gammopathy in normoproteinemic and hyperproteinemic ..,ra. and providing a mor~ ac<:uraU ~stimation of [:dbumin] wh~n globulins interfere with the BCG .... ay. A""lysi. of .. rum proteins by SPE is not common in clinical malicine. but understanding SPE r.. ules . id. in understanding routine .. rum roncentrations of total protein. albumin. and globulin .. 2. Th~ prot~in bands on th~ allulose a,,~ate or ararose mould be examin~d in addition to th~ densitom~ric findings. ba::.use th~ tracings do not alviays demon_ strat~ th~ abnormalities in th~ prot~in fractions. 3. Th~ calculot.d concentrations of the dectrophoretic fractions ..~ >t ~st <:stim. tes. Th~ true value is usually within 0.3 gldL of the calculatal v:due (assuming proper marking of fractions). The calcul>tal con",ntrations a.. frequently not nttd.d to classiry- or interpm dy>proteinemi. patterns. 3.

Th~ pe=nt"i:~ det~rmin~ th~

v.

Immunoelectrophoresi. A. This is a method of id~ntiJYing the pr=n'" of specific proteins or protein components. It can ~ used to id~ntiry- th~ pr=n'" of immunoglobulin cl..... or sulx:l...... h....vy chains. or light maim. B. Th~ .. rum prot~ins are first .. p..>tal by dearophor~sis. Appropriate antibodi .. are addal to long trough. cut paralld to th~ dearophoretic separation. Th~ antibody and the dearophoretically "'Parat.d .. rum proteins diffu.., toward ....ch other and form precipitant arcs if th,,~ is a r....cti"" antig~n {e.g.• he. '}' chain oflgG} for the antibody.

VI.

Sia euglobulin test A. A (uglohulin is • protein that doe< not dissol"" in pur~ water. Th~ Sid fllglubulin .ttI is simply adding 1 drop of serum to demin~raliRd H,O; formation of a precipitat~ or flocculates is a pmiti"" te
Table 7.2. Serum protein. Ihal conlribute 10 clearophoretic region. Rceion

PlOIcins

M, lin thousands)

NO{ ucoogniud in routine SPE of

Pre-albumin

Albumin

Albumin'

.,

"

a, _LipoprOlcin

180-350

a ,-Antitf}'l"'in'

54

a, _Anlichymotryp5in"

"

a.-Macroglobulin"

'"

,

Funct ion and oIlier inform~lion

H3IHoglobim" Tr:u"fe,rill'

80-160

~Lipoprol (in

2400

Complement (C3;I)"

,so

76

IgM and 19A

19A.: 160 IgM: 900

'gG

'50

C-reactivc protcin"

'"

:rnim:d ... t:I: indudcs thyroxine_ binding albumin and ~inol_binding prot';n Map- cont ributor 10 ona)!ic pressure; lrmspGm C.>+, .\1g". unoonjug~1ed bil irubin, fatty:acids, Ihyroxinc. and m:my ot~r mbst:ma:. T ran.port. lipid< (esprcially lory prolCln In:octivales proteasoes, incl uding chymolf}'l"'in, :rnd Ihus i. an :rnli_inf!:tmJrullory prorcin I n:OClivaus prole...,. and Ihw i$ an anti-inlbmmal0ry pror cin Bind and mlll! pon fm. Ilemoglobin Bind$ and nampam iron; measured as total iron_binding capxity in chernial assay. Trampom lipidJ (cholesterol and lrigl)'urick): alKi caUed LDL Promot.. inH;unm~lion; memot:lClic .ubouncc Bind to specific anl;Vns; conanmll ioll5 an tOO low in htahh 10 be _n vi a routine SPE Bind. 10 . prcific anl;g..M; many different i..,typos and idiolypc:s of IgG gin a broad and umally indi.tiner gamma region POJilive :KUlc_ph_ protein: rarcl)'_n in lYWlIm:dian SC't:I via rouline SPE

• N ptoIciN, Pro ..iN in domesI-ic mamauJ pl..",>;w ... umed '0 migra,. in sima.. ~ON, n.... are hundtedo of olher plasma "",..iN of c~ nical.ignificanu. bu, ,bftr """",ntrationo ..., "'" to.. in pIIpioIogic and ""thoIogic ... _ to 01 .., tho rlcctrophomic ""oom in cdlut.- ...,.... . lrctrophoR.is, [f pI»ma is .l«1rop1o.or-d, 6bri""ll'n (M,: 341,000. a pooio", :lCIJ1rpluM rr-in) .bould migrue in tho: carb.od:ol ..,d of tbr ~on, Sotfmr. Rinmrnn and Dani<:Js ''''

3"

7/ PROTEINS

379

w .. a scrttning t~n for macroglobulins. How~nr .• nal~. of human sua indicat~ that th~ positive Wt Im}' remit from. vuiety of monoclonal and polyc/o",,1 gammopathies and thus is not diagnostically useful. " A modified Sia test (using weak ela:trolyte solutions) for mocroglobulins resulted in few", f.l", positin. but more fahe negatives. B. Rerogniz.i"l: the precipitont propeny of immunoglobulins is important in two .itu._ tions. First. this propeny am b. used to .. p",at~ immunoglobulin, from oth~r ",rum proteins; the precipitoted immunoglobulins con th~n b. dissolved in saline. Second. the precipitate. can imerfere with the a""lysi. of ••,," or blood sampl..; for a;;ompl~. fabely inc",ased ",rum phosphorus ronumratiom (Chapter II). falsely incrrased blood [Hgb] (Ch "pt~r 3), .nd fahely incr~ased .. rum bilirubin concentration (Chapt~r 13). HYPERPROTEINEMIA {INCREASED TOTAL PROTEIN CO NCENTRATION IN SERUM OR PIASi'JA} Th~

I.

di"'.... and conditiom that couse

hyperprotein~mia

... listed in Table 7.3.

H.mocon"",mration i, a rommon couse of hyperprotein~mia. A. Pathogtnesi ,; Hyperprot~in.mia results nom the ronumration of pl.. ma prot~ins co...ed by the loss of plasmo H,O. The pi..."... H,O 101< and result.nt decreased ECF volum~ moy b. due to vomiting. diarrh ..... impairal ",,,,,I concentr.ting ability. ,~ting. insensibl~ loss via respiration. incrrasnl vascular perm .... bility. or decr .... sed H,O intake rombined with normal losses. B. If prot~in' _'" th~ only 101ids in pl"'ma. th~n p!amta would contain .bout 93 % H,O and 7 % prot~im and the [total prot~in) would b. about 7.0 gldL in h.... lth. If dehy_ dration led to a 10 % dec",... in plasma volume. the [total prot~in] would inc=-< to about 7.8 gldl (7.0/0.9 = 7.78). C. All prot~in. ar~ concentrated by lost of pl"'ma H,O; ther~for~. h~moconce mration r.. ul .. in • nomela:tiv. hyperprot~in~mia. Concentrations of albumin. globulim. and fibrinogtn are proportionatdy incr~ased if dehydration is the only cou.. of th~ dY'pro.. in~mia (Pl.t~ 12B). D. Oth~r expected laboratory finding. l. Erythrocyt<>lis 2. P.. r~nal azotemia 3. Hypemh~nuria. if .. nal concentrating mech anisms ar. functional Table 7.3. Disca.... and conditions that co use hyperproteinemia • H~moconce ntration Inc..ased prot~in .ymhesis InHamm.tory diseases 'Infection; I>.ct~rial. viral. fungal. protozo;ol ' Noninfa:tious di",... ; nec""i,. noopl ..ia. immune-mediated dise-ase B_lymphocyt~ noopl .. ia Pla.ma cell; multiple mydomo. p!amtacytomo lymphocyte; lymphoma. lymphocytic I.uk~mia • A lel1t;vely common di ..... 01 condition

Note; AU of me.. di,..,,,,, or conditiOn! m.y= wiU c..,.., conrun.nt hyp
hyperglobu~ ... mi ..

but only hrm<><x>nc
300 II.

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY IncrtlKd prot~in .ylllhe.i. A. inHammation i. a common cau.. ofhYP"rproteinemia but does not necessarily cau", hyJl"rp rotein~mia. I. P.rhogtnesis: InHammation (,,!Used by infections or other proce=) stimul ates the .ynthesi. of ceruin globulin. by hepat<> 1.0 gldL. and [haptoglobin] may incr..,... from 0.3 gldL to 0.9 gldL}. The ron""lIIrarions of oth.. APr. are relativdy much less in h.... hh. :md thm a marked incr .... sN in an individual protein con""mration adds littl. to the [total protein] {e.g.• [C-reactiv. prot~in] may incr....., from 1 mgldL to 50 mgldL. and [.. rum amyloid A] m.y incr..,... from 0.2 mgldL to 100 mgldL).6 b. Drcreased con""lIIrations of the neg.rive APP. {I} Nq;>Iiv. APP. are proteins whose plasma or ",rum con""ntr>tions decr..,... bn:;,,,,,, of dec",....:! production by hepatocyt .. during inflammation. Thi. group indud.. albumin and transferrin. {2} Due to the plasma half_life of albumin in various .nimal. (e.g .. .. 8 d in dog. and .. 19 d in ho .... ) and the variable degre,,, of rwuced produnion. an infl.mmatory h}'llO"lbumin.mia may not be ...,n ulllil inflammation has !"'rsi..ed for at least sevtral daY" in dogs :md .. lean 2 wk in ho ..... The magnitude of decre.", i. typically mild {i.~ .•• deer..,... by < 30 %; ~.g.. from 3.0 gldL to 2.4 gldl} if inH.mmation is th~ only re.wn for th~ hypoalbuminemia. {3} The plasma half_liv., of transferrin ..~ not firmly mablished in dom~"ic .pro... However. decr~asN toul iron binding capacity (as • m.... mre of transferrin concentration) may not be =n until inflammation has Jl"rs.istw for at least a wtt!<. c. Dd.yed_respon", prouins {I} Th... are protein, who .. plo.ma or serum con""lIIration, incr..,... 1- 3 wk aft .. the on",t of inflammation; ch~ inc ...... i, caUS«! by increasN production. {2} Thi, group includes all immunoglobulins {IgG mo.dy) and complemelll {C3}. Increased .ylllhesi. of a variety of immunoglobulill! by many don .. of B_lymphocyt.. produc.. a polybmal gtlmmopl"hJ. {3} Th~ m"l:nitude of increase; in [C3) (as d.,ectw by ~-globulin inct ...... ) is typically < 1.0 gldL The magnirud~ of incr ......" in [immunoglobulin] em be mild « 1.0 gldl) to marked (:> 4.0 gldL) 2. Expected dY"protein.mi. pattern,

7/ PROTEINS

381

a. Acute_ph..., respon~: hy!",rproteinemia ",u...:! byacuu inflammation lasting 2- 7 d (I) Mild hy!",rpror.inem", causN by hyp..globulinemia (inc..=<:! Cl,_ andlor a._globulins and hy!",d'ibrinog<:nemia) (2) Possibly mild hypoalbuminemia or low_norm:d ",rum .lbumin oonc:<:lIt.. tion b. Dd.ya! respon~ hyp<:rprouinemia ",usN by infummation lasting mo", tru.n 7 d (I) Hyp<:rprouinem", (mild to mark«l) i, causn! by hyp<:rglobulinemia (incr .... '«1 po,itive . cur._ph..., .ndlor deL.y«i respon~ prouins). A poly_ e10nal gammop>thy mayor ""'y not b. d,,=«1 by SPE (Plar. 12C-F). (2) Mild to mod ..a" hypoolbuminem", ""'y b. p"''''IIt. {3} The net m.ng<: in protein con",mrations may produce"' dysproteinem", with a lower [:dbumin] and gr.... t.. conc:<:lItratiollt of some: globulin fractions. (4) A polycloruol g.mmopathy with. remicta! migration has b..::n calla! .n oupdo""l g"",,,,qpamy {pt.", 12D}. [n human m«licine. it is not consid_ ..«I • monoclon:d g. mmopathy because its proteiru do not m..,t the criur", for monoclon:d pror.iru (i .•.• ina.."" in K_ or A.-light main,. not both). Th. "monodoruol" gammop>thi.. that lOme .uthon have described in animals with inf=iou. dioe;;o..,. (•. g.• ehrlichio,i, .nd leish/lUllia,i,) may have b.c:n oligodonal gammopathi.. or compact polyelon:d g.mmopa_ thi ...'''''' They were co",id..«1 monocloruol gammopathies because of the narrow ,pike in an dectrophoretic pattern. and the .uthon enabli,h«l that the gammopathy WlU primarily cau...:! by one class of immunoglobulin (i.e .• [gG) via immunod=ropho""i, or radial immunodiffi.. ion. Howt""r. the gammopathy could indude mor. than one immunoglobulin subclass and thm not b. monoclonal. and light..m.in analyw; we .. not done to suppon e1onality. (5) A bidonal pattern (dectrophorotic ct,- .nd ~,_spike.) ",usn! by an inn.a=! [[gG,] was found in • ho .... Th. g.mmopathy di .. ppeara! after the hor.. was tr .... t«l for a "rontyl. infection." [gG, i. also ",fe,,«1 to as IgG(D. c. A monoclonaleIpan,ion ofT_lymphocyt.. has b.c:n found in dogs infect«l with Ehrlkhid ranis that ooncurr.mly had gammopathi.. with n.rrow d=rophoretic ,pikes. We a.. not aware of = in whim. clonal ""pan,ion of B_lymphocyt.. could b. producing. true monodon:d g. mmopathy. 3. Oth.r laboratory.Jau .uociata! with inflam""'tory hyp<:rproteinemias a. Anemia of inflammatory di5U.le ""'y devdop if inflam""'tion !",nists. b. Inflammatory neuttophilia or n.... tto!"'nia may devdop. c. Infl.mmatory lymphocytmis or lympho!",nia may devdop. d. Inflammatory monocytO!is may devdop. 4. Concurrent pathologic proces.ses may complicau imerpreution of the prouin .Jau. For example. the .. am b.. roncut",m ino..... «1 fibrillOg<:n production becau"" of infl. mmation and increased fibrinogen oonmmption becau~ of illtraVll5CllI.. ro"l:"lation. Or. the .. can b. concur",m inflamm>tion .nd hemoconc:<:ntration or infummation and protein_losing sUtes. B. B_lymphocyte neoplasia I. Pathogenesis: Neoplastic B_lymphocyt.. ""'y produc:<: larg<: quamiti .. of an immu_ noglobulin; typically. the .. i, one neoplastic c:<:JJ line or one e10ne of neoplanic

3"

FUNDAM ENTALS OF VETERINARY CLINI CAL PATHOLOGY

••

n

~

Hnyy chain

~a.T.6."or~)

'!1 ~L'9h1 ch~'n -,<"a"l

--

Fig. 7.2. &h.m .. ic "ructu.. of :on jmmunoglobu~n. [mmunoglobu~n consis .. of two h..vy cb. ins of th< & for IgD. £ fOf IgE.:and" fOI IgM ) and two light ins (.ithe, ,, or A but not both). n.. rombin:n;on of • ~ght clu in .00 tb. ,Ian"d Rg""'Dt of • MOVY chain i.s • fr>glllffit (F) tbot ron..i"" an rntigon_binding .it< (ab) (F + .b = Fob). T h< toil of tho Y (wnic:d >egm
.om< d . " (a for [gAo yfOl IgG,

lymphocytes. Th~ .ingl. don. of lymphocytes prod""". ~n d«:tropho",",jcolly. structurally. and . nti/:"nically hom~neou, immunoglobulin or a compa"bly homo~neou, immunoglobulin subunit. Th. resulting d",prot.in.mi. is calbl a momxUmll1 gllmmopathy. 2. Prot.in> produe«l by B_lymphocyt. "roplasi
7/ PROTEINS

J83 h~:IV}'

chain,. or abnormal fJ:lgm~m,}.17 Two da:trophore{ic~.ks wer~ d~ta:tM in a dog', .. rum m..t had an 19A.-producing mydoma; th~ two p<"lILa we~ d~t~rminal to bt rompl.,; .. of 19A. (dim~rs. trim~rs. or Utr:l_ m~rs)." Similar biclonal pnks w~" found in • Glt with 19A.-producing mydom • • but only on~ ~.k (thu, sugge indicotM that two don .. of 19A.producing pl• .m", cdls we~ pr=m.JJ {3} Con""mratioll5 of immunoglobulins oth~r than th~ monoclonal prouin frequ~ntly ar~ da:r .... ~. b. Mild to mod~rat~ hypoolbumin~m"- may bt couKand or albumin p<"lIk) in ~ith~r P-globulin or y_globulin region. aft" .. rum d=rophomis i, frequ~ntly natal to rq>" .. m. monoclonal g. mmop. thy."·'....., How~u. findi"i:' am bt du~ to . ither on~ immunoglobulin or • group of prot~ill5 th.t are migrating in th. sam. rq;ion. &rum da:trophor.. i, i, "",lUI to =""n for pot~ntial monoclonal gammopathies but frequently I""La .uffici~nt sJl"Cificiry to reliably diff~ .. m"-te a monoclonal gammopathy from a restrictal or rompact polydonal or oligodonal gammopathy. (a) A narrow globulin spi~ in th~ "fglobulin fmction rould bt. monoclonal gammop.thy {consa! by B_lymphox:yu nooplas"-} or.n oligodonal {or =-trictal polydonal} gammopathy (cousoal by . n immune =pon ..}." {b} A narrow globulin spi~ that is not in th~ y-globulin fraction i, prob_ ably a monoclonal gammopathy ,ina. mrn !>ands.~ um..!ly due to ~ith~r 19A. or IgM and not to IgG. High ron""ntration, of 19A. or IgM: a", not exJl"Clal in • nonneoplastic immun~ ""pon... {3} Comparal to cdlulo.. . ""m. m~thod,. :ogarost d=rophoresis or high_ /"<SOlution da:trophomic m.thods will impro"" d~=ion of prot~in !>ands; that i•• what 'p~'f1 to bt on~ band on cdlul"", a""tat~ may bt ...,n .. two bands on "Cam... How~er. th. pl"nltn"" of . single b.nd on agaro,. dO<::! not confirm th. pr=n"" of . monoclonal g.mmopathy.lO b. Immunoda:trophor..i, {I} If. monoclonal g.mmopathy is SU'Jl"CtM. th.n immunod=rophor~,is with 'Jl"Ci"-'pa:ific ami_lgG (induding subd ..... ). :mti_lgM .• nti_lgA.. :mti_{K-chain}. or . nti_(A.-ch. in} amibodi~. is n""dal to diff~remiat~ monoclonal .nd polydonal gammop.thi.. by prot~in analy.;'!'

=.

sum

384

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

{2} If immunod,""lrophowi. results indic;;ole Ihat nearly all of the gammopalhy is due to IgM or 19A. Ihen the g.mmop:llhy very likdy is monoclonal. If nearly all of Ihe gammopalhy is due 10 IgG. then il could keith" mono_ donal or polydonal with multiple da .... of IgG; .uch • polydonal state WlL! found in a dog with canine ehrlichiosis." If the gammopathy is due to only one IgG .ubclas... Ihen it it probably monoclonal. {3} If th"e is a monoclonal gammopalhy. then Ihe inc"as.d oom,.ntulion of one heavy_ch.in dass {or subcl ....} will k "-"OC",IM with eith,,!C_ or A.-chain! kc.use a lymphocyl< done produce" eith" !C_ or A.-ch.in •• not both!' {4} A potent",l monoclonal g.mmopathy a.ss<>
7/ PROTEINS

385

7. Associat«ilaboratory or dinial probl~ms a. B~nce Jon .. prouinuria (BJ prot~ins = light ch.ins of immunoglobulins) (= Chapt~r 8) (Plm 1lJ.2) b. Hwrvise;o,ity syndrome {I} High immunoglobulin concentntions may au"" the pl"'m. to ba:om. viscou •. Hyperviscosity .yndrom~ is =n with high coneeJ poor ptid.. within th~ amyloid had .mino acid sequences very similar to 'equences of human 1..-chains. 8. Amplification of variable regions of immunoglobulin ~n.. by polym~ra .. chain reaction is being u.snl to ch..""t"iu the donality ofB_lymphocyte neoplali . ... HoW.-vtr, analysis of small bio!"'y ,ampl.. from .. ""tive lymphoid ti.!U~ may yidd what appear. to be a monoclonal prolif~ration b.cau.. of donal exp.nsion within a ~rminal center or b.ca...., of random amplij]ation of ,mall amount. of DNA"·" P....,dodonality is present if .mplifiation produce. one or mo .. di,tinct bands. but the r.. ults.", not rq>roducible.

HYPOPROTEINEMIA (DECREASED [TOTAL PROTEIN] IN SERUM OR PLASMA) The di"'.... and conditiofl1 th. t au", hypoproteinemia ..elist..,j in T.bl. 7.4. I.

Ina.....d protein I"" from vascular 'I"ce A. Blood I"" (primarily at
,,.

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

Table 7.4. Di..,a..,. and conditions thai ca u.c hypoproteinemia inc,",..,M prouin loss from v'l5cul"

'1"'''''

'Blood loss ·Prol~in.Josing

DqJhropathy: g1om~ruJonq>hritis. ~myloido1is ·Prot~in.JosiIll: ~nt~ropathy: .mall intostinal mum,.,] di..,=,lymphangi«tasia, immill
blood loss Prol~in.Josing d~rm.{opa{hy:

burns, !:""rr:diud nud . tin skin dis ...... pw.m. loss: ptritoniti., pleuritis, v>sculiti. D=. ..«I protein synth. ,i, and/or incr.,..,..,j protein atabolism • Hq>.tic insufficiency 'Malabsorption or maldigenion: int.. tinal mueosal di.., ... , exocrine pancreatic inmfficicncy 'Carna:l;c ,(;;II." chronic di~, na>plasi., malnutrition, survation

lymphoid hypoplasia or ' pl,.,;. Failu", of passi"e transfer {FPn Hemodilution Excess administration of intravenous Huid Ed: congtui"" hem f.ilure, cirrhosis, Dq.hrotic .yndrom~ Excess ADH ,~""tion: SIADH • A ,.t1tively common dio.... or condition Noto: T ot:d prot';n OOO«nttotiOlU in h..Jthy P"P", kittens, a1v"" rnd fuals m>y b. ],0-2,0 gldl Jes. than tho>< found in ([UtilI< rnimili,

of th~ glom~ruL" b"",ment m~mbran~, Eith~r or both l",ions allow largc:r and mor~ neg. tivdy cl,..gnl prouin' through the glomc:rular filtration Nrrier, When prot~ins ent~r th~ filtrat~ at a rat~ gre;;ot~r tNn proximal tubule. con r..orb, then prot~inuria occurs, Wh~n th~ rate of protein loss excttds prot~in production, hypopro .. in.mia occurs, The largest prot';ns (~,g" a,_ma.croglobulin :md P_lipoprot~in) typically at< not lost through glomc:ruli, .nd thw a od«:tivc: hypopro .. in.mia occurs, B«auso dog, and cots normally have: very little P_lipoprot~in, the p,_globulin fraction typically is not .datively iner.....:! :as it is in pmpl~ with a PlN, 2 Di....." a, Animal, with a PlN (as ddin.d in thi, sa::tion) h.ve a glom~rulop.thy, Glom~r_ ulonephriti, or renal amyloido'i' may b. .ppar~nt hinologically, b, Soft_coat.d Wh...t~n t~rri",. have :m incr ......! incid.na of PlN . nd PlE," 3, Major laboratory findings a, Mild to mark.d hypoprot~inemia with hypoalbumin~mi •• nd normoglobu_ lin~mia (occasionally hypoglobulin~mia) b, SPE results: Th~ .. i. a .. l~tive: hypoproteinemi a p.tt~m with a,_globulins WRI or only mildly d~re;;o..d (but appear rdativdy incr ...,.d), Oth~r protein fraction, 'l< typically d efinitdy da:r.....:! {Plat~ 12L I}, Th~ dogr.., of d~re;;o.. of the rglobulin fr.ction wiU d~pc:nd on the poro'ity of the glomeruli, e, Modeme to mark.d prot~inuri a dominat.d by albuminuria (Plat~ 121.2) d. P",haps evidencr of ""nal inrufficiency (.zotemia or isosthenuri.) if the di ...... has destroyed enough nephron,

7/ PROTEINS

387

e. Hyp"molesterolemi•• nd pthog.nesis a. Intestinal =:mions. whim are rdatively protein rich, typically are digtned :md absorbed in the small intestine, .nd th.n tra"'poned to the portal syst.m :md lymphatic v.... ls. Wh.n generalized small intestinal murosal dis.a ..s or lymphatic diseases prohibit the absorption or transpon of the pro_ teins, the proteins.re Ion in f"""s. When the ..e. of proe.in los..! exCtt,!. the capability of the liver and lymphocyt.. to produ"" proteins, hypoproteinemia occurs. b. In some disord.rs, inHammatory au""tion and decr•• sed protein intake contribute to the hypoproteinemia. c. Intestinal blood loss beca"",,, of po.rasitism is on. form of PLE. 2. Di ...ses a. G.neralized small intestinal mucosal di"""",s: lymphoma, histoplasmosis, and lym phocytid pl •• macyt id rosinophilic .nteri tis b. Horses with acut •• nt"iti. c. Lymphatic di .... se: lymphangiect.. i. or lymphoma d. Intestinal blood loss: hookworms, whipworms, or neoplasia 3. M.jor laboratory findings a. Mild to marked hypoprotein.mia with hypoalbuminemia .nd hypoglobulin.mia (or normoglobulinemia) b. SPE results: The pattern is usually nonsdecti"" but may be .dective. c. Oth" findings may indicot. or suggen the inciting pathologic nate (e.g., HitroplMma organisms or neoplastic lymphocytes in biopsy s;omples. or mel.n. or oth" top>thy l. P>thog.nesis a. Thermal or chemical bums allow pl"'ma proteins to exude from cuUneous l.,ions at a rae. greater than th. rate of protein production. If the animal is not sttn soon aft" the injury, the dy.protein.mia will reHret a mixture of cuUneous protein loss .nd:m acute-pha .. inlLommatory respon ... " b. G.neralized au""ti"" skin disease c:m cau.. a hypoproteinemi, 'W', but the dysprotein.mi . prob. bly ",Hects • mixture of protein loss .nd :m inH. mmatory dysproteinemi .. 2. Major labomory findings a. Early: non ..lecti"" hypoprotein.mia b. Loter: nonsdecti"" hypoproteinemia masked by either an . cute or mronic inlLommatory dysproe.inemia E. Pl.",n. loss caused by p< for extravasation of protein_rim Huid into the pleural :md p
,.

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

2 Major laboratory findings a. Early: non,d. ctin hypoprouin.mia b. Lot,,: nonsd
Decrnlffi protein synthesis .ndlor incr.aKd prouin caubolism A. H.patic jmuffici.ncy (or h.patic f. ilur.) I. P>thogtnesis: A mark«i reduction in functional h'1"'tic /IUSIl {< 20 % w"ainingj d""rraoes th. synthesis of nearly all pla!ma prouins nctpt immunoglobulins. Normal protein cotabolism rombined with da:reasffi protein .ynth.si, product, the hypoproteinemia. 2 Disorder> a. Cirrhosis b. HqJ.tic na;rosis or inHammation (not acuu ) c. HqJ.tic atrophy =ond" y to ponosys{<mic shunts d. Neoplasm. that damag< th. Ii"", at.nsive/y 3. Major laboratory findings a. HypoprouiMmi>, hypo:olbumin.mi>, and normoglobulinnnia or hypoglobulinnni. b. SPE results: Th. pm .m is f~u.ntly nonsdecei"". but eh~ .. Jruly be, ~ rd . ein aces< in Pr or r-wobuliru becau.. of on. of two ehalries: (I ) The"" Jruly be, • compc:nmory incr~asffl .ymhesi, of immunoglobulirn to attempe to m. imain • colloidal osmoeic (oncoeic) pr.ssur. in the vascular 'Y"um. (2) Th. linr ",mov.. 'lIIig~nic Jrult ..ial .nd IgA from the ponal blood. Wieh hq>. eic insuffici. ncy. th•• lIIig.nic Jruluri.ol gaillS .lIIran"" to pc:ripheral blood and induces. sY"umic immun.... pon.. (increa"':! IgM: . 19A, or IgG ). Becaus<: 19.\1 . nd IgA migrate in th. IJ,-wobulin fraction . nd IgG in th. r -globulin &:><:tion. th ... may not b.. a de.. dinillaion bffiv..,11 IJ,- and ri:lobulin fractions (c:dlffi ~ ima-ga"'mIl bridg~). 4. Oth~r ch~mical finding> ofhq>atic di ...... or dY"filllction: illC""asffl hq>at ic errzyme activiti... dec ....."':! urea cona:mratioll. or increa"':! bile a.cid or ammonium conO::lllratiollS B. M alabwrptioll or maldigtnioll I. P.thogtll.. is: A mal . bsorpei"" or maldig.sti"" sme r.. ults in • d.fici. m imak. of b",ic body fuels (G1rbohydrat ... proui",. or lipids) to "pl. "" fuds used by m.ta_ bolic p:uhwaY" for daily energy. Ono:: d rpleted. protein G1ubolism and th. 'ISr of amino ~cids for glucolleogenesis lead co a d~ficiency in en.rgy and amino .cids for hq>atoa:llular . nd lymphocytic protein sylllh..is. When anabolism a CNds produc_ tion. hypoproteinemia occurs. 2. Disorde.. a. Malabsorption: Small illl..einal di ...... with gtlleraliud mucoul inmlvem.m may G1U", malabsorption of digened proteins. G1rbohydrates. and lipid •. b. Maldigmion: fuocrinr panc... tic irnufficir ncy (becau.. of chronic pallcreatiti, or p. ncreatic .trophy) creates deficiencies in prot........ lira", and amyl. .. and thus maldigtnion of proteillS. lipids (f.e). or carbohydmel (starches). 3. M.jor l. boratory fillding> a. Hypoprotein.mi •• hypoalbuminemia. and normoglobulinemia or hypoglobulinemi.

7/ PROTEINS

389

b. SPE r~,ults: Th~ pm~rn typically i, nonsd'""tin (Pl.t~ 12H). c. Oth~, findings d~~nd~nt on th~ primary p.thologic stat~ {~.g., d,"",,,,snl trypsin_lih immunor~activity with noc,in~ paner ... tic imuffici~ncy, or POO' xylo,", ab,o'ption with malab50rptiv~ nat~' [,..., Ch apter 15]} C. Cach~ctic stau. I. Pathog~nesis: Wh~n ch~ rat~ of prot~in cat.bolism aads prot~in production, th~ negativt prot~in naN, caUst, hypoprot~in~mi •. B.fore hypoprot~in~mia d~nlops, gluoo", and most strum prot~in ooncentration> {espl .nd muscl~ m.... 2. Di""rd~ .. •. Chronic di......,. such a, chronic inf..aion> and malignant na>pl...ia {Plat~ 12J.I} b. Ma,hd malnutrition 0' starvation 3. Major J.boratory findings a. Hypoprot~in.mia, hJ'P""lbuminemia, and no'moglobulin~mia or hypoglobulin~mia

b. SPE results: Th~ pm~rn typically i, nonstl'""tiv~. c. Oth~, findings de~ndent on the primary pathologic stat~ D. Lymphoid hypoplasia or aplasia l. B_lymphocyus produce immunoglobulin> and not othe, major plasma protein .. A mild hypoprouin~mia pountially can be, cr~.tffi by lymphoid hypopla!ia if oon""n_ trations of othe, prouim a,e WRI. 2. Exp«:tM dysprot~in~mia a. Th~ [total prouin] i, WRI to slightly d,""r.ase
FPT A. Nwnat.. who fail to ingtn or absorb oolo,tral antibodi~, will han low....rum 0' plasma [total prot~in] becaust of lower [lgG]. How",.. , inflammation 0' dehydration con iner ..... the [total prot~in] in mch .nimals and thus nust.: th~ FPT. B. Thi, ,dationship becw...,n [lgG] and [total prouin] has b...,n aplo,ffi and ,esultffi in the following findings in stra from 1_ to 8_d-old calvt. when comp..ffi to an [lgG] deci,ion value of 1000 mgldL" l. With a [.TP...J of 5.0 gldL as a d,""i,ion limit, 83 % w..~ rorr,""tly classififfi reg ..ding p ..... i"" tramf.. naN,. 2. With a [.TP...J of 5.5 gldL as a d,""i,ion limit, 82 % w..~ rorrecdy cl ..... ififfi regarding p .... i"" tramf.. naN,.

IV.

Hemodilution: iner~a ..d ECF volume A. By itstlf, iner ...,ffi ECF i, a nry unrommon cou .. of hypoproteinemia, but it may low.. prot~in ooncentration, chat were alr~ady d,"",,,,snl because of anoch~r problem.

""

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

B.

Di""rd~n

or ronditions I. Ex~ss ~dmillim.{ion ofimrannous fluid (too fasr or too much) 2 Ed~matou, diKlrdus (congestin hra't failure, cirrhosis, and nq.hrotic . yndrome) 3. Ex~ .. ADH 5<'Cfffion (SIADH) 4. Us< of plasm. expand.",_ when eJ[travaocuJar fluid is pullal into pt.",,,,, soon . fter adminislration {may not result in incr
H¥PERALBUMI NEMIA

Th. di",ast. and conditions that cou,.

hy~ralbumin.mja.",

linal in Tabl. 7.5.

I.

H.moconctmration (d.hydration): d«r•• sffl ECF volume A. ~r.,.sffl plasma H,O Iud. to gr
[I.

IlIdu""d '}'IIth..is by g1uoororticoid thuapy A. Glucoconiroid thuapy might caust mild hYP"r>lbumill.mia ill dog.'~" :md ill cm (ullpublishal S.LS. dau). III dog. giV<1I predlli"",,~ {0.55 mglkg. q 12h} for 4 wk, th~ [albumill] :md [toul proteill] illansal mor~ ill th~ pralllisoll._treatal dog. thall ill, oomrol group." III alloth.. study with dogs """,ivifll: p..dnisone (0.55 mglkg, q 12h) for 4 wk, [albumin] .nd [toul prot~in] inc",=!aI by subtr>ction} did IIOt."

III.

Fal",ly inc..=tioll d.t .. min
T able 7.5. Di..,a.... and conditions that ca use hypcralbumincmia • H.mocon""ntr>tion [ncre.sed albumin synth .. is inducffl by g1uooconicoid drugs or hormones • A rel1tively common di...... or condition Noto: If lolbuminJ ;, ~.,mined by the BeG method, the BeG dy< m.y bind to proteim other thrn :albumin rnd tbw yioo..byper:dbumi ... mi • .

7/ PROTEINS B.

391

Th~

. nom. lous [albumin] 1lU1 not k r~rogniud in hypoalbumin~mic sampl..; th.t i., is mo .. sn"~r~ than indiQlted by th~ mrasured oonctntr:uion. For ~xampl~, in ",ra of dog. with PLN. th~ [albumin] from SPE may k oomid.. ably less than th~ BCG valu~. th~ hypoalbumin~mi.

HYPOALBUM[NEMIA I.

This rommonly occurs with hyp<rprot~in~mia and hypoprouinemia but al", am k >:«n when the .. is normoprouinemia.

[I.

The di",a.S<s and ronditions that cou'" hypoalbuminemia ar~ listed in Table 7.6. Pathogtn_ est::! of the hypoalbuminemic nates a.. described in the appropriate dy.proteinemia sections (s« the Hyperproteinemia and Hypoprotein~mi. sections). A. Hypoalbuminemi. may k found in hyperprotein~mi" stat.. not rdated to hemooon""ntration. l. [nHanlmatory hypoalbuminemia occur> kcou,", albumin is • negative APP. The following two major concqm are rdated to infummatory hypoalbuminemia: a. Th~ hypoalbuminemia d""s not d~dop until th~ inflamllUtion h", persisted for dap 10 wffit:., .. peciaUy in horses kcous<: equine albumin has a plasllU half_life of nearly 3 wk. b. [f inflammation is th~ only cau.. of th~ hypoalbuminemic state, hypoalbumin_ ~mi. is npected to k mild. 2. The p:lthogtnes .. of hypoalbumin~mias ... n concurrently with B_lymphocyte neoplasia vary and may relate to inflammatory cytokines, .. sponsc: to inc....,ed rolloidal osmotic (oncotic) pressure cau ..d by hyperglobulinemia, or damaged tissues as liv.., intestines, or kidney>.

sum

Table 7.6. Disca.... and conditions that ca u.e hypoalbununcmia Decrrased albumin synth.,is • [nHanlmation • Hq.atic insufficiency ·Mal.bsorption and maldigntion 'Cachectic stu., Hyperg:munaglobulinemia Increased albumin loss • Blood lms • Prouin_ losing n~phropathy: glo merulonq.hr itis, .myloidous 'Prouin_losing ent~ropathy: 'lIUll intestin.l muros;ol di..,..,e,lymphangiectasi. , inlenin.l blood I"" Prot~in_losing d~rllUtopathy: burn., gen .. alizro exudative skin di ...... Hemodilution Ex""ss .dmininration of intravenous fluid Edematou, diKlrders: congestive hra" failure, cirrhosis, nephrotic syndrome Excess ADH secretion: SIADH • A ,el1tively common dioe ... 0' condition Note: [Albumin] in healthy pups. ki""",. cal~, and foal. lJUy be OS_ I.O gidL less th:m thos< found in mature :mim:d,.

392

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

B. H)'pO'dbuminemi. is commonly found concurrently with hypoprouinemi .. AI; d=rikd in the Hypoprouinem;" ,~jon, hypoalbuminemia,.re usually muoN by d,",,"'a<M production of "'bumin, incr.aKd lou of plasma "'bumin, or both. HYPERGLOBULI NEMIA (.... the Hyperproteinemia =:tion)

I.

Hyperglobulinemi<> is typically pr... nt ooncurmuly with hyperproteinemia. [n fact, th. t
[I.

Th". at. four major concq>ts to consider: A. Th. major au.., ofhyperglobulinem;" """ hemocon"'ntn tion, infl:munation {•• pt_ cially chronic}, and B_lyrnphocyu na>pla,ja. B. By way of routine clinic;o[ cheminry mffhod" the globulin ooll""mralion is determi"M by subtraction; dut is, [total prot~in] minus [albumin], Thus, ~rrors in thou values can result in urontous globulin ron""ntrations, C. ~Ust th~ routin~ [globulin] .. pr=nt' th~ rum of the conctntr>tions of all proteins oth" than .lbumin, hyp"globulinem;" may r~SlIlt &om th~ incr~ conctntration of on~ or mor~ diff"ent globulins, D, SPE r~mlu typically will diff"entiat~ the hYP"rprot~inemi .. ofh~moconctntration from hy~rglobulinemi .. of infl.mmation and lymphoid ntopl .. ia, but th.y IIUy not be able to diff~rentiate inflamllUtory &om lymphoid ntoplasti, hYP"rglobulinemi ..,

H't'POGLOBUU NEMIA (... the Hypoprotein~mia KCtion) L

For .nimal, oth" than ntonms, hypoglobulinem;" commonly O<'Curs concurrently with and might!:" Sttn with normoprot.in~mia, wh..... it is not exp«tM with hyp<rprot~inemia if referen"" int"vah at< appropr;"te, hypoprot~inem;"

[I.

At with the hypoproteinemic di",rd ..., the IIUjor alUstS of hypoglobulinemi. fall into two altegories: A, Globulin production i. d~c""M, Ikmem!:..r that most globulin. oth" than "(globulin> are productd by hepatocyt~., B, pu,m. globulin loss is incre. tffl b.caUst of blood loss or loss via dam3i:.d glomeruli, intestinal mucosa, or skin,

POSITIVE ACUTE-PHASE PROTEINS (APP.) L

G~nu:d

roncq>ts A, M.jor "p«ts of the scimuutM production of po.itiv~ APP, .. ~ descrikd in e..li.. KCtions of this ch apt.. {Ott General Concq>ts for Tot:d Prot~in, Albumin, and Globu_ lins, Sect, [I.q, Ph)"iologic "'!=ts of positive APP, and clinical application of po.iti"" APP assay> in domestic IIUmmal. have oon reviewM,'" B, Ess.ntiaUy any injury that product. an arut~ inflamllUtory reaction can inc...", conctntration, of the "",itive APP., Th"", injuries aln be due to infections (e,g" bact..ial, vir:d, fungal, or protozoal) or noninfectious condition! (~,g" phy.ical trauma, burns, or na:rosis), As a group of protein!, the incr~"M con<:entration of positive APP,

7/ PROTEINS

393

con incrtlSt [total

prot~in]

and [globulin]. IndividuaUy. the

magnitud~

of incr ...... v"ies

oonsid~rably.

C. Positin APP mult, typically .. ~ valu abl~ for two r~ason" l. Oth~r m~thods of d~t=ing inflammation {i. • .• pyraia or neutrophilia} may bt too in .. nsitive or nOfl!pecific for the pathologic nate of interest. 2. Thq provid~ .nother method of monitoring.n inflammatory process .ith~r for thenprIlower positi"" APP conctntmiofiJ. b. DiKlrd.", that activate pl.. min or thrombin cm lower [fibrinog~n] by fibrinogt. nolysis and ro.gulation. c. IntnVlUCul" h~molysis will lower [fuiptoglobin]. II.

Fibrinogtn A. Physiologic process l. Fibrinogtn is a pl",1IU prot~in that i, produced by hq.atocytes. 2. When enzYllUtic processes in plasma conven prothrombin to thrombin. thrombin then promot.. the con""rsion of soluble fibrinogen to inKlluble fibrin. Interactions of fibrin. platdets. and ~ndothdial ctlls hdp prennt blood loss from blood nsseb. Fibrin formation it limited by plasmin.induced fibrinolysis. 3. Becau.. fibrinog~n is a positive APP. plasma [fibrinogen] is up«ted to incr~a .. during inflamnl. B. Analytirn conctpu l. H.".t. precipit:mt method a. Principle: The diff.",nct in the [TP..J in • sample btfore and aft .. removal of fibrinog~n via heat precipitation {56-58 oq and ctntrifugation estimates the [fibrinog~n] .

b. Unit conversion: mgldl X 0.0] = giL (SI unit. ne"est 0.1 giL)" c. Comments {] } It is a semiquantitati"" technique used to scr..:n plasma for hyp
3"

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY {ru~ valu~. In mon s~j .. , th~ Jow~r .. f~"'n"'" limit is 0.2 gldL d. Outljn~ of the h.~{_p=:ipitan{ method

~i{h"

0.1 gldL or

{I} FiU two microhematocri{!Uk. (at 1...1 thIN-quan.", full) with EDTA_ amiCOI [
=nd

tube,.

(b) Exampl.: 7.0 gldL - 6.7 gldL = 0.3 gldL 2. Thrombin tim., von Clauss modification a. Principl~ A [fibrinog.n] can b., dcterminM from the time r"'luirM for fibrin formation aft" the :addition of a high concemmion of thrombin to dilutM citr>{M pt.",,,,•. This time primarily depends on the [fibrinogtn]: the lower the [fibrinogen] i•. the longtr will be the time umil dot formation. b. See Chapter 5 for detail, pertaining to measurement of fibrinogtn concent"'tion. 3. Some people have attentptM to G1kulate a [fibrinogtn] by determining the difference betw..,n the [pTP...J ofEDTA.plasma .nd the [.TP...J from a ",rum sample. Such a method i. not recommended beau.. solut.. other th.n fibrinogen alt .. the ref",ctive index and thus cause diffen::nces becw..,n plasma and serum [total protein] (Itt the section on Analyticol Principles for Total Protein. Albumin, and Globulins). Erron em ari .. when two diffi:n::nt sampl.. an:: collectM and compan::d. C. Hyperfibrinogtnemia (plarm.) (Table 7.7) l. Two major em.." a. Hemoconcentration: decreased pl:wn.. H2 0 b. Inflammation: increasffl fibrinogtn production by the liver 2. The [total protein] is usually also incr",sM with thc .. disorde .. but may be WRI. Unle.. fibrinogtn ronsumption is increased enough, the [fibrinogc:n] incn::ases n::htivdy more [han the [total protein] in infl.mmation. With dehydration, the incr ...... in the [totol protein] :md [fibrinogen] mould be n::latively the same; for example. they both incrUK by 5 %. Th.". concepn 1M to the development of the PP: F mio {Eq. 7. I.}." Th. "'tio was simplifiM • few y..... l. tcr by not rubmcting the [fibrinogen] from the [TP...J {Eq. 7.1b}."

Table 7.7. Di",a.... and conditions that co usc hypcrfibrinogcnemia Incn::asM fibrinogtn production • lnfl:mlmation • Hemoconcent"'tion • A ,.t1t;v
395

7/ PROTEINS

· =-,iPC'"=:=::;,,, TC Pic-"iC fi·bO",, ·":O "" ="i PP:F "no [fibrinog~n

.

.

(7.Ia.)

1

[pWJII.1 TP J

(TP:F,b) r:lUo = i · i • fibnnog~n

(7.l b.)

Exampl.: [PTPJ = 8.8 ydl & [fibrinogtn] = 0.8 ydl PP:F ... tio = "C ·'O,,/= 'L::--:;',,·'O,,/'=L , 8.o g/dl 0.8 gjdl 0.8 g/ dl (TP:Fib) ... tio = 8.8 g/ dl = II • 0.8 g/dl 3. PP:F ... tio or (TP:Fib). ratio a. Th~ .. ratio. hdp in diff,,~miating

hypc:rfibrinogtn~mi..,

of inflammation and

d~hydmion.

original int~rpmin guides and ",f~r~n"" im~rval, were establishM with th~ PP:F ratio ...." The PP:F ... tio. in h~althy mamJIl.1Is ",riM among s~ies and by ag~ group, mostly kcoUK of diff,,~nt total prot~in con""ntrations, .., (I) Cattle: If the ratio is :> IS, hyporfibrinogenemia is probably c;ou...i by dehydmion, If th~ ...tio is < 10, hyporfibrinog~nemia i. probably c;ousffl by inflammation, (2) Hor...: If th~ ratio is :> 20, hypc:rfibrinog~nemia i, probably c;ousffl by dehyd ... tion, If th~ ratio is < 15, hyporfibrinogenemia i. probably c;ousffl by inflammation, (3) If th. simpl~r calculation, (TP:Fib).... tio, is u...i, • "I " should b. add M to guiddine ",lues to b. con 16 in c;otde is probably dehydm ion), c, Guiddines are OOsM on the assumption< tbat a h.aJthy animal'. [total protein] and [fibrinogtn] .re WRl, and th~n one of two things h'pp mor~ difficult. Incr",sM fibrinogen consumption in tbe pathologic state will also doud the issue, (2) Oehyd ...tion by itsdf will cau.. only minor incre.... in [fibrinogtn], (3) The .fo"'m~mionM "tio guidelines are not .ppropriate for calv.. and foals bn:.usc: tbe ... tio ",f~renct int~r",b a.. for mature :mimaJ values, Thry also ar~ not appropriate for amle :md bones th at han a ooncurr~nt pathologic state that c;ouw; hypoproteinemia, b.

Th~

".

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY Table 7.8. Di..,a..,. and conditions thai ca u.c hypofibrinogcn.cnlia inc,",asM fibrinog<'11 consumption • Intravascular cn;;ogulation (locali=:! or di"".minat«i)

inc",=<:! fibrinogtnolpis D'"".....d synthesis of fibrinog<'l1 Hepatic insufficiency Afibrinogenemia (oongenit:d or inhcrit.d) • A rel. ti",[y common dioe ... 01 a",dition

(4) Th. accuracy of ..fracrommic v:du .. i. at 1><51 ± 0.1 gldL Thu., the accuncy of Ih. calculated ratio is prob.obly wIler with a high" [fibrinogtn]. Even then, howen., th" QUrtw in a Bernese mountain dog {Ikrner .. nn~nhund} {note: sometime! translatw .. Saint Bern. rd)." (c) Hypofibrinogenemia or dysfibrinogenemia h ...lso been m~ntionw as occurring in Utasa apso,. vizsJ ... collies. and bonoi, {W.J. Dodds, unpublished}. but the laboratory data to ch. racterize these nates have not betn published. lll.

Other acute_phase proteins A. An:dyticaJ com:qm' l. like mo,t plasm. protein.. the positive APr. are rdativdy ,table an:dyt~. when stored fr"",n. Except for fibrinogen, .. rum is the p",ferred lample for mo,t APP,. Antiooagul.nt. (EDTA, heparin. and citrate) have been reportal to cou.. a variety of effects on APP remh •. Th. effects of Hgb. bilirubin, or lip<mia on the .....Y'vary with the . nalyte and the assap med. AI would be exp«led for a h aptoglobin .....y b:ued on Hgb binding, the use of he moly=:! samples must be avoidw.

7/ PROTEINS

397

2. A v.riety of analytic.! method, ar~ u~ to m.... sur~ inc",aW oonctmration. of sp«ific positi ... APP,. Som~ of th~ immunologic method. a", 'pacies spa:ific. In health. the con""mrations of som~ po,iti... APP. are n... r the detretion limit, of the a=1'. '0 d~t.-cting drc ..asffl ronctmratiom i. difficult or impo.. ibl~. a. Srrum C_r ....cti ... protein it usually m.... surrd by immunologic m~thods: immu_ noturbidimetry. rITzym ... linkrd immunosorbe,nt a=y {EUSA}. or !ata .gglutination. b. H.ptoglobin assay, typic.!ly .r~ ba~ on haptoglobin·, binding to Hgb. The binding i, detrctrd eith.. via 'pactral differena::s of bound ... rsu, unbound Hgb or by dimini,hed peroxidase .ctivity. Breau.. of the spaci.. diff.rences in haptoglobin·, amino .cid rompo,itions. inununo=1' d.. ignrd for human haptoglobin mayor may not det«l. domestic mammal·, haptoglobin. c. Srrum amyloid A i, m...surrd with immunologic a=ys: immunoturbidimetric ..u1' or .nzym~_linked immunowrbe,nt :assays. A=1' using ami_{anin. SAA) or .nti_(fdin. SAA ) antilxx!ie, have been d ...eloped. but the .. is sufficient homology in the canine and fdine proteins to cro.. _rnct with some anti_ {human SM} antilxx!i ... d. a,_Acid glycoprotein i. m.... urrd with .pacies_spacific immunoassay" .!ingle radial immunodiffusion :assays and immunoturbidimetric '''"Y'. e. Ctruloplasmin assay. are typically .pactrophotometric :assays basrd on th. oxidation of 'pacific submates. f. F.. ritin: c;.,nerally. ,paci....spacific immunoassays are nttdrd to m...su", ",rum or plmn. [ferritin]. A=y. ha... been developed for canin •• 65 fdin . .... nd equine" f.. ritin .pacifically. O<:casionally. the cro.. _reactivity i, sufficient to m.... ur. f~rritin conctntmion, of .pacies oth .. than the one for which the :way w;o. developed: for aampl~. the equine f. rritin a=y can be, u~ to measure con""n_ tration. of rhinoceros. tapir •• nd l.mur ferritin. When polSible. the ferritin u~ in calibrating (st>nd",d) solution, ,hould be, isolated from the 'pacies of int..esr. for aampl •• the u", of lemur ferritin to calibrate.n assay for l.mur ferritin that us,," an anti_{equine ferritin) antibody. B. Inn.. ~ ronctntratiom of positive APPs oth.. than fibrinogen l. Exampl .. of potential incre.>es.re providrd in Tabl. 7.9. It should be, noted that the iner ...... in [h aptoglobin] may be, sufficient to produ"" . recognizable inc",..., in the ",rum [total protein] by it,elf. wh ...... inc",...d conctntmioll! of the oth.. APP. typic.!lyar. not. 2. Conditiom oth.. than inflammation can cau.. incr ... ~ po,itive APP con""ntrations. a. Pregnancy in bitch.. will inn.... [C-..""ti... protein]." b. In dog•• glucoconiroid therapy an au", inc",aW [h aptoglobin] .imilar to haptoglobin con""ntration, Sttn during infl.mmation. '''·' Also. ""rtain .nthd_ mintic compound, {potassium mdar><>nyl. l.... misol. hydrochlorid •• and milbe,mycin oxime} can increa.. [haptoglobin]." c. Phenobarbital therapy in dogs an inc",... [a,_acid glycoprotein]." d. [C-re.ctiv. protein] incr.....' in cattl. during l""t>tion. Th~ highm ""lues w..e during the fim 4 mo oflact. tion. n • . If.n animal does not ha... an inflammatory di ...... a plasma [f.. ritin] t.nds to reflret iron storage in the animal. Thu •• if iron ,toral:" is inc",aW {e.g.•

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

'"

Table 7. 9. Enn'pl". of changes in aculc_phuc protein cofloenlnllion.

Acute_ph .... Protein

CoII""mration in

M>gnjtud~

he:dthy dog

re'ponst (x b....,linrj

(mgldL)

Fibrinogen C-reaclin protein Strum amyloid A Haptoglobin ct,.Acid glycoprotein

uruloplasmin

<300

« <0.4 <300 <60 <,

" "" "

OS, 800x

Time to p...ak

of FirS! deuc{ion of iller""", (h)

,4

" "

OOflctntralion

ldi

,

3-4 4

No«: MOl< info,m1tion (acq>t fOr fib'inogmj is .... ibb&. in 1 ,.view mid•• bot """ .... ..,""'" of th. d .... Not. th" units we", ron.... "'d from "fl' to "idL' .0 they could b. mo .. _ily romp>n<J to oth.er protein conam trations (report«! .. gldL) in this cb"l" <J. Sou,", Oron .. . t.'

hemochromatosis, hcmmidcro'is, or chronic hcmoiy.blishffl but ap~ars to ~ rd~ast from tissuo; oth.. th.n li",r. IMMUNOGLOBUU NS The mon common r... son for m~~.uring [lgG] i. to det~rmin~ wheth~r th~r~ has oon .uccessfu[ p"ssivt tran&r of IgG from a moth~r (rna .. or cow) to h~r foal or calf via colostrum. Aa:ord· ingly. passive transf~r is the focus of this Sffiion. Immunoglobulin con~ntrations may also ~ m.-asurffl when ",",,"l""ting cong<'nital :md acquirffl immunoddici~ncy nates or when :assessing potenti. l monoclonal gammopathia .

I.

Failu", of p .... i'" transf.r {FPT1 A. Physiologic con~pts l. Pl.~ntation in horses .nd cotde prevents in utero transf.r of immunoglobulin, to the fffus, so namatal ho .... :md ruminant. must inge;t colostrum soon aft .. birth {~fo .. "gut clo.u "'"l to obtain mat.rnal immunoglobulins, espaiIlly IgG. Aft .. ingestion .nd absorption, the half. lif. of maternal IgG is about 2()'" 30 d in foals'" and 20 d in colvt •. " Inadequate immunoglobulin transf.. for a particular environ· ment inc ...... s the ri,k of infectiou. di ... .... nd deer ...... the rat. of weight g.in. 2. Neonatal foals, prior to ingestion of colostrum, h""e """nti.lly no IgG in their plasma, and cal"... havt vtry low ron~nt .. tions. On~ ""poW to .ntig<'ns, neon.tal foals . .. nimulatffl to produ,,", IgG and protectivt immunity in .bout ]()"'J4 d." Synth.,is of IgG by calves can ~ detectffl by 8- J6 d." 3. Fetal foals and calves havt limitffl ability to produ~ IgM, 50 neonatal foals ~nd calves should ltavt low ron""'ntrations of IgM. 4. Intestinal absorption of colostrum rapidly inere..es plasm . [total protein]. Most of the increase is due to inereaKd [l gG], but rolonrum .lso contains positive .cute·

7/ PROTEINS

399

pha.. prot~inJ mch at amyloid A that i, producal by mamm ..y gland ~pithdial edls." 5. Immunoglobulin uptak~ is medi.ted by th~ namarn Fc r«:q>tor on epithdial edit. B. Cau",," of FPT I. Lock of colostrum in~tion {the nwn>le i. too weak or ~u .. of oth~r f. ctonl 2. In.d,",!u .t~ IgG in the colostrum {a da:reastd con~ntration or in.d,",!uat~ vol"m~ of colostrum} 3. Fail"", to .b..orb ~nough in~ted IgG could occur if the colostrum i. in~ed.fw "gut clo,"r~" and h.. bttn ' !5OCiated with o:nain haplotJ'P'" of the neonatal Fc ~ptor in calves!' C. Est.blishing the pr=no: or ab .. nce of passivr tran.f~r I. Appropriat~ da:ision thresholds to ddine FPT v.ry with th~ assay b.cau.. of • ....y bi...,s {inaccuracies}. Despit~ these bi...", gen~ral r«:omm~nd .tions have bttn made. Loboratory_.p«ific da:ision thresholds should be used when available. 2. Foal guiddines: Blood .amples are collected bet",..,n 18 .nd 48 h aft~r binh, wh~n antibody absorption .mould be .... ntially compl~t • . a. For """ral J="', [lgG] :> 400 mgldl was consid..al rodence of passivr tran&r. How<:v.., [lgG] :> 800 mgldl llUy be a bett~r crit~rion for ad,",!"at~ passivr transf~r .nd is cur"'ntly rerommendal,"'10 although .d'"'!uacy may vary with diff..ent IlUnag<:ment .nd environmental foctor>. b. [lgG] < 200 mgldl is consid..al evidence of complete FPT. c. [lgG] beIW~en 200 mgldl and 800 mgldl i, consideral ~id~no: of partial FPT. Such con~ntration. may not provide adequate humoral protection, esp«ially for foals with high .. risk factors. 3. Calf guiddines ar~ not firmly e!lablished; two published guiddines follow. For ...ch, blood sampl.. a", collected betw.~n 1 . nd 8 d aft~r binh. a. On~ r«:ommendation"'" (I ) [lgG] :> 1<XX> mgldl is con.id~red evid~nce of passive transf~r. {2} [lgG] < 500 mgldl i. colI!id..ed evid~n~ of complete FPT. b. Anoth~r =omm~nd .tion"'"' {I } [lgG] :> 1600 mgldl is con.id..ed ~idence of passive tran.fer. {2} [lgG] = 800-1600 mgldl is consid..al partial pas.livr tran,f... {3} [lgG] < 800 mgldl i. con";d~red FPT. c. When ",fractometry was used to estimate ..rum [total protein], decision thr .. h_ olds of 5.0 gldl and 5.5 gldl correctly classified th~ pa"ivt trall!f.. !latus in 83 % .nd 82 % of th~ calvr., r~sp«livdy." 4. Results for blood sampl.. collected from old.. fool. or calves, ,sp«ially if ill. will be mor~ difficult to int .. pret ~use the half_life of m. t. rnal IgG and a nwn. t.', respon .. to antigtns . ft .. birth must be consid..ed. D. Analytical con~pts for methods of m~asuring or estimating [lgG] in foal. and calvts: Because of the lock of nandardization of assay methods and ref~ .. no: standard sera, ther~ may be IlUrkal variations in [lgG ] mn.mrw by di/fc:"'nt IgG :usays. Th~ .-valuation of various analytical methods i, frequ~ntly comparal to [lgG] determined by RlD. How.:ver, a RlD assay might be inacrurat~. " Thus, the int..pr~ti"" guiddin~s will vary. I. RlD t~st for foal or calf .. rum a. Principk IgG diffu"" in a gd containing anti-{,",!uin~ IgG) antibodies or anti_ (bovin~ IgG) antibodies for 18-24 h. The diamet~r of a precipitant ring is proportional to the [lgG] in .. rum.

400

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY b. R..mh.: RID js consid~rffl to b.. quantimin '''''Y for m~... urjng [lgG}. Assays for m=mring bovine [IgG,l are .lso .vail. bk c. Comment' {I} Usually RID is colI!id.rM too tim. consuming (24 h diffusion time) and ""!",n,i",, for routine clin;",! ""'. {2} RID h", bttn comid.rM the gold standard method in veterinary l.bomo_ rie •. Howe""r, some commercial RlD """Y' overestimate [lgG], espa:i<>lly.1 [lgG] ;> 2(xx) mgldL" [n one study,. commorcial RID 'lU)' for bovine IgG had a posit;"" proponional bi"", thus indiating the provid.d standard solutions had lower IgG oon~mrations than labd«i." 2. Glutar:dd.hyde c=gulation 1m for !WI or calf .. rum a. Principl~ GJut:arald.hyd. (IO %) promotes the fonrultion of Dlolt'Culor cross_ ljn~ to ooaguJ.te b...ic proteins such :as immunoglobul;", and fibrin"!:,,n. B«aust fibrinogen is .b..,m in ,erum .nd ""I}' linle IgM is prestnt in n
=pJ • [lgG) "" 800 mgldL if .. rum form, gd .. to min, • [lgG) :> 400 mgldL if .. rum form. gd .. 60 min, • [lgG) .. 400 mgldl if .. rum did not gd by 60 min, {b} Excdl~nt ag=ment with th~ RID assay wh~n [IgG) < 400 mgldL and v..y good ~gr..,m~nt with th~ RID a=y wh~n [lgG) :> 800 mgldL" {2} For ""timation of bovin~ [lgG) {a} S.miqu.ntit.tive value." • [lgG) :> 600 mgldl if the .. rum form. a firm opaqu~ dot by 60 min, • [lgG) .. 400~ mgldL if the .. rum forms a ",misolid gd by 60 min, • [lgG) < 400 mgldL if the .. rum did not gel by 60 min, {b} Comment. • To det .. min~ th. [lgG) .t the deci,ion th ....hold. for FIT (eith.. :> 1000 mgldL or:> 1600 mgldL), the ",rum would n.ed to "" diluted prior to analysi', • Th~ =ult, of the glutaralddtyd. coagulation tost (Gamm._Check_Bl w~'" unreliable wh.n whole blood was used. r1 c, Th. te,t is ""I)' inal"'n,i"" and .impl~ b..cou", it jun requi ... glutaralddtyd., r.:.ction tu""., and pipettet, It does requi .. that .. rum"" harvested from blood, 3, Lota agglutination (Foakheck) for foal ",rum a, Principle: Lota ""ad. coated with anti_{.quin. IgG} .ntibodies will agglutinate in the prestn", of .quin. IgG, b, R..mlts: [t i. a semiquantitativ~ assay that I:"nerally agr.." with the RID .ssay ..sult. but i, not as good a, the glutaralddtyd~ coagulation un," c, Comments: Th. test i, mod.ratdyapen.i"" but tak"" only about 10 min to compl~te aft .. the .. rum is coll.cred, 4, Znsa, turbidil)' t""t for foal or calf .. rum a, Principle: Sulfat~, sd.crivdy precipitate cationic protein •• uch as immunoglobu_ lin.; other protein, ... n.... tral or nq:ativdy charged, At. con",ant [ZnSO,.], a

7/ PROTEINS

401 great~r

turbidity correspond. to • high~r [immunoglobulin]. Sin"" th~" is very 19A or IgM in foal or c;olf .. ra. th~ amoulll of turbidity r~Haots th~ [lgG] in a sample. b. The results can ~ .......d visually or turbidimetrically. Turbidim""ric ,.,...ssm~1Il ""quires a 'P'""trophotom""" and ,undard ",lutions to esublish a nandard litcl~

curv~.

(I) In fools. visual turbidity occurs when the [lgG] i. n~ar 400- 500 mgldL. which does not match with the curr~nt daoilion thr.. holds for FPT." (2) In calv.-.: (a) With a ZnSO, .olution of 208 mgfL, suffici~nt turbidity to obscure nrw.print occurs when [lgG] ;> 1600 mgldL." In thi. nudy, th~ ZnSO, turbidity tm provided a good estirruotr of the [IgG] but was not consid"ed to ~ .. u.~ful ., th~ Na,SO,-prmpitant t<St. (b) In .noth" study, the results with a ZnSO, solution (208 mgfL) wer~ comp"red with RID :way results." Inad~uate turbidity woo. found in "'mples with [lgG,] value> [hat ranged from 0 to 2825 mgldL (m ... n, 955 mgldL). Ad~u.te turbidity (nrw.print not legibl~) Wa.< found in "'mples with [lgG,] valu .. [hat ranged from 1085 to 4305 mgldL (mean, 2219 mgldL). The rruorked variatiofl! in [h~ results ...,U"'t th.r the assaY' were too inaccurat~ to enabl~ confidelll daoisions reg:ording Jl"SSive transf~r in calv... (c) With high" con"'lIlrations of Znso, (250-400 mglL), low~r [lgG] produa:s the same degree of turbidity as high" [lgG] when 200 mgfL Znso, is used." c.

Comm~lIls

{I}

Znso, r.-ag~nts may ~ made from KrlItch or purchased in kits. Stock

solutions need [0 ~ ....led to prevent carbon dioxid~ .bsorption. (2) For foals. turbidity tests hav.- ....lIlially bttn replaced by mor~ .ccurate and convenielll """Y'. Th~ ZnSO, turbidity trn trnds [0 und~renimate [IgG] when;> 400 mgldL " {3} Th~ p"'~n"" of Hgb from hemolY'is will falsely increase th~ measured [lgG] if turbidity is =sed by .pectrophotom""ty (.r 660 nm): Hgb_ induced inc"m~n" are about 200 mgldL a[ I % hemolysi, .nd 1300 mgldL a[ 5 % hemolysis." 5. N .,SO, precipiulll test for calf .. rum a. Principk Sulfit...daotivdy precipit.te cationic prot~ifl! lUch .s immunoglobu_ Ii",; oth~r protrins.re neutral or neg>tiv.-ly charged. Higher con"'ntra[ions of sulfites h.ve gre.,,, c;op"bility of praoipitating IgG at lower ooncelllr>tions. Sin"" th~r~ j,; v.-ty little [gA or [g.\1 in calf .. ra, the amount of turbidity rdlaots the '1ualllity ofigG in [h~ sampl~. b. Guiddin.. for the illl~rpreution of ",ul" with 14 %, 16 %, .nd 18 % N.,SO, .olutions."' {Note: Estima[ed ron"'lIlratiofl! do not match . 1500 mgldL, precipitates m ... n in 14 %, 16 %, .nd 18 % solutions. {2} [f [lgG] .. 500- 1500 mgldL, praoipitat...re...,n in 16 % :md 18 % solutions. {3} If [lgG] < 500 mgldL, precipiur. is , .. n in th~ 18 % .'lOlution.

402

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY c. In ano{h~r nudy. results w,,~ compa'.d with Ih~ [lgGl from a RID :assay." {Not.: Th~ markffi variation in thest ,",suit, highlighu (h~ pot~nti:dly poor accuncy or b el< of p,a:j,;on of ..... ch >.<Says. ) {I} [lgG] nngffi from 0 to 2400 mgldL with no prrcipim • . {2} [lgG] n ngffi from 645 to 2450 mgldL with prrcipi u te in the 18 % solution. {3} [lgG] u nge
d. Comments {I} Th. Na,sO,_precipiunt tell is rd.tivdy innp<nsive .nd quick. Th. IIUjor advam~ 0"'" ZnSO. is th. cap:!bility of ellim. ting the [lgG ] in a bro.d rangt.

{2} Com"",..! to the RlD .ss.y results, the prrcipiuni em is at btu a ..,mi_ quamiutj"" ...... y. {3} The:way d~ not work wdl foal ..,rum. 6. Changt. in y-g1obulin oone;.,lIIr>tion that .'" determina! by protein d«trophoresis: The difftrene;., bet",,",,n y-globulin oone;.,lIImion in pr«olostral ",rum and p
rot

II.

Immunoglobulin deficienci •• A. Other than dir«dy or indira:tly .....,ing [lgG] for th. pU'l""e of deta:ting FPT (= Immunoglobulin" =to I), an immunoglobulin cone;.,mration, "'" measura! to dora:t or confirm the p",sene;., of congenital immunodeficiencies.

7/ PROTEINS l. Ho .... ·~ ..

a.

~~'"

combinffl immunoddiciency in Arabian .nd Appaloosa fo;;o/", Aff=ffl foals do not produ"," functional T_ or B_lymphocyr. •• and thus th~y succumb to inf=ions .hordy aft" mor.rnal antilxxlies obtainffl by the colostrum a.. degndffl (.. 6 d for [gM. and .. 2- 3 wk for [gG). Other than the ab",n"," of [gM in premdde .. rum. oth" diagnostic f~.tures include ~rsinent lymph0P'nia and markffllymphoid hypopl.!i. in lymphoid organs. b. Primary agammaglobulinemia in thoroughbreds. standordbreds. and q""mr ho ..." Affectnl foals do not produ,,", [gG or [gM beau.. th")" lack B_lympho_ crr. •• but th")" do have T_lymphocyr. •. r..,uckJe .. rum lach [gM •• nd mat"""l [gG .nd [gM obtainffl from colonrum disap~ar over time to yidd the agammaglobulinemia. c. Sd=ive [gM deficiency: Aff=ffl fo.ls (Arabian and q""ner hors ..) have a marknl decr..... in .. rum [[gM] but do produ", other immunoglobulins. B_ lymphocyte con"'ntratio", are not decr.. ~. d. Sd=ive [gG d~ficiency: Affected foals h.ve • morked decr .... in .. rum [[gG] but do produ", other immunoglobulins. e. Combinffl variable deficiency: An aff=ffl adult horse had very low .. rum [[gG]. [lgM]. [[ gA]. and [IgG{T)]." Blood. marrow. and spl..,n lackffl B_lymphocyt... but lymph nod .. containffl OCCl.
404

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY •. Sd=i"" 110 ddiciency in ,h>r_~is: AIf«t«i dog> han d=~ .. rum [lgA]. f. Sd=i", IgA ddiciency in beag/." Aff,",,'M docs had d.crrasffi .. rum [lgA], but [leG] and [lgM] wm eoch WRI. g. Sd=i", IgG and 19A deficiency in W.imann.",: Afftct«i pu!'" ha", d~r.asal . in heohhy animals repr=nt about a third of the total [globulin]. a oompl~•• b..,n"" of immunoglobulin, might cr....t. a hypoglobu_ lin.mia. How."", th... are many orhe, more common '<'WOflS fur hypoglobu_ lin~mia (..., Hypoglobulin~mi. abon). 2. s..caus~ mon of th~ r-globulin &:>etion ofSPE is compos.d of IgG. an IgG ddi_ ci~ncy can b. s"'ptct.d wh~n th ..e is hypog.mmaglobulinemi .. Howenr. th~ smpicion should b. confirm.d with a sptcies-.ptcific quantitati"" assay for IgG or IgG subcl~. B="",, 19A and IgM .. pre.. nt only a sm:.!l .mount of the ~_ or y-globulins in hrahh. a deficiency in tho .. immunoglobulins cannot b. detttl.d by routine SPE.

Ill.

Immunoglobulin ex<=; A. M"'t hy~rglobulin~mia1 are caus.d by. chronic infttlion or d~hydr.otion, but • f~ are du~ to exct..in production of an immunoglobulin by nwplastic B_lymphocytes. If not caus.d by dehydration (hemoconctntr.otion), SPE dffermination of which immunoglobulin or immunoglobulin. are contributing to th~ hy~rglobulinemia will provid~ diagnostic <"Vid~nce for either a chronic inflammatory staU or B_lymphocyu nwplasia. In such cast', it typicaUy i. more imponant to ch ....cteriu the ty~ of immunoglobulin pres~nt than to measure immunoglobulin concentrations. B. An ~arli .. stCtion {HYP"rprot~in~mia, =t. lll.B} of this ch apter d"",rib.d diagnostic mffhods th.t can b. us.d to attempt to differ~ntia t~ an inflamm>lory oligoclon. l gammop.thy &om • B_lymphocyte nwplastic g.mmopathy. Mon of thost diagnostic target the hravy chain of the immunoglobulin dOls or subcl ... and do not pron clon. lity. Documenting that a gammopathy is caus.d by only one IgG subclass, or 19.\1, or 19A v..ould b. better <"Vid~nce of. monoclonal nate bernu"" it would b. unlikely that th~ conctntration of only on~ would b. incrras.d in an inflammatory nate. Establishing that • n becom~ more difficult .nd unctnain when th ..e typical associations are not found; for example, possible monoclonal gammopathy in an animal with. chronic infection. Th~n, mo .. definiti"" ch aracteriI.ation of the inununoglobulim mayestabli,h th~ pathogenesis for th. gammopathy.

=y'

7/ PROTEINS

""

COLLOIDAL OSMOTIC PRESSURE (COP) (O NCOTIC PRESSURE) I.

PhpioloCic proce=. and con~pts A. COP i, al"" calla! oncotic pressu",; th~ prdix OruYl· com~' &om th~ Grttk for "bulk" or "swdling," which, in thi, context, ",f~", to 'po.a-. cont:aining rolloidal partid.. that .wtll with fluid. A mor~ common contat refu. to nnlpl ..Jns; th>l i" oncolocy· B. Hydraulic pressure g,..dients and COP g,..dients for~ the movem~nt of protein.poor fluid, out of and into copillari.. as described in Surling'.law (Fig. 7.3 and Eq. 7.2). In health, th= gndients a", imporunt for th~ maintenan~ of blood volum~. In ""'em•• touo, transudative, and audati"" disorders, alteratiom in the gradient, cous< pl,mill. H,O to move into intemitial.p:oces (..., Chapt~r 19).

Pressure gndient =
{7.2.}

C. COP i, cou=\ by con"'ntrations of rolloidal.olutes or panides. C~lloiddl pamdts in blood a.. IlUCfomolecul.. that a.. too small to ~tle out due to gravity but too large to ~rme.te intact va,rular membran ... They.re g~nerally I nm to I ).lm in diameter, with M, ;> 30,000, though colloidal propmi.. a.. affect"'" by facton othu than size (e.g., .urn.", .rn.). In normoproteinemic pl",ma, albumin contributes about 75-80 % of total COP. l. COP develop' whenever two fluid, with different con~ntrations of coUoid po.rtid.. are separated by a .. miperm.. ble membrane. 2. Of a total capillary COP of25 mmHc in human pl",ma, about 17 mmHg is directly ",Iat"'" to plmilll proteins :md about 8 mmHg i, due to the Gibbs.Donnan equilibrium." a. Th~ major cona-pt' of the Gibbs-Donnan equilibrium .'" illunrata! in Fig. 7.4. The proteins "tr.pped" in the intravasrul.. 'po.a- have. negative charge. For dectroneutrality, there must bt more diffusible cotions (~.g., Na+) in the plasma to balana- the necati"" charges of the proteim. In th~ Gibbs. Donnan equilibrium, the product of th~ major diffu,ible cotion charge con~ntration .nd IlUjor diffusible anion durgr cona-ntration of the plasma equal. the product of the major diffu,ible cotion charce con~nt .. tion .nd major diffusible anion charge con"'ntration of the inte",tit;.,1 fluid (Eq. 7.3 .nd Fig. 7.4). {7.3.}

[lIUjor Qltions+]_ X [lIUjor .nion,-] _ = [major cotions1 .............. X [lIUjor .nion'-] ~ .... b. This protein.inducnl imbalana- of ion, couses • creater osmolality in blood and augment. the direct colloidal osmotic pressu",. In health, plasma protein, contribute .bout 0.9 mmoUlq:, and the Gibb,.Donnan effect contributes .bout 0.4 mmollkg." D. [Total protein] and [.Ibumin] may ,uggest alt ..«i COP, but COP is inlluen=i by pH and the .pectrum of prol
~_~I~~~_ Blood

A. Peripheral capillary, arterial to venous blood -J~~ ,_, ----- ----- - - ------ ---- • • •• • • •• - - . •• ----- • •• • • :'

p

p

~

(30 ,~tHg)

':

,

( 10 ~gi

... (28I1Wi1Hg) ...

~lj~"ip;.~~·c-"-~--iA-P' ~ - - - -~ -(~ -~~). ' . - - - ~~ _~M~=it~-~~;~-c--~\c-;;) (-3mmUg) +

13nwnHg

= 33mm Hg

AP

~ ___• __ •

Blood

All = 20 mmHg

'

(- 3~)

AP = 13 mmHg

B. Average pressures for peripheral capillary bed

• • • •: _ \

.- - -. --- --- -- ••• • • -. . ......... .. ...........

"

:

300 mmoII1qj \'

~

p : (17.3::lHg)

_.' _..

_7 mmHg

---

... {28""" mrioHg)

-;---~~------;---~ --------;;,. ~~ -------. --. \~ ... (- 3 omlHg)

(8 rnmI-4g)

::;£f:,':':::"203~H9 M.20~Hg QOP . ~I~ry

O.31M"1Hg

"

'.

~,:;g<,>

No lfansudale if {AP - All) _lymphatic pull

= 0 nvnHg

if - rn.n1i1i~1 ftuid Fig. 1->. 1I1....... ion of 8uid mo..,,,n.,ltJ boa .... of Surling·. law in. rypial .. piU..y bod. A. p.npb..r.d apiU..y, _ri.t.I to....., ..... blood. o Hydrau~c P""""" iD b..olth • TIw: plum. hrdnulic: preuur< iD .ho arteriol ";do .. tho capillary bo:f6A>'" in ,b.. in .... itill f1uN!. A idll .,.....,tic) pial..,.,. in bellth. • TIw: plasma ""axi<; 1""''''. is greu.. tban .... in .... titiol ",,«>tic p.am.. t-:a .... the pwm. [t<Mal pro .. in1 i.o g""'" than tbe i", ...titiallluid [total protein]. Th. [t<Mal protrin].....ui.olly doors.clung< in tho e>pillary bNs, and rhw tho """""ic plalure g.-.diu" (A1<) •..,wru.t.....". fn:om tho anu,ol .ide <0 Ihe ...now "do of """ capiUory bed. FOf mOil apilluia. """ All ill near 20 mmHg. o M_ of tho """""ie plalU~ (both in pWma and in intmcicill 8uid)" duo <0 oIbumin. bu, globulin. do "",td>u... S- tho ~ (0. """'" infurmation .bout pWma oncotic f"<"'WlO (a>lloiclal osmotic p, .......) . o Th. diffe<en« ioo .bou, 13 mmHg, and th ... ftuid Ie:rva d.. capillOly and en .... ,h. in'...,;aol.,.. .... On ,t.. ... nou •• ide. ,t.. diffi:,....... u .bou, - 1 mmHg. and tbw fluid Ie.- the in<ena,ia[ 'f"<'" and .n.. n the .. piU..y. B. A~ p ......... ix peripb..r:d capilbry bo:
pmo''''

dilk.,,.,,,,,

406

-u

.ide.

"'7

7/ PROTEINS

Plasma

Interstitia l fluid

12 Na'

,

~:

,

ClIl>l ory wall

(neao1y ImlHH"moobio to I>lISma pmleln.) Plasma osmolamy > Inter;;titial fluid osmolality Fig. 7.4. Concq>t\UJ 'oprnncilibriwn, .lK:troneutrality <>CO.UJ on . :ocb ,ide of th. c:opilhry wall: fo, =mpLe, t2 corion cborg .. :and t2 rnion d u "i:'"' in tho phuru, and 9 carion chuges and 9 .rnon cborg .. in in«ntitial /tuid, Bec...". ofth. neg .. ively chuged pro«in moJ.o.OO, ther. :or. mo .. ",tiorn and fewer di/JwibJ. rnio"," in pw.n. (12 ond 6, '<'f'<'Crively) thon in in«"'itial /tuid (9 :md 8, "'f'<'Crively), Osmotality (de«,mined by the numb... of ",1Il« mot.c..ks 0' iom) is gre .... in m. pt .. m. (24 p:uticks) tb:an in int .... titialfluid (t 8 particles), Tb. COP is g<'nttlt I nq;1tivo clurg., m. Gibb .. Donnrn effett is

proportion ...ly g""«' •• g'e:o«, pro«in <x>nc
II.

Analytical ConcqJts A, Th. osmotic df«t of prot.im i, UsM to m.~m'" COP with a roJJoid osmometer '" illunmal in Fig, 7.5, B, Th. COP of blood, plasma, and .. rum will . ... ntially "" the ... me, Blood ""lis :or. not colloidal partid •• and thus do not contribute to cop, In thwry, blood .nd pl"'ma COP values will b. slightly gr<.t.. than .. rum COP values b.c.u.... rum bch fibrinog<'n, s.rum ot pWJruI is typically the pr.fm'ffi .ampl. b.caus< frtt Hgb cm b. more .",ily det«tM; allO, whole blood may contain un...,n SJlliIll dots th.t am pilli: tubing, C. Uniu: I mmHg = I torr, t mmHg X t33,322 = t "".cal D, Int.rf... n""s L Hgb in the sample incr
Fig. 7.) . <'Imri~ UI!"

• k Long as the lymphatic system '"""""" tb. int .... ritial /ruid, • «:mn"'... d"", no • •crumut.«, • EY
408

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

A

,

.....m

.,

.-.

•••• """"' ••• ••

•• • ........ .. .....

~ :: e ••••••••

/

.... moperm .... bIo momb< ...... (p
B

...'-{

Diffo<~"""

"'

C

d"" \0

00!m()(ic 1"........0

""' " ~

••• ••••

\

••••• •• •• • •• onm"". ..) '.: ; ••••••••

-----

•••

••••• ----;+ -•....... -.-

-.......

membrane b9cau .... 0( coIoidal osmoIic pmssum

in

....m

~nt

••••

soIvool m"""" through

ooIut~

/

........ .......

.:

....m'P"'"m
Fig. 7.5. &h.matic rep",.. nution of d.. p'incipks of colloid ounom< ,ide , ..ull> in a bighv<mv<m
"'0

This

.,0

..,Ivm,

",,,,,wl.,,.,,,,i,

osmotic p, ..... '" is m,,,•••,,,,d by . pre.mre tJ=ducer.

2. Pl""m. npand~n (~.g .• h...:""c.rch .nd dextran) iner"".. COP. In ... mples cont.ining plasm. np"nd~rs. the COP does not represent th. conctntration of colloidal protS
Increa=l COP A. By i..df. an increa=l COP is not. common d"'gnostic probl~m for two ",asons: ( I) it typically will bt du~ to h~'prot~in.mia •• nd thi, condition is mOl< ...ily d.ttttffl by oth" means; and (2) COP i, ra,dy me;uu'N in ... mpl .. that might ha"" an increas.d COP. B. Increa..d COP am occur with inne""'" in pl""m. colloidal p.nicles. J. Inc",=d pt.sma or .. rum Hgb conctntration a. In plasma. fiN Hgb from in vivo or in vitro h~molysis am incr~ ... plasma COP.

7/ PROTEINS

""

b. Infmion of Hgb_ba=! oxyg~n carri~", I~.g., Oxyglobin and Biopur~} quickly increases pl,uma COP. [n on~ study, th~ Hgb_b~ oxygen C
[v.

D=-~ased

COP A. D~r~a=! COP values remit from hypoproteinemia. B. The primary valu~ of m.... suring COP is for the clinical managtm~nt of sev~rdy hypoprotein~mic animal.. Becau.. mark«l hypoprot~in~mia (especi
I. Het",rarch administration incre..". COP, but the duration of its df~ts dq>
410

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

4. Ikumioll of Na+ .od H,0 j, largdy cirrhosis. PLN, and h~an f.ilu",) (=

"'pofl5ibl~ Chap{~r

for mon {ransudatin Slates (•.g., 19).

References l.

rn.o..

FI, Maw .. PH. 19H. !-Wf·t;,.", of k.....1op>w ........ aIbwnino in 0
83.187_ 288.

o....p. JR. 19<>2. Dimibution ond .."""'" of iodin,·) }l~ Ixnin< attk Am) V .. R.. 2J,8}7--342. M.nb«u.. DR. Ka.rto Jj, 1.., RG, Coa.di., CE. WJ,,,,, JD. 1966. COm ........... "1iuti.on and """"",.of OJ· l-J..l>1o.tn.I imm~1ino G and M in .....k", foab. Am J V .. Reo SlhIS9S-160}. Pollod PI, r.,nd.~ M. Sd..umoc .... j, IIdI'DV' CR. 2005. E«.ct. of """Y on "'" aoot< pIu.o< "'I""'"" in dioblly DOnnal...d di......! Vrt Rrc IS60 S}3-W. c...,.. II, E,tIio"'. [~ , Fddman BF. Zin.I.1 IG. I';~ NC. odo. s.h.s!m' V.., ...... ., Hmwto'-o- hl odhlo~ . 1l9--1)4. Ph.iI.ddpbia. uppincott Willi ..... 8< Wilkin •. Tho", .. IS. 2000. ~of pl .. ma pro";",. 10' Fddman BF • .l.in.U IG. I.in NC. ed •. s.h.s!m' V.., ......., Hmwto'-o- hl odhlo~ . 891-1!~. Ph.iI.ddpbia. uppincott Willi ..... 8< Wilkin •. sa",,,,,,,, L\l. Cl.oiom>"", RH. I ~O . Amino acid. ...d p«>,d",. 10' B..rti. CA. Aok--l E.R. odo. TUt. T <>tb.o ~ ofOi,w,J 0-.;"", 2nd editioo... 62s.-7H. Pkiladdpl.ia, WB S.""n RH . 1976. 1h< .d', .CID""trk d .... ",in.';"" of ob, ptotron.j",. [0' Ilwtio CA. AaI.....,.[ fR. ed •. r",. T,........f am",1 ~ ltd edition. 477- 500. l'biJoddphi" WB s.""",, ~ T dnxIi YD. Saun. I. Siddiqv.i MU. Q.,j", MA. 1997. [",,,,,,tioon ofb""""",I. pttO ...itb difft-otnt ""'''' . nodied by a...".,,,,,,,,, q~ Bio. U. 2000. c...in, "'.... _ i n patttm • ...;..,; hi&b-"""lution doctrop/oo... • j" (HRE). V .. I 159,154-160. Rittm ..... SE. WolfRE. La"""", MC. Hut IS. kin we 1969. TIo, Si,,~ ttot, A ,,-a-alua';"". I Lab aio Med 7M9li-705. '''1''''''« to inlIammoti.on. N EncJ I Med G ....r C. Kwhnto I. 1999. Acu~pI. .., pro
2. C..mdiuo C Eo Bol .. NF. Kan,b, Jj, , " om", io...,.nuJ

J.

t. S. 6.

7. 8.

9. 10. II . 12. [J.

10. 15. 16. 17. 18. 19.

20.

&nd

paca ~ ciud

""""lin

""n...

Sa."""'..

tot"

.".",in.

.lad".

Zoo.".

H(),448-t5J.

.,.......it

21 . M""E..... EG. H....inAI. 1977. Di.~...d m.... """'" of monoclonal pnm<>patbi DI. Roo,do.k RA. O·NdJ SP. SdruItbtiu !'C. 1995. Monodo .... pmnoopatb, in • "'-I;...iob cIo.onic py<>
'"

7/ PROTEINS

26. Ktp.,bulin ioooyp<. in......, ca ... Y" Padool l7d064- .l69. lJ. Ram ...... SK. Struin IdA, Car....ik HF. Raokio RE. 2002. Biclooal pnmopadt, ..."ci"od ..ru. immw«>p.bulin A io. doc...idt m.ltipI, mydoma. Y" Oin P.thol JhSH9. }to R",I"dilld MA. 0",," M . Sd",a". 1988. St. .... albumin. H......,t.o&Y 8,.J.8S-401. }5. ~. KS. Fo.-.. SD. 2000. M~nocl.onal pnmopa"',. 10, Fddman BF.l.ink.l JG. J.in NC. ed •. ~; V.......·""'" H __ 5th oditioo. 9J2-9.J6. Ph.il""'lph.i., Lippinnt<% W. PIa", SJ. llnkr RP, llnkr W, H'J"pic d.a.>et<.iuti.on,,( _mmcdull • .,. p~ in";'" ca". Y" P.tboI. .((),;M9-25l. "J. As .... B""", W, H=aano. W. 1996. Imm"""ltitoodttmiad aami ... tioo of Ji&h.,-d..in ",,,,,,..ion (lombdJ kappa ",ti.o) in "",in" fdio,. oq";"", t.... ... and po.rtn. pl ..m. «lla. ZnrtcalbI Yrttrinatmod [AJ H,S7l- 576. ..... Kim DY. T ayIo. HW. E.J.. SC. a,., DY. 2OOS. 5pt<"'ic AL ....yIoid<> .... ...,.;at«! ..i ... m..!tiplt ...,..Ioou. in • "".". Y~ PatboI. .. 2,81-8 .. . .. 5. C&rotl..t. MAo}o/u>_ GC. DiBatroIa SP, !.i'l"'kL J, Bnuoo MD. 1989. En.omodu]....,. plaomacyto.... and ;mmwt<>p.,bu!in_...ooci•• ed amyloid<> ... io • ca •. J Am Y" Med A""" 195d 59}-1597 . .. 6. W,m" JA. WOO JC. Y...... W, G ....... ps, G.ahn RA. L1"'" L\., M""t< PF. 2005. CJ.acactp.,bu!in d.ain .poo p>« fo. ... , moktul.. di."",'" of B-cdJ nt<>pl ...... Y" P.thol 42,596--607 . .. 7. [ijim. T. lnadom, y, N<>pi M. 2000. Oooo.! proIif"....". of B Iymp/>ocyt<. in .... "anirud ""t<.. ,,( humm '''''''''' lymph oodt" P.-ibil-..,. of o"",di."", ... of B «H clonal po<>lik ..ti .... Dia", Mol P.... ol 9dJ2-I}6. .. 8. Zltou XG. £ooxlv<j K. G."""o N. Hamilroo·Dutoit SJ. 1999. D",cti.on,,( dono.! B «llt in micJ.odi ..«tod '''''''''' lymp/top ..lifn.ti.ooo, P...-blt dia~ pitfalb in I'CR analpi. of immwtop>buJio h,"'T d ..';o ~o, ,n",,,&,m,o •. Mol P.tboI. 52')()"_ 110. .. 9. !.ittm.o MP, D .... b.cl. D'\1, Yodm. 51., Gi,,,, U. 2000. Familial po<>tt
ss.

"o-

,.t<,'7

';". vw,,...,;,

ami"./A,,....,.

ss.

t...,.,

,...iabI, •

412

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

SJ. H.....,. JW, W," CL 1987. P...IniooDo-ic.duc.d iocre..",. in.,......lpha-l_p,bu\io...d haptoclobj" 00"" ...... _ St .

no... iD doc •. Vrt Pathol 24090--92. ,\1""", GE • .\1.... "ffi,y EA. H<>
1992. H<>natoI"Ck ...d ....... b••d"•.,icd ,ff«t. of 10..,.,.... odmini".. _ no.. of anti..wJ....m..,.,. do",. of p' ..... ioo"" in doop. Am J V.. Rr. Sl,IOJ}-IO}7.

"P'''''''''

55. RctIudilld MA. 0 ..... M. Sdudb", 55. I~O. Abwnin 1m RrY pIoy.ioI2Ia49--Zlt. S6. G.mpbYin< and equin, .. rum pobuIino. Y" Rr. Com ...... 8,1}-}2. 58. G",,,fuo .. IE.. 1976. [m.,..,....J .p«i&ity of ........ .u..",in dmrminatio .. and ,rtim.tion of"antt< ph ... ,,,.ct.,,,,· I., .... of tho broona,,01 u= "",';"H.. Clin 22,616-622. 59. Sod..).. OW, Smido R. K.,,,,k. JJ. 1970. PI ..m. protrtrulibo1"'T'" ,otioo. in do"" catti< and 1.0..... Pu, I. ].,/I.,DC< .f 'V "" noon.] vat.- ...d ""pIanoti.on of .... in ttiD ioa,uod by ...thdmintk lOt D; ..jlom.. ;,.,,,.;tis iD ital at • th",apnotic douc' •.pn... in dop. I V" I'bannoool 10" 2M21_ 127. 72. Ut We. H,;'" HC. W. YL. linlH. Ut Y1'. Fun, HP. 0, ... HH. Choo YH. Ck. RM. 2OOl. s....", C,,"'.;,.., ptot<>,';"n ,,(bo.i... ........ • myloid AJ (SAAJ) by manuaa
=

1"""""'"

oat'"

dru,.

oooct"'..';"""

'"

7/ PROTEINS

B2. Hopl1", FM, 0. ... DF, Gn,o, W. 198ol. Fail.", d r-i« ,..mf.. io ~ , Compad"'" of 6dd d.i.~ mrthod •. Mod V.. Pract 6S.62S-428. BJ, Di T .. lizzi R. Stodd..m Sl., ~ R. W,u..."", M). 2OOS. Com"";"" of p."",in «>n«n0';0' "". uio.,; .pectropbo""",trk. ,,n.co,,,..... ic. duli"" Com"";"" of . in~ ,.dial immwoo<\iffw;""",;o. rulfat. nubidity...... '" ,100""1'1."... ;.. and .d'ractom"" mrthod •. Am) V.. Re • .J.Il<>9}-M8. 8S. a.I>o.", Dl, c....boy HS. RDbert. MC. 1989. Com",,,,,,,, of fouc """'minc; tod.oiq." "" the di."",tit of «J.";"" n«><>at>l kypocamntap,buJi......; •. ) Am V.. M«I A..oc 194, 1717_ 1720. 86. Be.t"", SA. Hilbert B), MiU. IN. 19o! S. Th, ...., of the rl.,. "kkkyd, ooopdatioo . - foe <>, DS. 1996. Optimirin,; pnI'onoutee of • qu.~taci", ,;"" ...If"" nubidity t<" foo ~ of imm"",,~in G in ""Jv.. •. Am) V" R.. S7,1711 - 171}. 90. Monio DD. Mon. DA. M=yman GS. 19o! S. P"';", .......... faiIu.., in boo .... Inci.d,<>« and "'.",;.." 1><""" on, bmodi", f...... Am) V" Ret 46.2294--2299. 91 . Dni. R. Gir<>' s. 2OOS. Ed ....... of 1M rooom..dally , ...ilab.l, ""Y' and m u • .." ..,,'" d """'" total po<>t-I60S. 92. TtzanllR. 2000. r.;.....,- i",mwt<>d,6ci0 .... I Am

",_,n'

""".f..

V............,.

9S.

96.

97. 98.

V.. M«I A..,,, 22h 11%--I}02. 1267. Gaodo .. RlI. Hut KA. Stolol T, Di", .. T). FJ.",inio MJ. 2006. FdJ Pon, oyodrom, in • 1""" in No.th Am ..;". ) V.. Int..,. Med 2O,19S---20}. F.. oco< D, Lapoint< 1M, W,ll,"' .... V, o.""d,.. A. C. ....ll)l, S.. t< 11.. DuboruiJ P. 2004. I",,,,~lin G2 de1'.ci.ncy...ith tntuiId Hot.odn ltdn. ) V.. Di..,;o I..,... 16,.jJ2-0S. D., M), P"", .. C . Oktt.ko I, R.... M. 1997. Lo.o """'" imm.ooV>I>uli0 ronccntn.tioo. in.dated W,imatant-. do" . ) S.....u Anim PtaCt }S,}II _} IS. R.... BD. Poo' TW. 200 l. elmioc'" ~,. loo ofN,J.a.,. .wi £1.",..11" Du..J. ... Stb «Iition. N"" Yo,," McG....·HilJ.

99. S.tado.i .. B. s-J.J. .... S1.. Moo", LE. 2004. Th, "1P'R • .ioo and ""J"V"
So"""" •.

"0

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Chapter 8

URINARY SYSTEM Physiologic Processes ..................•......••......••.••......••........ Chronic Renal Insufficiency or Failure .....•.......•..........•................ Acute Renal Failure .................................... • .. •• ...... • •. . ..... Azotemia and Uremia ................................. . •. . •• .... . . •• ... .... Urea Nitrogen (UN) Concentration in Serum or Plasma .......................... Creatinine (Crt) Co ncentration in Serum or Plasma .............................. Urea Nitrogen (UN) Concentration versus Creatinine (Crt) Concentration in Serum or Plasma ........................................................ Creatinine (Crt) Clearance Rate ............................................... Abnormal Routine Serum Chem istry Results in Azotemic Animals ....... . . • . ...... Major Urinalysis (UAI Concepts .................................... • •. . . . . .. . Physical Exa mination of Urine .....................................••........ Chemical Exa mination of Urine (Qualitative or Semiquantitative) ..........•....... Urine Sediment Examination ....................................... • • • ...... Quantitative Urinalysis............................................. • • ... .... I. Basic Concepts ......................• • ......••.••...... • •. . . . . . .. II. 24 h Excretio n Studi es ........................••.• • ...... • •...... . . III. Analyte Urine to Plasma Ratios ....................•........•....... IV. Quantitative Urine Total Protein Assays .......... . .. .. ..... ..• ....... V. Urinary Prote in t o Creatinine (Prot:CrHu Ratio ....... . •• ... ... . • ... .... VI. Microalbuminuria ............................... . •• ... .. . .•.. . .. . . VII. Fractional Exc retion (FE) Ratios or Percentages ........................ VIII. Urine Bile Acid to Creatinine Ratio ................................... H20 Deprivation and Antidiuretic Hormone (ADH) Response Tests in Animals with Polyuria and Polydipsia (PU/ PDI ........................................

416 425 427 429 433 436 437

438 439 440 441 452 469 475 475 476 476 477 478 480 483 485

485

Urolith Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 487

415

416

FU NDAMENTALS OF VETERINARY CUNICAl PATHOLOGY Table 8. 1. Abbccv:i.tioo l and oymboJ. in

(PIOI:C n).

Ur;lI~ry

[~l

I

,hi, cbaplet protein 10 crulin iM

concrntration (x '" aruLlytc) 1.25-Djbyd~holrodciftrol

\,25-DHCC ADH

Amidju~ic

AMP

Adenoline monophosplutc

AMS ATP_

Amrl~5C

hormone (2Iiininc v:/'sopreuin)

",

Mmo.;n. triphosphatase Conj~{td bilirubin UnconjugarN bilirubin

a."

C:.Icium

",

en

C~.tininc

ELISA

Enzyme.linked immunosorkm aSS:l.Y

FE

F",cr ioruol ncr..rion

Ie,'

1M,. GFR GGT H CO ,-

hpr Ipr

LPS M, NH, NH,

PD PG,

PTH

PV

.

RPF

Free ionized calcium (not bound or oomplntd) F.... ;nni=:! magnesium (nol bound or oomplnni) GlomcroL.r fiJrl1uiOIl r:lle y..Gl II lam yl t ",nsf. 13k' Bicarborute High-power field (4OOx magnification ) Low_pow..r ~Id (100x rrugnificalion) Lipm

Rehtivc molecular mass Amrnotlia

Ammonium PoIydipsi> Phosphate including PO/'", HJ>O;.... 01 H, PO.Par.nhyroid hormone Polyuria lten.] plum. Row St:mdard dn'iation

SDS·PAGE Sl

Sodium dod~ lulfarc-poJyacryI:un;m: gd

SSA

Sulfosalicylic acid TOI:ll akiurn Uri n:ll)'sis (urine an:llysis) Urra nitrog~n Urra niTrogen 10 crealin; ne Urine sp«ific g ....vity lkf'r.Ktorn..r.ic urine sp«ific gravity

,a.' VA VN UN:Cn VSG USG ...

Srsre~ I nr~r",lI ion:ll

.~trophor~lis

d'Un;r&

PHYSIOLOG IC PROCESSES

(.

Thm: ma)o. processes coAlrol ren:!l a C'-':Iion of H ,0 ~nd KIlu.e:s: glomerular fill ..... ion (pm;ve), rubul~r resorprion (~Clive or pm;ve), and rubulu l«m:io n (XI;ve or pusive), The nel re:s.ults of Ihe ren:ll fimcrio ... on plas"", in M:IIthy ani"",is "'" sumnwiud in Table 8,2.

8/ URINARY SYSTEM Table 8.2. Net result of nonnalrenal function on plasma analytes ConstfVffl

H,O

Excmffl

u"'

Gluco .. Amino acids Protein,

Creootinin.

N,'

H"

ClHCO,Co" •

NH: LaCYt. ~t",,=ate

M,"

~.Hydroxybutyr;.r.

PO,

K'

Bilirubin H.moglobin dim", Myoglobin 'Ren1l """",tion of H+ may mult in oither ;ocidi" or albJj"" urin<.
[I.

Glomerular filtration A. A major rour. for wlul< ~nd H ,0 excmion from .n anim;;o[ i, through ..""I glom. ruli. The glom. rulu filtration l>arri.r i, compo..d of copillary .ndothdium. l>a.stment m.mb"ne. and glomerul.. cpithdial e.U, (podocyt..) with foot procc!MS (Fig. S.l). Th. fill 3.4 nm pass through. b. Electrical CMq:'" Positivdy ch ..gffi and dectrically neutral mbsYne., p"" through better than negatively chaq:ffl .ubnances bccau.. the glomerulu baICment m.mbran. conYins ncgativdy chargnl mola::ul... 2. [n many.p«i .., albumin (M, .. 69,000; 3.5 nm "diu. ) it n.ar th. threshold ,ize of the filtration l>arri.r, its ncgativ. charg' a1K1 imped.. transit. Albumin i. not apectnl in th. urin. of cats, hor .... or con!.. A small IDlount IIUy be found in th. urin. of app ... ntly h.a1thy dog,. Thi. albuminuria IIUy represent a subclinical disease or a 'peci" variation. B. GFR l. GFR is th. ralC fluid moves from pi"""" to glom.rular filtrate and i, mrasurffl by determining the rat. a substane. i. cl'~rffl &om pla,ma. 2. GFR d'rends primarily on th. rate of RPF . nd vari.. proportionatdy with RPF. RPF dep<:nd, on blood volum., cordiac output, number of functional glom.ruli. and constriction or dilation of .ffi:rent and .ffi:.. nt glomerular art.riol ... Oth.. f. cton that a!fa:! GFR indud. illlra.c:op,uiar hydrostatic pram .. in Bowman', 'p"e. (if pres.su .. i, increased. GFR i, decrra'ffl ) and plasm. colloidal osmotic (onrotic) prcssu.. (if pressur. i, dcarastd. GFR is increased).

418

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

Glomerulus

Glomerular FiHralion Barrier

..,.,.

----r-

Bowm~n· •

pI~"",~

~,

,

(_oll"aloo CII~I~ry

.,"

1

ba ... mQOI """"twwith ooga~"" charge cell per..

,

Fig. 8.1. Glomorulu filttloon b:on;". n,. glo"",ru~ filtr:ooon bam.,. colUis," of tb. e>pilury .ndoth.elw ""U. the glomerulu b....nent m<mbr:u><. and tt..
.. nat tubule.

3.

Th~ id...J ",Iut. for mac. ti...., compound. (",tal scintigraphy) or COntr.., mal;" (contr.., n. phrography).

III.

Function> of ..nal tubule. are reflretal by th~ rat~ of urinary acretion of a pWIIU mbstance comparal to th~ rat. of inulin auetion. A. [f acretion of a solute i. greater than acretion of inulin, there i• • n", solute srcr.,ion by tubules. Ren. 1 tubular KC"'tion involves proces.ses by which solut~. from pl.. ma, interstitia! fluid. or tubular cell. are tran>f~r..d to tubular fluid. B. [f acrffion of. solute i. I. .. tru.n acr.,ion of inulin, th~", is a nff solute resorption by tubules or th. ,ub",ance does not frffiy pa.. through th. filtration barri...

[V.

Function> of tubules renaining to major solutes' (Fig. 8.2) A. N. + J. About 7S % of filtrat. N. + is resorbed in the proxima! tubules down a oo"""ntration gradient establi.hal by th~ N. +.K'".ATP ... pump (basolateral mnnbrane) .nd a Na+. H"" antiporter. Na+ resorption i•• nhanced by an d«trical gr;od~nt <:Stablishal by co"",,,,,,· tion of HCO,-: N. + is cotransponal with glu=. amino acid.. :rnd pboJphates. Angiot~min II .timulates t~ proxirrud tubular resorption of N • ., a -. and H,O. 2. N.' i. p" ..ivdy resorbnl into th. d. ... nding limb of th. loop of H~nle to IIUintain N.+ in th. count~rCUrr.nt .yst~m.

'"

8/ URINARY SYSTEM

3. N.+ .ccompani.. K+ .nd Cl- th.t "'" resorbnl in th~ thick a=nding limb of th~ loop of H~nle vi.> • Na+. K+.2CI- cotranspomr in the lumin..! m~mbrane. The rat~· limiting factor i, Cl- ddivrry to the loop. Furo..,mid~ diumics block thi, process. 4. ADH stimui.tes Na+ .nd ct resorption in th~ medullary thick limb of the loop of Henl~ through the Na+.K+.2CI- cotransporter {a minor role for ADHj. 5. Aldosteron~ nimul>l.. th~ activr r=>rption of Na+ in coUrcting tubules by opening Na+ chann.!., ~nhancing N.+. K'.ATP ... activity in the b...,latI.id~ diuretic> block thi, cotransportthway.

B.

ct l. About 75 % of filterM

ct i. ","",rbnl in

th~

proxinui tubules down a concentr>tion gradient cre.tM by Na+ .nd H,O resorption .nd through a format~.Cl- exchan~r.

2. Cl- i, r=>rbnl vi •• Na+. K+.2CI- cotranspomr in the thick ..""'nding limb of the loop ofH ~nle. 3. Cl- is p..... ivdy resorbnl in the disc.t n~phron by an dectroch~miao.l gradi~nt ~nablishM by Na+ movrm~nt (through . ldos{
"",n,

.. ......

,,. ..". "",. .".,

"'.

,'"

'"

...... _. ....n

PO,

.on .on

..

"""i

'"

~OCOM

amino.odt

Collecting

Distal

,,.

tubu.

~r t "·

.~

",0

",.

J

--_""..

""

Loop of Henl

•

•

" ,0

"

,,'

''''}-,,o-

u

",0

~~-~

m

fC,.,·

..".

"'

1*)·' -. 1*1 -

}" " 0,

I., ... "

HCO ,

",0

,-

""

w-

Na'

"' " "'"

CI-

,,,, -

..

,

UHWIinoo

"

".

""

"',' PO,

."""

> '''''

: _ _ Ie.g.,

0-

ON

urea

J

.",

_

(+) ..

tubule

"'"""

> '''''

)l)l).eool_~

"' _ _ 'PKIO _

.... i . . . .

_

.

11.1.;0. pbyoioIogiC P' : " , .. of _01 rubulos dw ~ to ooIu... and H,Q. n.. ..lute _ .miom.,., psic faaon . Fig. 8.1.

."

n_ 8.~).

."

8/ UR INARY SYSTEM

thm a con<:tntration gradi~nt is m. intainal. Th~ mov.m~nt is ralucal wh~n th~ uriruo'Y flow rat. through th~ tubul~ is low kc.UM K+ nays long~r in th. tubular fluid. and thus th. con<:tntmion gradi~nt is diminishal. 2. ADH promot~. K+ ,,,,,retion in th. conical coll<"Cting tubul •• ~rhaps by o!",ning K+ clunnd, in the luminal m~mbr:m~. This process rom!"'n .. t.. for th~ ralucal K+ "",mio" that occurs with d""",as
Fig. 8.2. ",",ri~ UI!" o The ",molality of th. pl:wru and th. ultt:dilm.t~ ar • • q..w (""ar 300 mmoUkg) .. H,O and aoaproteia ",lut'" p ... tluough the glomorular filtmioa bar';',. o la the proDmol tubules . • m. jority of th~ H,O and ""lut.. tbot the tubuJ.. ... ,esorhed through :octi¥<. b cilitoted, and p. "i"" proo<sses. The osmolality of the tubul .. fluid leoving the proximal tubuJ.;' .. ill ..... 300 mmollkg, but the fluid volum< is gr •• dy diminisb.d. o la the descpe<;". n.. 1500 mmoUkg v:due is probably ' pprop,iote fo, hol5e5 and cattle, .me",.. the ..,lute roaceatmion ia "'''' m.y he > 2400 mmoUkg. o la the """"ading limb of the loop of Henle. wlu"," (mostly N . .. 0-. and K,) p .... vely Ie.".. th. tubul., fluid vi •• N. ' · K'"·20- corrier (energy provided the by N. +· K'"·All'... pump in th. b...,htent memo brane), but H,O ",,,,,,ins. Thus. tbe tubul:u fluid becomes dilute, and tbe fluid Ieoving the diluting "'"J!"'ffit h .. 10 osmoWity ""., 100 rrunoUkg. P... ive fCa" :md IMg'+ ,esorptioa depeads oa :m elect,ic:d gndien' promoted by the N. +· K'"·2CI- rotronspo,te, and the ",cyc~ng of K'", whe,e .. . ctive fCa" and IMg"" resorptioa is promoltion of N. · :md 0- and the .oec,etion of K' and H+. Tbe ",molality of tbe uri... typically;' > 600 mmolllq; and moy be > 2000 mmoUkg. o la moot he:dtby dome"ic m:unm>.ts, the net funcrioa of . nephron is to exc,ete ure •. C,~ K+, H· . N I·V. and po. and to oonserve N . · . cr. HCO,-. fCal+ . IMg"", gluro>e, amino.ad.,:md H,O. n.. "lui"" nephron -1.034) were tletennined by using . ",fJ:K1omel<'" u,ine specific gnvity sale. Tbe true specific grovity of tbe pt..m. """,Id be ""He, l.OI 1>-1.022, n, :md rum • ,:mil" h .. little to no c~nical relevance. ®, ATP ... pump: and 0, tr""'poner 0' ,hunle.

ea",

422

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY F. Co.>+ l. About 80--85 % of filtr>te Ca'+ j, resorbM ill th. proxinul tubule. and loops of HenJ~ through • ",,,in proct::U that deptnd. on N .+ .nd H,O resorption. Co'+ is passivdy re50rke[ivating .n adenylate crd,..., p.thw"}" ill the conical chick asullding limb of the loop of Honle, the disc.! tubule, .nd the conna:ting stgmem be{y,,",,11 Ih. dinal tubule alld Ih. colla:ting duel>. 3. Viumill D promot.. 0..'+ resorption in Ih. dinal lIq.hron by inducing production of a colcium_binding protein. Cakit,iol (1 ,2S_DHCq formation in the proxim:d ",nal tubul .. i. stimulot.d by incr~ PTH .ctivity and hypopho.ph . temia. 4. Thi+ in the disul nephron. G. PO. I. About 85- 90 % of filt"te PO, is r...ork
H. Me'" l. At physiologic concentrations. nearly aU filt"te Me i. resorbed (mon in the

I.

J.

conical thick ascending limb.. oflooJ» of Henle. but also in the proximaltubul.. ). P."i"" Mg" r..orption it mo.dy down an dectrochemical gr:ldient esrabli.hed by the Na+. K+.2Ct cotranspon...nd a r«ycling of K+. Th... also may bt .pecific transporters or crunnds who .. activiti .. depend on blood [Mel. 2. ADH. PTH. glucagon. calcitonin. and p-ad.. n..gic agonists .timulate Me" =orption in th. cortical thid: ascending limb. Gluco.. l. Typically. glucose pa"" freely through the glomerular filtration barri ... and all filtrate glucost i. re;orbed in the proximal tubules •• N.+ is ..sorkresent subdinic;ol disease or • species variation. 4. Meg.lin, aminionless, and rubilin, receptor proteins exp.....d in the .pic;ol pm of proximal tubular epitheli.l edt., are imponant in mediatiIll: endocytosis of a wide variety of prouill1. Th... indude vitamin D- binding protein, which i. r«lui=! for ",nal formation of 1,25·DHCC. These proteins .lso mediate intrinsic fa.ctorcobalamin uptake in the small intestin•.

8/ URINARY SYSTEM K.

U",,~

l. About 60--65 % of fihr>te u",,~ i, t ..ork
tr:l.tion g"di~nt crl Huid to fluid in th~ loop of Henl•• nd th~n from maluU.ry coll.aing duct Huid to ma!ullary int.",titi<>l Huid. 3. Ur
en

at. ,,,,reta! by th. proximal tubules.'"' Crt secretion does not . ppear to occur in hones or c:ou. ~' 2. In peapl., Crt secretion may occur through ~ pathway ili ..ed with org.nic c:otiom. s"rum [Crt] incr~ases wh~n oth~r organic cations lsuch as cimetidin., trim~thoprim, and quinidin.} int"r. .. with Cn secretion. M. Resorption of H 2 0 by tubules I. About 30 % of RPF b.roIm' uhr:l.fih"r.. About 75 % of the uhnJiltrar. H,O is passivdy resorkl cdb of the roll"'t. ing tubules, r~",lting in activation of a prot.in kin. ", th.r phosphoryl.r.. aqu. porin 2 .nd .ruobles it to be tnmpona! from v.. icl~. to th~ .pical membrane. Lack of ADH triggen ~ndocytosi, of aquaporin and its return to cytoplasmic vesid ... Water mon o into cells through th. po« at th. c;.,nter of the prouin,. Water '>Capes to the inr.rstitium through por.. formal by "'Iuaporin 3 in th~ basol.r~ral membr:l.nes of the prin<:ipal cdl,. l. In som. dog" small amounts ofCn

v.

R~nal

A.

conc;.,ntr:l.ling ability and ",ruol diluting ability

D~Jinitions

I.

CD~,mrating "bilif):

the .bility of kidn..". to «sorb filtr:l.u H,O in exa:ss of fihm. solutes; .bility to conc;.,nt"re solutes 2. Diluting "biuty: th. ability of kidn..". to rrsorb Jihrar. solut~ in uc= of filtr:l.le H2 0; . bility to dilute ",lut.. B. T "m, usN to describ. th~ concentr:l.tion of urine I. Many authors describ. th~ solut~ ronc;.,ntr:l.tion of urine with th~ t~rms hyponh~nu. ria, isonh~nuri<>, . nd hypenth.nuria (."l1m. mnn, "'t""ngth"). How.nr. th~"" is litd~ agr«n,.nt on th~ d~Jinition' for the .. terms. lsosth~nuri. should m.an same {iso- j strength (."l1m.) utin. (.uria).

424

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY 2 Our ddinitions (.~ Fig. 8.8 for rdationship of USG"" .nd urine osmolality) a. iumhmuria (working ddinition) j, the nate in which urine OIDlOJality is th .... me ~. plasma osmolality, wheth.r pJ..,/ruI osmoWity is [ow, normal, or high. W. ddin. "th. sam." as "-ing within [00 mmoUkg of pl",,1IU osmolalitY"' dut i,. osmolality. = osmolality, ± 100 mmoUkg. In mon domestic mammal._ such urine typirnlly will han a USG ... from UX)7- 1.013. inclusivdy (unpublishtd .uthors' dau). b. Hyplllfhmurkl is the ,We in which acretII osmolality that is I... than the isosth.nur;c vain ..; that is, osmolality. <: (osmolality, - ]00 mmoUkiI. In most hyponh"lUric .nimab, USG""wilJ be, Jess th an 1.007. Such urine is dilute {the fihme has bttn dilural by ..nal processes}. c. To be, co,,,isr.m with ' .nd hy~rsthenuria.re rardy usa!, Physiologic p~ for com:entrating or diluting glomerular filtrate I, Function, of renal imemitium a, Provides. bri~ ~tw..,n tubules .nd blood v.",.ds to focilime the movement of H,O .nd ",lutes b, Hdps maimain medullary hypenonicity to en.ble the con"",ntr.rion of tubular Huid 2, Functiom of tubules a, Proximal tubule {proxintal convoluta! tubule} {I} Approxim.rdy 30 % of the plasm. that enters glomeruli becomes glomeru_ lar filtr.re {USG... .. 1.010; osmolality .. 300 mmoUkg}, {2} About 75 % of the H,O in the ultrafi]tme is passively resorbM by the proxintal tubules {ind"l"'ndem of body n..,Jsj, The remainder passes into the loop of Henle, Th. solute conctntmion doel not change much in the proximal tubule, but Huid volume decreases markedly, b, Descending limb of the loop of Henle {I} The osmolality of tubular Huid inCfu"", b«:au.. of the passin resorption of H,O and the secretion ofNa+, ct-, .nd urea into the filtrate, {2} Fluid em..ing the loop of Henle has a USG .. .. LOlO and .n osmolality .. 300 mmollkg, Tubular fluid at the dinal end of the descending limb has a high conctntr:ltion {USG... :> 1,050; osmolality:> 1500 mmoUkiI, c, Ascending limb of the loop of Henle (the diluting segment of the nephron) {I} The a=nding limb is rdatively impermrable to H,O but activdy pumps el- and Na+ {al.o K+, fC.'+, .nd fMg'"'l from the tubular Huid to the interstitial Huid, Thus, tubular Huid 10..,. solute and the interstitial Huid becomes more hypenonic, Fluid l",ving the a=nding limb for the dinal tubule is dilute (USG .. < 1,007; osmolality < 200 mmollkg), {2} A functional """"nding limb is n=ry to mainuin a hy~rtonic interstitial Huid in the medullary rrgion '" that H,O may be p""';vdy resorbed in the descending limb of the loop of Henle .nd in collecting tubules, {3} The a=nding limb .nd the closely :lSSO<:iared v:asa recta are the major structure> of the ooumercur",m system that maint.in a hy~rosmolar medull.ry interstitial Huid,

,h.

c.

."

8/ URINARY SYSTEM d. Dinal tubul~ (distal convolut~d

tubul~)

{I} Th~ disul tubul~ has minimol H2 0 ptrm ... bility, and thus nry little H 2 0 is resorbtd in this sq:ment, {2} The r~50rption of N.+ and Cl- in this .rgment low~" the tubular fluid osmolality, ~ , Coll«ting tubules {I} The coll«ting tubules of the disul nq>hron contain the concentrating process.. of th~ nephron, {2} ADH controls ptrm ... bility of th~ epithdium to H2 0, H,0 is r~50rbtd in th~ pr=nce of ADH if there is .n osmolar gradient becwttn th~ tubular fluid and mfflullory intef5{itial fluid, An osmolar gradient is cre~t.d by high roncent .. tions of urea, Na+, ~nd Cl- in th~ intemitial fluid thot ~r~ pro_ duced .nd mointain.d by segm~ntal functions of th~ nephron, {3} The H 2 0 rnannds through the epithdial cdls are c.. >I.d by m~mbrane_ :u5OCi<>t.d protoins call.d aqu"p"rilf', When ADH binds to th~ renal ~pithdial cdl basolateral memb .. nes, it activ,u .. a cyclic adenosine mono_ phosphate (cAMP}-d.".,nd~nt 5«Ondary mess~nger .ysum by which aqu aporino are transported to th~ apiml membranes, .nd thus membranes ba::ome ptrm ... ble to H 2 0, ADH :dso 'P!""''' to inc ...... production of aqu'porino,' 3, For kidneys to concentrate the ultrafihrat~ , th~ following are necessary. a, ADH mmt bt p.... nt, Stimuli for ADH =",ion include hyptrosmolality, d«reastd cardiovascular pressures as =n with hypovol~mia, and, to a I=r d~grtt, increas.d [angiotensin], b, Epithdial cells of th~ dinal nq>hron must bt ... ""nsin to ADH, c, The.. must bt • con""'ntration gr.di~nt; th . t is, the o.mol:diry of the int..,;titi:d fluid of th~ renal medulla must bt gr ... ter than the mmol:diry of the fluid in the tubules, 4, For kidneys to dilut~ th~ uhrafiltrate, the following are n=ry. a, Na+ .nd ct must bt .ctin ly transport.d from th~ tubular fluid to th~ interstitial fluid by q>ithdial cell, of th~ ascending limb of th~ loop of Henl~, This prace.. r"'luites ddivrry of Na+ and Cl- to th~ loop of H~nle, b, Very little to no H 2 0 is remov.d from th~ tubular fluid by the distal n~phron, D, The osmolality changes that occur in a nq>hron during th~ formation of urin~ a.. illunmed in Fig, 8,3, CHRONIC RENAL INSUFFICIENCY OR FAILURE L

Wh>l is chronic .. n:d imufficiency or f. ilure? A, M.ny chronic diseases moy dam"l:e kidneY' suffici~ntly so that functional .. nal tissu~ i. iruod"'luate to mointain h... hh, Th~n, the anim.1 ent~" the pathophy.iologic state of chronic renal insuffici~ ncy or failur~ , B, Ther~ a.. no uni"""ally a.c:cept.d definitions or criteria for .taging impairrd r~nal function, On~ .yn~m seems to oorrdate with dinical finding> seen in many domestic mommal,," I, Diminished .. n. 1 .."'tv<; The GFR is about 50 % of normal, .nd the animol is clinically h~althy, Aror.mia i. not p.."'m, but the kidneys are less .bl~ to tolerate ooddition:d insult {~ ,g" disease, dehydration, or poor ptrfusion},

426

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

~

>2400

-

, ."

r

--maximal coooonlration

;. J--""' ~----l---------~--"':-:---------!!>~"::"""'" --Plasma

PCT

Collecting Tubulall

Glomerulus

Fig. 8.). OsmobJjty ch1Dll"' in > h../tby momrrul'. nophron. (D.t:lils of tho movomont of ",Jut< and H,O in nepluons "'" dosaib.rW. In tI..:oboenc< of ADH octivity, H,O ..maim in tt.. coJL.cting tubuLes. rnd .bw th< nephron produ= hyposthJLecting tubules •• ~ portion of th. H,O is resorbed to fonn muim:d ronomtr._ tion. Th< mrnmal ooncentr:lting .bi~ty van .. among dom.stic momm:d.. n,. right y-axis is probably . pproprutr for ho,... and cat'" (i.~ .• mHimum USG .. .. 1.050). wh<", .. om.. y_"""" .,., need.d for dog. (.. lBOO mmoUkg or USG", .. 1.0(0) and cats (> 2.wo mmoJIkg or USG .... > LOBO).

2. Chronic r~nal insufficiency: Th~ GFR i, approximatdy 2Q.-50 % of normal. Azot~mia and an~mi. ' Pl'='. Polyuria occun btall5< of da:r~ ron""",rating .bility. 3. Chronic rtnal failur~: Th~ GFR i. < 20--25 % of normal. Aro .. m;" and impair~ rono::ntr.ting ability (thu. polyuria) .'" prestnt. The kidnoy. connot regul ... atracdlular Huid ""lume or dectrolyt~ bal.no::. and thus «i~ma. hypocal<:<:mia (genually not in hon~) •• nd mHaoolic acido,i, will dovdop. Overt uremia with iu neurologic. gastroint.stinal and cordiovasrular romplicotiom may dovtlop. 4. End_suge r~naJ di..,.",: The GFR i. < ~ % of normal. The .. rmin:d stagc:. of ur~mia are p....,'" with eith~r oliguria or anuria. C. A:J a gen~ral roncq>t. m. ny authon write that mo .. than two_thirds of functional renal m .... must be lost bc:fore kidn~ 10.. th~ ability to rono::ntcatr urine. and mo .. than thrtt_fourth, must be lost before an animal bc:rom •• awtemic. Sum fractions hdp

8/ URINARY SYSTEM devdop m. jor con~pts but do not represent firmly establimM facts fur aU .p«ies. In on~ nudy in which r~nal function WlI.! rwucM by n~ph=tomy and ..,l~ctiv~ art~rial ligation. c;;ots ba:am~ azot~mic with only a 50 % rMuction in functional /IUS.S, and mon maintainw r~nal con~ntrating ability (USG ... ;> 1.(40) with an estimatw 83 % loss in ",n. l function! II.

Why do animals 10.., r~nal conctntrating ability in chronic ",nal failur~? A. Mor~ solut~ than usual is pr=ntM to th~ r~maining functional nq>hrons, and th~ high solur~ ront~nt within th~ tubules rontribut~. to 501ut~ diur~.
1lI.

Polyuria in mammals with chronic r~nal in!Uffici~ncy or failure A. Th~ dhrons will ev~ntually c;;oUst ~ith~r oliguric or anuric renal failur~.

IV.

Figure 8.4 illustrates th~ devdopment of azot~mia and abnormal urine volume caused by a lOS! of nq>hrons.

progressiv~

V.

Eviden~

of chronic .. nal insufficiency or failure A. Eviden"" of insufficiency or f.ilur~ l. The", i. azot~mia (inc",ase<\ [UN] and/or [Cn] in .. rum or plasma) ba:.Ust of inad~uate renal . bility to .. move m
ACUTE RENAL FAILURE I.

Acuu rmillfoil,," is rennible or irreversible .. nal dysfunction resulting . bruptly (within hours to dap) from a renal dist..., or insult th . t markedly d<er~...,. GFR and l~ads to azot~mia. Umal c;;ou..,s are toxicants, .. nal ischemia, or inf<etions.

428

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

Stage 1

Stages 2 & 3

dea&a... d renal ",...."'"

renal insulficior.cy/

..

stage 4

""'~

,_I f8iur~

renal d>se3SO --------- ~ --

,,

polyuria

~

/

~

"'-- , ,

, ,,

.....

~

\

,'lIZoIomia

\ \

"./

X

,, \ azotem,a. ' patienfs ""rum [UN) ...

-----------------

J9':!l _____ _

---

~ oliguria \

~;:,----------------,:c:--------L-----------------------,:c,\''-' ' nuria 100 %

50 %

<5 %

Percentage of remaining renal functional capacHy Fig. 8.4. Gr:ophic:d ,.pr=nution of ..... ffi.c .. of chronic Rna! di ..... ond prog=siw <Jy.fun<1ion on:on .rum,r. uri... voh"". >ltd soerum ooncnlttooom of UN or Cn. ShaMd K"Y ""as "'p,.",n' th. ",fer~"" in.orval. for urin. volu"", ("'I ba,) and fOr the [UN ] or d.. [Crt] (bo""", bary. Th. !",imt', uri"" ",twn. ond [UN] OJ [Cn ] "" rep"""""" by "'ONg kiNti N_. A .h.o",tic.l _i_I ~riw ""I~"" liM i.s .110,"", to illustm. tit .. .ru"u" .. itb polyuric rmal f.tilur. do no' produc< muimaJ uri... volum<. • In ''''C' 1 (diminim.d ,.nn ,...,vo), progrossi"" "nal di..... is
de_« is inruflici.nt (azotemia is pre.. nt). • In ,fag< 3 (renal f.oiJwe). ate rontrol of H,O baLon« or .t. ctrolyt. oon=>tr.lOo"". n.. rnumJ b .. c~nic:d .igns of ur.mia rnd abnornul .. rum oon« ntr.ltioru of N.'. K'. 0-. Ca". po•• H\ or HOOJ-. • In ,fag< of (.nd""ag.ren..t ru.e.... oligurie or rnurie rerul f.oiJwe). only. &... n
II.

A:r.o!~ mia

A. Th. magnitud~ oftb~ llOt~mi. d"", not diff~rent"'t~ acut~ from chronic ..nat failur~. Both c;on have mild to KVU. 37.0t~mias. B. Th. rates of inc=-< of th. [ure~] .nd [Crt] .re in 'C\It~ renal failure than

gr,,,,,

chronic renal f.ilure; that i, •• mod.m. to mukrd arot~mia m.y drvdop within do"., in acuU f.ilur~ but tah Wttks to month, with chronic f.ilure.

8/ URINARY SYSTEM

'"

Ill.

Urin~ mlum~ and USG ... A. Ikcaust of the abrupt .nd ~e" d~"..,e in GFR, with no tim. for com~n,atory hy~rtrophy of healthy n.phron,. the kidneys may filter linl. blood and produce linl~ or no urine (oliguria or anuria). B. Th~ USG .... am varycon
[v.

Acid_b.st lIatu, and l.mia or acidemia).

d«trolyt~

concentrations can becom. acutdy .bnormal (•. g.•

hy~rb_

AZOTEMIA AND UREMIA I.

Definitions A. Azolrmid i, increased nonprotein nitro!:"nou, compounds in the blood that ",. routinely d~tttted a, incr• ...d serum [UN] andlor serum [Crt] B. Urmtid is clas.ically cofl!id~red to mean "urinary con'titu~nu in blood·' but now typically r~fers to the clinical 'igfll reHttting "nat failure k.g. , vomiting, diarrhea, coma, convul,iofll, and .n ammoniacal odor of br~ath)

[I.

Azot.mia dassification, .nd di'iOrdul A. D«re.w urinary acretion of urea or Cn l. furrndl flZOtf",id: The initiating cause of abnormal urea or Crt acretion invol~, rMuced r~nal blood How. a. DiKlrd~rs (f.bl. 8.3) b. Pathog<'neses {I} Any P"""'"' that dimini,h .. RPF will dir~dy d~re ... GFR and thu. d~rease dearance of urea and Crt. Th. volum~ ('tretch) ra::q>tors in the juxuglom~rular .ppamll'l of the aff~rent aneriol~ "stnse" reduced blood How and tri~r the angiotensin_renin .yst~m. Angiotensin II con'tricu the afferent and efferont g1om~rular .neriol.., which limher rMuce. g10llYrular perfiuion and thll'l GFR. {2} Hypovolemia enhancc" resorption of Na+ and H,O in proximal tubules, which in turn promotes pa .. i~ proximal tubular resorption of urea {but not Cn} ba:ause the low~r How rat~ provides more tim. for resorption. {3} Hypovolemi. alKl tri~rs rd ..... of ADH, which enhancc" resorption of urea (but not Cn) by medull.ry coll~ting tubules. (Aquaporin 2 tran'pom urea, .. well.., H,O. ) {4} Azotemia occurring with a prot.in_lo,ing n.phropathy and marked hypoal_ buminemia may be prorenal and rdated to d~reased GFR caused by hypovol.mia KCOndary to d~reased colloidal osmotic pressure. {5} Azotemia is common in clinical hYl""'drenocortici,m. Aldon.rone defi_ ci~n
4J()

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY T able 8.3. Di..,a..,. and conditions thai ca u.c azotemia'

D'""....«I urinary excretion of urea or Crt' Pre",,",! dise=. or rondit;on 'Hypovolemia: d.hydration (including hypoadrenoconic;,m), blood loss D,""",,~ cudiac output: cudiac insufficiency, ,hock, hypoad .. noronic;,m 'Shock (hypovolemic, caroiogenic, :maphylacric, "plie, n..... rogenic) lUna! di,.,.= or condition.

mode.

'lnft.mmatory: gloD",ru/oncphril;s, pyelonephritis, tubulor.imcrstitial Dq.hritis

•Amyloidosis "Toxic ncphro .." hy~rcalcrmja. ethyl.ne glycol, myoglobin, gemamicin, phcnylbut37.0nc 'R..rud j,chemia or hypoxia: poor ",ill p."fusion, infarction Coog.ni{:o/ hypoplasia or aplasia Hydronephrosis Nwpl.,ia {renal or metastatic} Pourenal di,eases or conditions 'Urinary tract obstruction: urolith;"";., urethral plug. in co[ •. n~pJas",. pJ'OS{atic di=st 'Lukage of urine from urinary tr.ct: trauma, neoplasia lnc",ased ur~a or Crt production: intestinal hemorrhage, incrraoed dietary urra or Cn, increased protein cat>oolism • A lel1tiYlves • dec",...d glom
loss of Na+ reduces reruol ron ...""'tion of H,O and sometime. re",h. in hypomlemia, [n some animals, there may be inc",asffi intestinal loss of H,0 {i,e,. diarrh~a} along with decreaoed H,O int>k~ to acantuate hypovolemi .. Thus. a rombin>tion of facton can decr.,... renal blood flow and produ,,", prerenal .zotemi .. {6} The prer~nal azotemia developing in :rnociation with KV~" intestinal hemorrhage is partially due to hypovolemia, which lead. to decr~a oed renal acretion ofCn and urea (5« Azotemia and Uremia sect, lLB4), c, Decreased RPF th.t is seve", and p<:rsistent may l~ad to renal hypoxia, acute reruol dam"i:". and thus acute renal failu"" [n such animal •• the azotemia may be reruol and p",,,,nal, 2, &nal azqf(m;a: Th~ initiating cause is any renal di~ that cau..,s • major decr~= in GFR. Any of the following m"y contribute: los> of nephrom, decreasffi vascular patency within th~ kidney. decrraoed glomerular p<:rmeability, incr~",«i r~nal interstitial pr=ure, or incrraoed intratubular pr=ur~, a, Di,.. "" or diwrde", (Table g,3) b, p.thogc:ne.." {I} Reruol d~= {acute or chronic} causing the loss of at lrast 65- 75 % of nephron functional capacity reduces the GFR rufficiently to product azotemia, Reduced GFR causes inadequate renal excretion of urn and Crt

8/ URINARY SYSTEM

."

from plo,m. (without .uffici~nt com~II!:ltion by intestinal pr=), and thm ",rum [UN] and [Cn] iner ....., {2} Proc..... dut contribut~ to. pr~renal.zot~mia may also bt prestnt, 3, P~ffrnUll =tfmid: Th~ initiating cau.. of d~fa::!i"" u..... or Cn acrffion is distal to th~ n~phron,

a, Di,~ases or di50rd~rs (T.bl~ 9,3) b, P.thogenesis of obstructiv~ azoumia {I} Urinary tract ob"truction causes th~ rd~..., of vasoactive subnan= (prosu. glandi", and .ngiot~nsin) that constrict the glom~rular amrioles, thus r..ducilll: RPF and dimini,hing the GFR: r~ducrd GFR imp. i.. cl~...n~ of u"'•• nd Crt'· {2} [mpairM outflow causes a trans.i~nt incr~.'" in intracopmlar hydrostatic P"'"SU'" that d«",...,. th~ GFR. Th~ pres,m ", diminishes with tim~ as tubular fluid diffu..,. into mbular ~lh and less ultrafiltrau i. formM, c, P.thogen~sis of .zot~mia caUSM by l~.kag~ of urin~ within th~ body {I} [f ther~ i. l~abgt into the p",iton~.l cavity, uru and Crt ~nt .. th~ plasJlla after passi"" .bsorption through th~ ~riton ... l ~pithdium, Th~ ~riton..aJ [UN] ucretion, th~n the .. rum [UN] will inc",... , 3, Th~", i. umally . n ad
432

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY 7- 37 mgldL in individual doC" P~.k rona.mration, occurrffl4- 7 haft .. f=:ling and re{urn~d to bas.!io. value, by 24- 36 h. Ikmoving from 52 % to 76 % of the blood volum .. of stYen dogs while ,estricting ,.ccc,., to H,O d=uKd blood pressure, and cau.al concurr.m .wtemias. Con""mrations of UN w... inCf.a~ 2- 25 mgldL in individ,",J dogs nom 12- 24 haft" bl=ling. Th. combination of blttding (II....' 50 % blood ,dume) during ,.vern hou", and fNding blood (near 30 % blood ..ulum.) au=! greater illau.." in [UN] {2Q.-67 mgldL incr.as.. in ten dogs}. Th. author> ooncludal that hemorrh:og. cousing dec",=<:! blood pressure, fttding , l~ .mount of blood, or a combination of these p"",", .. r. can cr"ate azotemia." c. In a re{~tjn srudy in which 52 dog. with uP!>'" gastrointellin:d h.morrh~ w... comp"ffllo 52 dog. without upptr g.moinustinal h.morrh~, [h. malian [UN] WlU 14 mgldL gr~m in the hemorrhag<' group. AI"". the ..,rum UN:Cn ratio was ~~t" in the hemorrhag<' group. AI the autho .... pomd. how"""r. thq did not determine whether the hydration nall1. of the hemorrhage group v..a, similar to that of the rontrol group. Thus. this study did not esubli,h whether the owtemias associated with gastrointestinal hemorrhage were caused by incre~..d or .... production. d=~..d or .... excretion. or both. In the data provided. some dog. with hemorrhag<' were not owtemic."

Ill.

Guidelines for >zotemi<> diffi:r.miation (Table 8A) A. Th. cause of owtemia flUy be multifactorial. Both p .... nal dilOrd... (e.g .• hypoml •• mi.) and ]><>Iuenal di""rd.", {e.g.• obstruction} em em.. xute .. nal di ..... and thus

Table 8.4. Major criteria u ..d to differentiate th e three type! of azotemia caused hy d ecn:ascd GFR Historic:ll. physical exml. or T YP' of '7.0temi<> EJ'!,«ted USG ... Urine mlume oth" infOrmation P.. n::nal

:> 1.030"

1.007- 1.013'

Pomenat

D«..a..d GFR due to dehydration. xut. hemorrhag<'. ,hock. or d«r ......d cardiac output Oth" UA findings. dectrolyte ch an!:"". or anemia that an:: suggestive of .. nat di ....... {acute or chronic} Dysuria. enl.rged or rupmr..! urinary bladd". urine in abdomen

• This cri",rion is for dog, and is ..... ming th.re ar. not ""tnJenn disaders th .. "'" aJJecting the «n>l conomtl1ting :IDi~ty ond uri... volume. USG", guidelines for em thot b.... oom:en 1.040 i",,,,od of:> 1.030: for bon><> and cottl. :> 1.025. • Assuming th.,., ... no' mI.".""", (mrn .. prot<>lwne wiU be i",,,,,1>«I. Not<: p,. ..n>l. «n>l. and postr.".] disorden mayocrur independently or m:oy oerur in combinations.

8/ URINARY SYSTEM acute "n. 1 f. ilu". Animals with "nal failure m. y .Iso b. hypovolemic. Accordingly. at the time of p".. ntation. an .nimal·s awtem", may b. the product of both renal and extra"nal factors. B. Th, major J.bomory crit"ion for diff"enti. ting awumi:u involv." the USG .... Ho"..""r. the diagnonic",n must ronsid" the "nal .nd atra"nal f. ctors that moy infJuenct an .nimo]', .bility to oon<: 1.030 (in dog.). :> 1.040 (in alU). or :> 1.025 {in cotd•• nd horses} ba::."", the kidn')" ..., b.ing stimu!'talto oo"",,rv. H,O. 2. If the USG ... is b.low th= values in awtemic animals and th". is no ",id.n<:< of incr .... sed u". production (•. g.• gastroinuni",,1 hentorrh"l:')' there is impairal """I oon<:tQu}. a. Rrnal di ....", {I} Th. USG ... is oft.n 1.007- 1.013 ba::.u.. of impaired tubulointmtitial function associated with n. phron dam. g.. {2} Th. USG ... may b.:> 1.013 but still inappropriatdy low if. {a} Th. """I disr ... imp.irs glom.rular funcrion mo" than tubuloint.mi_ tial function (.specially in aUs or in any .nimal with acut. r.nal f. ilu,,). {b} High urine ron<:
Phytiologic processr. or ron<:
II.

Analytical oonctpu A. Turns .nd units

4J4

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

"

-'.

Tissues pmteins

----+

NH/

Muscle -=_~cmati""'PO?--

_!

U"8~""

""",linine

=

"" "", t \

NH +

Liver

'

uraft

'

""':'. '

" eye. . . "'-

./

Fig. 8.5. Physiologic pro<J<SSeS or 00"""1''' ooncaning UfO. md Crt. Ur.,. .ynth.si, occurs in MpatOCJ"" vi. ,he w e. <:ydr, which is on< mtocyt.., it h.. two pouibl. f..tes. • U,.. passe> fr ..ly across the glomeruhr filt",ion b:urier:ood is «1 in uri"" or resorbed by reM tubul ..: 50-65 "" of ur •• p,.,.."t in g1omorulll film'" is in proxim..l . nd ooI]",ting tubules. U,.. r=>rption in pro:riaul tubuLos i, ."bonO«! by H,D ".sorption in p=inW tubul.. and by incr"""! ADH .ctivity in ,he medulhrycoJLecting dOCh. • Vr.. rnte,. tho intestinal t<1ct of monogastric mammols (vi. tho blood or th< bj~ary systom), ..I..", it is degDpkLL~LL' :md in uliv:o of conk!" AJimontuy tr1<' ""a.tion is .mp«:ted to ocau in dog •• """. md h""",•• as Cn i. diffusibk . cross most ""U membnnes.

=,..

,...,rW

="''''''

J. Current clinial.i = p m~",ure [u",~] in ",rum. plasma. or whole blood. and some laboratories «port [ur..] dir«dy. In /lUny. though. th. clinial.i custom is to ""press [u..~] in t .. ms of nitr~n con .. m in the urea (i.•.• urea nitrog<'n). a. Urea has a M, of 60. so it w.ighs 60 glmol. It is romposed of on. corbon. on. oxygen. two nitrogtn. and four hydrogen atoms. Th ..efor•• I mol of urea contains 28 C of nitroC.n. Accordingly. a [urea] of I mmoUdL = a [u rea] of 60 mgldL = • [UN] of 28 mgldL. b. Th. [urea]. reported as the roncentration of UN. i, commonly ",ferred to as the BUN (blood urea nitrogen) con",ntration, .lthough usually .. rum. not whole

.35

8/ URINARY SYSTEM

B.

C.

D. E.

blood. i, ..... y«l. Brcam. u",~ is ~ frttly diffusible mol«:U.J. fur most cdl m.mbran ... the exmOOlu!.r [UN] and imracdlular [UN] in blood wiU uSll:olly b. the same. Th..efu",. the [UN] in .. rum = [UN] in blood = [UN] in pla,ma (SUN = BUN = PUN). 2. Unit conversio,n a. mgldL o,f UN X 0.3570 = mmoi/L o,f ur.... (S[ unit. nearest 0.5 mmollL)" b. mgldL of u..... X 0.1665 = mmo,llL u..~ Sample l. s"rum o,r pla,nu may b. u..d in most 'pffirophotometric 2. Ur .... is ,ubi. for I d at room tem~rall1"'. ,,,,,er:ol doys at 4-6 ·C. and .r l.-as! 2- 3 mo when fro7. 80 % 10 ..) and thus ,ufficiently dec ........ ure. ,ymh.. is to dec ....... [UN) and correspondingly iner ....,. [NH:]

=,...

Table 8.5. Disca .... and conditions that cau.. d ecrea..,d .. rum or plasma [UN]

Disord." that cau,. d.er ....-d u..~ ,ynth",i, 'Hepatic insuffici.ncy: hep.rocdlular di, ...... portOlystemic shunts Ure. cycle enzyme ddicicnci .. Di50rders that cau,. iner ....,cd ",na! ""cr.cion o,f u... • Disorders that caus<: im!",ir..:! proxima! tubular re50rption of ur..: gluco",r;" C.ntral or n.phrogenic di. b.t.. insipidus • A "1"i",,ly rommon di..... or rondi.i""

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

{2}

Porto.yn~mic

(a) {b}

munt {con!:"niul or .cquirMl

u .. NH' i. ddinr«l to th~ hq.atocytes &om th~ intestin....

Thu~ is less uptak~ ofNH' by hq.atocytes kcau .. of da:r.-astd functional hepatic mass du~ to atrophy. na:ro.is. or fibro.is. b. Ur rption of H,O cr
CREATININE {Crt} CONCENTRATION IN SERUM OR PLAS"fA I.

PhY'iologic proco=. or concepts rrgardiD{: Crt {Fig. 8.5}

II.

An:dytical concepu A. Turns .nd units l. Cn has • M. of 1l3 •• bout twice that of utea. 2. Unit conv~rsion: mgldL X 88.4 = j.lmoUL {51 unit. n<arm 10 j.lmolfL}" B. Sample l. Serum or plasm. may k ustd in most spectrophotometric =ys. 2. Cn in .. rum i, nable for up to 4 d.t room tem]>"rature .nd longer {for 1- 3 mol when "or«l at - 20 oc." C. Principl.. ofCn =Y' l. Dry reagent slid. on the Vitro< instrument: Cn is enzymatically hydrolyud to creatine. v..hich then enters a ",ri.. of ...ctions th.c result in H,O, r"Ctrophotomerer. 3. In some ..... ys using J.ff~·s ruction. non_Crt chromogen! int~rfere with the .....y. How<"V~r. modem modifications h. ... r«luch:dosporin •. 17 4. In a nudy using domestic .nimal .. ra in a kinrtic Jaffe assay •• ooon~. cq>h:dosporins. and glu= caust
s/ URINARY SYSTEM prot~im. How~~t,

this corr~tioll f:>ctot is b. ~ on an HlUmptioll of a normopro_ t~in~mi .. If th~re i, a hy~rprot~in~mia, th~ rorr~tion factor would ~ inoo:l«l""t. ; if th~ro it a hypoprot.in~m;". th~ corr«:(ioll f:>ctor would ~ n=si"". Ill.

[lIcr~

[Crt] ill serum or plasma (not.mi. ) (T.bl~ S.3) A. [Cn] i, typically incr~a~ by pathologic p r = that cou"" d~r~a ~ GFR; th~ init;"ting process may ~ preronal, ronal, or postrenal. B. Inn~a.ffl Cn production and rd~ ... &om damaca! myocytes could contribut. to incro.,a! ",rum [Cn] wh. n r~lIal function is impaired (e.g., myoglobinuric lIephrosis ...rondary to rhalxiomyoly,is in hom.), but Cn is quickly clnred from plasm. if r~nal fUllction i. ad«\uate. B....,lill. ",rum [Cn] may V:lfy amollg individ""ls ~""'" of var;"tiom in totallxxly muscle mau or m~at imak., but th.", f""tors are not npecta! to cou"" moro than. mild notemia. Grqhounds have a grnt" mean .. rum [Cn] than the mnn valu~ for dogs in the general popul.tion, .nd "'m~ greyhounds h. ve values mildly abo"" the Crt up~r refer~n"'" limit." C. Neonatal foal, con have an illcreased .. rum [Crt] if born to dams with dysfullctional pla""'ntas chat pr....,ntNllormal clearan"", of fetal Cn by pla",,"tal blood. In rontrnst to oong~nital r~lIal f.jJur~, thi, '7.0t~mi. should diminish quickly .fm binh . nd resolve over """'!":II day. becou"" Crt will ~ nn~ta! v;" th~ urin~."

[v.

Decr~ .. a!

[Crt] ill serum or plasma A. A da:rnsffi [Crt] in serum or pla.ma is not dinicolly recognizal or dinicolly signifi_ cont. Animals with a d~reased muscle ma ... would tend to have • lower [Cn]. alld th~ pr.sen"", of a hypoprot.in~m;" could yidd • slightly lower [Cn] {Ott Croatillin~ Con"",mration in ~rum or Pl.. ma, sect.II.C.5}. B. [n most speci~s, th~ lower referen"", limit for .. rum [Crt] is nnr th~ detection limit of Cn assays, so documenting a tru~ d=~.", would ~ difficult.

UREA NITROGEN {UN} CONCENTRATION VERSUS CREATIN[NE {Cn} CONCENTRATION [N SERUM OR PlASMA I.

Concepts A. In most mammals, illcr= ill [Cn] and [UN] generally paralld each other. and thus the 5ame informatioll crill usually ~ gailla! from .ith~r value alon~. In hones, [Crt] t.nds to ~ mor~ ..",itive thall [UN] to d~re.... in GFR, probably ~use of ur~a excrotioll into the .limentary t!":let. Similarly, ill ruminant' with renal f.jJur~, urn excrrtioll into th~ alim~ntary t!":let may couse disproponiollat~ incru.e< in [Crt]. B. [n theory, [Crt] is • bett~r indicotor than [UN] of d~reased GFR becou"" th~ q""ntity ofCn presenta! to the kidlley. is more collstant . nd i, not roKlrbed by the tubules, wh~re., urea is teKlrbed. Oth .. factors that may a!fa:t [Cn] alld [UN] includ. the following: l. Hypovol. m;" increa .... ur~. resorption in th~ tubules ~use of d~ro.,a! How rat~ in the tubules, which allows more tim~ for ur~a diffusion, . lId ADH promot.. ur~a resorption in th~ distal nephroll. 2. Increased protein ill imeJIin.1 contents {high prot~in diet or massive intestillal hemorrhage} leads to increased generation of NH: alld SlIb"
438

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

Table 8.6. UN,Cn ... ti ... in azolcmic dogo and caU P"'r~lIa1

Numkrof =

6 A",,~

",.

R.:.ngc:

" '"

[UN] (mgldL) [Crl] (mgldl) 0.4-6.4 ]0.-260 UN:Crl mio 55.3 Sou," (.
n.

A",I'3f:~

'"

4.9 29.8

POS{renaJ

"'"" "

R=~

3»-470 1.2- 11.1 7- 102

A",rag~

'94 9. , 30.5

" Rang<: 85 340 1.3-20 12- 128

Strum UN:Cn mio A. Clinical ob.. rvations hove 'UggestN that a ",rum UN:Crr mio cm hdp dilf",miate pr... ruol .nd r.nal not.mias, and the following ,UUmenU han b..n mode: l. An iner""...! .. rum [UN) and concu,,,,m normal strum [Crr] is most Jikdy a pr...nal >wlemi .. 2. If .. rum [en] is inCl.,."N propo,tionatdy more dun .. rum [UN], th.n the awlem", is probably renal or posIrenal. B. Publishal .. rum UN:C'I mios for 37.0temic dog'''' .rrlistal in T.bl. 8.6. The authors' condudrd the following: l. Th. most ~"e >zotem; .. occur with ..nal or postrenal 37.0um;"" wh.reas Ih. ~.{.n

.. rum UN:Cn r>tK>s ~r~ found in pr~rena! >7.Ot~mi ... Ho~r, r~nal awtem;" cannot b. co",ist~mly diff,,~miata! from extr>r~""l awt.mw by me;om of th. serum UN :Crt r>tio b.cause of the onrlapping range". 2 Con""mrations of UN and Cn in ..,rum should b. regardal a. crud. indi"". of renal function b.cau.., both lock di~no,tic 5tnsitivity and spa;ificily for renal dysfunction emstd by renal di..,ast. CREATIN[NE {Cn} CLEARANCE RATE

I.

Crt rk"raru:r ralr, which i. th~ rate Cn is d~ared from plasma by th. kidn~., i. a good e'timate of GFR in domestic animals but i. not aJuivalem 10 GFR. A d=rastd Cn clrann"" rale (if a valid result) indicote:s the animal has a d= ...a! GFR. However , th. COU5t of the d=ea..M GFR can b. prerenal, renal, or po'lrena!.

[I.

Crl

cle~nn""

rate formuJ. (Eq. 8. I)

.. cI.... rana: "te = [Crt l, xvo) C r.... WHn. ume. . ... lJme ... bw

[Crtl [Crt]. = Crt con""mration in coUecta! urine during a timal coll=ion pc:riod [Crt], = Cn con""mmion in serum from a blood sample collecta! during the tima! urine collection period volum~ = urine volume (in ml) collectal during a tima! collection period time = l.ngth of tim. (in min) during th. urine collection period bw = body weight (in kg) of animal unit, = mUminlkg = ml of plo,ma thai were de.red of creatinine/min/kg

(8.1. )

8/ URINARY SYSTEM

'"

Ill.

Endogenom Cn d~~ranc;., m~ A. Indication, l. To ousess GFR in noruo7.0t~mic, non..dehydrated animal, that are .usp«:t~d of having ,""ruol di",ast. mu:dly ~l1St thq :U~ polyuric 2. To obuin a mo", objective """ssm~nt of th. d]' E. Endogenous Cn demo.na. rate is g.nerally mot< ",,,,itive than .. rum [Cn] for det«t. i"i: d«r~ased GFR ~u .. it i, ~ dir«t assessment of ",mol acmion of Crt. &rum [Cn] d"P"nd, on Cn production, r~nal acretion ofCn, and intmimol Crt act
IV.

Exogenous Crt d .... ranc;., rate'" A. Ba,ics of the procedure: It is the same as the ~nd,,!:.nou. procedur~ ac;.,pt that Cn i, inj«t<"d into th. patient ~nd th~ urin~ ooll«tion ~riod i, routindy ,hon. B. Advantages: Th..~ is inn...."'" pla,/IU [Crt] and th~ .. for~ incroas<"d chall~nge to the kidn.,-., 10 it i,. b.tt.. a=ment ofGFR. Th~ procedu.. minimiz,,, .ffects of assay int..f... nts. C. Disadvantages: It /lUy incr ...... th. p..c;.,ntag< of acr.c<"d Cn from tubular KCmion. Th~,"" i. a lack of standardization of method,.

ABNORMAL ROUTINE SERUM CHEMISTRY RESULTS IN AZOTEMIC ANII>1ALS I.

Strum [inorg. nic phosphoru,] A. In dog., cat" .nd hor""" hyp<rphosph' l<mia occu," when r~ruol d~mo.nc;., of plasma PO. is subnantially r<"ducnl du~ to decr.....M GFR caus<"d by .. mol di"" .. (acute or chronic) or oth.. pathologic nat .. (~.g., p.... nal or pom.nal a7.0t.mic .tat..). B. Some 37.0l<mic hones h. ve a low.normal [inorg. nic phosphorm] or hypophosphatemia du~ to deer.......:! .. n:d ron"''''. tion of PO. or to atrarenal acmion of PO•. C. Cattl~ /lUy or may not have hyp<rphosphatemia. Remol ac .. tion of PO. is minor oomp"red to excr.cion via .aliva and th. rum.n." ...

II.

Strum [tCa""'] A. The [tC,'1 m. y b. b.low, above, or within .. f~ .. nc;., int~",:d, in all 'p«i...

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY B. Dog•• em, .nd cnd~ usually han .low_norm. 1 [cC,'+] or mild hypoc:d"" mia in chronic r~nal f.ilur~ becau.. of drcrrasal ",nal functional m.... and chor~for~ drcr~.sal r~nal formacion of 1,25_DHCC. Hypercal""mia oo:urs in • minority of C;;O"'". In c;;onl~, PO. acrHal in ... Iiv. may bind with Cal+ in th~ alillYntary tr""t .nd mak. ic 1= av:lil.blo for .bsorpcion.'" C. In dogs wich chronic r+ rich, ho""s ar< oft~n hypercal""'mic in arut~ or chronic r~nal di ..... becau.. of da:reaM+. hor.... c;;on also ~ hYl"""lctmic during r~nal f.ilur~.

III.

s.rum [K+] and blood pH (or [W]) A. Dog., c;;ocs, .nd horses l. Arut~ drc",,,s.,, in r~nal funccion may"'''''' hy~rkalomia and .cid~mi. becau.. of impairal renal acr~ion of boch c;;otions. Aho, inorganic acidemia a"""muat .. hy~rkal~mia chrough ch. ralistribution of ions ~tw..,n im",,,,,lIular and ac",,,d_ Iular romparcmonts. 2. Hyperkalemia and a.cidomia ar~ ...,n primarily with oliguric or anuric r~n al failur. and am ~...,n wich ~ichor acuU or c~rminal chronic r~nal failur~. 3. Many other bcto.. influon"" .. rum [K+] and blood pH, 50 apectal chang.. may noC oo:ur. B. C,nle'lJ' l. Cattle und to han hypokalemia du~ to .Iblosis, drcrrasal diotary potassium intake, or inc",,,,al pota.. ium ner~ion via saliva.'" 2. Th~ .nimal·s m~tabolic .Iblosis and ronrumm hypochlor~mia .'" oft~n ronoid~ral cho resulc of H+ and CI- s"lu..c"'tion in the abomamm duo co aboma.
[V.

s.rum AMS and LPS .ctiviti.. A. Dog" C,nine kidn~ provide. major rout~ of AMS and LPS acr~ion or inactiv.tion. [f ",naI p..fuoion or functional r~nal m.... is drc",=<:!, there is I.... ",nal in.ctivation of A,\{S and LPS, and thus their long half_lives ..~ iner~" sal. With tim • . mildly to mod~"'tdy in"",=<:! .. rum AMS and LPS activities may develop (Itt Chapter 12). B. c,ts: Tho invol""m~m of fdine kidneys in th~ clraran"" or inactivation of AMS and LPS i. not wdl docum. mal. Of 32 """. of fdin~ renal failuro, hype"'mylasc:mia {.light to thredold iner....} WlI.! pr~ .. m in 10."

MAJOR VRl NALYS[S {VA} CO NCEPTS I.

Compon~nu

of a routin~ VA may vary from ono laboratory to . nOlher; how<:vor, aU procalures ,hould ~ don~ on fre,h urin~ « I hold). Be,id.. the pot~ntial for dmrio",_ tion of ""II, and cast" dday of 6- 24 h in completing tho urinaly>is may allow in vitro crystal formation, especially if tho .. mpl. is Sloral at 4 'c."

[I.

Procalur.. for. routine urinalysis have: been descriW .......

8/ URINARY SYSTEM

.41

Ill.

Common compon~ms of mon routin~ urinalyses A. Physical ~umination: color. darity•• nd USG ... B. Ch~mical namination by r~agtm mip methods l. Common """y" pH. protein. gluoos<. ~one. heme (occult blond). bilirubin. and urobilino!:"n 2. Oth~r """1" nitrit~. USG •• nd leukocyu me~ C. Sedim~m aamination: microscopic aamiruotion to id.mity erythrocytes. lalkocytes. bacteria. com. crystals. epithdial cdh. and other nondiuolved Jrulterial (may be done with nOllStained or with stained urin~ ...dimem p"'p"'.tiollS)

IV.

Urine compo.ition i. determined by thr .. Jruljor factors. Becouse of th ..e factors. the urine composition is .ffected by th~ emire urinary system and by other body .ysums. A. Quamity and composition of the plasma prestmed to kidney. B. Renal functions. including filtration. tubul", stCretion. :md absorption C. Material (ch.micals .nd cells) .dded to th~ glomerular fihrat~ •• it How. through kidney•• ureters. urinaty bladder. urethra. :md prepuce or ~ruoIvulva

V.

It is importam to know the method of urin~ coll=ion (i .•.• voided. cynocem ...is. cotheterization. or "off_.urface") ",-h.n th~ UA results.re being imerpreted. Voided umpl .. may have more bacteria. ~pithdial cdls .• nd leukocyu. from the dinal urethra and !:"nital tract. Cystocemesi. sampl.. m.y be affected by iatro!:"nic h.morrh>ge. but they localiz<: abnormaliti.. from the kidnry"S to the proximal urethra. Cathet~rized .ampl.. may have more epithelial cdls. blond from iatrog~nic h~morrhagt. lubricam. and Nct~ria. And 'off_surface" sampl.. m.y be comamin.ted with a variety of microJCOpic paniculat... Results upected in healthy dogs. cots. hoJ"Sel •• nd cottl~ ",e lined in Tabl. 8.7.

PHYSICAL EXAMINATION OF URINE I.

Urin~

color (pigmems) A. Physiologic processes I. Th~ normal yellow 10 amber i. due to urtX"lmtmtf. a group of poorly ddined urine pigm~nu of which on. i. riboHavin. 2. Pale yellow urine is usuaUy less roncemrated th:m dark yellow urine. but not always (plate llA [for all plat...... the rolor sectiou of thi, book)). B. Aualytical roncepts: GlOSS :wessm~m of urine color is typically don~ on &~sh. wdl_ mixed urine. C. Abuormal urin~ color ip;gmmruri/J) I. An abnormal color iudicot.. the presence of abnormal pigm.ms in th~ urin~. Other parts of th~ uriualysis or other assays .re nttded to determiu~ which pigmem or pigm~uu are pre",m. 2. Although concurrem p~menturias Jruly alter upected colors. the following.re common abnormal colors and the substance. that create them: a. Red: .rythrocyt~ •• h.moglobin. :md myoglobiu b. Red_browu: erythrocytes. hemoglobin. myoglobin. or meth~moglobiu c. Brown to black: meth.moglobin from hemoglobin or myoglobin d. Ydlow-oran!:": bilirubin e. Ydlow_grec:n or yellow_brown: bilirubin:md biliverdin

442

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

Table 8.7. Expected UA resulu in health):: dog.,

Cilt.'i,

ho"",", and caltle

DOll;

c.,

YdJow"

YdJow"

Cl~r

C[ra, 1.035- 1.060"

Ho=

CanJ~

YdJow" Hazy_turbid

Y~Uow"

1.020--1.050"

1.025-1.045"

5.5-7.5

7.5-8.5

7.5-8.5

N" N,. N,. N" N"

N" N,. N,. N" N"

N" N" N" N" N"

Physical Color Clarity USG""

1.015- 1.045'

CJ~~r

Ch~mjcal

pH

5.5- 7.5 Neg- l+'

Pro{~in

Glu= K.:tonc

Heme

Bilirubin

N" N" N"

Neg---l +'

0.2- 1.0 0.2- 1.0 Urobilinogtn {EU} Salim.nt (from ~ntrifugation of 5 mL of fre;h urine)

uukocytc5lhpf Erythrocyteslhpf Roctcrialhpf Casts/lpf

Epithdial cdJ./lpf Crysl:lls/lpf Olh.r

<5'

<5'

<5

<5

Non.

Non.

Non.' NOMiO

Non~

Non.' f~

Non. to Non~

0.2- 1.0

0.2- 1.0

<"

<5

<5' <,

Non.

Non.

Non.'

fow'

Non. to Non.'

few'

Non.' None to

f....l

Non"

Mucm

• The in..nsity of y
voided mel .0.... c1thrteriud ..."ples. , Pbosphat<
3. Horse urine may tum rrd or brown during "o"'!:, or when exp<>« i, reponed to b. cau...! by pyrocatechin (pyrocata:holl ," which i. the aromatic ponion of catecholaminn. A p .. hologic ,ute is not :associated with thi, pigmenturi .. [I.

Urin. clarity A. Physiologic p~ I. Clear urine is expected, but it may h."" mild turbidity due to sus~ndrd partid.. {e.g., .pithdi<>l cdls and crystals} 2. Equine urine is frrquendy turbid or cloudy b.causr of th. prestn", of mucoprot.in {produced by kidn.ys} or calcium carbonate crystal •. B. Analytical con<:qlts: Gross ....... m.nt of urine darity i, typically don. on fresh, wdl. mix.d urine.

8/ URINARY SYSTEM C.

Cloudin~ss

cryst:ols, III.

Solut~

or turbidity indicates th~ pr=n"" offormal casu, .nd lipid droplets in the urin~,

d~m~nts,

tuch '" ""ll"

bact~ria,

con""ntration A, Physiologic proc= I. The solutes in urine . ", the dissolval ions and molecule., Most of them, including dectrolyu, (Na+, K', ct, Ca'+, PO., and NI-Lt) and mffabolic products (ur ... :md Cn), are being excretal by the kidneY" 2. The con""ntrations of filtrate solute. are modifial by the tubular resorption or =mion of solute. and by the resorption of fihrat~ H 2 0. 3. Urin~ solute con""ntrations are upectal to incre-are when the kidney,; are con.. rving H2 0 and decre> .. when the kidne,.. are not coJl5.. in solute ron""ntration {i.e., the refractive index inc=ses}. Specific gravity:ol"" incr ..., .. proportionately to the ,olut. con""ntra_ tion, so .pecific gravity corrd. tes with refracti"" index if the types and propor_ tion, of the solutes remain similar. If urine ha, rdativdy norm:ol ",lute composition, USG"", from a good-qu;o]ity refractometer rorrdates very wdl with osmolality. b. Th. r~fractive index is highly d~ndent on thr.., facto",: solute con""ntration, chemical composition of the solute, .nd temperature. Refractomffe .. m=re the refracti"" index of the soluble K11id, in the fluid {i.•. , solutes in the solvent}; ,uspendal particl.. (e.g., cdl" casts, and most crystal,) do not refract light and thu, do not :olter the refractive index of urin . ... lkcau.. the suspenda! panid.. interfere with light tran,mission {thereby cau.ing cloudiness or turbidity}, they may make the demarcation line in the re&actometet more difficult to read. The following are USG"",v..!u .. for ten cloudy utine ... mpl.. (direct r...ding! ,upernatant r...ding): 1.043/1.043, 1.054/1.054, 1.028/1.028, 1.016/1.016, 1.083/1.083,1.021/1.022,1.05111.052,1.048/1.048,1.06111.062, .nd 1.05]/1.052 (author<' unpubli,ha! ","to). c. Most temperature-rompen",ta! refractometer< have USG"" sc:ol.. that are calibratal for th~ norm:ol composition of human urine (Fig. 8.6). d. How<:ver, there are r.fractometets th at a", calibrata! for constituent, of canine, feline, and lar!:" animal urine {Fig. 8.7}. {I} The calibration scale for dogs and large: animal, i, slightly different from the calibration sc:ole for people. {2} The calibration .cal~ for cats i, mod~ratdy different; for example, a 1.010 on th. canine sc:ole i. about a 1.008 on the fdine scale, • 1.025 on the

444

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

SERUM PLASMA PROTEIN REF RACT.

;."OO ml

URINE S PECI FIC

GRAVITY ,,~, .!).I5

:

, ,!).IO

:

, ,035 : I,03(l

:

, ,025

:

, ,02{J

:

1,015-: 1 ,O l~

, ,005

=

=

, , ~-=

.11

12T 10

!

J' -t-7

INOEX, no

=-:: 1356 ' 357 :: 1 355

iL'1S-l

~1 353

:; 1.352 ~1

351

i:::1.3SO

= 1 349 ;-1.348 <=--1 347

'-i- 0-""

':111:

p~

R.O.TIO

".

1-1.345 i-1.~4

..... 1.343

E'342 1 341

1:11 340 339 1 338

r' 337

(1.336

1 335 ,,~

--'333

Fig. 8.6. lll .... mion of >coo in . L.i", TS~oo hand· held ,m.ctomet<'. n.. ,.fuctom«ltD ",tinure USG", or toul protein con",n.mioJU, mp<'Ctively. Th< ,m.ctomet<' ocaIes .. on calib",«l for bum:on sampks. llJUll"....d with permission from Lei", Miaosy".."., Bufh/o, NY. PRIN ... io 6.54. convm;;on f>
canin~ scal~ is .bout a 1.022 on th~ fdine scal., .nd a 1.040 on th. canine scal. is .bout • 1.036 on th. fdin. scale. •. Th. ~mount of error in th. fdin~ USG ... is mild wh~n • r~fractom"'~r calib.. tM for human urin~ is u..d. If ",fractin indices of human and ftlin~ urin~ "'.. r~ 1.3365. th~ USG ... would k 1.0lO .nd 1.008 .... p«tinly. If th~ ..fractin indices w~ .. 1.3420. th~ USG ... would k 1.025 and 1.021, respeerivdy." Thu., if a ref""'tom"'" calibratw for humon urine i. U.ffl for aU urine, th. urine .p~a" .lightly more ron""mratw than it really is. f. Non.t~m~ratur~-com~nsatw ",f""'tomet. " und"estimat~ the USG v:llue wh~n anlbi~nt tem~ratu .. increa ... abo"" 68 OF (20 ·C ). Th~ .mount of error increases :u Ih~ t~m~ .. tu'" inc",ase •. g. When the USG. , is greater than Ih~ upper end of Ih. USG seal. of the ",fractometer, a dir.-ct r~adinc cannot k obtained. If. reading am k mad. on the .. &active index .ale, som~ labs ron""n th~ .. fnctiv~ index v:due to a USG b..s«i on tables g.n~r:llw ex~rim~ntally. If th~ reading i. also gr-..t" than th. refractive index .ale, the "'mpl~ moy be dilutw 1:2 with di"ilJed WlIler, r...v:dualw, :md rq>Onw afm ror=ting for the dilution (. 1.026 result wilh th. dilutw sample would k r~portM •• 1.052). How~.., diagnostically, it is usually

.45

8/ URINARY SYSTEM TE M P~RATUI't £

DOG

URINE

COMPENSATEO

S.G. CAT

..... RGE M
-=

1,000

: 1 050

: U)4O

H

1=,

: 1 03(l

1=5

1 ,Q50:

1,04(1 :

1030 :

: : 1 (120

1 ,02():

=-

E~O

'-.

1:' 1-3 1:,

: 1 010

:

1 ,000

13

1=" 1="

1060=-

1010 :

f'

-= 1 000

SERUM OR

"""~ PROT EIN ~m"'\OOmI

1 L.i", VET 360 band·MId ",terinary rd'ractomoter. n.. .. fnctom
Fig. 8.7. Illu ..... ion of >coo in

1

not n=a,y to know how con~ntrat.d th. urin. is wh.n it is too ron~ntratM to br .ud di=dy ITom th. ,~fra.ctom., ... 3. Osmololity (smoles of solut~ panicles p<. kilogram of .ol""nt {<>smoUkiI o. in mol .. of solu,. J><'. kilogram of solv.nt (mol/kg): I osmol is I mol of osmotically acti"" panicles. For a mbscan~ thot d""s not dissociat~ in solution, I mol '"'Juals I osmol. Fo, • substan~ that oompl.,dy dis.tOciates into two ions J><" mol., uch I mol of th. substance: ~n.rates 2 <>smol of panicl ... c. U,in~ osmolality m=ut.m~nU at< usually limital to .p«ific cu.s wh~,~ accurat~ 1SSCSSm~nt of .~nal oonce:ntrating o. diluting abiliti~, is critical. Th. gold scandord for =sing urin~
FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

..roo 1.025

.."" J 00

1.015

~

1.010

..000

..000

•

Urine osmolalHy (mmollkg)

Fig. 8.8. Comparison of osmoWity:ond USG",in unm. uri... sampLes with USG",S 1.025 (n = 185). n •• ", is . high oonel"ion (, = 0.91) b.tw...a tM oro mrthods of ......ing u,m. ",Iut< conc<:tiv<: ind.:x. Fo, ...... umpks. tM me"" Oun.:USG .. _ 33 with • .d of 5; tIM: "",:on ± 2..d (Of 95 ~ intti"",ted rono
b. Conclu.ions from the comparison {I} In most urine umpJes. there i, a good linear relationship becw,""n USG ... and o.molality. Thu •• the USG ... i. typic;;dly:on aocur>te reflection of the urine .olme concentration. {2} Because of thi. good rorrelation. the Osm,,:USG ... mio i, rdativdy constant {Eq. S.2}. In IS5 canine urine "'mpJ.. with USG ....... 1.025. the Osm,.:USG ... ratio w>s 33 ± 5 {mean ± sd}. In 37 fdine urine sample. with USG ....... 1.025 {using non_feline .udej . the Osm,,:USG ... mio Won 35 ± 5 (mean ± ,d}."

(8.2.)

Osm. :USG... Example: If urine oomol:dity _ 600 mo.m/kg .nd USG ... _ 1.020. then: Osm. :USG... =

600

600

1000(1.020 1.(00)

1000(0.020)

OR Osm.:USG ...

- ;c::;

600

- = 30 20

8/ URINARY SYSTEM

the USG .... ""lu~ or urin~ osmolality. it may!>' impor_ um to r«ngniu th~ following r~latiofl!hips: (I) If urin~ I has an osmolality of 300 mmoUlq: and urin~ 2 has an ounolality of 600 mmollkg. th~ ",lut. cona:ntration in urin~ 2 is twia: th~ ",luI< cona:ntration in urin~ I. {2} If th. USG .... of urine 3 is l.OlO .nd the USG .... of urine 4 is 1.020. th~ ""lut~ cona:ntration in urine 4 is approximatdy twia: th~ ",lut. cona:ntra_ tion in urin~ 3. d. Wh~n th~", i. ~ith~r a IlUrked proteinuria or m ..ked gluco.uria. the USG .... ov~ .. nillUte. th~ cona:ntr:nion of urine ""lut~ and thus n:nal cona:mrating .bility {Fig. 8.8}. Th~ Oml,.:USG .... ratio will !>. decreased (in dogs. below 20). (I) A [prot~in) of I gldL adds .bout 0.00}-0.005 to USG .... but has .lmost no .ffect on O!molality. (2) A [glucose] of I gldL adds .bout 0.004-0.005 to USG .... . nd only slightly incr..,."" urin~ osmolality (about 5 mmollkg). 5. Ikagtnt mip for ~tim.ting USG (Bay~r Di"i:no,tics) a. The r'""i:ent strip m~thod is not =mm~nded for estim.rion of urine solute cona:nrration or USG of domestic m. mmals. b. Principle: Th~ ionic str.ngth of urin~ is r~lated to total ""lut~ cona:ntration. Th~ rrag<:nt 'yn~m h", indicators that produa: diff~ .. nt colors wh~n dipped in urine ,ampl~ of diff~ .. nt ionic str~ngths. c. Th...miqu antitatiw ""Ie i. 1.()()()""1.030 at 0.005 int.rval,. with be" accuracy wh~n the urin~ pH is < 6.5. Add 0.005 to th. r~.ding for. pH:;> 6.5. d. Falsdy low r~mlu occur in .lkalin~ urin~: for uanlpl~. most urin.. with a pH ... 7.0 and a USG .... of 1.025-1.035 had • dipstick USG of 1.015." Mod.m. quamities of prot~in can cau.. falsdy high ""lues. and glucosuria can cause falsdy low valu ..... C. Expected USG .... I. On. must h.w knowledg.. of.n anima!". H,O bal.na: .nd medications to pro~rly ....... an anillUl'. ability to cona:ntr.t~ or dilut~ urine. a. An .nimal that i. d~hydrated or h .. r",ricted a{Uss to H,0 ,hould uc"'t~ a rdatively small volum~ of concc:ntr.ued urin.; that i•• with. rdatively high USG ... b. Aft.. diumic or fluid th~rapy. an animal should excre"l~ a rdatively high ""lum~ of l~ss cona:ntrated. isonh.nuric. or dilut~ urin~; that i •• urin~ with a lo~r USG .. 2. Htl!thy animal, with norllUl or .dequat~ ..nal function am ucret~ urin~ with a broad USG .... ran~. deptnding on what th. kidn")",,,, !>.ing challenged to do. a. Maximal urine dilution in dom.stic IlUmrnah as .......d by USG.,.: n... 1.001 b. Maximal urine concentration •• ~ by USG.,.: cats:;> 1.080. dog. n~.r 1.060. and ho=. and catrl~ n.... r 1.050 c. Usual USG .... ""lues wh.n H,O intal.:~ i, adeq""t~ .nd hydration status is normal: dogs (1.01 5- 1.(45). caU (I .035-1.065). hor"" (1.020-1.050). and cattl~ (l.015-1.(45). How~wr. th. USG .... may be lo~r or higher in . nimals with normal ",nal function. D. Im~rpreution of USG .... ""lues I. A USG ... is usually neffied for th~ ....... m.m of r~nal rona:mrating .bility wh~n animah .re awt~mic. polyuric. oliguric. or anuric. Because of th~ many v.ri abl~ that chang. urine ",lut. cona:ntr. tions. it i, difficult to formulate firm guidelines for th~ im~rpreution ofUSG .... in all cas ... c.

Wh~n im~rpre{ing ~ith~r

.47

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

T able 8.8. Major p"chogcnic mechani.m. of polyuria Solut~

Chronic ""nal f.ilur~ Acut~ ""nal failut. Postolmructivt diuresis' Di.!>'tes mdlitw Hyp Canin~ pyometra Hypokal~mi> Hypoodr~noconicism

+. + +

diuresis

.J.. Tubular respon ..

.J.. Malullary

toADH

tonicit!

. ADH

+ +. +.

+

-'

-'

+ + +

• • -'

+' +

1<

LiV
2, Ani"",J, with impairal ",,,,,I conc;.,nt.. ting ability will hav~ on~ or more of the following d~f«" {m«hani.ms for the resulting polyuric "at~s ."" listal in T.bl~ 8.8}:

a, ADH ddici~ncy is prestnt (c;.,ntral d i> i>.tes insipidus), b, Epithdial c;.,1I. of dinal nqJhrons ",e not rrsponsivt to ADH (nqJhrogenic dia!>'tes insipidus), c, Solut~ ovtrload ~ presc:nt (too much ""Iut. enuring th~ loop as occurs with osmotic diuresis, r~",,1 failu"", or incrrasal GFR), cau.ing. high flow m . and d«=sN rr50rption of tubular H,O, d. D=~..ed medullary hy~rtonicity (I) Prolonged hyponatremi> or hypochloremi> (2) D.f«tiv~ Na+ .nd ct transpon in the loop of H~nl~ (~,g" loop diumio) {3} D«""...d urra production bernusr oflivtr di",... (4) Soluu ovtrload or prolonged diur .. is 3, G.:n~ral guiddinrs for USG ... int~rpreution are in T.bl~ 8,9, 4, USG ... in various disord." or conditions a, USG ... ;> 1,030 in an oliguric dog, ;> 1,040 in .n oliguric cat, and;> 1,025 in oliguric horses or cow>

Table 8.9. Guidelines for inlerpretation of USG...- values in a dog Co..., infOrmation USG .." Im~rpmalion Nothing known

1.001-1.060

Dehrdr:al~

> 1.030 1.014- 1.030

1.007-1.013

< 1.007

Polyuria

;. 1.020

1.007-1.013

< 1.007

Oliguria

Glucoluria

;. 1.030 1.014-1.030 1.007-1.013 > 1.020

1.007-1.020

Hyposufremia and hypochloremia

> 1.0 20

1.007- 1.013

Could be found ;n a clinicaUy heahhy or sick dog ReAeclJ rem! anemplJ 10 conserve H,O appropriately Sugg.m impai.~ .em! cooormraling ability and pouibly rem! F..ilun:; could be 5ftf\ wilh g1uooouria. hypon:mmtialhypochlon:mia. parlial n:naI diabeles imipidus disorders. h)"p'»d rtllocon:icism Sirongly indicates defKlive renal oonuntt:ning abili()~ if arolrmic. then ",nal insufficiency or f.tilure until pro""n OIherwi.., Strongly indicates defKlive ",nal oonuntr.ning .bility bm nOt d~ 10 renal f. ilure. as kidneys ha"" ability to dilule ullrafi!rr:ate; consider cetltr21 or ",nal diabetes insipidus disorders Reflect> n:naI altemp" to conserve H,O and dm, not in ren:ol imuflicitncylfailun:; could"" £rrn .... i.h g1Ua.:4Uria, hyponatrmtialhypochlon:mia. partial cmtr21 or ",nal diabetes iruipidus disorders Strongly indicates d.r.ai"" ",nal oonuntrning ability; if u.otrmic. then ",nal insufficiency or f.tilure until pro""n OIhrrwi.., Strongly indicates dd"K1i"" ",nal concen!t:uing .bility bin nOI d~ 10 rena! F..ilure. as kidneys h.;ave ability to dilule ultrafiitrate; consider cetltral or ",na! diabetes imipidus disorders Reflect> n:naI aI.cmpts to conserve H,O :approprialdy Uncommon: suspect oCUle ",nal f.iJu", Typiol fOr oliguric renaJ F..ilure; acute Of chronic Reflect> n:naI concentrati ng ability but rnay be pmialJy impair~ by ..,Iute diun-sis or d«rcasft! m~ulJa.y hypertonicity (mirffi co"""mrating abil ity ouscd by solute diu .... is or mrdulJary w:ashout but could h.;a"" conrurrmt ",n.1 inlufficimcy/F..ilure Reflect> n:naI concentraling ability but nuy be impain:d by loop of Henle failure as may occur ",ilh hypoWm<>OOrticism M.y reflea greater impairment of concentrating abil ity beau.., of the loop of Henle f.ilu", but also mu,t comid" insufficiency/failure Reflect rena! diluting ability and thus nOl renal imufficiency/failu",; defective ADH sca<1ion probaJ.Iy

re.w

< 1.007

• A-.uning th ...... USG... v...... ;w I>Of bl.. ly .. ~au.
ODd canl< wou.Id ... f1«t bon conc
<~ or

in hnJt.h.

449

450

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

b.

c.

d.

•.

f.

{I} Nomenal p"""'''''' (~.g.. hypovol.mia and da:r~ cardiac output) ha"" led to da:r~=d r.nal ~rfu,ion. Hypovol.m", or pl .. ma hyperosmolaliry ,timulned th. rd• ..., of ADH. {2} ADH promoted th. resorption of H,O in th. colla:ring tubules. thu, con""ntrating th. tubular fluid and thus urin •. USG ... < 1.030 in an obviously d.hydrated dog. < 1.040 in a d.hydrated cat. and < 1.025 in a d.hydrated cow or horse {I} Such finding. indicat~ a renal con""nrrating d.fa:r that could be caused by ",nal or ntrarenal disease. {2} P.rhogtn=. of the .ptcific ca ..... vary with th~ pathologic ,tat.. (Ott polyuric disord." in the nut stetion). USG ... = 1.02()....1.03S in. polyuric animal {I} Diabem mdlitus: Glucosuria cau..".n osmotic diuresi. by inhibiting th. passin ruorption of H,O in th~ proximal tubul ... If it ~ni
8/ URINARY SYSTEM

{3}

{4}

{5}

{6}

{7}

{S}

{9}

45'

{b} Cortisol may inhibit th~ ... ponsinn.,.. of renal tubules to ADH. and thu. I.,.. H,O i. resorbal. How...~r. some ,tudi.. indicat~ that cortisol dot, not int.. f~r~ with r~nal activity of ADH."'" Hyp..aldosteronism: Th~ path~n .. is of. dog', polrurie .me was not firmly establi,hffi, but the ... id~no:: supportffi an impairffi r~sponst to ADH . nd deloyffi ADH rd~.st aft~r hypertonic nimulation." Plasma rortisol cono::ntrations w..~ within ref~reno:: interval. in th~ basal state and during. low-d= dexam<'thasone suppre:uion ten. Me.mrement of oth~r adrenal steroid con""ntrations WlL! not mentionffi. },'fpItrDgrnk dill"''''' insipidu, is a group of ren . l .nd ext""enal di"",,.. in which ADH is pr..ent but renal tubules.re not ""'ponsin to it. Th~ group includ.. hy~rcal""mia, canin. pyometra.linr failure, and hypobl.mia. Hypercal""mia {a} [nc....ffi [ICa><-] inhibits ADH activity via dysregulotion of aqu. porins. Aquaporin 2 tr.nslocation to apical membranes appears to be decreased. In .ddition, ""Uular "'Iu. porin 2 .p~." to be decreased becaust of degradation by a calcium_sc:nsitin prot ...... "'-" {b} The.. is also ... id~no:: that c,'+ rffiuo::. resorption of Na+ . nd ct in the a=nding limb of the loop of H~nle, which reduces th~ OJmotic gr.dient nttdffi for H,O reKlrption in th~ disul nephron." {e} A persistently incr..... d [IC?] may emsc: min.ralization of tubular b... m~nt m.mbrane>, which results in • calcium nephropathy:md thm r~nal insufficiency or failur • . Hypoad.. nocortici,m {Addison', diseast} {a} The USG""wiH be this low in only. ,mall minority ofhypoadrenocor_ ticism pati.nts. {b} The pathogenesi, i, not wdl dorumentffi. but th.re may be failure of N. + and ct ddi""ry to th~ loop of H~nle and thus failure to mainuin meduJlaty hy~rtonicity. {e} 0«......:1 effecti"" plasma OJmolality (becaust of hyponatremia .nd hypochlor~mia) will rffiuo:: the osmotic stimulus for ADH synthesis and rei...... Thu" ADH activity might be rffiuced {ronv"..ly, hypovol.mia i, probably stimuloting ADH rd~a ..}. C,nin. pyometra: The specific pathogen..i, i, not cl .... but the kidn..,.. ",e ..&actory or poorly responsive to ADH. On~ potential mech.ni,m is that bacterial .ndotoxins initiat. the ..fractory stat •. liver failu.. {a} 0«......:1 urea ,ynthesi' m.y lead to. deer.... ffi mffiullary [ure.] and thus a decr ......:1 mffiullory rono::ntration gradient {mffiullary washout}. {b} Other possibilities h. "" been sugg..tffi: psychogenic polydipsia, defect, in portal v.in OJmor=ptors, .nd impairment of renal wncentrating mech:mi,ms becaust of increasc:
(a) Hypokalemia makes wHeeting tubul .. I.,.. respon,i"" to ADH. ~rhaps becau.. of reduced generation of cyclic adenosin~ mono_ phosph .t~ (cA.,\tP)" .nd down_regulotffi expression of aqu . porin channels. 4"

452

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY {b} K+ is liNda! for N.+ alld CI- ruorption in th~ ",,,,,"ding limb of th~ loop of H~III~, and thus hypokaJ~mia rruoy imp.ir counurcumnt function,"" {IO} Hypopuachyroidism: Th~ path~nesi. i, not wdl und~mood, {II} Fdill~ hy~rthyroidi,m: The pachog~lIrsis is not wdl undmtood {12} Psychogenic polydipsia: fu;c;., .. iv. H,O ronrumption I...d, to npand I ,020} with hyponm.mia and hypochloremia {I} B.G1U,. Na+ and ct a" rruojor comributon to th~ hypc:nonic im~mitial fluid in r~n:ol m
CHEMICAL EXAMINATION OF URlNE (QUALITATIVE OR SEMIQUANTITATIVE) L

M.jor concqJts A, So:miqllre u..d to det<et ot rnancteriu: pathologic ren:ol and ntrar~nal nates ot to monitor respon .. to ther:lpy, Th~ colI""mr:ltion of a solute in urine will d~p'nd on two ""jor factors: (I) th. amoum of solut~ ncreta! ill th. urin~ onr time, alld {2} ch~ amoum of H,O excmr~ r...d by visual n.minatioll, distingui,hing betWttn. 1+ ,,""tioll :md • 2+ reaction, or betw",n 2+ .nd 3+ (~tc,), m"}' ~ difficult, Thu. , th. true ooncrntr:ltioll of a ",Iut. ""y ~ consider:lbly different from th. "pon
H H

-, . ,

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"n.o.-_ ,"

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,

., W W

<0.01)0>0.061

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<,

d.

,~

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<0.< ,~o..

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.

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.......

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"(_"'_ ~

.,.

,,~

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,

, ••

o.!-I.O

, •• "

rt . . . . . . . . ;..,. .. ......... ~ . . . . . . . . _ _ ~"'.Loc-- ... _ .. ....,. ........... . .......... _;... .. ' n. ___ "-<10 ..... _ ...... - " ..... n. ,"- .. a.. . .... _ - ' " w ... ' ; 1"(-elL). n. ........

...............

"n- ...... _""""_ ... "-
...... .......

..... -

454

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

.. :agents in the p ads. Routine u.. of commerci. l urine comrol ""lutions is ..commended to """"rtain dUI th...actions are v:did 2 Automat«i r.Hecton"" photomete", ... available for photometric a"...sm.fit of oolor clunges (e.g., the Cliniuk 100 Urine Chemistry Analyur). A major adv.nt"l:' of th. instrumems is that thry .. mon the person_Io_person v.riation in th.oolor clu nges on the p ad. a. In a .lUdy involving emin. urine samples. th. authors report.d good :ogrttmem becwttn visual estimation md autoJrultic mumrem.ms for most analytes. Th. USG ..:agtnt p:od v:due. ~ .. unrdi.bJ .... b. In another romp" ison of 40 canine urine .ampJ.., thert w:as ""I}' good to ncdJ.nt agrttllYnt for g1u= and hem •. lmtrum.nt ,radings fur protein und«l to b. slightly [OW"" and ketone :md bilirubin d<'l«:tion was slightly high" (i .• .• •om. in'trum.1It r~jom pmi!; ... wh~n vimal "".ding> w~r~ nq;ati ... )." 3. Rehtionship of urine solute con~ntration. urin~ volum~. :md daily urinary acretion of solute. {Eq. 8.3)

w.'"

Daily urin.ry solut~ excretion . [",lute]. X urin~ volume/day For exampl~ fOr prot~in excretion: Daily urinary prouin a cretion = 70 mgJdLx I<XX> mLJd = 700mgJd

(8.3.)

a. If th~ urin~ solut~ concentration ""mained comt:mt (e.g .• 150 mg/dL) from one day to th~ nat. but th~ urin~ volum~ doubled (~.g .• from 100 mUd to 200 mUd ). then th~ urinary acretion of th~ solut~ doubled from on~ d~ to the next (from 150 mg/d to 300 mg/d). b. If th~ urin~ solut~ concentration doubled (from 150 mg/dL to 300 mgldL) from one day to th~ nat. but th~ urine volum~ remained (X)fist:mt (100 mUd). th~n the urinary excmion of the ",lure doubled from on~ day to th~ nat (from 150 mg/d to 300 mg/d). c. If th~ urin~ solut~ concentration doublal {from 150 mg/dL to 300 mg/dL} from one day to th~ nat. but th~ urine volum~ halved {from 100 mUd to 50 mUd}. then th~ urinary acretion of th~ solut~ r~m>ined constmt from one day to ch~ next {150 mg/d}. 4. Rd ationship of urine ""lume and USG. ,valu.. a. If th~ animal it not in ren. l inmfficiency or f>ilur~. urin~ volum~ is inversely proportional to USG ... b. If th~ urimlry acretion of solut.. re"",ins constmt (~.g .• I g/d). but th~ urine volume doubl.. from on~ day to th~ nat. th~ USG .. is apn:ted to "hal..." from one day to th~ nat (~.g.. from 1.040 to 1.020). c. If the urin. ry acretion of solutes re"",ins ronstant (~.g .• I g/d). but th~ urine volume halves from on~ day to the next. th~ USG .. i. apected to 'doubl~" from one day to th~ nat (~.g.. from 1.020 to 1.040). 5. Use of conc~pts to int~rpret urinalysis r.. ulu a. Dog I with • urin~ [g!uoo..,] of 500 mgldL and a USG .. of 1.015 is rypically acreting just'" much glUOOK per day as dog 2 ""'t has a urin~ [g!urosc:] of 1.0 gldland a USG .. of 1.030. b. Dog 3 with. urine [protein] of 50 mgldL and a USG .. of 1.040 is probably not prouinuric. H~.lthy dogs may h a... urin~ prouin con~ntrations of

'"

8/ URINARY SYSTEM

4--65 mgldl;"" USG ... is typically 1.020- 1.045. Ho~ver, dog 4 with. urine [protein] of SO mgldl.nd a USG ... of 1.010 is protein uric. c. c.t I with a urine [protein] of 50 mgldl .nd. USG ... of 1.0lO is typically excreting just'" much protein ~r day as cot 2 th at h", a urine [pro,"in] of 200 mgldL and • USG ... of 1.040. Both cots are protein uric. 6. Conclusions a. In results &om rourine urinalpes, the USG ... helps determine the significance of estimoted solure concentrations. b. A po.itive chemical ",. ction .hould first ~ considered. qualitative result (substance present ), and then the .ignificonce of the positive .. action can ~ weighted according to the smngth of the rraction .nd the USG .... B. The physical .nd chemical pro~rties of urine may ~ different .fter centrifugation if enoUJ:h particles sus~nded in the urine prior to centrifug.tion. I. The .. following valu .. m.y ~ the same prior to and afier centrifugation (unle .. the heme pigment in erythrocytes interfer.. with the rrading of color changt.): a. Color (if due to pigmented solute .nd not pigmented ceUs or ouspended particln) b. USG ... {bur the line m.y ~ mo .. difficult to read if the urine is cloudy} c. pH d. Protein (unless hemoglobin i. prestnt in erythrocyt.. ) • . Glucos. f. Ketone g. Heme (if the positive reaction is due to &.., hemoglobin, fr.., mrthemoglobin, or myoglobin rather than erythrocytes) h. Bilirubin 2. The following values may ~ diffe .. nt prior to and .fter centrifugation: a. Color (if due to pigmented cell. or other sus~nded partid •• ) b. Protein (if hemoglobin in the erythrocytes is re.cting with the pad) c. Heme (if intact erythrocytes are present)

"r,,,e

II.

pH (neg.tive logarithm off= [H1 ) of urine A. Physiologic processes I. The pH of urine it affected by mony renal and extrarenal facto ... Carnivo... usually have acidic urine, where", herbivores usually have .lkalin. urine unless they are on milk dim. 2. Typicol urine pH in healthy mommal. val}' .mong .p«ies: dog. and cou (6.0- 7.5), and horses and cows {7.')-8.5} 3. Much of the W that is excreted by kidney>< is incorporated into other mol<"CUln (e.g., NH:, H.PO,-, .nd H,O) B. Analytical concepts I. Principle: basal on the double indicotor synem chat is .. nsitive to changt in [H+] . Th. indicotor does not detect H+ ~ing acreted in NH' or H,PO,2. Th. reagent strip has. pH indicator pad with a rangt of 5.0-8.5. R..oulu are rq>Oned to the n..... t 0.5 unin (acept 5.5 r.-action with some "'ips). Th • •ynem is d.. ~ned to provide .n mim.t. of urine pH .nd do.. not r""iace the more p=ist pH met .. wh.n quantitative .nalysis is needed. " 3. Abnormal urine color (pigmenturia) moy int.rf... with vimal interpretation of the color changt in the reagent pad.

'"

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY Table 8. 11 . Major di.ord ..... o r conditio"" which cau se abnonnal chemistry results in a rouline UA

Aciduri<>

• Ex~{ed in he:dthy cunivo,eo, omnivore., and herbivores on a milk dirt 'A<;ido=, rome m<'l.bolic and potentially with r.. pimory 'Associ.tffl with hypochlo,,"mic metabolic alkaJo.;. (J>=Idoxical.ciduri.) Hypokalemia W production by Meteri. Proximal tubular acid",", (if HCO,- i. dq>Jered) Alkalinur;" 'Ex~ted in hr :dthy hrrbivor .. and .fter meal. in monog:lStric mamma], (:dkaline tide) 'Ur.,. degradation: spomana>u, in older ",mpl.. , initiated by ur.,.~ntaining bacteri. AlkaJ05e:'l, .ome meubolic and potent;"lly with respiratory Proximal tubular acid",;. (e.rly) Prouinuria r .. renal (ov
F:d... po,iliv~ r~,ction (..., th. tnl) H~m~ poJiliv,," ' H~m.turia lpathologic, i' lrogc:nic, ~m:d} 'H~moglobinurio

Myoglobinur", M~th~moglobinuria

F:d... poJiliv~ r~,ction (..., th. tnl) Bilirubinur", ' Exp«IM in ",bnanti. JJy con""ntrat~d 'H~molytic di ..,..,. 'Hq.atobiliary di..-ases F:d"'p",ili..., r~.ction (..., Ih. tn l)

urin~

of h....hhy dogs

, A r.tlliv.ly common dio.... or condition 'In most ,..gmt 'J""'m~ the hem. t..l;' c:dltd either the "blood' "" or the "ocrult blood" ""~ .JthOllgh the ....y ;, d<>ign«! to d
4. Contaminalion with buff.. &om an .dja""m prol~in r'""l:ent pad may f:d,dy drcr ..... th. urin~ pH result. C. Aciduria mgg<::s{' . n inC,.asM KCmion of H+ (T.bl. 8.11) l. It i, ap«tM in G1rnivom;, some omnivom;, and herbivores on milL:. H+ i. produ""d from prouin di.".

8/ URINARY SYSTEM

457

2. Acido=. respir>tory:md som~ m~tabolic: Th~", is a n~t incr...... in H+ =retion (H+ and NH') by proximal and dist:d tubuJ.r cdls: the =retion is ~nhancffl by drer .......! atracdlular pH. 3. Hypochlo.. mic meubolic alkalo,i, (= p. thogc:nesi, in Chapm 9: Bicarbonate Con~nt"'tion and Total Carbon Dioxid~ Con""'ntration. =t. III). 4. Hypokal~mi.: H+ i. =ret«i. and typ<: A intm"lat«i ~JJs =orb K+ through H+_K+_ ATPast pump. that apprar to be: most actin when th~ .. i. a nat~ of K+ d~plffion. 5. Furosc:mid~ th~rapy: Th~ H+ =mion ""'y be: increa..d bc:cau.. ofhypokalemi<> or incr .......! by other factors (SOn~r in th~ loop of H~nl~ i, block«i by furosemid~).' 6. Proximal r~nal tubular .cido.is (if HCO,· dq>let«i): A dre",ased con.. rvation of HCO; by proximal tubule. provides mo", HCO; to buff.. mo .. W in tubular fluid. and thus th~ urin~ pH is g.... t~r th. n apect«i in an .cidotic :mim. l {which may be: alkalinuric}. How"""r. wh~n pla,ma [HCO,-] drer ........ th~ r~maining tubular function may be ~n0"l:h to conse"", th~ filt~red HCO;. Then. th ..~ will not be: ",flici~nt HCO,- in th~ tubular fluid to buff~r th~ H+, . nd aciduri<> may be pr=nt. D. Alkalinuri<> SlIgg~m • drereased acr~tion of H+ (Tabl~ S.II) I. Ur .... splining ot hydrolysis: Br~akdown of ur.... rd~~, two - NH, group' that ~ach quicklyaccq>ts H+ ion to form NH'. Rc:moval of fiN H+ from urin~ makes th~ urin~ mo", alkalin~. a. I t may occur with th~ sponuneous degradation of ure;, that occurs with dday«i complffion of urinalyses. b. It may be cou...! by ur~...,-containing bact~ria {~.g .• Staphyloaccul . nd Prouu,}. ~ith~r in vivo or in vitro. 2. Respiratory alkalosi" Prob.obly less W i, =reted by th~ distal nq>hron bc:ca"", of l~ .. nimul.tion of th~ H+_ATPa .. pump. 3. Distal ren:d tubular acidosis: Drer......d H+ =rffion by th~ di,t:d n~phron con leod to an inappropriatdy high urin~ pH {> 6.0} in th~ fa~ of acidosis. Th~ pH ""'y not

be

alkalin~.

4. Proximal III.

r~nal

tubular acidm.is (ste

th~

aplanation in

th~

pr<effling .
Prot~in in urin~ A. PhJ"'iologic proc=:es l. M:my small prouin. {mu:dly Mr < 68.000} am p"" through th~ glom~rular filtration b.orri~r. In most h~althy .nimal •• the prot~ins ar~ resorbed in th~ proximal tubul ... and thus nry litcl~ to no prot~n i. dffreted in urin~ sampl~•. 2. Urin~ of healthy dogs may conuin a m.... surabl~ [prot~inl without cl~ar md~n~ of urinary tract di, ....... a. Most of th~ protein i. albumin. b. Dogs (n = 145) with con""'ntrat«i urin~ (l.020-l.045) and without evid~n~ of urin. ry tract di ....... hood neg.tiv~. tra~. or I + r~.ctions with . dipstick r~ag~nt p"d: 4-65 mgldL with • CooJIW.!i~ brilli<>nt blu~ method and 4-95 mgldL with a trichloroa~tic xid mffhod. " 3. TIlmm_Honfoll J»'Dtdn i. a mucoprot.in that appa .. ntly is ,re.. ted by th~ thick aw::nding limb of th. loop of H~nl~ . nd part of the distal tubul~ and collecting ducts. It i. 1Olubl. abov. pH 7 but insolubl~ below pH 7. It is • major rompon.nt of hyalin. cam .nd thought to be part of th~ matrix of granul.r casts.

458

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

B. Analytical

OODcq.ts

l. Ikagtnt mip method

a.

Principl~

Th.

r~.m

p"d contains a coJorim~ric pH indiclor {IHrabromph._

nol blue} at :ocidic pH . Amino groups of nrgativdy charCftl proteins bind the dye .nd change th. pad', color. b. Chang., in the oolor of the pad oorropond to .nimatM [prouin] (Tabl.8.10 ). c. Abnornul urine 00101 (pigm.ntmia) =1 jlu.rf• .., with r.~nt p.d oolor .nd th".fore with estiJrultion of [protein]. d. R may be, f. [,dy incrnsnl in highly buff.r<'d alkaline urine {i.• .• :> 8.0), in mod.mdy alkaline urine if highly OODctmm.d," or in uriM th.t oonc.im quatern.ry :nnmonium ullS or chlorhaidin•. •. Analytical .. ruitivity and ,!=ificily

{I} Th. m",hod d",=, .lbumin !>HIe, than globulins, which""" Jess nrgat;vdy clurgffi. Protein in cdl, (• .g., .pithdi.] cdls and leukocytes) ",.el1 vur poorly with ttagenu.

oon"'ntntions nNdal to gi"" a tne;., to I + r~~ction: albumin {14-21 mgldLl. ct-globulin (20-30 mgldLl. ~_globulin {40-50 mgldLl. 11:lobulin (> 1,000 mVdL), light..roain prot~in' (26-52 mgldLl. and h~moglobin (5- 50 mVdL}""" 2 SSA turbidity a. Principl~ Proteins u e d~naturrd by acids and form a pr«ipitate that is Sttn as incrrasrd solution turbidity. Urine that i. hazy to cloudy should be e;.,ntrifugrd prior to SSA turbidity testing. b. Remit. may be apressrd on a visual turbidity ""Ie (I + to 4 +1 or vi.u..tly comparrd against nandard solutions to interpolate cone;.,ntrations. There ~re ..tso spectrophotometric SSA methods that provide more quantitati"" results. There i, a lack of interlaboratory nandardization for reporting SSA test results. c. SSA re~m with ..tbumin better tru.n globulins (reportrdly 2-4 tim .. as well) ~nd will det«t Bene;., Jone! proteins if cone;.,ntrations are mfficient. d. Fal.ely increased r.,.ding. con be causM by X_ray rontrast malia, tolbuumid~. penicillin {mas!ive dose}, mlfisouzole, tolmetin ,odium, and turbidity COusM by coprecipitation of c'}"uls bea"", of the low pH ofSSA. e. Falsely da:reased r.,.ding. con be causrd by highly bufferal alkaline urine."'''' C. Proteinuria (Tabl. 8.11 and Fig. 8.9) l. Prerenal (overflow, overl~, and preglomerulu) proteinuria a. A pathologic ,ute incr.,. ... the plasma oone;.,ntr.otion of a """II protein that p..... through the glomeruur filtration ru.rrier. [f the amount of filtered protein aettds th. ability of proximal tubules to resorb it, the protein is ncretrd in th. urine. Examples: paraproteinuria (light-chain protein. including monomers with M, " 23,000 and dime" with M, " 46,OOO), hemoglobin uri<> (dimer M, " 34,000), myoglobinuria {M, " 17,OOO}, and postoolostral proteinuria (include! P_uctoglobulinuri<>) in food animal •. b. Light_chain proteins, hemoglobin. and myoglobin molecule! are d..r«trd by routine urine protein assay•. c. O""rflow proteinuri.. do not produce hypoproteinemi<>. 2. Glomeruur proteinuria a. Glomerular disease damages the filtntion barrier and d«re.... .d«tivt permeability. The glomerulu. become! inc",..illl:ly permeable to largu proteins or to {2}

Prot~in

45'

8/ URINARY SYSTEM

Blood Pro<~",,1

.., ""

.

L:'~::'~ '-::'. '. :: ....

Kidney"\

............ ,. Tubular'.·,: '. \ Hemor. .'

•'

~

'.: •••:':&101.

"-

-""

.:.

"""'"

& In[

Urinary bladder

..

~

& In[

"""'" j

& In[

Fig. 8.9. Four mojo' types of pro«inwi .. • In p",nn:d prot';nuri.. (P"",,,,,f). ,moJl protr;", (r.g.. hemoglobin dimen. ~ght cluj",. oad my<>globin) p"lent in tbe pl .. m ... incr""'! ooncrntr:otiOllS :ore """"trd in the urine bK..,.., they p ... through the glomrrular filtr:otion b.rri .. :uxl ..e inc.omple«1y ,rsorb.d in tubules. • In glomeruhr prot';nu,i .. (G., .... ). glomeruhr di ..n l . pro«;"'. Th"", pro«;", p1S' tbrough the defecti"" filtmion barrier :uxl .. r inromplr«ly =orb.d by tubules. '" thry .. e ="'tOO in uri .... • In tubular pro«inww (TrJ,,,},,,), prorim:d ",rut tubulr. ... defectivr, '" pro«;", tlut nomully :orr ,,,,orW from uhn filtr:o« (e,g,. 'om< oJbumin :ongic 000 inJl:unmotory prot';nurw (H",,,w, 6< Inf.), phrm. prot';", 0' hrmoglobin rnt .. tb. wi ... brcru.. of hrmonh. gr or in80mm1tion involving thr ",noJ tubules, 'rrW prlvir, we"m, wir=y bbddrr, wethn, or g
neg .. ivdy chargal protrins, Prolongffi mild or r:lpid ~e" g1omerulor protrin. uria will cau.. a ,d~ivr hypoprotrinemia (loss of plasma protrins rxcq>t for the lorg.. t forms) (... Chaptrr 7), Incrra,al amounts of albumin and In!:"r protrim a.. ",,~al on SDS·PAGE of urine from dogs with glomerular protrinuria, ..... b, In people, a tramient protrinuria that occurs .ftrr rx ..ci.. is oonsid..al • form of g1omrrulor protrinuria, 3, Tubular protrinuria a, Proximal ",nal tubul .. a", drf~ivr, '" protrins th at normally arr r=>r~d from ultr:lfiitmr {e,g" ",me albumin .nd mull .. globulins} .'" not and thu, a.. rxcreta! in thr urinr, Inc",asrd num~rs of protein band, rrpresrming proteim with a mol«uiar mass Ie" chan that of . Ibumin a.. rxpretal with SOS·PAGE of urine from do!}! with tubular proroinuri., ..... b, Tubul .. proteinuria.< arr usually a"ociatal with acure rrnal di, ...... {toxicoses .nd hypoxia} but can ~ rongenital, They do not produ~ hypoalbuminrmia. 4, Hemorrhagic or inHammatory protrinuria (aho callal ..cretory or post.. n. 1 proteinuria, but hrmorrhag<' .nd exudation may occur in kidn,>" .nd are not =rrtoryp~)

a, Hemorrhagic: hemorrhagt' into the !:"nitourinary tract due to impaired hemosta. ,is. including blood """",I "'' 'Ilag<' by inHammation, trauma, nwplasia, or other n=o,,"

4611

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY b. Inft.mmatory: exudnion of pl.. m.

prot~ins thro~h ves,d w:d[, imo th~ genitourinary InC! due to inflammation c. Th. I""lrenal proteinuria, '" the most wmmon prOieinuri ... The qu:mtity of protein lost is usually not sufficient to cau.. hypoalbuminemia, but ""'1 bt mild hypoalbuminemia COUIM by inflammation or h.morrhogr. Mon proteins d.t""t.d in th. utine enterrd filtrate from the pl:asm .. Proteins from leukocytes and epithdial edl, are poorly d~<'Ctffl by utine protein :assays. d. Evidenct to support this ty~ of prouinuf;" is usually found in OIhu urinalysi, ..suits: inflamm.tion (pyuria) :md h.morrh~ {hematu';"} . • . One must oonsid" reproductive tract .our""" (e.g., pro,utili, and estral bl....:!_

th.""

ing), ~cially in voidrd utine .ampJ... D. Other prouinuria cW..ifications: Th. afo .. mentionw d ..,ification 'y,um is not th~ only spurn. Som~ divid~ prot~inuria, into thr"" ty!"" (pror~nal. r~nal. and postr~nal) in which th~ r~nal ty!'" includ.. both glomerular and tubular prouinuria •. Anoth~r cl ...ificotion sch~m~ w..d on pr~renal. renal. :md pomemol divisions includ• • fimc_ tional and pathologic subdivi,ions of the .. mol proteinuria co!egof}'. with glomerular. tubular •• nd int.rstitial subgrouJ>5 of th~ p.rhological renal cotq;ory." [n this ""h.m •• function'! proteinuri.. are consid~r.d to k mild and tr.nsi~nt proteinuri.. COusffl by phy.iologically altered renal hmdli"l: of notmal plasJrul proteins in the aboen'" of reruol l.. ions (~.g ..... n with nerci .. or f~er ) . E. Prot~n_lo,ing n~phrop.thy and r~nal faiJur~ con""pts (Fig. 8. 10) l. Ure •• nd Cn are sm.!l mol.cul .. th at p .... freely through the glom.rular filtration barrier (.i~e;) . Some fihrat~ urra is resorba! by tubule;. Th~ reJrulining u",•• nd Cn ar~ ncr~t~d in urine .nd thus do not .ccumul at~ in blood. It has bttn pr~_ suma! that albumin does not paM through th~ glom~rular filtration barrier in hrahh and thus remains in th~ blood (dogs may k an ncq>tionl . 2. With a protein_losing nephropathy, the glomerular · .i~.." krom. more porous, and larger protein> or charged proteins that usually are ..".lla! ~nter the remol fihrat~ via glomeruli. [f the ability to ..,orb prouins is nceeded, th.n a proteinuria will be pr~sent. Th. continu'! 10.. of protein willlrad to hypoalbuminemia. As long as the numkr of functional glom.ruli is .dequatr, ure•• nd Crt will k adequately ",mova! from blood .nd notemia will not develop. 3. [f the glomerular dise= destroy. more nephrons, then renal failure ocru ... The f...... "'maining functiomol glom~ruli connot ",mon urea .nd Cn fan enough from the blood, so .zotemia develops. Proteinuria continues kcouse the "'maining function'! glomeruli .'" !",rmeable to proteins. Th~ """,rity of hypoalbuminemia iner..... because of continued albumin loss, but d.fective ncretion of H,O (associated with N .+ .nd H,0 rff~ntion) m.y contribute to hypo.!buminemia. F. Benc~ Jon .. prot~inuria (immunoglobulin light..ffiain proteinuria) I. Analysis of urin~ for Ben", Jon .. proteins is not part of. routine urinalysis. [t may k indicated to clarifY the type of proteinuria or to investigate a possible lymphopro_ lif.ratin disease. 2 Bmcr jon" protr;ns ar~ light chains (~ither I( or Aj of immunoglobulins that havr unique therm.! propenies: th~ precipiuu ktw«n 40'C and 60 'C, return to a solubl~ sUte at 100 'C, and then pr~cipitate again when cooled. The thermal properties of B.n", Jon .. proteins .'" due to the variable portions of the light-chain prouins." As d.scriba! by Ritzman and Danids," these prot.in> were first recog_ nized by WJliam Maclntyre in 1845. Dr. Maclntyre..,nt th~ urine .. mpl~ to Dr.

Healthy Animal

'-/--,,-;;-;,;-..-;;-,!----1 Kidney

Protein-losing nephropathy

Kidney

@@

,:::::::::::::"~,~ ~"A':>":::" "'" A 0 "~c :--;j'I' ..A,. ",",~I(d'
-:>., •• !p."/", ., • c

U

, " A" , , A

....

: u:/

•

:".,:

•

"..}J.'

..... .",.

•••• y ••.•

. .

.I> • 'c'

:7":" ii,:c

cu

•. .~ .....'u:

.....

A

,·······u Au C"c A" ,. C A "c U

.'

c

A

Fig. & 10 . Pro .. in.]"";"!t nrpbrop. thy and I'ftW foilu",. iHUiIr:l';O ... dm and riT... The 1000 of albumin from the \aU co>=< b~bwninomi. and protoinuri .. n.. r«rUinill£ Iiltft"l .,., sufficien. to ktq> urea and Crt ...,vwd from th.taU. ond tb .......mi. does not drwlop. • In p""'oin.looins ncp~hy . nd _:01 &ilure. 80 ... of ,1>0 fiJ« .. (and ncphro ... ) ~ boon deauuy.d. The mnaininS 20 ... are damognl. mou porou •• and allow o.Jbumin ro en«, the dam and ma. The .... of albumin from the \aU co ...... b~bwninomi. and prowinu'; .. ini"i IiItft"I.,., i..... fficieo, to Jo.ep Ufe. and ~ from.he lake. and th", "",«mi. ~ Aloo. the r«rUining ""I'luono connot adequ. tdy..,.,..,..... H,D . and thuo polyuri • ......,.,,1".

e ..

en

Ii'"

n.. .......

46'

462

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

3.

H~n'Y Benet Jone,_ who confirmffllh. findiIli:" Th= protein, are known as "Benet Jones protein." .nd not "M.clntyre proteins," ~rhaps b«aus. Bence Jones publishffi the cast dna fim (1847) and Maclntyr.'s publiation WlI. in 1850. Th. unique therm:'! pro~rti .. of light-chain protein. are .....d as the basi, of the B.nct Jones Ietffl to 56'C for I 5 min and ob",,,..! for floccul.nct or pra:ipitat... (3) If f1occulen"" or plmpit.t", are present (the test i. lIegat;", if thry :lr' absmt), th. urine "'mpl. is placal in a boiling waW bach for 3 min and n.:Iminal for a danasal amount of p=ipitat•. A d=_ in floccuJ.no: SU~, Iknct Jon .. prot, inoli .. (4) Fill« the hOI urine through a funnd with fihr paP" imo a tub. contoining a thelmOnt.uf. If Ikn"" JOII" prot~illS ~'" prestm, they should p=ipitat~ .r . bout 60·C and rali...,ln at about 40'c' Vuiatio", ill th~ hrat tes{ ""idiJYillg solutiolls, filtratioll', and tim~ im~rvals h. ve bttn d=iW, b, Th~ colI""mratioll of Bell"" JOII~' prot~i", IIttds to 145 mgldL to get a positin mult, alld r..gulatioll of th~ pH i. very important,'" c, Oth~r proteills m. y p=ipitat~ durillg h~.tillg {e,g" fibrillo~1I precipitates at 56--58 'C}, which m:d.:es imerpretatioll difficult without th~ filwillg alld ..... ssm~nt of p=ipitatioll durillg coolillg. Othu methods of d<:ta:rillg light-<:h.ill prot~illuria a, Th~ ikll"" JOlles prot~ill5 r~""t ktt~r ill th~ SSA proteill method thall they do ill the proteill r~~m p. d method of routill~ urill. lysi. proceduro;,'" The reag~nt pad tm Jruly fail to detect light-chaill prot~illuri. if th~ prot~illuria is millimaL b, If urill~ prot~illS u~ colletmrated prior to decrropho ... is, 1I0rmal light_chaill prot~ill' COli "" foulld ill the P,1:lobulill fractioll (pl. " 12J,2), c, Immullodectrophoro;is for K_ or A.-light-ch. ill prot~ill' i. the pr~f~rred m~thod of docum~nting light-<:haill prot~iIlUri. , but species_specific ami",ra u~ not .. adily .vailabl~ for illdivid""l species, Re",lu from immullohistoch~mical studies "'gge'lt that .ntibodies against human light-dtaill prot~ins cros.s-",,,,,t with light_chain protei", of domestic mamJrulI. (Stt Ch'pter 7), Bellet JOlles prot~inuri . may occur kcause the formatioll of K_ or A_light-<:haill proteins by lymphocytes or plasJrul ""ll. i. illcrrasal, a, Typicolly, th~ir prestllet is associated with B_lymphocyt~ or plasma cdl nalp!...i ... On~ study .. poned that .bout 30 % of th~ dog> with mydom. h.d a Bell"" JOlles proteilluria, though th~ .uthor> did 1I0t S{at~ how th~ prestn"" of th~ B~lIet JOII" proteins v..... deta:red or collfirmed," b, B~lIet JOII" proteinuria COli also "" cou=! by B_lymphocyt~ h~rpl",ia. By m~.'" of SDS_PAGE . lId immunofixatioll methods, light-chaill prot~illuria, w~ .. foulld ill dogs with l~i,hmani.
"";>

4,

5,

6,

IV,

GlucoK in urill~ A, Physiologic p~ L Glucost i. a rdatively small mol«:lll~ (M, = 180) that passes frttly through th~ glom~rular filtratioll b.rriu and enten th~ ultrafihrar.,

8/ URINARY SYSTEM 2. Glucost i, resorbal in the proximal tubules vi. a Na+_glucost cotr:msport .ystem that dq><:hlorite. Such rontamination may occur when samples are obtained from an namination ubi. or cage Hoor. c. Falsdy decreased r...ction, may be cau...! by aocorbic acid, ketones, and very con""'ntrared urine s;;omples. Cold urin., .. peciaUy when r... ction times are very bri.f {e.g., 10 .}, and m ..k<-d bilirubinuri a may also inhibit r<:action<. 2. Copper_reduction method {Clinirestl a. Principle: Cu""' r<:acts with a reducing subsuno: {•. g., gluoo .., fructo .., lacto.. , galactoso, maltose, or pento..} to produ"", cuprom (Cu+) oxide and cuprou. hydroxide and thus a oolor chan\:". b. Semiquantitative results for the "andard method are negative, .. 250, " 500, .. 750, .. I(xx), or " 2(xx) mgldL The method is prob. bly mo .. aCCII .... than the reag.nt nrip method but requires a gr ..... r ron""'ntration lOr detection; that is, the detection limit i, higher. c. The method may be used to confirm questionable positive reagent mip result, such as v..hen urine color interferes with interpretation of the r.-agent pad oolor. d. Fal.... positiv...actio", Jruly be am...! by ""'phalosporins, formaldehyd., and .>corbic . cid (if rono:nt .. tion, are high enough). Reactions with sugars other than glurose might be ronsidered false_positive r...ction,. C. Glucosuria {glycosuri..} disorders (Table 8.11) l. Hypergly""'mic glucosuria a. T ..",ient or persinent hyperglycemia re.ulu in more glumse in the uh ..filt .. te than can be resorbal by proximal tubules. b. Hypergly""'mia is typically concurr.nt with the glucosuri. , but • tr:onsient hyperg!y""'mi.. and delay in bladder emptying may JruI,k concur.. nce. 2. Renal glucosuri.. {normogiy""'mic glucosuri..} a. T ra",i.nt or persin.nt glucosuria resulu from d.fecti"" resorption of glumse caused by damaged or abnormal proximal tubules. b. Tubular abnormalities am be acquired or congenital {I} Acquired; proximal .. nalmbular toxiro.is or i.chemi.. {sometim....f....d to as acquirrd FalUt",i rynd,.,,,,u} {2} Con\:"niru; Fanooni .yndrom. and pu.. primary renal glucosuri a (basenji, Norwegian dkhound, and Shetland ,h..,pdog}7<

464

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY 3. GluCClSt in tubular fluid wiU em", osmotic diu ... is (H,O i. "hdd " by th. g1uco,," in tho tubular fluid. es~;alJr in tho proximo! tubules) and thus au.. da:rn=! ",ill coD"'ntr>ting ability and increastd utin. volum. (polyu'"').

V.

Koron .. in uriM A. Physiologic proc=es l. AcrlOacttatr, ~_hydroxrbutyrat., and action.... !.:elon. Ixx/ies. bur only acttooc_ .taU and .",tono han tho ch.mical struClur. of keton••. Act,,,,,,,,,{;C acid and fl.hydroxybutyric acid at. ketoacid. that a.. productd by h'r:nocyt •• bur dis.wci.t. >I phy'iologic pH to th. ir :mionic form and W. 2. Keton. bodies our not exptct.d in tho min. of h •• hhy m:munah that hav. an

ad«J.Ut~ .nd ~_ hydroxybutyrat~ ar~ nonr ..orbabl~.' B. An:dytical con<:qlts l. Ikag.nt mip m.thod {fu,..,r Diagnonics} a. Principl~ Ac.t""e..tat. {moody} and ."",tone (.bout \0 % as .... tiv. as ae..toac_ ~tate) form colored complex.. with nitropru ... id~. Th~ "",ount of color chan!:" rdla:u th~ amount ofkaon.. present (T.bl~ 8.IO). Th. reag.nt system dox. not .. act with ~_hydroxybutyrau, the kaone body that d.,.. not h.v. a krton~ ch~mical structure. b. F:d ..... positivt r~.ctionJ may b. caused by highly pigm~nted urin., l.-vodo"" metabolites, .nd ",me compounds that h.vt sulfhydryl groups {e.g.. captopril .nd C)"tin~l ." T .."", ..actions may occur in urin~ with high USG .nd low pH. c. In theory, ~_hydroxybutyrat~ can b. ronvtmd with H,O, to .e..toacttate so that nitroprusside methods can b. used to da..cr ~_hydroxybuty.. t~. How.-v.., urine cone..ntrations of p_hydroxybutyrate must b.:> 200 mmollL {using 3 % H,O,} or:> 50 mmoUL (using 30 % H,O,) to produe.. mor~ than a tract ...ction. At such ronctntrationJ, th~ [acttoa=at~l would probably b. great .nough to b. d.tect~d routinely.'" 2. Acrt~st tablet m.thod {fuy.. Diagnonics} a. Principl~ It is the same .. with the =t:~nt strip method. Color chan~ is ...,i~r to detect, so it has • low.. detection limit than som...agrnt pads (about 5 mgldL in urin.) and thus may b. used to confirm tr.e.. or questionable .. actiom on r~ag.nt ""ds. b. The m.thod may b. used '" a qu:ditativt ..... y for blood, plasma, urin~, and milk. C. K.tonuria (Table 8. II I l. Krl,muriil oa:urs wh~n the mobiliLotion oflipid. i. inc",asnj ba:;ou", of. shift in .n~rgy production from cubohydrates to lipid. {~.g., in di.b.t .. mellitus, starv.tion, and hypoglye..mic disord ...}. E"Jrct ... ivt ~_oxidation of fatty acids in h.""tocytes grn~rates mo", acttyl~nzym~ A than em b. used for gluconeogen .. is .nd triglfC"rid~ .ynth.. i•. Exe..ss ~-<:o<:nzym. A stimulates hq.atic kao!:"n.. i. and thut increased formation of krtoacids, leading to incr ... sed ketone lxxIi.. in blood (Jmonnnid) . Krtone lxxIi.. ar~ e",ily cl~.red from blood .nd are ncr~ted in urine.

8/ URINARY SYSTEM

."

2. D,""",asaj insulin a.ctivity and increasnj glucagon .ctivity promote ketog.n ..is. Such ch.nges may result &om .ither phy.iologic or p:uhologic proc:.=e:l. D. The p>thologic stat. cou=' by acess ketog.n .. is is coned Imods or, if labomory data indicot. a concumm acidosis. k,lrJIU:u/o,u. In ketosis, prim..ily P_hydroxybutyrat. (which is not d.t,""t.d by the .....y) is acmed in urin., but aceto.~Ut. and .C~Ut•• nd P_hydroxybutym. {both of which are anions} oblig.t.. acr.tion of cotions (• .g., N.+ or K') by the kidn'Ys. Prolongnl ketonur", may co .... N.+ or K' depletion that comributes to hyponatrem", .nd hypokal.mia. VI.

Hem. in urine (tm frequ.ntly labelrd blood or IKCUIt blood) A. PhJ"'iologic con~pts: H.me-ronuining compounds .'" nOi apectrd to be pre.. m at det,""ubl. amounU in the urine of h.althy mammals. B. Analytical concqJts l. Ik>gl'm mip m.thod a. Principle: In some syst.m., the peroxidase activity of hem. cotalyus the oxid._ tion of .... toluidin. to • blue compound. In other syn.ms, the peroxida.. . ctivity of h.m. catalyzes the oxidation of a chromog.n (e.g., t.tramethylbenudine) to • colorrd compound. b. Heme may be from h.moglobin, methemoglobin, or myoglobin. Ifimact erythrocytes . '" pr=m, the cdls .'" ly=' in the "'>gl'm pad to produ~ .ith.. specldrd or solid color changes in the pad. lksulrs m. y be described in .. mi_ quamit.tive terms or as .n enimatrd [h.moglobin] (T.ble 8.10). c. Falsdy increased reactions may occur with oxidiz.ing oompound. mch •• hypochlorite (bleach) and microbi.t or leukocyte peroxida... d. Falsdy d,""reased reactions may occur with. high USG or with urine that comains coptopril, aocorbic acid, or formald.hyd•. Erythrocyt.. may not be d.t,""tw if th'Y are not suspendrd in the Wt sample. 2. Hemat.. t ubl.t method a. Principle: It is the .. me •• with the r"'1:em p>d, with modificotions. b. The m.thod may be used .. a confirmatory test .nd al"" to test for fet"al occult blood. 3. Unfortunately, Iher. is not. widely av.ilabl.labo.. toty .....y for diff",.miating myoglobin from h.moglobin in dom.. tic mammals. Diff..emialion i. umally a.ccomplimrd by clinical drductive r,""",ning {s,"" T .bl. 3. l2}. a. A simple pr,""ipitam test using ammonium sulf. t. is oonsiderw umeliable. In theory, h.moglobin, bUI not myoglobin, pr,""ipiut.. in 80 % ammonium mlfa,. solution, wh ...... myoglobin pr,""ipitates in . 100 % ammonium sulf. t. ""lution. Howev.., d.natu",d myoglobin will pret:ipita,. in the 80 % souhion and thus be incorrectly classifiw .. hemoglobin.'" Also, a !imil.. erronellu. cbssificotion wiU "",ur if the urine pH is nOi adjustrd to 7.0--7.5." b. Immunologic methods ",quire species-specific amibodi... El,""trophomic and spectrophotometric procedure; may be .mployw. 4. Th. hem......y is more .. nsitive than the protein • ....y. Therefo"" when smaU amoum, of blood are in th. urine, the", may be marked hu"" positivity without the hemoglobin cou,ing. positive protein ",.ction. C. H.m.... positive ",xtion in urine (T.bl.. 8.11 . nd 3.l2) l. Hem.turia

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

'"

a. Erythrocyt~, ~nt~r th~ urin~ vi. h~morrh"l:~ into th~ urog~nital tract. H~mor_ ,hag<' may ~ ",uK
P''''''''' (•.

'''''If

,.dimom. 2. Hemoglobinuri.

a. During clinirn intnvaKulor hemolysis, pl •• rna hemoglobin dim." (M, " 32,000) Jruly form from hemoglobin (M, " 64,ooo), pas> through the glomerular filtration barri."., .nd .m.r the uh",fiilrate when haptoglobin i, .,tuntal {,'"" Chapw 3}. If not completdy resorbal by the ren.l tubul .., the hemoglobin dim.... are acreted in the urine. b. IntnVll.lCular hemolysis of sufficient severity to causo hentoglobinemia .nd hemoglobinuria an bt aused by. variety of di,orders that au...nemia {_ Tabl. 3.lO}. Hemoglobinuria may aho bt creatal by lysi' of erythrocytes .fw they enter urine. In such <:aW;, hemoglobinemia mould not bt present (unless erythrocytes were lysed during blood collection or handling). 3. Myoglobinuria a. Myocyte net:ro,i, or dam"l:' can rei.... myoglobin {M, " 17,<XXlJ from the myocyte into the interstitial fluid, lymph, .nd firuolly blood, from which the ,mall protein easily passes into the glomerular fihm •. If not rompletdy resorbnl by the renal tubul." myoglobin i, acretet! in the urine. b. Myoglobin i, rapidly deuet! from plasm., .nd thus pink plasma is not npeclet!. Myoglobinuria is associatet! with acute myopathies caused by trauma, =ive exertion, or e"'rtional or paralytic rh.lxlomyolysi, ofho ..... {azoturi.}. 4. M.th.moglobinuria a. Methemoglobinuria may rontribute to • potitive urine heme reaction in the following situations {concurrent hemoglobinuria i. apectal}: {I} Wh.n hemoglobin {or hemoglobin dimer} molecules.re in the urine, some of the ferrous heme will undergo .pontaneous oxidation to ferric heme {forming methemoglobin}. {2} When there is an intraVll.lCulor hemolytic .nemia aused by an overwhdm. ing ""pcuur. to oxidants, Klme of the lysed erythrocyte! will rd .... methe. moglobin 10 pla,ma. Then, methemoglobin dime", can enter the glom.rular filtrate. b. Both metheme .nd heme have peroxidase .ctivity, '0 either mol",ule will give a potitive heme (occult blood) r..ction." VII.

Bilirubin in urine A. Physiologic processes I. Bilirubin i. not npeclet! in the urine of domestic mammal, other than the dog. 2. Bu furms from the degradation of heme, primarily from hemoglobin degradation in macrophag... After conjugation in hep"tocytes, Be i, ""aetet! via the biliary ')"ltem.

8/ URINARY SYSTEM

.67

[f rrl>ffl h~moglobin of hemoglobinuria} to Bu .nd then Be, which con be eICreted in th. urine, 4, Usually, Bu is thought not to be in urine bec.Ust it is bound to pl""m. albumin, which is not filt~=' by mon glomeruli, Ho~""r, become app'lf~nrly h....hhy dogs nuy hove mild albuminuria and glomerul... di..,.= con couse prot1) and may be ustd to confirm trace re.ctio", on ~nt lIict.:.. Reag.nt p.d ..",h. mould be r~port~d wh~n th~ [ctot", confirms positivity. 3. Bilirubin mola:ul.. are drgr.ded to biliverdin by exposure to ultraviolet light. Bili""rdin is not det~ed by the bilirubin ...ction. B.fore .nalysis, urin~ should not be dira:tlyexpostd to ultraviolet light for more than 30 min. e. Bilirubinuria (T.ble 8.11) I. Ex"""ive bilirubin formation (in hemolytic sut<.) or imp.i=' h~patobiliary excretion of Bc inCJeas~s plasma [Bc]. Be 1"'''''' e""ily through the glomerul.. fihration barrier .nd i. excreted by the kidney•. 2. [n dog., a positive bilirubin r...ction must be int~rpreted b.sed on knowledge of the USG ... Concentrated urine ( 1.025-1.040) of h~althy dogs frequently produ"". a small bilirubin re.ction. Moderat< reactions occasionally occur with more con""n_ trated urine (> 1.(40). 3. BreaUst of the low renal threshold for bilirubin in most animals, .. pn;ially dogs, bilirubinuria may be det~ed befote hyperbilirubinemia or icterus is deta:ted in hemolytic . nd hepatobiliary diKlrd~rs. Hyperbilirubinemia without bilirubinuria suggests. falst_negati"" urine bilirubin r....ction or the dq:radation of urin~ bilirubin before sample .nalysis. VI[1.

Urobilino~n

in urine A. Physiologic pro==! I. Urobilinogrn, a colorless compound fotmed from the dq:radation of bilirubin in the intestine, is pa.<sivdy.bsorbed by intestinal muC<>S;L 2. Urobilinogen that is not removed from portal blood by h~patocytes ente.. the syst<mic blood, from which it is excreted in th. urin~.

...

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

B. Analytical ooncq.ts fur l.

Princip[~

th~ r~m

Urobilinogen

r~~CU

'trip m",hod with p-dimechyL.mino~nzaldehyde to form a red

pigment via a modifi.d Ehrlich', alddtyde ..action. The dq;<'"" of oolor change ""f1«:u the [urobilinogen] (T .bl. 8. IOJ.

2. Ullin: Ehrlich units (EU) or mgldl; I EU = I mgldL 3. The:may connor be, u...:! to d.I,",,{ d,""r•• ~ urobilinogen ooD""ntntiollS ~Ust the lowest r.~ding is 0.2 mgldL (Muhinix) or 1.0 mgldL (Chern'trip), the conetn_ nation exptct.d in urine of healthy ~njmals. Th". i. not a nq::atj"" r.action p~d on the ""~nt strip, 50 [hi. m",hod cannot be, ustd to det«! d.cr•• 1ffl urobilinog<'D coD<:entntions in urin•. 4. F:dsdy incrrasnl ,<suIts may bt causal by aminosalicylic acid, sulfonamide;, .nd aminobtnzoic .cid. 5. F:dsdy d<'C.rasN ,.xtio", /lUy be, cal1!lt'd by fornulin. [n .ddition, urobilinogrn degrades easily when urine is n pooffi to uhnviolet light. C. [nne~sffl urobilinogenur", I. Hemolytic di ..~ .., will caust the increased formation, biliary ncmion, and degndation of bilirubin. Thus, incrn!ed urobilinog<'n formation .nd excretion are nptttet!. However, incrn.. d urobilinog<'nuria i. not ron,istendy found. 2 Mammal. with hepatic or biliary di,.....,. Im)' ocauioruolly ru.v. increaset! uriruory excmion of urobilin0c<'n, perhaps from the degradation of bilirubin in the uriruory

,ysum. 3. Mon vet ..inari.ns do not find the ........ ment of urine [urobilin0c<'n] to bt diagnos_ tically fruitful. IX.

Nitriu in the urine A. Physiologic proce<ses: Ctnain Gram_neg>live bacteria can ret!uce nitrate to nitrite. B. An:dytical concept' for the reagent strip method I. Principle: Nitriu rncts with p-arsanilic .cid to form. diawnium rompound tru.t roupl .. with N_l_naphthyl ethylenediamine to form. pink product. 2. F:d.. ly incr ... offi ",action! =y ... ult from an:dysi. of nale urine rontoining con_ taminant bact""'. 3. F:dsdy decrrastd reaction! =y occur if the", is insufficient time (< 4 h) for bacterial ret!uction of nitrates. Asrorbic acid {especially when USG i, high} Im}' produce: f:d .. ly decr ... offi "'.ction! in dog and cat urine." C. Nitrite-positiv. "'~ction l. Suggrni"" of. Gr:lm_neg>tive Mcter",l infection in the urin:ory tract 2. Detection of nitrites in urine i, • screening procet!ure for Gr:nu_negati"" Mct..i:d uriruory infectiOn!. Significant bacteriur", Im}' be pre"'nt but undetectet! with the nitrite ....y. D. Man v.terinari.ns do not find the urine nitrite test to be diagno,tically valuable btcau", it d~ not d etect .ignificant bacteriur", ron!istendy.

x.

Leukocyte este.... in the urine A. Physiologic proce<ses: Leukocyte, in the urine rde= an est..=. B. An:dytical concept: The ....y;. consid..et! to be inadequau for the chemical detection of pyuria in dog, beau.. of f.l",_negative ",.ulu," and f.l"_p<>!iti"" reactions are a problem in cats."

8/ URINARY SYSTEM

'"

URINE SEDIMENT EXAMINATION I.

Gen~ral

A.

B.

C.

D.

E.

F.

II.

roncqm using the .am. urine volume for sedim~nt prq>.ration will ~nabl~ • mo", critical a"e"ment of the Rmiquamitativ. r~sults. Th. quamity of urin~ used may v"'}' betWttn labontori.. but is usually 5-10 ml. About twice the amount of sediment would be expected from 10 mL m..n from 5 mL. Expected findings in thi. chapter are b.... d on th~ sedimentation of 5 mL of urin •. The cont. nts of urine are not suble. so urine should be analyzed within a few hours (id.ally within I h) of rollection. During sample storage. cells .nd casts deteriorat •• crystals may di"oln or form. and Mct.ri. may di~ or proliferat • . The method of urine collection should be considered wh.n urinal)";' and especially urine sediment results ar~ imerpreted. A voided .. mple may contain cdls and Mct~ria from the genital tract. A catheterized sampl~ may conuin mo", q.ithdial cells or . rythrocytes beause of urethral traurruo. A <}"tocentesis .. mpl~ may conuin blood beaus. th~ n«dJe .ra_ tions Jruly be hdpful. Con
Leukocyt.. in urine ..dim.m {Plate lIB. D. and E} A. Physiologic processes: A ftw leukocytes (ftv..er th.n about 5/hpl) may be found in the urine ofh.althy mammals. B. Analytical roncq.ts l. Leukocytes in urin~ sediment are enumerated as the rang~ or mean ofl.... kocytes ... n in mon 400x fields (hpl) 2. Leukocytes.re not routindy differ~ntiated wh.n found in urin •. On suined slid ... prq>. red from fresh urin •• most of the leukocytes will be n. utrophils and macro_ phages. though eosinophil. m.y OCClSionally be prominent. Small q.ithdial ceUs may look .imilar to l.... kocyte.. 3. Leukocytes d. teriorat. in urine within. few hours: thus. numbers diminish with tim~.

C. Pyuria:

in"'~ • ..d O~ukocyt'l

(number/hpl) in urine sediment cr.ble 8.11}

Table 8.1 2, Major disorden or condilion. thai cau.e abnormal findings in urine scdin,enl a aminalion. Pyuria (Plm lIB) 'Urinary trael inflammation, inf..crious:md noninfeclious ~nilal lracl inflammation Hem>luria (PI al' IIC) 'Urinary trael hemorrh~ pathologic {damaged vessel. or coaguJ.lion defecl}, iauo!:"nic ~nilal lract hemorrhage: p.thologic. estrus ~teriuria {Plate lID .nd E} 'Infection in urinary or !:"nital tract 'In vitro growth or contaminalion Cylindruri a (Plm I IF-H) C.n be found in low number> in healthy.nimals AMociated with glomerular proteinuria {especially hyaline cast.} 'A<-,i,~ l~ll~I'UI",] .. cd] Jq;~Il~["jOll jlllLtJJllll~ljOll, O[ [,<JllO,,[,"l;"" llq>]"""", llq>]"j,j, Epithdial cells in sediment {Plate III and J} 'Cm be found in low numbers in healthy.nimal, 'Inflammalionlhy~rplasia in !:"nitourinary lract Neoplasia in genitourinary tract Crystalluria (urine pH in which crystals tend 10 occur) Ammonium {bi}urate {pH usually s: 7J (Plate II K): .... n in h..althy mammals: common in D.lmatim dog. and English bulldogs: suggest liver dysfunction and porto.ystemic shunts in dog. and calS 'Bilirubin (pH <: 7) (Plm IlL): Sttn wilh bilirubinuria 'Calcium carbonate {pH:!: 7} {plate l3A}: h..allhy herbivores; not seen in dogs and calS Calcium oxalate dihydme (pH usually s: 7) (plale l3B): .ecn in h..allhy mammal" suggest possible hy~rcalciuria or hyperoxaluria {including Clhylene glycol toxicmi, or ingestion of oxalate_rich pI.nts such as HIllqgmm and Amll1'llnthu. in ruminants} Calcium oxal.te monohydrate (pH usually s: 7) (plale l3C): suggest pos.sible hypercalciuria or hy~roxaluria, especially ethylene glyrolloxieosis Calcium phosphate (pH usually:!: 7): Sttn in h..allhy dogs, dogs with calcium phruphate urolilh" or dogs wilh ~rsistent alkalinuri a Cholesterol (pH s: 7) (Plate 13D): uncommon; Sttn in healthy dog>; su~st hy~rchol"lCrolemia and prolCinuria as scen with protein_Io";ng nephropathy Cystine (pH usually s: 7): ra ..; ... n with cystinuria and suggen li""r di ..... Drug crystals (sulfa, ampicillin, conl",,' media, primidone) {Plate l3F}: reflect p.... nce of compound in urine Hippuric acid (pH usually s: 7): very ...., a.. confused with calcium oxalalC monohydrate crystals Leucine (pH <: 7): rare; SUggesl liver di ..... 'Magnesium ammonium phosphate {struvi,.} (pH usually:!: 7) {Pl ate 13E}: common in dogs and caU with alkalinuria, may be associated wilh u..... _producing bacteria Tyrosine (pH <: 7): ra ..; 'ugg<:Sllivcr di ..... Urate, sodium (pH s: 7J or ammonium {pH variable}: Sttn in h..althy mammal.; common in Dalmatians .nd English bulldogs; suggen li""r dy.function .nd portosystemic shunts in dog> and caU Uric acid (pH <: 7) (Plate 13G): .. me .ignificance •• urates x..nthine (pH :s; 7): ra .. ; may reflect treatment with allopurinol or mCiabolic defect Other organi,ms Fungi, yean (Candida 'P" B/asromyrtO 'P" Cryprou«w sr,) and hyphal forms (Pl.te l3H and I) Nematode ova (DiqfflJphymd 'milk, Capil"'"" plira) (Piau 13J) Microfilaria (wilh hematuria) • A ••J.ti_1y COHUDOn
470

8/ URINARY SYSTEM

'"

l. Urin.,y tract inHammation

a. In!L.mmation of mucosal or submucosal ti ... ues or renal pa"'nrnyma m.y cau.. leukocytes {moody neutrophils and monocyt ..} to migrate from blood to urin•. Inft.mmation can be caused by infections (by bacteria, fungi, or po.rasit..) or noninfectious proc.....s (neoplasia, urolithiasis, or necro,is). b. Th. method of sample collection (voided, cystocent.. is, or cathetem..tion) may help determine the site of the in!L.mmatory proce ... ; that is. leukocytes might be from anywher. in th. genitourinary tr.ct in a voided .pecim.n, bur their p.... nce in a cystocentesi, sample indicates the source i, 5Omewhe.. from the kidneys to, :md induding, the proxim:.! urethra. Other dinical information may .lso help determine the ,ite of the inft.mmation; for .xample, pollakiuria and stranguria would suggtst lower urinary tract involvement. whe",,,, fever, n.... tro_ philia, or azotemia would suggest nephritis. Leukocyte casts indicate inHamma_ tion associated with ",n:.! tubules. 2. Genit:'! tr.ct in!L.mmation a. Leukocytes may enter urine from the male or fem:.!e genit:'! tract befo", (•. g., pro,Utoj or during (' .g.,. prq>utial or v>ginal 5OUrce) micturition. Th. chance of contamination during micturition is reduced if the "'mple i, collected midway through micturition. b. The inflammatory proce ... mayor may not be of dinical signific;once {e.g., mild posthitis}. Ill.

Erythrocytes in urine sediment (Plate IIC) A. Physiologic proc= A ftw.rythrocytes (fewer than .bour 5/hp/) may be found in the urine of healthy mamm:.!s. B. Analytical concq'" I. Erythrocytes in urine sediment a", enumerated as the range or mran of .rythrocytes ... n in most 400x fields (hp/). 2. Erythrocytes may crenate in urine and can be confused with leukocytes in non_ stained sediment. They may .... ell in hypotonic urine. 3. Erythrocytes may lyse in urine either before or :ofter urine is collected :md appear as ghost cells. lysis tends to occur in unconcentrated or mildly concentrated urine (USG"" < 1.015) or very alkaline urine, especially if analysi' i. dd.yed. 4. If .rythrocytes a", Sttn in the urine sediment, the hem. rraction should be "",iti"". C. Hematuria: incr.... ed [.rythrocyte] (numberlhp/) in urine sediment (Table S.l2) l. Pathologic hentorrhage a. VascuJ.r damage c;oused by trauma, inflamntation, renal infarcts, or other pr~; common in animals with urinary tract infection! b. Thrombocytopenia, thrombocytopathia, or \IOn Willebrand disease and the ..,ult:mt poor "'pair of sm:.!l "".... ls c. Coagulop:"hies (acquired or congenit:d) d. Abnorm:'! erythrocyte ,hap" h"". be.n reported in human pati.nt. with glomerul., hemorrh>ge. bur diff",.ntiation of glomerular .nd nonglomerubr hemorrhage by urine erythrocyte app"'rance h .. not oon reported in ""terinary SpecIes. 2. Iatrogenic hemorrhage a. Blood vessel. may be damaged during bladder palpo.tion, cystocentesis, or catheteri7.;Otion.

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY b. Animal, with h~mOS{"'is d~f~u or urinary muoos;ll di,,,,, .. may to iatrog~nic h~morr~. 3. w.nital mct hemorrhage (associated with <:SIru.) in voided .... mples

~ mor~ pron~

IV.

B30Cteria in urine sedim~m (Plate 110 .nd E) A. Physiologic processe;: Urin~ furmed by the kidneys should ~ st~rile. Low num~n of bact~ria may a = th~ urin~ vi. th~ distal uriruory tract or g~nital ti .. u~,. B. Analytical conart' I. Bacteria in urine sediment are enumerated a, • rdative demity of bact~ria .... n in mon 400x field, (hp/). 2. Bacteria {~cially cocci} Im}' ~ difficult or impo .. ible to different"'te from small particulate d~bris by routine sediment ex;;omiruotion. Rods and chain' of cocci ..e mo .. ~a,ily identified with catainty than are singl~ cocci. 3. If bacteria are of clinical .<>rted '" present or a~nt. Routine c;.,ntrifugation of urin~ does not roncentrate bact""' .ppr~iably in th~ sedim~nt beams<: they do not 'spin down."" Thi, is in contrast to other JUspended p.nicul>le:, such as ceUs. 6. Th~ use of stain<"d air_dri<"d utine pr~parations for microJCOpi, e""mination has bttn suggtst<"d as • ~mr WlIy to det~t I>.cteriuria dun the us<: of routine wet_mount p"p"rations. "' C. Bact~riuria{Table8.12) I. Det~rmining the clinical signifiamc;., of bact~riuria involves comid.ration of th. pr=nc;., or absenc;., of pyuri •• the SOutce of the .. mpl~. th. conc;.,mration of b3oCt~ria. and oth~r evidence of. utinary tract inf~tion. 2 Th. absence of detectabl~ bacteria in urine sediment doe. not exdude the p"'-'ibility of.n in~ion. On. mould u.. quantitative utine culture: method. wh.n"",r a urinary mct inf~tion is SllSpected. As many as 10.000 rodsImL or 100.000 ooccilmL may ~ required fur det~tion by routin~ utinalysis. but as f= as lOOO bact~rialmL Im}' be .nt in • cyst<><:ent .. is sampl•. The decision thremold (I>.sed on quantitative: culture) betw..,n significant bact~riuria and rontarnination dq>ends on the species of animal and the urin~ rollection method used. OJ

V.

Casts in urin~ sedim.nt (plate II F- H) A. Physiologic processe; l. Casts are cylindrical concretions with round. squ .... irregular. or tapered ~nds and of v.rying widths that mirror the tubular .. gm~nts in which they formed. They ... furmed in renal tubular lumem from proteins. intact c;.,lIs. or cdlular debris. Most are thought to have:. matrix composed ofTamm_Horsfall muropro_ teins secreted by ~pithdial cdls of th~ loop' of Henl •• distal tubules. and roll~ting ducts. 2. A few casts may form during th~ normal sloughing of tubular ~pithdial cdls that occurs daily. C",,, .... n in healthy mammal, typically are hyalin. casts or fin. granular cam. Typically. only. few hyalin~ or granul .. casts.re found (< Zllp/). but a .hower of casts may occur after phy.ic;ol 3oCtivity.

8/ URINARY SYSTEM B. Analytical roncqm l. Casu in urin~ Kithdial ""Us}, erythrocyt~, leukocyte, fatty {lipid dropl<'t1 or lipid a.ccumul>tion within d<:terioma! cdls}, wny (p<:rru.p1 det~rioration of gr.nular cast), or mix..:! (combinations of the pr""iouuy mention":! types). 3. Casts d"erioral< in urine (espo:cially .lkaline urine) and thU1 a.. best det"'t..:! in fresh urine sampl... 4. Casts may be pigmented by bilirubin, hemoglobin, or myoglobin. C. Cylindruria (cam in urine) rr.ble S.l2} l. Casts m.y form in ",nal tubul .. but may not be flusha! into urine, or th")" m.y be discltarg..:! imermittently in sho_n. Th..~fore, th~ absc:n"" of cylindruria does not exclude the possibility of .ctive renal tubular di~. 2. Hyaline ca.m m;oy be found in h..althy aninuls .nd occur more commonly in animals with glomerular proteinurias. 3. Epithdial or fatty casts typically ",fleet active tubular dq:en~ration or n",rosis. Th")" may be ""iden"" of toxic nephrosis (e.g., gentamicin induced, or hemoglobinuric and myoglobinuric nq>hropathies) or renal ischemia. 4. Granular =ts m.y similarly form from cdlular degtn~ration and refl"'t tubular degeneration, n",rosi., or inflammation. They may alro form from the pr"'ipitation of plasma proteins. Th")" can be found in the urine ofh~..tthy mamnuls, possibly ",bta! to normal ""II turnover or to entrap""'" prot~in .. 5. leukocyte casts refl"'t inflammation involving renal tubules {e.g., in tubular nq>hro'is and pydonq>hritis}. 6. Erythrocyte casts refl"'t glomerular or tubular hemorrhage:. 7. Woxy casts;ore uncommon and ..en primarily with chronic ",nal disease. 8. Hemoglobin and myoglobin casts are r.d_ to brown_pigm~ma!. granular cam and may be ...n with hemoglobinuria or myoglobinuria. D. Pathogtnesi, (pr"".iling theory) l. Hyaline cam form from the conglutination of the T.mm_Horsfall mucoprot~in tru.t is secret":! by tubular cdls of the loop of Henle, distal tubules, ;ond roll"'ting ducts. The rrason for the ronglutination is not und~rstood. 2. Granular, lipid, and cdlular casts form when cdlular d~bris or ""lis are trap""'" in the Tamm_Horsfall mucoprouin. Granular casu may also be forma! by th~ incorpo_ ration of plasma prouin1 into the T:unm_Horsfall mucoprouin matrix. Waxy cam are forma! by the d<:terioration .nd 5Olidification of granular casts. VI.

Epithdial cells in urine sediment {Plate III and Jl A. Physiologic proc= I. Epithelial cd!. are connantly sloughing from the urinary tract muco .. and are repl a=' by n<'W cdlt. Thus, epithdial cell. are expo:ct":! in the urine ofhrahhy animals. 2. Typ"s of q>ithdial cell, v:lry within the urinary tract. Renal tubular epithdial cdls are cuboidal in 1itu but may be round in 'lUp"nsion, transitional q>ithdial ceU, lining the mucou from the renal p"lvi, through most of the umhra app"ar round, and "'!u. mous q>ithdial cell, lining the dinal urethra .pp<:ar round or polygonal.

474

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

B. Analytical

OOflcq.ts

l. Epithdial cdh in urine ~iment "e "numeratffl as the rangt or mean of q>ithdial

""U, «ilhdial cdls that ha~ angulor bord .... b. Round transitional q>ichdial ""lls " .. difficult or impossible to diff... m;"te from Ih. round ""lis that oa:ur in the intermaliat. loy"" of sqlIamou, q>ithdium. c. In nonsuin«i ...!imen!, individual .. n:.! tubular q>ithdial cdl.... diffirult or impo.,ibl" to differentiate from ffi3oCrophog., or .mall transjtional q>ithdiaJ cdls, and dum!" of renal tubular q>ilhdium may look .imilar to lran,ilioruoi

.pithdium. 3. All cells d.uriorau in utine and thu, ,,~ best a.min.d in fwh utine. C. Clinical significmc;., of ~pithdial rell, in urin~ I. Epithd",l cdt. m"y ~ found in th~ utin~ of h~ahhy animal" ~lIy in cath~w. iz.d .ampl... 2 Mor~ tr:ln,itional qJithdial c;.,1I, m"}' slough &om inHam.d or hyp<rpl:ostic mucos;>. 3. Nwpl:ostic epithd",l cdt. truly ~ detaot.d in urin~ s.dim~nt. Diff~"'nt"'tion of malignant from nonmalignant <:til, i, ~n aocomplim~d by microlIDpic a.mination of,tain.d, air-dri.d prqJat"atiom of c;.,lls rollaot.d by traumatic ath<'terization or nedle biopsy, but pr~p">lion' of centrifugal, fmh, n~ly form.d urine truly also ~ useful. How"""r, diff~"'nt"'tion truly ~ impossible. Dyspl>Stic and proplmic (re"C1ive) cdl, ar~ rommon in utine from animal, with cynitis and rextive hyp<rpl:osia. VII. Cryst:d, {plate. II K and L .nd 13A- G} A. Physiologic conc;.,pt, l. Crynals rqJresent the praoipitation of .<:dt, (ation + .nion). 2. Some.<:du tend to form .t .n alkaline pH but dissol"" at an a.cidic pH, whe= othu. tend to form at an acidic pH but dissolve at .n alkaline pH. A .alt that is solubl~ .t • ceruin pH truly form crystal. if the conc;.,ntration of th~ ion, i, high enough (",turation point) :md the trult,,",l, ar~ present to begin ct"}"ul formation {nudtltion}. B. Analytical concq.ts l. Crynals are enum~rat.d :os th~ num~r of cryst:d, ""n in most I 00 X /idd, Upl) 2 Enhmctd cryst:d fortrultion may occur in nor.d utine beau.., of ~v:lporation or beause of changes in temperature {especially r~fric<'ration} or pH." Conversdy, in othu stor.d .
8/ URINARY SYSTEM

'"

3. Pr..en"", or abs.n~ of crynaUuria i. not • rdi.bl~ indicotor for the pres.n~ or ab"'n~ of urolithi,,,i, (= th. Urolith Analysi • ..,.;:tion), bur crystalluria is. risk f.ctor for urolithiasi •. VIII. Organisms other than bacteria (Plat< 13H- J) A. Yeast structures {•. g., CandidJl, B/IU"'mymj, hyphal structures {'.g., Alpn-gillu, :md various rontominants}, .lga.. {Prowdm:il}, :md p.rasitic seructu... (e.g., DiDcwphynw, Cap;!"',.;." :md Strpmmurus ova; :md Dirofila,ia microfilaria) con b. found in urin. (f.bl. 8. 12). Id.ntifiauion of lUfii:al structures may raJui .. culturing m.rhods. B. P....itic "ructures may b. pr=nt b.au"" of lample rontomination with f«:.l mat
Oth.. 'ffliment findings th.t ... of linle or no di'4:nostic signifiam~ A. Lipid dropl'" {pl. .. 13K} l. Prob. bly from renal tubular
QUANTITATNE URINALYSIS I.

B",ic ron~pts A. Quantitative urinalysis indudes pr<><::fflur.. in which ch.mirnl or phy.icol proptive UA i. to quantity the amount of a .ubst:m~ th.r is <XC"'tffl by kidnqs in • ddin..! tim. p<:rnSionally oth.. individual prot
416

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY l. To mu.mr~ urin~ [Crt]. th~ urin~ .. mpl~ n~' to b. dilur.d to get the [Crt] into

the assay'. aruolytical r:ml:"'. This PJ'O(:e;S can CUM ina.ccu,,,,,ies." Non-Crt chromo_ gtm may give positive int.rf... nct with some en ...... Y'. Kinetic en = p are d.. ignal to minimiu the inu,f.""nct of non_Crt chromoge",. 2. To m •• m,. urine [tC."]. the urine .. mpl. maS! bt acidifi«i "'fo,", :malrs;, so that Ca>+ i, in a soluble form and thus .bl. (0 react with rtag<'nu. 3. To m.am", [K'] in aJuine, bovine, and fdine urine, a f1.m. photometer should b. u...d. Th. urine of tho .. sp«ies contains a . abuan"" th.t inhibiu th. reoction of K+ with ion_sd<'CIi"" d.ctrod ..... E. Th= orh" f""tors may nttt. ,ef.ren", inurval. for your patient? For some analyt .., body weight, di~t, ~_rdat..d fuctors, or tr~>Im~ms am .lr.r urinary acmion of subnane<:s. 2. h an .nalyr~'. urin"'}' acr~tion d=~aMd b«:au.. th~ kidn~Y' are trying to ro""'rv~ the an.lyte or b«:ausr Ie .. analyr~ i. king p.."'nt..d to the kidn~rs-'

n.

24 h Excmion studies A. This is th~ most ddinitin m..rhod of d..r~rmining urinary acrrtion of an .nalyr~ bur is not f""lu.ntly u..d in vet~rinary m..dicin. b«:ausr of "",.ral f.cton. First, it requir~' oomplff~ and tim..d urin~ ooll=ion that is difficult in most domestic mammals. &cond, ref~r~n"" im~"",h are not alwaY' .... iJabl~ for each spa;ies, for diff~'""nt assay mffhods, .nd for diff..~nt dim. B. Procalur~: Bq;in ch~ urine oollection period by romplffdy ~mprying th~ urin.ry bladd.. and discording th~ urin~. Collrcr all urine chat i. produ=i in 24 h. End th~ ooll=ion period by ~mprying th~ bladd~r. Mea.mte th~ total volume of urin~ ooll«t..d in 24 h. M~asute the analyr.'. oon""'ntration in ch~ rollrct..d urin•. Finally, calculat~ th~ quantity of subsuno:: acrrt..d {•.g., Eq. S.4.}. B=us<: of ch~ mark..d ... riation in th~ ,izes of domestic mam"",ls, results are usuallyapressal per kilogram of body weight {Eq. SAb}.

Amount of .nalyr. acm..d in 24 h = analyt~ oon"",mration)( 24 h_urine mlum~ Enmple: analyt~ oon""mration = 50 mgldl; 24 h_urin. volum. = 300 mL Amount of .nalyres exCtet..d in 24 h = 50 mg/dL X 300 mL = 150 mg

{8Aa.}

Amount of .nalyr. acm..d in 24 h per kg body w~ight = analyt. oono::ntr>lion ... kg X 24 h_urin. volum~ ... lxxIy wgt {SAb.} Example: analyte oon""mmion = 50 mg/dl; 24 h_urin. volum~ = 300 mL; lxxIy wgt = 5 kg Amount of .nalyres aC'""t..d in 24 h per kg body w.ight = 50 mg/dL X 300 mL ... 5 kg = 30 mg/kg C. B«>usr a 24 h acrffion study is difficult, mor~ ronnni~nt mrthod. to ...... urinary ex
Analyt~ urin~ to pl"'ma rati05 A. Theory I. Th. rat~ of urinary excretion or cl •• r"""", of • subsean"" con k ao.kulat..d by ming an acmion rate formula (Eq. 8.5al .

a/ URINARY SYSTEM

[ Analyt~L 1 x volume.

Analyt~ ncr~tion rat~ = [

Analyt~

. ncreUon

rat~ ~

2. [f urin~

Analyt~1p

[Analyt~L

[

,

Analyt~ ...

-t-

tim~

-t-

bw

(a.Sao)

(a.Sb.)

volum~, tim~,

and body ~igbt ar~ consid~rM const:mts for a ginn .nimal, to pl>smo ratio is proportional to th~ rat~ of urinary nn~ion of a subnance (Eq, 8.5b), Thos<: factors ."" not con!lants, but th~ mio does t~nd to ""flttl the urinary ncr~ion of a sub,cana (ana!yt~) or solutes, B, Analyt~ urin~ to pum... ratio. for differ~miation of pr~r~n.1 azot~mia .nd r~nal azotemia 1, Th~ urin~ ounolaliry to pl.. ma o,molality ratio reflttl. th~ kidnqs' . biliry to conKrvr H.O, a, A high ratio rrflttl. an .bility to conantrat~ solutes, An .nimal with prer~nal .zot~mia cau...! by dffiydration ,hould h. n a high ratio, b, A mio n... r 1.0 indicat~, i90Sth~nuria and thus failure to ronantrat~ or dilute th~ wtrafiltrate solut .. , [n an animal with rr",,1 .zotemia, th~ ratio should be n~ar 1.0, c, A mio much below 1.0 i. not n pa;tM in m""t .zot~mic animal, kcau .. th~ ratio would indicat~ r~nal diluting ability i, p" s<:m, Azotemic animal, usually ~ith~r form hyp
azot~mia),

Th~ urin~ UN to pUlma UN and urin~ Crt to pwmo Cn ratio> asse.. th~ r~nal ability to e:l[crrt~ nitrogenous W>s{e., a, Higher rati"" r.flect th~ kidnq.' ability to acrete nitrog<'nou, wast~ via urin~ and thus ar~ mdena of .d"luat~ rrna! function, b, Lo~r ratios r~flttl d«r~aseJ r.nal ncretion of urn or Cn {i. e" a drcr ...,ed GFRI and v,ould suppon the conclu,ion of rr",,1 azotemi., but thq:dso may occur with prrr~nal and po'trena! azot~mias. 3, Publi,hed ratios for horst. iJJuurat~ the applicuion of tbe ratios (also called uri",,? indkn) (T.ble 8, 13), Tb~ ratio, would vary betwttn 'pa;i.. , but th~ g<'n~ral concqJts apply ""ross .pa;ies.

2,

[V,

Quanticatin urine total prot~in ......Y": Tbese ."" typically not part of. routin~ urinalysi, but ar<: used to det~rmin~ a mor~ accurat~ urin~ [prot~inl for protein to Cn ratios, for urine dectropbo ... is calrul.tions, or for 24 h ncretion studie;, A, T richloroaatic acid method 1. Principle: Prot~ins ar~ d~natured by acid, .nd form a pr«ipitat~ that i, "",n >S incr ......! solution turbidity, 2, Th~ trichloroacetic acid method i. Ie.. af&cted by th~ :dbumin to globulin ratio in the sample tban is the SSA method, The trichloro.cttic acid method is t~m~ratur~ stmitin and nttd. to be completed at 20- 25 .c," B, Coom... i~ brilliant blu~ assay 1, Principle: Th~ .mount of d~ binding to amine group" of amino acid, is propor_ tional to th~ quantity of prot~in present,

·78

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

Table 8. 13. Urinary indica fur differentiation of prerc.nal and renal azotemia in horses

Numkr of hors .. Osmolality. (mmoUkj:) Osm,.:Osm; ratio [U Nl.:[UNl : mio [C,tj.:[C'tl; mio FE ofNa+

6 727- 1456 2.5- 5.2 34--100 2- 344

6 458- 961 1.7- 3.4 15--44 51 - 242

to 226--495 0.8- 1.7 2- 14 3- 37

0.0--0.70

0.0--0.5

0.8- 10.1

So""", (. ... pled from): Gro,m\all " :d. LL ' o Uri... osmobJity to p1:wru ,,"m,,".lily • Vrin< oonCffitr>tion of UN to pI .."", co~ntr.ltion of UN < Urin. ooncmtr:ltion of Crt to pJ .. m. oonc='<1tion of Crt

2 Thi, assay is mini"",J]y .ff«:t.d by .Jbumin to globulin rat;m in th~ urin~ :md will d.t~ Iknct Jones prot~ins. C. B.nzemonium chlorid~ "'''r l. Principl~ B~nZ
Urinary protein to creatinine (prot:Crt). r:llio A. Theory l. Inc",asffl prot~in 10... via the urinary '}'Stem is be;t determined by a 24 h protein excretion stud,.., th>l is, determining the millign ms of prouin lost ~r day per kilogram of body weight. However, sum a study r"'luires a tind .nd complete urine collection. 2. Cn cl~aranct via the urinary sY'tem is considered to be ",htivel.y constant in hnlth. In . ddition, if urinary Crt dnranct is d,""reased, then th~ rate of glomerulu protein loss should be da,... 1ffl beatusc: Crt paues mo .. ~asily through the glom~rulu filtration Mrrier th. n do protein molecules. How",""r, if more protein ente", the urine through dam"l:ed glomeruli or through other processes, then the rate of protein excretion compared to Cn excretion will be incr....ed. 3. Considering th~ afo",mentioned conctpts, comparing th~ rate of urin. ry protein loss to the Crt excretion mould reBect true chang.. in prot~in lOll via the urinary 'Y'tem. Thi. conctpt can be =n in the compari",n of urinary excretion formulas and the derivation of the (Prot:Cn ). ratio (Eq. 8.6). volume... ... tim~ ... bw (8.6.) ... time ... bw

8/ URINARY SYSTEM

'"

For a r:mdomly coll,""t..d urin~ sampl~, som~ factor> in th~ num~rator and d~nomi_ nator formulas "'~ ~ithted as fullow>, [Prot L. Protein acretion rate Crrat inine a cm ion rate consr.nt B, Analytical concepts L Unle.. th~r. is se,.,,~ prot~nuria, urine prot~in concrntration. a", bdow the analytical rang~ of .. rum protein a=ys, Thus, diff have oon shown to underm~alU'" urin~ [Cn] (~'pe<:ially at high concrntration.) and thus producr higher (Prot;Crt). ratios, " C. Published d.r. L When dogs with potential nonr~nal prouinuria we", aeluded, th~", Wll good corrd.tion b.tw..,n the (prot;Cn). ratio and th~ quantity of urinary protein acreted per day {~ithtion guiddines for the (Prot;Cn). ratio baKd on the>< studies a, Healthy dogs; (Prot;Crt). ratio < 0.5 b, Borderline values; {prot;Crt}. ratio = 0.5-- 1.0 c, Dogs with glomerular prouinuria; (Prot;Crt). ratio:> 1.0 (Not~; Other furms of proteinuria could give similar results but w
FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

Table 8. 14. (rrollCn). ralio. and 24 h urinary protein ncrction . Iud;.,. in dogs Grau~r et :d .... Itl Whil~ ~I :d.d v c.nt~r ~I :d.d" Grou~ H~ ..hhy 0.08-0.54 (8)" 0.02-0.17 {IG} 0.01-0.38 (19) Prouirmric" 1.09-8.63 (10) 0.48- 15.1 (14) 0.4 7--46.65 (38) 24 h Uriruuy prol~in Huhhy 1.9-11.7 (8) 0.6-5.1 {IG} 0.2- 7.7 (19) excmion {mglkil Prouinuric 32.2- 27 1.1 (10) 12.2- 287.5 (14) 7.5- 533.7 (38)' • Protein m
(Prol:Cn). ralio

,,,,di<..

' Tho dog .. itb , protrin Jo.. of7.5 mglkg .... incltxl«l in tb. pro«inwic group boa .... it bod gIom
1. Th~ (Prol:Crt). mio ,hould k inc",~ in :my .nimal wilh prol~inuria , induding pr~..,"" (overflow), glom~rul .. r. IUbular, :md inflamm"lorylh.~morrhagic prol. inuri .... Ochu dinical infonrullion i. usod 10 diffu~mi"l~ Ih~ prouinurw (Stt Ch~micol Ex .. mination of Urin~, =:1. III.C). 2. Glom.rular prolOinuri", I~nd 10 bo mor~ RVu~ and cous< hypoalbumin~mi .. or hypoprol~in~mi ... How~ver, ",rliu nageo of glomerular dam"l:~ m..y cou", only mild prolOinuri ... In ch~ .b",n"" of hyptrprolOin~m;" and inc",=<:! ""II con""mralion. in Ih. urin. , lar-g~ incr....... in Ih. (pror:Crl). ratio (e.g., :> 5.0 ) .uggesl g1om~rul .. r prolOinuri ... Th. ralio, in = of ren ..1 amyloido.i. typicaUy .... high~r chan Ihe rali", in COlO; of glomerulon~phrili ., but Ih~ obsc:~ valu~. overlap romiderably! ' 3. Eni""'lffl (Pror:Cn). ralio a. A ",miqu.ntil"li"" method of eoli""'ling a (Pror.Cn). ralio is availabl. (Petstix 8 .. ag.nt mips). b. Umil crilical ,lUdi. ..... ..vailabl. Ih ..1 assess Ihe prffliclive valu~. of ch~ sysl~m wilh velerin .. ry .,mpl~s, ch~ method probably mould bo ron.id~red 10 bo anolher subjeclive assessmem of urinary prol~in exc"lion :md simil .. r 10 interpreting Ihe rd .. lion.hip botwttn urine [prol (Stt Ihe nnl =:Iion), st:mdardius Ih~ ",Iut~ concemralion 50 Ihal prolein con""mralions con k romparM wilhout using calculalions 10 ronecl for diffe .. nee. in urine roneent ralion. VI.

Micro .. lbuminuri .. A. G.n~ra.l cona-pu I. [n Ih~ drnic vi~ of r~nal functions, m.. mmalian glom~ruli ar~ nol ptrm~. bl. 10 albumin bec:.u", il h.... strong nq:arive ch .. r~ .. nd Ih~ .Ibumin molecular di .. met~r

8/ URINARY SYSTEM

."

{36 Al is clo,", to th~ di.m~" of a g1om~rular por~ (42 A)." How""~r. th~ glom.ru_ lar filtration b.rriu do.. allow ..tightly uruo]J" proteins to I"'ss into th~ ultr>filt .. t~, from which th~y are re.orkd by the proximal tubul~ .. If th~re i. minor domag.. to the barri" (. reducal neg.ri"" charge or damaged podocytes), then .lbumin .nt~'" the tubular fluid. Inromplete resorption of th~ .lbumin caus.. albuminuria. With incr..sing dom~, larC"r protei", (various globulins) also p:w into the tubular fluid. 2. In ",ndom urine umples from heohhy peopl., renal excr~ion of .lbumin result. in a urine [albumin] < 3 mg/dL or < 20 mg excreted per doy." In this oontext, micmolbuminuria is ddined .. a urinary albumin 10.. gr...ter than those values but less than 20 mgldL " Using rq;ular urinalY'is rrag<'nt strips, mrn .lbumin concent.. tions might cr...te tr.ce protein r..ctions. 3. For many ~ars, the protein r...ction of. typical urinalysis Wa5 expeeted to be negative in the urine of healthy am, horKS, .nd c;ottl~. Ho~ver, a trace or I + re.ction in concent"'ted c;onin~ urine was accepted •• a .peeies variation and not .vidence of a pathologic stot . ... Moraw.., b.nds corr.. ponding to albumin are oft.n pr=nt in dectrophore.is gds of urine from dinic;olly normal dog.......," How""" more recently, som~ ha"" ronsid,,~d th>t the mild proteinuri.. in dinical healthy dogs represent ""idence of subclinical glomerular dise""' ... Consequently, assay. have been developed to m....... re low urine albumin concentrations; that is, to detect micmol bumin uri.. 4. Th~ current working definition of mimJll/bumi..urla in dogs and alU i. urine albumin concentrations from I mg/dL to 30 mg/dL in urin~ .djuned to. USG""of 1.010.";'' ' Concent"'tions:> 30 mg/dL are referred to as albuminuria or Dvm albumi ..uriil. It is imponant to remember that a urine [albumin] of 30 mgldL in urin~ with a USG ... of 1.010 would have :m [albumin] of 60 mgldL if the USG"" was 1.020, or 120 mg/dL if the USG ... was 1.040. 5. Som. authors sm~ that 30 mg/dL i. the decision threshold for microalbuminuria ba::..... common urinalysis m~hods do not detect protein concent.. tions < 30 mg/dL Ho~r, the Ch~mnrip p.ckag~ in",n nates that, in 90 % of urin.. t.. ted, protein concent"'tions ~ 6 mgldL producal a color ch anC". For the Muhinix system, • t",ce r..ction corresponds with an estim.red [protein] of to mg/dL When turbidity standord 5Olution. are u",d with th~ SSA t.. t, increastd turbidity am be detected wh~n [prot~in] is 5 mg/dL B. Analytical roncepl5 I. Quantitative immunologic ....y a. Th~ immunologic microalbumin .....Y' .'" .peeies .peeific ba::.u", of the .peri.. diff..ences in albumin molecul ••. Enzyme-linked immunosorbent .... y. (ElJSAs) :md nephdom~ric assay' h."" been d""doptd for c;onin~ .nd fdine urine." b. The stondord 501utions for the assays are canine or fdine albumin. For on~ ELISA, stondard solutions w..~ diluted to cr..te.n analytical "'''i:e of 1.930 ng/mL (0.19- 3.00j.lg/dl)." Urine .. mpl .. ~'" diluted (up to I ; 3200) to obtain urine .lbumin concentrations within the analytical rancr of the assay, but surn marked dilutio", c:m be a source of .nalytical error.'" 2. Semiquantitative immunologic ....Y' a. Comm~rcial""'Y'.re marketed as ImmunoDip C.nin~ and ImmunoDip Fdine; both are call~d E.R.D._HealthScrttn Urine Tests (E.R.D. is:m .bbr""iation for

482

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

"'''Y' .""

.... rly renal di..-a,.}. Thest similar to an ..say denloptd ror human urine (Irnmunodip, Diagnonic Chemieob). h. Aft.r the urine solute oonctntr:u ion has bt.on adju,eM to a .ptcific gravity n"", 1.010, the d~ict j, dip","", into the urin., and then the r.action is gnd.d

visually. A nrgatin ,...."cion is npectffi if the [albumin] jt < I mgldL. Higher oollctntration, . '" grad,"", from low posjtive to vuy high positive b ..al on color chan~ in the drnce. 3. Albumin to creatinine ratio a. B:I}"" Diagnonics mar~, a r~1II pad method of estimating an "'humin to en ratio (Clinitek Microalbumin ). Th. albumin .""c{ion is • dJ"'-binding method with oolor c.... nges .. pr=nting 0, 1,3,8, 15, and 50 mgldL. Th. Crt ,,,,clion is a ptrm:id..,,,,lih a=y with oolor rn:mge; ror ... ponding to 30, 100, 200, and 300 mgldL. When th. oolor change; are ,....d by rdl=anct photom~tly in a Ihy~r inS{rum~nt {Clinitd'J, an ~imated albumin to Crt mio is alruJ.ted. Th~ report.d units of th~ ratios v.ry: 300 mg/g = 300 J.l{;lmg = 0.3 mg/mg (or just 0.3). b. Th~ albumin as5ay is not s~fic for :oIbumin. Many oth~r protein! will r.-act with th~ dy~ including light chain" immunoglobulins, IJrmicroglobulin, T.mm_ Horsfall prol 5/hpf,:> 1+ heme reaction), and hemoglobin an bind to the reag.nt dye." Thus, "'m~ discrepancies may ha"" b.:c:n due to prot~in! oth" than :oIbumin binding to th~ reagent in the dip.nid,. 4. Comparison to other urine protein :waY' a. [Prot~in] mimated by rourine protein :u.s.ays of urinal,.. .. (i.e., the reagent pad method .nd SSA turbidity) rep...,..nu the rum of albumin and globulin con~nt"'tion'. Both method, detect .Ibumin better than most globulin" bur globulins do rontribur. to th~ reaction,. The detection limits of these: .....Y' vary (f.bl. 8. IOJ. b. Th~ SSA .ssay is :01'0 all.d the h"minlm beause it deteas :oIbumin better than globulins. %~n a .ample', r~,ult i, compared to .nandard solutions, turbidity can be detected .t .n [albumin] of 5 mgldL If. ""ry mild prot. inuria is aused by glom~rul .. diseas., albumin will be the dominant protein in the unn•. c. Th~ result. of these: routine prot.in assays are typic:dly not normaliud to • 'paoific gravity of I.OJ o. How...", their rerults ,hould be interpreted with knowledge of th~ .. mple', USG ... so that an estimated adjustm.nt an be mad •. In a ron""'ntrated urine ..mple in which microalbuminuria is detected aft .. norm:olization to 1.010, it v..ould not be unusual for common protein dipnick

8/ URINARY SYSTEM rractions to produce a trace or I + r~.ction or for th~ SSA :way to ha"" 1+ turbidity. C. Canin~ microalbuminuria l. Thu", h:IV~ bttn ~~ral pubJi,hal .bsu:>ets ptn:lining to th~ ""'" of th~ ImmunoDip Canine ten, but ""ry f~ pnr r
Fractional excretion (FE) ratios or percentages

A. Thm ry l. In. random

urin~ s;;ompl~, the FE of mbst ance X will reflect the rdati"" rate of urinary excretion of sub"ance X comparal to Cn. If substance X p..... fredy through the glomerular filtration barrier and is neither secreted nor r~sorbed (like Cn), the FE would b. 1.0. If some of subscance X wer~ ..sorbed .fi~r frttly enming the filtra ... the FE would b. < 1.0. Therefore, for solut.. th at freely p .... the glomerular filtration barri~r (e.g., dectrolytes), FE is th~ fraction of the solute ent~ring the filtra .. or tubular fluid that i, ultimately ""creted. Sub!lance X ",n b. an d ectrolyte, an ~nzym~. or anocher solut~. For the derivation of the FE formula (Eq. 8. 7), sub".n"" X is Na+.

Urinary Na' excretion rat~ U rinary c..,atinin~ excretion rate

[N'· ~ [Na+J xvolume. + tim~ + bw [Crtl. xVoumt. I . + bw + lJme [Crt].

(S.7.)

For a randomly coll=al urine S:mlp[~ • Urin~ volume. time the oyer which urine formed, and the body w.ight value< a.., the .. me in both formulas and thus ",n""l out. • Serum [Na+] and [Crt] will prob.bly b. nrarly th~ s;;om~ but ",n vary. • Urin~ [Na'] and [Cn] are expecral to va'y beca"", of multiple f""tors. Considering these factors, ch~ rdati"" rat .. of N. + and Crt urinary excretion ",n b. expressed as follows:

484

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

Uriruory N' acr~{ion Urinuy crra{inin~ an~ ion

[Cn ]. [Cn].

1

Th,,~for~, fr. ctional excretion ofN. ' (F.E. of N. +) = tN.: x~ N. [Crtl. [Na'l

[Crt]

Or up .......:! .... "",crlllag<'. ",,= m excretion ofN . ' = r;:r::+Tx - - -' X ]00 LN. ' J, [Crtl.

2. A major adv.nt"1:e of the FE nudy 0"'" a 24 h acretion study is th at the :as=sment can b. don. on a random single urine .,rupl,. How~er. th. urine and strum (or plasma) .ampJ.. for the ,....ssm.m should bt collect"'" nUl th .... me tim • . A FE ""Jue typically will bt • btmr ass ... mem of renal functions dUll a .impl. ratio of urine .na/yr. collcrntration to pl" ma analyte rollctntn tion breau.. FE ron,id." nri. tions in renal acretioll! causal by . Jurffl GFR. B. Interpretj"" conctpts I. A FE "tio provide. the rdati", m e of acretion of an .nalyte romp""'" to Cn. For a FE ratio to ..f1«t the 24 h urinary acmion of an .nalyu accumdy. Cn acr~tion n~ to k within the ",f~ren". intav:d .nd rda tivdy constant. 2. An incr ...snl FE r.ltio may r~H""t in"'~=<:! urinary acmion of.n analyee. a. Plas= .n:dye~ con""'ntr.ltions :or. inc",=<:! (..",lting in inc..=<:! filterall""d) • • nd th~ kidn..,.. ..~ attempting to acme the a"",,,," This may occur with increasal dietary inc.h of the an. lyee. b. In,,,,,ased tubular =retion of the .n:dye. c. Decr.ased tubular resorption of the an:dyu 3. D«",=<:! FE ratio of an .nalyt~ may result from the opposite P"""'''''''. a. Pl .. = .n:dyr. con""'ntrations :or. d""",ased (resulting in d"" ..=<:! filtered lo.d) • • nd th~ kidne,.,. ..~ attempting to ron .."",, the analyee. Thi, m.y occur with decreased dietary inc.h of the analyee. b. Decr.ased tubular =retion of th~ .n:dyr~ c. lnc",ased tubular resorption of th. an:dyte 4. An incr ...snl FE mio may al"" occur with d"" ....ed Cn acmion. a. An increased FE of K'" would occur if the .. nal acretion of K+ i, maintained through =retion of K+ but Crt acmion is d"" ..=<:! beau.., of d=~asnl GFR. b. FE of PO. =y be incr ...snl bea"", of d""",ased GFR (thu, I... filtration ofC" .nd PO.) and d«r ...snl tubul .. resorption of filtaed PO, beause of increasal PT H .ctivity. C. Th..... Y" used to mea", .. an:dyt. roncrntr.ltiom in ..,rum or plasma =y not provide accur.lte ""ules fOr urine samples. l. Urin~ am ronc.in an inhibitor that interf."" with the ion_sd""tive d«trod. :way for [K+J ." 2. Urine may ntt
.85

8/ URINARY SYSTEM

D. Clinical UIO; of &:>etional excmion studies l. FE of Na+ a. In a hyporuot"'mic animal. an incr.~ FE of Na+ indicates that ",nal excretion ofNa+ is contributing to th. hyponatr~m""- Such a process may r~fl= d"""""",d .Ido,t~ron~ .ctivity, increas«:i . trial natriuretic pq>tid~, or r~nal tubular di.u",. b. In a hyporuot"'mic animal, a d""reas«:i FE of N. + indicates that extrar~nal factors ar~ causing th~ hyponatremi<> and that th~ kidn~Y' ar~ att~mpting to conKrv. Na+ through th~ actions of :ddost~rone, :mgiot~nsin II, or ADH. c. FE of Na+ inc",as<:S in ren:d F..ilur. :md d~cr~alO; with p",renal :rz.<>umia, wh.""" GFR i. d""",as.d in both condition •. 2. FE of PO, a. In a hypoc:dcemic animal .• n inc",:ased FE of PO, su~sts inc",~ PTH . ctivity as m.y occur with nutrition. l or renal s«:ondary hYP'rparathyroidism. b. In a hypoc:dcemic animal, • d""reased FE of PO, mgg<:Sts that d""rused PTH activity =y ~ contributing to th~ hypocak..,mic ,(;>1<. c. In a p.ti~m with hYP'rc:dcemia caused by primary hy~rpa"'thyroidism, th~ FE of PO, is incr~sed. 3. FEofGGT a. GGT is p:m of r~nal tubular cdl memb",n .. , '" mo", GGT it excm.d in urine wh~n thrr~ is ",nal tubular cdl dam"l:~. GGT .ctivity in a r:mdom urin. "'mpl. mayor m.y not ~ incr~ased with .. n:d tubular di,.,ase. GGT activity should ..fl= tho [GGn in the urine, which i. deurmin.d by rdati"" IDlounU ofGGT and H,O in th~ uriM. Thu. , dilute urin. would ~ <xpecta! to h~~ lowrr GGT .ctivity than does concem"'t.d urine. b. An increased FE of GGT indicates .cti"" r~nal tubular damage or n""rosi t. It d".,s not d~t~rmine wh~ther th~ .. i. or is not ",n:d insuffici~ncy or f.ilu"" and it does not refl""t GFR.''' E. FE studies are not commonly used in vet~riruory m.dicin~ h«:aUK information obtain.d nuy not ~ raJuir.d for diagna.i. :md cas< managtm~nt. AI"" appropri. t...f~"'n'" intervals a", difficult to obtain for ea.ch 'peOes, especiaUy wh~n on~ ronsiders that ""P""'u ",fe",n", intuvah nuy ~ nNda! for diffe"'nt :usay .ynems :md diffe",m dim. VIII.

Urin~ bil~

.cid to

cr~atinin.

ratio

[Htumining th~ urinary excr~tion of bil~ .cids rdative to Crt excretion i, used as a m~thod to det""t .bnormal bil. acid metaboli.m by th~ Ii""r. D.tails of this di<>gnonic m.thod ar~ p""ema! in Ch' ptu 13. H,O DEPRIVATION AND ANTIDIURETIC HORMONE (ADH) RESPONSE TESTS IN ANIMALS WITH POLYURIA AND POLYDIPSIA (PUfPD ) I.

R~nal

con"'nt"'ting ability i, a"'.JMd in • routine urinalysis by deurmining the USG ... T ypicaUy, [he USG .. along with oth~r at.>< information (historical and physical finding. and other laboratory data) ~n .ble:! vetuinari. ns to identify th~ prob.bl. cause> of a PUfPD disorder (Stt Table:! 8.8 and 8.9). Wh~n a more critical :usessm~nt of ren. 1 con"'ntr>ting ability i, nNd.d, and .. pecially if ",nt,..] diabetes insipidus is ru.pect.d, ~ith~r H,O

..

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY d~prjvatjon or ADH ... porut {~m may k oonsid,,«I. B~u .. im"p'etation guiddjn~' =y diff~r wh~n sp<'Cific o.s:ptclS of the cl...ll.nge, V'ry. th. following provide! th. IIUjor oonctpu (,.f...n"" .rtid.. or tats should k oonsult.d for 'P'cific im"pretation guiddine.):

n.

Abrupt H,O depriv:nion ten" A. General oone to COII""ntm. th. ul{rnlil. traU by inducing an .brupt nimulus {hypomlemi. or hr~rosmolality} for renal H,O ~.mion. The lest moy bt usffl to
=rely hypovolemic. B.

a...i"" of th~ proudure l. Baseline information is rollrcted and may include body w.ight. USG .... urine and

""rum osmolality, and ""rum [Na+]. 2. A=SIl to H,O i. abruptly removed, the aninul is monito ..d, and th~ findings are romp.red with w.dine inforJrultion until they indic;;ote one of the following: a. The aniJrull has become dangerously dehydrated. b. The kidneys am concentrat~ urine. c. Th~ aniJrulI was .deq""tdy chaUenged, and its kidn.,.. did not .dequ>tdy concentrat~ urine. C. Ba.ic interpreutiom l. [f th~ animal demonm>tes ability to concentrate urine {the USG"", or urin~ mmolal_ ity criteria are a CNded l, the PU/ PD sUte i. a primary PO di"')f(I~r. 2. [f the animal d"". not demonstrate ability to concentrate urine, """eral pos.shour} may be present because of p
Gradual H,O dq.riVlltion test" A. The pur"""", indications, and contraindic;;otion. are the same as for the .brupt H,O dq.riVlltion test. The gradual H,O d~privation test i. indicated if medullary w>shout from prolonged PU is aptcted or if the .. i. f.ilure to concentr. te urine after abrupt H,O dq.riv.tion. The gradu.al d=~aK in H,O inuh will enable the kidneys to reestabli,h a medullary concentration gr.dient. [t tends to be a Ie.. dangerous procedure in """~rdy PU aniJrulls. but .nimal, nill must be monitored thoroughly for drvdop_ ment of dehydration. B. Th~ major diff..~nce ktw..,n th~ grad",,1 and the abrupt procedures it that H,O intake and urine output a", me:arured and r<"COrded o,,",r 2 or more daY". Th~n, H,O availability is drcr....ed by 10 % earn day. Lih the abrupt t.. t, th~ animal must be monitorai for evid~nce of dang~rous dehydration and ability to concentrate urine.

8/ URINARY SYSTEM

.87

C. Advantagt. of th~ gradual H,O d~privation a.. that it t~nd. to ~ less dang~roll'l and allows tim. for r..,nablishm~nt of m. dull:ory hypmonicity. Th~ m.jor disadv~ntag~ is the tim~ r"luir~m~nt; it truly uk. onr ~ w..,k to romplet~ th~ test.

[v.

ADH (vasoprnsin) r~n'" test'-' A. Th~ b..ic purpo"'" ar~ to asses! the ability of th~ kidn~ to ~nd to aogenou. ADH and to confirm a complff. or p:mial.b .. nc< of ADH (dia~tes in.
v.

Modified H,O d.privation test'" A. Combination of H,O d.privation test .nd ADH respon .. t~n B. ProcMur. I. Abruptly depri"" th~ .nim.l of H,O and th~n m~•• ure urin~ OJmoblity hourly until the incr ..... in urine osmolality ~tw..,n oon=uti"" hourly umpl .. is < 5 %. 2. ADH .dmini!l~red and urine collected 1 h .ft~r injection C. Ex~ted results l. H~althy dogs: urin~ osmolality ~ft~r H,O d.privation;> 1000 mmoUkg, and. 0 % incr ..... in urin~ osmololity .ftu ADH adminimation 2. Panial c<mral dia~t .. insipidus and panial renal di.kt .. insipidus = (including dogs with hy~radr~noconicism): urin~ osmolality .ft~r H,O d.privation < 6(X) mmollkg, ~nd a 20-50 % incr~= in urin~ osmolality aft .. ADH administmion 3. C.mral dia~tes insipidus =s: urin~ o,molality aft~r H,O d~privation < 3(X) mmollkg, .nd a ;> 100 % incr.... in urine osmolality aft~r AD H administration 4. Primary polydipsia Cast" Th~ respon .. should ~ simil ar to that of h~ahhy dogs if the kidn")', h."" medullory hy~rtonicity, but th~ .. truly ~ a poor respon'" if "medullary washout" is pr ...~nt.

UROLITH ANALYSIS I.

A u,."lim (or urinary nlle,.I,.J) is • solid concretion ('ton~) that forms within th~ urinary tract and typic;;olly is found in • renal pelvis, urinary bladder, urethra, ot voided urine. lr.!rning the composition of a urolith provid.. ~id~n"" for th~ po.th~n ..is of its form •• tion and .id. in d~doping th~rapeutic plans to r~mo"" or to reduc< chana:s for recur· r~n"". Urolith, are found most commonly in dogs and ar~ rdativdy uncommon in cau,

...

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

Table S. lS. lnorganic compo.ition of urolith .. Grou~

Ch~mical n.m~

Formula

Min~raI0l2c n.m~

Carbonat~

c.lcium carbon>t~

Caco,

c.Jciu,

aragonit~,

v.{~rite

Cynine Oxalate.

C.rbonal~"P.{i{.

S CH, CH{NH,}COOH eac,o•. H,O eac,O • . 2H,O Ca, (PO.},(OH) Ca,. (PO.Jo(OH), Ca,. (PO" CO,OH).{OH),

c.lcium hydrogrn pho,!,hou

CaHPO,.2H,O

Cystine

c.lcium oxalau monohydm. c.lcium Olul.u dihydm.

Phosphat.,

B.,ic calcium phosph>te Hydroxyap"titr

Cystine Wh..w.llite

Wald.Uiu Apatite Hydroxyal"tiu Carbon 'l~aJ>'l{il' Brushite

dihydrate T ri",lcium phosphate Ocucakium phmpru.te M'4:ne1ium ammonium

Co.,fPO,}, CaH (PO.}, .2 . 5H,O MgNH,PO,. 6H,O

Whitlocki ••

MgHPO.·3H,O

N<'Wb.ryite

Sio, C, H,N,O, c:,H ,N,O, .2H.O c:,H,N,O,NH. c:,H ,N,O,N •. H,O

Silia

SUllV;!O

phosphat. hexahydrate M"l:n .. ium hydrogtn

phospha,. trihydm. Silico Uric .cids

Urates

Silirone dioxide Anhydrom uric acid Uric a.cid dihydrat~ Ammonium acid urat~' Sodium .cid ur.u. monohrdrat•

• A uro~th;, dusi&d .. to . >pe oomposition do<> not m
j,

compoo<"
[f it

• Aho known .. :ammonium hydrogen

0 . . . . Of

ammonium

bio ....

horse; •• nd cotd•. Most urin"y tract obnruction, in cats .re not cau,aI by urolith, but by plugs con,isting of phOJpru.t. cry,tal, .nd mucoid m. terial. Urolith. at< not J.tg\' urine crystal. but con>ist of aggregm·d crystal, .nd an organic matriL Crystal, form with a uniqu. thIN_dime",ional ,tmetur•. The ch.mical compositions of urolith, .re linal in T.bl.8.15. [I.

Two iss .... of the Vrtrrinary Clinics ~fNQ"hAmrrira comain valU
Ill.

Analytical conctpu A. For many yr.n. urolith. _re analyzrd ch.mically with ...ri., of qUignificom amoum, of 10m. ion, _re missrd. (4) they lackal method. for detrcting >ilico and <}"tin •• (5) false_pmiti"" results occurral fur 1Om~ compounds. and (6) mixal urolith, could not b. cl""ifial. m

.nt.

8/ URINARY SYSTEM B.

08'

Th~ ch~mical

m<'lhod, han !>ten r~plaad by physical analytical method,. Th.,.. off",ed by a ~ spa:ialiud labomories {e.g.• the Minnesota Urolith unt~r. the Urolithiasis Labor.tory.t Baylor Colleg.. of Medicine. the Uri,",ry Ston~ Analysis Laboratory >I the Univ",sity of California- D avis. and th~ Canadian V<'l~rinary Urolith c.ntr~.t th~ University ofGudph}.I ....ll. l. Optical crystallography: Ground urolith mu.rial is aamin.d with. polariz.ing microscop<: aft" th~ mat~ri<>l is pla.c.d in a ..,ri.. of oils with diffe"nt "fracti", indice: •. Each crystal is id~ntified when its "fractin index i. Jrultch.d with one of the oils {i .• .• it disapp<:ars). Diff"ent l.yc:rs of th~ urolith ." analyud to d<:{"mine the comJ>Olition of ~ach lay"r. 2. X.ray diffraction: Ground urolith material is bombard.d with X.rays. AI the X.rap hit a Cl}"ul. thq." diffracted (satte"d) in a unique way for each crysul. The diffraction. are =rd.d to determine the chemical composition of the urolith. 3. Oth" 1~1I common methods: electron microprobe. sanning d""tron micro=py. and infru.d SpectroKOpy a,",lp~ ar~

[v.

Pathog..n..is of urolith formation: Urolith formation is a complex process that involves many factors th.t promote and others th at inhibit formation. The major steps of urolith forJrultion .re as fOllows: l J j A. The cations and anions of the urolith ar~ pr..ent at sup<:rsaturat~d conce:ntrations in unne. B. The cations :md anions combine into a unique crystalline structure. Factors such as pH. temp<:ratu". and How raU inHuen"" crystallization. This process oa:urs more rasily when it occun on the rurfa"" of debri •• casts. or other urine particl~ •• proa:lI called hrtmlgtnnJus nuc~"t~n. These crystals may be...,n in urine Klion. E. During the formation of many uroliths. variable .mounts of matrix are incorporat.d into the urolith. In people. the matrix is compostd mostly of haosantine. which might be a product ofTamm.Ho.. fa]1 (uromuroid) deg..n ... tion.

V.

Predisposing or contributing factors fOr urolith formation: Factors that contribute to crystalluri<> typically also contribute to urolith formation. [n .ddition. incr .... K
""

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY (choroJ.t~. nuts, spin.ch, .wet! potato<::!, wh~at germ, and oulau-<:omaining plants ouch as halol:"lon). D. Ura{~ urolith, ar~ found primarily in D.Jnl kid"ry', but thry are . lso found in dogs with ponosyn.mic ,hum. or hepatic insufficiency. E. ernin. urolith, ."" found in dogs that han d.f«tivo metabolic pathw>)" that l
tiff., Aunr.[ian eml. docs, .nd Sam;,h d.."hound •. F. Xanthine urolith, ."" round in dogs trratal with xanthi"" oxid .... to CO[l{ro] ur:ltr urolithiasis .nd in dogs with x:mthinuria {caval;'r King Ch"],,, sp:mid, and dadtshund,j. G. Silica urolith formation in docs bttn .uoci
h.,

Refe re nces I. R.... BD. 19"9-l. Qi"",J l'Iryn.!.o ojhiJ.&zs, • ..J EJ"m,i,.. D.....J.... 4th «Ii';"". N ... Y~rb McG .... ·Hill. 2. O·ConndJ 1.\18. Romoo IA. M~ GH . 1%2. R..o..t tub.ot.. """'.tloo of "",tinin< in tJ.. doc. Am I P~

J. 4. 5. 6.

7. 8. 9. 10.

II. 12. lJ.

It. 15. 16. 17. 18.

19. 20.

21 .

2O},985--m. R<>binoon T. Huhioon M. s......, KC. 1974. [oIIupkpiolo" and mao.V"'"" <>l d.roni< , ..... faiJu", . In, a"o ICM. Gill III I •• cd •. K"Nhw] E"ktroIJl. DUorJ.". 457-489. Nnv Y""h a"""hi.II ~""". R...JA Fiol p nroin,,,cio..t t..u..,n"'v ..ru. inaeo.ood bI.ood "'." nitrovn and BUNI"",linin< utio io doc ~ A lit...."', , ....... "xl """"p«ciY< mod,. Y .. Clio P....001 270107_ 1 Il . Luodb.., GD. I,..."", C . R.du),",u G. 1986. N~.. ",d ,." HP. W'-n AD. 2OO}. C""tinin. in th, """ A""; .... Y" Clio P.tl.oI. J2d62-179. N""""", DI. P';'" CP. 1m. R.ou.I function and o j _ ro ... bollia. [0' Bu.';' CA. Aok.....,.J ER. od •. TNt. T,""-" tl bili.ubin in~ <><> .. nom ",,,tiDio, by th, linrtk Iaffi, ....aioa. I ain J..b AnaL IS, I 16-121. P........ WE lll. eo.. .. CG. Gn, TL 2OOJ. Sawn "",linin<
.,10.

""""'"tal

."

8/ UR INARY SYSTEM

22. FiDe<> DR. [)""",o IR. 1976. &al.atioon.,( blood """ nitrocOCn "f ""al dyofunction. A • ..d, of III cao<. and ........ of ",1..«IIlt....."," I Am V« Mod A.oc 168.S9}--6(JI. 2J. FiDe" DR. 1m. E..tu.""" of.."..) f........... 1m Oobom, CA.. Fin«> DR. «I.. c.. ... , ,,,,,J F,/;'" N"I-Ioo _, U..iorJ, I. oditioo. 216-229. llaItimo", William. &: Wdkino. 2",. 80- KC. loy« T. 1979. Clinia.I..-duation.,( ~ .. ..Ju f.DCtio~. z.j.J.ow ,.. ati..io, d,......., io <10". I Am Vrt Mod A.ooc 174 •.(88--ol9l. 2S. ~. KS. KomJ.n. A &o ..... SA., 1.«. GE. H~",""" D. Ru... EA. 1991. Com ......... offouc mrtbod. of .. cimati.., POm. Am I V.. R.. Sle61- 96-l. 26. G"""".tl R. I ~S. EHert .,( di."",. on ..,.;~ • .,. =mion ...d "",tinin< d.....Codw, b • ';mpl, mrtlo.od of m' ..... io, POm99. }to l.ulid..IP. o.t.o.n. CA. 0·&; ... TD. Pohio DI. 1992. F,tiDe""") failw" q.., ................. ~ . Comptnd Contin Ed"" Small Anim P= 1",.127_ 1H. l5. AJbaa.n H. With IP. 0 ........ CA. l.tkcb"""",uk C. Uldcl. LK. C&.ptut .. KA. ZOOJ. E1fetto of."",&< tim. ...d '''''ptra'.''' <>D pH. optdfic ""''Y• ...d «yO,,1 b matioon io ...int ounpl .. fro", <10" ...d at>. I Am V.. Mod A.ooc 22hI76-179. }6. G..If L 1983. A H,.,'.... "11.,,;,,, l'hiJoJdplO.. IB Lippi..-.. l7. Macdoupll DF. Cwd GI. 2000. V,;", «>lkction and romplttt an.ty.i •. In. &iob.i.dV I. Elliott I. od •. .IllA. VA M""oJ ojc.. ... ,.,.; F"'·_ N.,I-Ioo "'" U..iorJ, [" edition. 86-106. Ame,. 10... Stat< Vni...",;.,. P_. }S. Mdl';,d, 11. 1m. T.. II-J. ojU"-'"is .", &J, FIooiJs. l'hil.JdplO.. lippin=tt. Ra,",~. 19. 0 ....."" CA. Sttvtn. IB. 1999. U ....¥· A Qi,w.t G..NJ. '" c-.~p",;,", SIu....,,,, Miuion. KS.

um.+u.

-

c.. .....

. IR. 19I! l. q..;", U";"J a.. ......., "'" P.th.fby.;. iorJ, J!.onn." ~. KS. Vrttti""}" Medid~, . .. 0. Co/lina., .. I. Goldbtrr; HE. 1997. P,i",;,It, oj &1''''"'''"''''' BuIOolo. NY. lda.. .. 2. Wolf AV. 1966. ~ " '" s./..;"", .nJ JJ.J, R.Nk· "lim. c..",.",,.,,.;,, ~"i,,~"; C" "",,;_ T,J,i". N"" Y",b H~.

"J. Soodth.m SL ScoK MA. 2000. V~p.blid.cd data. ..... McC.ou;" T. Ro, LP. 1985. eo... ........nofloydro"""J'.~. 0""""""'J'. and Amt. N· M..Jtioti. SG i~ .. cimatioon.,( .........,.. 00DCt0'''''''''. All" Pacdi"", I 21. 185_ 188 . .. 5. Chu SY. Spout.. D. 198",. "-""",,,.,( • ...tid.ph .., ,,~o, foe wio''Y opocilK dtttm.i",tio~. Clio Iliochtm. 17.J-i-.J6. .. 6. IWF H. 1987. Gloe<>a>rtiooOd i....ibi""" of n<W<>lo.~.... .t '"'~ """''''''''. Am I Pkpiol2ShR6JS-R6.... .. 7. Di"&"",, IF. D..Point<. RH. 1960. Ad.tnal -=oid inlubition of ___ ";0 ,du" from tht orurol.."",ph,.." of noon.l •..bjoc" ...d patim. .. .,.;th Addioon·, dj"""," I Oin I..... " ljH851- 186J. .. 8. LiD1. E./foc, of """,j,d, on ... '" diw"i. and _ " , i n """ci';ty. I 0;" I..,... "lh)S2-IS8. .. 9. Rijobttt A. Kooioua HS, .. ~ V"""""" IK. Mol IA. V"",l.out G ... o Sluijo Fl. Ij_ I ...... """ lod> TS, Bo" P. Bot. WH. 200 I. ~ma in • llonint:; duct. of poIy...ic loypt.aktmk ,m. I Am Sot Ntphrol9.2IS 1_119'. SI. R.... BD. Poo' TW. 200 l. CIm",'" Pb,.iol.p ojki~B.u. .,.; E"'",!;,,. vu.,.;,,,. 5th edition. N .... Yo.tc MeG .....·Hill.

".vity

492

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

p«>td,.."

S2. I'uliyanda DP, W..! DT. Bawo MA. H........,nd TG. H ..... HW jt. 200J. c.Jpain-ta "; WD. Sj, .d. T_"'" oj' V... ,;""'7 l~"'-' )/""',"", Du-" ofth. 0., .nJ c"t, 2nd «Ii';"... 1592-161 • . Philaddpbi.. WB Saw.d, ... S6. Paqui"",n A, Tun G, P........ JP. 19'9'. E..!u.no.. of th, CIi..itok 200 on....,. ",._.trip "",,", in th, ....!pi. of doo, ...d ... "";0<0 in P",-dinic.J tozi..oIou . nod .... , U, Anim Zl,l4O--z.l6. 57. Wdkr."", MJ , Stockham SL 2002. A. •.-...:; 'r'''''''''' of ..... " of win, ,,"Uo' ..rtp _P' Vi .........."n'." vee .... Clioit ... )00 Uri"" 0,"";'"," Analyu •. ]'" T cocl.inc Clinia.l Patl.oloc:r, Ann.al Mo.tin& <>f th, Am ..bn So.i..ty fo. Vrt.rtna.y Clinkal r.tl.oIq,.. N .... Odcuu. 58. Ban..,'; JA. Fiooo DR. 1979. Protrtn """"",tration in ..,;..., of .,.,..mal """', Am J Y" R... 4O'ISU_ I5tiB. 59. H,""" Kj, Buffin" onCAT, a.... DJ. 19'98. Ap"m,nt brt.v. ... _ m,thod. b m,,,,,,;n, w;", pH;" at> and --m. 60. M""", PM. B""" SL. Bro.m L. 1991. U,.j", _ i n aykmid, p ,lectmpIt.."i. ~f an;'" ...u...y prot';", fo. ... , "".lyti. and dM"... tiation cf tub.ot.. and ~t.. di......, •. Y" ain P.... 01 18,9J-97. 65. Z;"i E. Boo .... ,; U . ZatoUi A. 200-1. rn."",ttk .dn'&tt« of qu.Ji"';", protmpuitoo..itb ,mal kistoJopo Iindinp;D dor;t. Am J Yrt R... 650964-971 . 66, l«. GE. Bro.m SA. Elliott J. Gu." GP, Yadon SL 2005. A"",m,ot and manaum,nt ~f prot,;m"j" in and ~ , 200.1 ACVL\lFon.m Con ....... S""m,ot (.....u ...;...,,1). J Y" In"", Mod 19,J77- JII5. 67. S""" MJ . 19I!1. M~nodooal Oinial"p
Elf"".

do,.

"""_thio,,

"0

69. MocE..... EG. H"";",Al. 1977. Di. ~ and m.... V""'" of ~ pnm<>patbi". Yrt Clin North Ant. 7, 1l9-lJ2. 70. Bonfamj U. Zini E. Minotti E. z..tdU A. 200 • . P..., U ~ •.du.in _irnuia and ""nnaI.....J and f.<><1 &h.u ,.nis. J Yrt Int<m Mod

bi.
1~.618-6z.l .

71 . Pddman EC. Nd.~n RW. 1996. Di ........ mdlituo. I", Go";_ "'" Fdi.u £"""""''''''0 "'" &jnwI.,.i"". 2..d odi';'m. lJ9-J9I . Phil.ddpbi", WB Sawtd", •. 72. Link 1lP. 19-(0, G........ toI .. aDC< ;n ..... " •. J Am Yo< Mod A.ooc 97,261_262. 7J. H_ ottk...AJl", RL. T.PPJ' L Blwn JW. 1994. I..... ;" ",iotanoe, byp..~ •. ",d ~"da in into"';""ly milk_fod ~ J Anim Sci nl60-ln 7'. _ KC. Joy« T, Bl .... _Y~" B, GoIdoobmidt MS, ~ S. 1979. Ch"act,.;,.,n.D of .... al WH. 200J. Simpli/iod mrtl.od to ....y ",tal pl .. ma poroxidar tcti,.;". and f
'0 ...,

.93

8/ URINARY SYSTEM

81 . 0 ....."'" CA. Sta.....1B, Lulkb IP. UJ.kIo. LK. 1Ii.d KA. Kod.k. LA. 5....... 0 LL 1995. A cli..ician·, ....Iy.i. ,,( ...inalyoi ~ [0' Oobono. CA. F;D
82.

s......o.. CI.. Boin-atAM. Kn., .. 1M. G;bbo.... B"""'"SN. 2004. &..I...ci.on of modified W,~,-naWn,; of on... ..dim ... , .. , m«Io.od fo. aawat< drtecti.on of bact,.;..,;. ;0 doc •. I Am V... I>W hooc 224, 12I!2-

1289. 8J. 0 ....."'" CA. 1.«. GE. 1995. Bao,,,,i..1 inf.rti.ono of "'" ""';". ",01 klio. o.u...,. to", .. 10, 0. ..... "" CA. Fioa. DR. ..... Cs";_.."J hii.uN;" doco..i ... protui.Jcoeatinin. ..... in doc •. Am I Y" Ro. 46,1665_1669. 91 . CookAK. c.....~ W . 1996. ClioiaJ and patI.oIoVal f, ....... ofp«>uio.~ ~ dioco.. in th, ~ A , ...... of ll7 """'. (19Ill-1992) . I Am Anim H""I' Ao.oc J2,Jll- 322. 92. !'up. MI. ktt ]A. CJ..k LW. Puke" DR. Wallo« IF. Willi. lW. 1997. Compacl"", of w;", dip";d,, w;th quantita';"" ",.tbodo fo. "'iaoo.Ibumio....; •. Ew I Q;" a.,,,, Clio lliod..... J5,693-700. 9J. 1'0 . ..... BM. V...... 51.. I ....... WA. 5impoon D. 2002. D_tion,,( anino~. u;''& .. ",iquon';.." ,;..., "'" .uip. d...i",.d foo uo< with kwaan w;" • . Vrt Clin P.tboI3h56-60. 94. ~ C. Ban ... R. c...""" F. T.p;. IA. Du..n E. Gonzako. M. Man. MC. 200J. Sl)S.PAGE and W ...... bIoot of ...inaoy po<>t<;", ;0 OW« of
=

(.bnoact).

100. Goao" GF. Ob.th ...... EB. Ba .... ba RI. LappiD MR. Sim"",n DF. J<""o WA. 2002. Dovdoopm.'" of "';.".. ' ''0",;'''''';.;0 doc. with k"""",,,,, di"'.... I Y" [m«n M«I16,J52 (.bnoact). 101 . Lu. GE. I .... m WA. Simp."" DF. Kaobtan CEo2002. P« ....... , .. bumiDoda ",«cd« on ... of <>Y«I _;""';.;D m .... <10", with X.Iinkcd ........ itaoy ......... p' ...y. I V... 10"'D Med I&J5J (...... "',). 102. G...,. AT. Cohn U. KrrI ME. I .... m WA. ZOO4. "Tho ,1Ject. of =«i"oo o.u...,. '''0''';'' =mi.on in <10",. I Y... [00 .. 0 Mod 1M2-55. Q.pon. B. 1987. E......,;.,., ofY--e!""",,1 u.~."._ 10J. Go""" KA. Twmookl GH. Kc..n.y MT. G.= a.";";" ...... f",m 'P'" "",pl<. of "';0. '"p
os.

494

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

108. liD, GV, ThwD>ODd MC. a..i YK. F...,'; C E.. Ruby AI.. loIuuoo DL 2OO}. C~ . i.. prop<>«i.on,,( ani"" ...inaoy aInoU «>m"",ed of calcium ""..tat< 0 • • tnnit< in .p<>D M, Rotnic.k Ml. 2005. U..n...y Utl.iati" Eti.oIoc:r. •., ED I., Wdn A) . edt. c-.~; U ..1.p, Bdo. cdici.oo, J229--}J(l5. Pkil.ddpbi", WB Sawtd .... Ill. G ..."" GF. Tltonu. C 8, E.ich. SW. 1985. E.imatioo of'l"aoti".;..., po<>t
Chapter 9

MONOVALENT ELECTROLYTES AND OSMOLALITY Basic Concepts for the Interpretation of ElllCtrolyte Concentrations ................ Sodium INa') Concentration ................................................. I. Physiologic Processes .............................................. II. Analytical Concepts ................................................ III. Hypematremia ..................................................... IV. Normonatromia in Dehydrated or Edematous Animals ................... V. Hyponatremia ..................................................... Potassium (K') Concentration ................................................ I. Physiologic Processes .............................................. II. Analytical Concepts ................................................ III. Hyperkalemia ...................................................... IV. Normokalemia in Acidotic or Alkalotic Animals ......................... V. Hypokalemia ...................................................... Sodium to Potassium (Na' :K» Ratio .......................................... Chloride (Cn Concentration ................................................. I. Physiologic Processes .............................................. II. Analytical Concepts ................................................ III. Hyperchloremia .................................................... IV. Normochloremia ................................................... V. Hypochloremia .................................................... Bicarbonate (HCO,-) Concentration and Total Carbon Dioxide (tCO,) Concentration ........................................................... I. Physiologic Processes .............................................. II. Analytical Concepts ................................................ III. Increased Bicarbonate (HCO,-) Concentration or Total Carbon Dioxide (tCO,) Concentration ......................................... IV. Decreased Bicarbonate (HCO,-) Concentration or Total Carbon Dioxide (tCO,) Concentration ......................................... Anion Gap ................................................................ Lactate Concentration (L-Iactate and D-Iactate) ................................. Il-Hydroxybutyrate (BHB) and Acetoacetate (AcAc) Concentrations................. Osmolality and Osmo. Gap ..................................................

497 498 498 500 501 503 505 509 509 510 511 516 516 519 520 520 521 522 523 523 525 525 527 529 531 534 538 543 545

."

Table 9.1. Abbreviations and oyn,ools in this chapter Na+:K+

1Mg" HCI

Sodium to potassium x cona:ntr>tion (x = analytc) Ae<:tooUiate Ae<:tyI-=c:nzym~ A Amidiumic hormon~ Atrial n<>triure{ic pq>tid~ Ad~nosin~ tripho.ph>le Ad~nosine triphmpru.taR ~. Hydroxybutyr.ue ExtncelJul" fluid Ethyl~nedi.mine{elr ... a:tic a.cid Fr,"" ionized calcium Fr,"" ionized magn .. ium Hydrochloric acid

HCO,-

Bicarbon.t~

ICF IV

lmracdlular fluid lmra""nou. L-lacme dehydrogena.., M ....... red anion charge M....... red cation charge Rdative molecuur ""... Nicotinamide ad~nin~ dinucla>tid~ Reduced nicotinamide ad~nine dinucla>tid~ Ammonium Calculated mmolalil)' M ....... red osmolality Diffcrena: ~tw,""n measured mmol. lil)' and calculated mmol. rity Part;"] prclSllrc of carbon dioxid~ - log [H1 - log (IC.) wh~re K, is th~ ioniz>tion connam for a partially ionized acid Phosphate, all forms R~nin.angiounsin ')"lum Syst~mc International d'Unite. Strong ion differena: Sulf. te, all form. T mal :mionic charge Tmal body water content T mal body potassium come", Total body ,odium com~nt Total cationic charg~ Total carbon dioxide com~m Unm ... sured anion charge Unm ... sured cation charge Within th~ refc:r~na: imerval

[' I A<.A.

AoCoA ADH ANP

ATP ATP~

BHB ECF EDTA

1\:,'

LD rnA-

mC' M,

NAD' NADH NH, O,m, O'm. O,mo. Gap P~

pH pK. PO, RAS

SI SID SO,
,C' tCO,

""oC'

WRI

40;

."

9/ MONOVALENT ELECTROLYTES AND OSMOLALITY

", Alimootary ," ,

'.0

""".

-------------. Ext",,:........ , ' Cells

Roo

"+ "',0 00,

---- --------

Skin

:'

• Third "1"'''''''

(1' ....... . . _

Fig. 9.1. Basic roDc
JDOVeIJ>ffitl into :and OIlt of pl .. m•. Ekctrolym rnd H,O . nt .. plasm.. from th. :dimentuy trxt, from ""Jl~ and by inj.crions or infuUons. In vitro lysi.s of blood ""It. . nd ph.. kt :octiv:otion m:oy.dd K+ to th. pl:wru or oerum. Electrolytes and H,O m:oy I..n the pl.",». rnd th. body via ltidn.ys, alimmwy m et, r'"'Pir1tory m et. or .kin, >nd tt..y m. y ~tc< loss (•. g.. to pleunl >nd peritoneal caviti.. ). In "'m< p. thologic . ..... involving muscle. H,O or .kttrolyt.. .run be""".n th. ICF :and ECF '1'>=1 (_ the ",," for d.tails). Fluids (•.g., pl .. m. Of urin<) crn ~tnd H,O wiU be . boorbed (_ the text for d.tails). PIt. pJ.t
BASIC CO NCEPTS FOR T HE INTERPRETATION OF ELECTROLYrE CONCENTRATIONS I.

Ela:trolyt~

cooctntrations in .. rum or pla<ma :lr~ th~ n.-t result of intak~ , ncr.-tion, and shifts bttw..,n ICF and ECF. Shift, n.. y occur in vivo or in vilro. Th~ basic ooncq>u for d,""trolyte and H,O movemml< in and out of plmrta :ore shown in Fig. 9.1. Ela:trolyu conctntration, in .. rum or plam.. esstnti 0",1 intake of food or fluids. HCO,Iypic:olly is g.n~m.d by carbonic anhydras< ",aclions in lungs, g:ostric mucosa, kidnqs, and ~rythrocyt~s. B. Out,id~ th~ VlI5Cular sy.um, m~t>bolic processes govern the mo"'m~nt of d.arolyt .. into and out of ""Us. I. In h.alth, ECF is rdati",,]y rich in N.+ . nd CI- but poor in K+, wh ..= ICF is rdalivdy rich in K+ but poor in Na+ and ct- (acepl in .rythrocytes of some 'P'""ies and brttds). 2. El,""trolyu shift. from or to Ih~ ECF will alt~r pl.,m. or .. rum ron""ntrations of Iho .. d~ctrolyt~s. C. Cd], within blood m.y :oil" plasma or .. rum d,""lrolyt. concentrations if th~y rdu.. d,""lrolytes. I. In vitro lysis of K+. rich .rythrocyt~' will remh in f. lIdy d~'lal plasma [K+) . How~.., ~rythrocyu. a.. K+. rich in only som~ 'P'""ies and brttds (Ott Potassium Concentration, Kel. 1Il.B.l.h).

.98

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY Th~ [K+] is slightly gr~t~r ill srrum than in pl""mo b«:.Ust platd~ts ,.I"",. K+ during doning. Th~ magnitud~ of Ih. iner . ... call be, clinically significant wh~n {h,,~ is • thrombocytosis. D. Elec{rolyt~ •• nd H,O a.. ""creta! or lOS! from th. ECF vi<> kidll«:orne hypotonic with loss of hYJ'<'nollic fluids or

2.

hy~r{onic with Joss of hypotonic fluid,. Drinking H,O .ft" th~ 10M of ECF wiU dilute the remaining ECF. E. HCO,- ronctntration. are all.ral by changes in the ooncrmmioIU of oth" dectrolytel and by ch~ in acid.bast b:danct .

n.

Abnormal dec{rolyt~ oollctntrations in pi"""" or .. rum '<::'lult from one or mo .. of thost basic process." involving dectrolyte, or H,O: A. D«ru=! or inc",~ im:oke B. Shifts to and from [eF c. [ncre.~ renal ,«.mion D. [nne~=' Joss via the kidnry>, :dim~mary mel, skill, or ~irw;;oys (HCO,- indi..ccly)

III.

El«trolyt~ ron~mration' ~nd

acid. b..... bnormaliti.. A. Th~ regulation of .cid.b. st !latu, in .n animal involves physiologic proc...ses that may alt~r pl:asma (strum) oono;,mutions ofN.+. Cl-. K+, and HCO,-. th~ major topics of thi, chapur. B. Pathologic stat.. th at first .Iur d«trolyt~ rono;,ntration, am cr~.t~ .cid. b..,., abnor. m:diti ... the m.jor topics ofChapt~r 10. Thm. it i, difficult to oomplffdy .. parate d«trolyu rono;,pc< &om acid. bast rono;,pt,. The e~rly stction, of Chapter to oom.in the m.jor definitions and d. ..ifianions of .cid· base abnormalities. The ~nd of Chapt~r to romains • section on SID. The SID .pproach u,,,, d«trolyt~ roncemrations to define metabolic acid. bast disorders.

SODIUM (Na') CONCENTRATION I.

Phy>iologic processe. A. Plo,m. [N.+) is ne~rly equival~m to ECF [Na+). which i, dq>end~nt on th~ ratio of tbN.+ to tbH,O (Eq. 9.I .... c). Th~r~fore. pla,ma or ",rum [N.+] must ~ imerpreted with knowlalg. of the pati~m·, tbH,O (i.~ .• .c.te ofhydmion) ~nd ron,idemion of the pati~m's ECF volum~ (i.~ .. normovol~mic. hypovolemic. or hypervolemic). Although not rompletdy accurate. this b",ic rono;,prual rdation,hip .... i,t, in th~ und~manding of mo,t disord~" that affect plasma or serum [N~+] . .

Normonatr~mJa =>normal

.

HypermtremJa => Hypomtr~mi.

t

tbN a' tbH,O

=>

norm:d nonrud

tbNa ' i => tbH,O normal

~r

,l. normal

0'

t

,l.

I

...

or .,., ~r

I

(9. 1a.)

normal,l. I 0' I I ... ......

(9. lb.)

nornul.w. or i ,l.

(9. 1c.)

(9. ld.)

9/ MONOVALENT ELECTROLYTES AND OSMOLALITY

."

B. A mo.,"" acrur>U rdatio.nship is mo.WII in Eq. 9.ld. in which Na+. and K". r~p",""nt th~ ~xdtang~able N. + and K+ in th~ body {io.m bound in mo.b:ul .. a.. not exchan~able}.' From this '"')uatio.n. it em b. =n dut the total body ",crungeoble K'" coment influencu plasma [N . 1 . Thus, when hypokaJemift Sodium Con""'ntratio.n, Sect. V.B. 6). Hy~rkaJemia tend. not to cou"". significom hy~rnammia for two reasons: (I) a marked hy~rkaJemi
b. Con",,,ely, hypoosmolality leads to. d", .. asrd H,O intake :md incr ......,j urinary H,O excretio.n. 3. Plasma [Na+] is .lso somewhat sdf.regulated in that hyponat,""mia promotes aldosuron~ secretion .nd thus Na+ retention. whereas hypernatremia inhibits aldosuron~ secretion and thus reduces Na+ .. untion. Howe",r. plasma [K'"] .nd alteratio", in .ffectiv. blood volume ar. the major facto." that control aldon.rone sec.. tion.' E. N .+ and ct resorption in the distal nq.hron also invo.lves an .ldosteron~.indepc:ndem N.+.Ct cotr:m.pon.r; th~ ..1Orptio.n via this cotr:m 'poner incr~...s with incr.......d Na+ deli""ry to the dinal nq.hron. Thiazide diuretic> block this ootransporter by binding to th. ct receptor.

500

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

F.

Th~

pathologic

sta(~

of d~hydra{ion is «!uiv:d.m to d=•• lffllbH,O.

l. D.hydrat«i .nimah have .ilh" a H,O ddicit or a H,0 and Na+ ddicit .

•. Cause" of a H,0 ddicit (without Na+ ddicit) include dec",,=<:! H,O inuk. and loss of fr« H,O (umral dia~{e'l imipidu., n.ph~njc diabtl •• insipidul, or in..,nsibJ. respiratory Joss..). b. Cau'ieS of H,O and N.+ deficits include alimentary [ = {",miling. diarrhea , ,,,,!ue;{ration, or ncess;n .,livat;onj, renal Jo~ (most polyuric ,ute.), and eulana",. lossr. (,~ating). 2. Types of dffiydmion a. H ypun. mmic, hy~rosmol", or hyp Na+ loss. b. Normoruuremic, iwosmoJar, or isotonic dehydration is au""" by net isoosffiolar or isotonic fluid Joss; thai is. H,O loss .. N.+ loss. c. Hyponatremic, hYJlOOsmolar, or hypotonic ddtydration i, c;ou=l by n<'t hyp
Analytical conctpu A, Turns . nd units L Assays m=ur. th. d«trical pot.ntial of N.+ by dir«t or indir«l potentiometry, and then th. d«trical pot~nt",l is conv~rt.d to conctnt.. tion uniu,' Na+ is mostly ~ fr"" ion in th~ plasma or ",rum H,O, a, In di=t potentiometry, Na+ ion activity (d«lrical pot~ntial) is m=ur.d in • nundiu.ud ... mpl~ by th. int~raction of N.+ with th~ surf.~ of.n ion_.d«liv~ d«trod~, Th~ resultant [N. +] r~pr=nts th. [N.'] in th~ """'pl~', H,O, [fth. sample contain.d I... H,O than normal b«au.. of incr~as.d prot.in or lipid, th. result would ~ th~ sam~ b«au.. only th~ . ctivity in th. H ,0 pha.. is m~asur.d,

b, In indi""'t pountiometry, th. sampl. i, diluud (sometimes gr....tly) ~fore Na+ ion .ctivity is me~mred by the interaction ofN~+ with the mrface of an ion_sd«tive d«trode, Th~ N.+ ion activity m..... ur.d d~nd, on th. [N. 1 in th. H,O portion of the dilut.d sampt.. The sample [Na'] is deurmin.d by multiplying the m.asured [Na1 by th~ dilution f. ctor, [f th. sampl. contains incrras.d lipids or protei", and therefore a lower pu~ntage of H,O , th. H,O portion will ~ dilutal more thon mual by th~ diluent .dd.d, Th. meamral N.+ . ctivity wiU th~refore ~ falsdy detr......d, Wh.n the m<'3.sur.d value is multipli.d by the sample dilution factor (rather than th~ H,O_pho", dilution factor), the ..stdt will also k a faJ.dy d«ru..d value for [N. +] , Results rell«t mmollL of klmplt rather thon mmolfL ~f H,O, as for dir«t pot~ntiom.try values, c, Flam~ photom<'tric methods hove th. sam. probl.m as the indir«l pountiomet_ ric methods in thot high conctntr:uio", of solid, in th~ """'pl~ th . t displa~ H,O {',g" lipid~mia or hyp<rproteinemiaj cau .. faJ.dy d«reased [Na+] , 2, Unit conv~rsion; mEqIL X I = mmoliL, and mgldL -t- 2,3 = mmoUL (S [ unit, n~arest I mmolfL)' B, Sample for [N . +] quantiution L Serum is pr.f.rred [Na+] is subl. for months if the sample docs not ddtydrate, 2, N a,EDTA plasma should not b. m.d b«au.. the anticoagulant'. N.+ will ~ in the plasma, The .mount of Na+ in Na_heparin is too small to cau,. clinically rdev. nt ch. nges in h.parini=! plasm. [Na+],

9/ MONOVALENT ELECTROLYTES AND OSMOLALITY

501

Table 9.2. Disca.... and conditions thaI ca u.e hypcmatren,;" H,O-ddicil group {d Na+ 10.. Rc:nal: 05molic diur~sis Alim~nury: osmotic di .. rh~. , osmolic ~uestration. phosph at~ ~n~mas. or paimball toxiCOlis Na+·rxcess group {increasN lotal body Na+} ExctSS N.+ with concurrent reslricred H,O inukc Salt poisoning Adminislration of hyperlonic .alin. or ,odium bic;ubonat~ D
or unknown mtth.ni,m ex~rcise in grryhounds • A r.tll;v<]y oommon di..... or condi,i"" N o\<: Sampl. ~.pon tion or mb~m.tion wiU

s.,,,,,,,,

CIDS<

'!'uriow hyperrutremi •.

C. As.ays I. 10n••elCCliv~ d<etrod. =ys a.. Ih~ m""t conunon. 2. F1:mt~ photomel~'" for m=uting [Na1 used 10 b.: the gold su ndard, but the inurum~n1s,,,, no lon!:"r uoed for routin~ pati~n1 scr..,ning. Ill.

Hypernatremi. A. This occurs when the ""io of IbNa+ to IbH,O is incr~ascd (Eq. 9.1 b) or with th~ shifting ofH,O from ECF 10 ICE B. Disord~", and patho!:"ne= (Tabl~ 9.2) l. H,O-ddicit group {dRS via kidnqs.lungs. skin, or intcnin~ may product dehydr:n ion. {2} Def. lor that Irif,!:"rs a thim r..ponst, or th~ thir.st ctnl~r ilself may b.: dam~ed. Hypodipsic hypernatremi. in minialUre ochn.uu:rs may b.: couscd by lob .. holoprosc:nctphaly .•

502

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY h. Purr H,O loss without H,O .. pJactm~nt {I} In .. nsibJ. 10.. ofH,O by panting. hypt"",milnion, or fenr: The animal ba:omes H,O d.pJeud by losing H,O via the "spimo!)' sySlem or ,kin. {2} Di.b.t.. insipidus {ctntral or nephrogenic} (a) [n the di.btt .. insipidu. dilOroers, diminish. d AD H activity or "'PODst in the roU'""ting ducts may re;ul{ in pure H,O loss {i .•.• urine with very low OOD"'niutions ofN.· .nd oth.r solutes: urine .p«ific gr:lVity app'''''ches 1.000). (b) Animal. with d"-"'I .. in'ipidul that h."" unrestricrffl :lCC<:SS to H,O may drink sufficiendy to prevent the hyptrnatremia. {3} Adrenocortical na>pl:asm socreting oonioonerone and :.!dost.rone in a dog' (a) This disorder is uncommon, and th.labor:ltory feature; of the ""'" w
hy""rnat",mi., hyptrchlo .. mi., hypokalemia, hyposthenuria, and abnormal ,esults of adrenoconirotropic hormon~ nimulation t .. u (iruod'"'lu .r~ incr..,... in strum [ooniKlI] . a~~rat«i incru,~ in [coni. ooneron~]. and ~rat«i incrUst in [:ddost~ronell. {b} This hyp.. natr~mia probably is rd.r«i to the .. nallms of &tt H 2 0 kau"" of th~ inhibition of ADH rdust by oonico.!l~rone. c. H,O loss;> Na+ loss {I} Osmotic diuretic ag~nt. {e.g.• glu= and mannitol} in .. nal tubular fluid inhibit passive H2 0 r=>rption. {2} [n the :dim.ntary .ystem: {al Accumul.cion of osmotic ~nts {.. occurs in K1m~ diarrh....} will inhibit H,O absorption. {b} A phosphat~ .nema will pull H,O from ECF 'p'= to the rolon. Concur.. m colonic .bKlrption of N.+ may .ugm~nt the hy~rnatremia. ' (c) [n ruminal acidosis (grain overload) •• ccumulation of K11ut.. {induding lactic acid} in the rum~n causes th~ OmIotic movem.nt ofH,O into the rumen to produce hyp<:rruotremia.' {dl Dog. with paint ball toxicosis {aft.. ingestion of paimb.Jls} may d~op hy~rnat"'mia kaUst th~ paintball. oontain osmotic molecul.. {e.g .• glycerol and K1rbitol} tru.t pull H,O from plasma to inte"ines.· {3} The H,O th.t move! during osmosis contains. srruoll .mount of N.+ kaUst of K1lut~ drag. but the net r....lt i, gr.at.. lOS! of H,O than Na+. and thus hyp<:rnatr~mia occurs. {4} Unremict«i access to H,O may prrv~m this hyp<:rruotromia. Al",. oth~r process~. in rurn ...... {e.g .. ketosi.} may inc ...... r~nal N.+ loss and roumerae! hypern.tremic t~nd~nci... 2. Na+-= group {incteas«i tbNa+} a. Ex= Na+ with concur.. m r.. trict~d H,O intake (r>te) {I} S.lt poisoning: Cattl. with a C6Sive Na+ (and ct- inta~) and with roncur· .. m =trictffl'=SS to H,O may drvdop an incr.... «i tbN.+ (and total body Cl- rom~nt) and thus hyp<:rn.r .. mia (and hyp<:rrnlor~mia) .' Ext.. m. hy~rnatr~mia .nd hyperchloremia ocrurr«i in • dog tru.t inge>l«i a salt.flour mixtu .. that was used as modding day." (l) Administration of hyp<:nonic salin. or sodium bicarbonate am l~ad to in~ tbNa+ .nd [hus hy"",natr~mia (and hypen:hlorc:mia or in~ HCO,).

9/ MONOVALENT ELECTROLYTES AND OSMOLALITY

503

b. D=.,.sa! ",nal acrffion ofNa+ {h~raldost~roni,m} (rar~) {I} fue;.,ssi"" aldosuron~ promot~. acessiv~ ",nal N.+ {and CI-} re{~ntion. Hyperruot"'mia (and h~rchlo .. mia) =y occur if th~r~ is H,O r~striction or d~f=i"" ADH activity. {2} Th~ Na+-maininJ: activity of aldostuon~ may play a rol~. but, beau", of a mManism ref~rra! to .. aldo.!l~rone =~, hy~raldosuronism d~. not typicaUy co"", hy~rn.mmia. One;., N.+ retention begins, .nd the", is corr~sponding H,O r~t~ntion, n.triuresis p=nts the developm~nt of hypernatremi .. The natriuresi, m.y be promotal by ANP.' 3. Oth.. or unknown mM.ni,m, a. Sev~", ""erei", (racing greyhounds}:u-IJ During .nd imma!iatdy alier a rae;." the [Na+] in pl"'ma =y be 10-20 mmollL .bo"" p...."" concentration •. Th~r~ is. concur",nt hypovol~mia and lactic acidosis; thus, the hy~rnatremia may be COUoN by th~ shifting of H,O from ECF to ICF. The accumulation ofL-lact. te in muscle fibers may cr.,.te th~ osmotic gradient.' b. £omple dehydration: EJrp<>!ure of ",rum or pla,= to air may allow evaporation that cau",. hy~rnatremia. This is especially true of .ir-conditioning syst~m. that blow cool, dry air over th~ sample proce... ing or . nal)"is ..."'. Sublimation of H,O nom frozen .. mpl .. may em", hy~rnat"'mia if the storage cont>inu is not . dequately •.,.la! or ifit is too large for the sample size.

[v.

Normonatremi. in dehydrata! or a!ematou, ani=ls A. Normonatremi. does not n=rily indicau that th~ Na+ bal.nc~ is normal {Eq. 9.1.}. Recogniz.ing th~ po ... ibility of altera! N.+ regulation in normonatremic ani=ls is import>nt in the =ruogtm~nt and t ..... tm~nt of these ca>t •. {That is, do you need to administer Na+ or restrict N.+ int .k~?} B. Disord~" and pathogtn= (T. ble 9.3) I. Net 10... of isotonic fluids that couses dehydration a. Aliment>ry loss of isotonic fluid =y occur with vomiting. diarrhe., or sequestration. b. Renal loss of Na+ and H,O may occur in "",eral situations. {I} Many polyuric r~nal di..,.... cou.., Na+ and H,O loss beaus< of def=ive tubular functions. {2} Osmotic diuresis imp.i.. r~sorption of H,0 in tubules. A high tubular flow rat~ also contribut .. to Na+ 10... Tahle 9.3, Dixa.... and conditio"" that cause normonatremla in dehydrated or edeo,atou. animal. Net loss of isotonic fluids coming dehydration 'Aliment>ry loss..: vomiting, diarrh.,., 'e<J..uestration 'Renal 1=: ren.1 disease. osmotic diuresi., diureti", Skin lou: ,,,,,,"ting in horses Net .. t~ntion of isotonic fluids cousing alema or transudau 'Congrni"" hean f.ilur~ 'Hq.atic eirrh",is 'Nephrotic syodrom~ • A ,.t.,i""ly rommon di..... or rondi,i""

'04

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY ~m, (~.g .• fi."os.mide or thi.z.id .. ) em", N.+ alld H,O loss through inu.fe""I1"" with C]- resorption.

{3} Mon diuretic

c. Cutanam, 10.. in horses: Th~ [Na+] ill swe~{ is ~bout th~ ... me as the [N~+l in ,erum or plasma. Thu.s, profu", ,~tjllg without incr..asnl H,O imak. could cau.. normon.t""mic dehydration. 2 Net raention of i50tOllic fluid, thai cau.., .dem. or transudation (Note: Ed.matou, dilOrders may crrate eithor normon.trem;" or hyponatremia, dep"nding on the

rd.ti"" mention of Na+

~nd

H,O.)

a. Cong.ui"" hean failure (causal by valvubr di ..as. or cardiomyopathies) {II "Forw..d" hypothesis: C"di.c di..,..., d,""",=, cardi"" output, which is ""mal by I>.rorN:tptors as decreasal df=ive blood volume, :ond {hut the .ymJ>'lth.tic nervous .)'Sum .nd the RAS are stimulated. If thest ..sponses do not ftt:'ltablish the dfn:tin blood volume. continual .ctivation of th~ RAS promotes ",n.l resorption of Na' and Cl-, which increa= pl",,,, osmolality. Hyperosmolality nimulates rd..,.... of ADH .nd th~ thirst "'nt~r, which =y calJ.5t mention of H,O .nd inc",a.o;al blood volume. [f the hypervolemia inc",ases venous hydraulic pre!lUre sufficiently, it promous mo""ment of isotonic fluid to extravascuJ:.r spaces and thus fOrmation of edema {pulmonary or d~ndent} or accumulation of H,O in th~ pleural or peritoneal cavities (transudation). {2} Retention ofNa' .nd H,O i. a compensatory procns th.t hdp" m.intain .n df=ive blood volume. [f df=ive blood volume is not achieved, the hypovol~mic stimulus =y promote thirst and thus iner ...... H,O intake. Mammals with. cardiac di ....., that decr..,....s cardiac output will Ita"" incr .... oed tbNa' .nd tbH,O ",""n without clinical edema. b. HqJatic cirrho.is with abdomin.l transudation {I} Three theories {und~diJJing. overflow, and peripher.ol anerial vasodilation} anempt to explain the peritoneal transudation that occu", with hep>tic cirrhosis.'~"

{a} [n the undafilling "1(ory, the initiating event is the IOSJ of plasma H,O caused by iner .... oed hydr>ulic pressure in hqJatic sinmoid. {caused by fibrosis or venous rongmion} and loss of protein_rich pluma into th~ of Disse {sinusoids a", highly perm~able to albumin}. This cau~ .planchnic pooling of blood and 10.. of H,O across the hqJatic cap"we to the peritoneal cavity and thus underfilling of vaocular .p• .".. Mo",,_ ment of fluid from ""... Is decr<"aWl the effecti"" blood mlume, which activate. the RAS, stimulat~. the rd~ ..e of aldost~rone, promotes the retention of N.· and, subsequently, H,O. The apandal blood volume a"",ntuates vascular hydraulic prelSUre. and thus promote. more 10.. of plasma H,O and mo", .ttempe. to rompens,ue. At the time of clinical transud.tion, th~ .nimal wiU h."" incr.....d tbNa' and tbH,O; inc",asaj ooncrntmions of renin, norepinqJhrine, and ADH; and reduced renal acmion of N.·. {b} In the ~"i1ffow [he initiating event is renal retention of Na' and H,O, but the f.cto", involvtd are poorly und~"'tood. {c} [n the "mph",,{ amri,,{ "",odilAtion thtory, peripheral vasodilation dec",,,,,,. the df=ive blood volume, which .ctiva[es the RAS. This

'P>""'

''''''ry,

9/ MONOVALENT ELECTROLYTES AND OSMOLALITY

505

inc""ast. hydraulic pressur~ in h~patic sinusoids, which l~ads to transudation, {2} H}'IX>"lbumin~m", .nd hypoprot~in~m", play =ndary rol .. in ascites furmation, {a} [n h~.hh, plasma prot~ins cr~.u colloidal osmotic (oncotic) pr..mr~ (about 80 % from albumin and 20 % from globulins) th.t hdps main H,O in v~ssds trull ..~ im~rm~abl~ to prot~ins. Wh~n hypoprot~in_ ~mia i. pres~nt with hq.atic cirrhmis, th~ increases in vascul .. hydraulic pressur~ h. "" mor~ df<'CI in promoting mo""m~nt of H,O from som~ v~ ... ls {h~patic or pulmonary} to atravascular spa"... {b} Hypoalbumin~m", alon~ will not cau", th~ tranmdation. Peopl~ with analbumin~mia may hav~ mild d
v.

Hyponatremi. A. This typically occu" when th~ ratio of tbNa+ to tbH,O is da:reastd {Eq. 9.lc} or with the .hifting of H,O from [CF to ECF. B. Disordm and pathogtneses (T. ble 9.4) I. N.+-deficit group: net N.+ loss;> H,O loss {hypotonic dehydration}. In the follow_ ing disord~rs, th~ [Na' ] in lost fluid is usually not gr~aur than th~ pla.ma [N .' ], but loss of Na+-rontaining fluid fullowal by drinkiIli: of H,O may cause hyponatr~mi .. a. Alimentary loss ofNa+_rontaining H,O m. y occur with vomiting, diarrhea, sequ.. tration, actss ..livation, canin~ whipworm infa;tions, .nd bovine h~mor_ rhagic bawd syndrome." For th~", conditions to cause hypon.trem"" ther~ prob. bly is • loss of isotonic ECF follo_d by drinking H,O and r~nal H,O retention {ADH r.. pon ..} that dilute th~ remaining plasma N. +. b. Renal loss {I} HYI""'drenocortici,m:' Adr~nal inmfliciency da;r ...... the [aldonerone], which cause. Ie .. resorption ofNa+ .nd mbsequently ct by th~ renal principal ctlls. Da;rrased Na+ .nd ct resorption l~..ds to d=~astd plasm. osmolality and d~cr~a...d renal medullary hypenonicity. The latter l~ad. to • da;rea...! ability to resorb H,O, so hypovol~mia ~",ues. Hypovol~mi. stimul.t~. ADH rdeast and thint cent~n. [ncr~a ...! ADH activity .nd incr ......! H,O intake dilute ECF N .+ . nd thus cause hyponatremia (and hypo<:hloremia). Hypoconisolemi. promou. the hypothalamic rdeas. of

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

'0;

Table 9.4. Di..,a..,. and conditions thai ca u.c hyponatremia

Na+-ddicit group: IIcl Na+ loss:> H,O loss (10 .. of N.+.ronuining Huid fOIJOWffl by incr~~sffl H,0 int:ok~. hypotonic dehydmion) 'Alim~lIt>ry Joss: vomiting. diarrh~~. sajueslr.otioll. Gl.llin~ whipworm in!ttiion, ex", .. ..livation, bovill~ h~morrhagic bowd .yndrom~ '!kn. J loss: hypo:od",nororlici,m, proJo~ diurn;i" ~onuria, N. +. waniJ\i: IIcphrop. thi .., hypoaldon~ronjsm Cutanams loss: sw
'Congeslin h....,t failure Hepatic cirrho,i, NqJhrotic .yndrom~ Exl"lIIdal ECF volume (bur without ed~ma) Syndrome of in .ppropriat~ ADH ..action Ex<:es.l administration ofN.+. poor Huid. Shifting of H,0 nom lCF to ECF • Hypugly""mi. Mannitol infusion (IV) Shifting of Na+ from ECF to ICF Acut~ muscle dunag. Shifting of Na+ from intra""",,ular to atrav ..cul .. Huid Urop",itoneum K+ depletion causing .hifts in N.+ and H,O • A "I. tively common 1l1to",ti. m.y b. uu...d by

displ~t of.«rum

or pl:wru H,O (... tb. ton).

ADH (muhipl~ th.ories), which cau... def"'tin acretion of H,0 (,.tention of frtt H,O in th~ pr=n"" of hypoosmolality) and a dilutional hyponatremia. U' In palpl. with hypoaldon~ronism :md l"d, of hypoconi. salemi. , normon. t,.mia i. maimain..:! by .nh. noM tubular resorption of N.+ ams.d by inc ........:! angiounsin II, d",reas.d ANI', and enh. nced pa.<sin "K1rption.' {2} Prolon~ diuresis (a) Prolong..:! osmotic diuresi. with Na+.poor Huid" or other forms of diuresi, k.g., caus.d by furo .. mide . dminim. tion}" t~nd to deplete Na+ and H,O, cau.. hypovolemia, and stimulat. ADH rd . ... and thirst ""nUrs. H,O drinking t. nds to dilut. th. Na+ in th~ ECF and thus ca ..... hypon. t.. mia. {b} Thiazide diumics cau .. N.+, ct, and K+ loss in ae=; of H,O lOS!, and thu, hypon. mmia, hypochlo,.mia, and hypokalemia may devdop. Urinary N a+ and ct lOS! is incr ... s.d b<e. ..... thiazide. inhibit a Na +.ctcotra"'pon~r in disul nephrons. Uriruuy K'" lou i. incr~as.d b<e.UK of an inc=s.d How raU in distal nephrons and hypovol. mia_induced rd~ of :ddost.ron~, which o~ns K+ channd •. The hypovol.mia also

9/ MONOVALENT ELECTROLYTES AND OSMOLALITY

''''

nimulat .. ADH rd~~"" which promou, r"orption of frN H,O and thu, dilution of th~ r~nuining Na+, ct, and K+, {3} K<:tonuria {a} During k~onuri., th~ aa~tion of ~on~ bodi~, (AcAc ~nd BHB ) i, incr~:asaI, Th~ p.... nce of thest non.bsorbabl~ anions in the tubular lum~n oblig. u. the acretion of cotion. and thus ina~~.M renal excretion of N. +, {b} Concurrent ounotic diuresis (&om glucosuria) may compound th~ Na+ loss :wociated with ketonuria in pati~nts with di.bete. mellitus, {4} N,+.w:lS{ing n~phropathies: Som~ renal di"' .... {especially tubular disea",. or pyelonephritis} cou", .n aces.> excretion of N.+ becou", of dec ....ed N. + resorption, Thi, is "'~n more in horses than other dom~nic mommals, {5} Hypm~nin~mic h}'ll'O'lldost~roni,m (Ott hyperkalemia) c, Cut;m~ous 10.. by sw~.ting {I} Among the domestic m.mmal., only hor= .wrat JUffici~ntly to cou", dectrolyu and H,O imWLonce" {2} Equin~ sweat is . Na+., K+., Cl-.rich fluid {concentrations.", gruter than pl:uma concentrations but ~poration may rontribut~ to the inc......),",· Drinking of H,O or th~ ADH.stimul.ted ""~ntion ofH,O .ft~r sw~~ting Im}' l~.d to dilution.! hyponatr~mia, d Third.space loss (typically loss to pleural cavity or peritoneal cavity) {I} ~.ted drainage of chylou. thoracic effusions (a) R~peated removal of ilOtonic fluid &om th~ thoracic covity probably r~sults in. N~+· and H,O-dqJleted nat~ that is followed by intake of H,O and.n ADH =pon,. to come dilution.! hyponatr~mia,"'" {b} This 'YJl<' of hyponatr~mi . would prob. bly also result from .. pe~ted r~moval of oth~r Qlviuty effusions, but such removal is not as common as it i. for chylous ~ffu,ions, {2} Acute int~rn.l h~morrhag~ or arut< exudation {al Acut~ I"" of ECF from th~ vascular space to other extraceUular sites in the body (third·.p.ce loss) cou... an isotonic hypovolemia, [f followed by inc.....ro intake ofH,O and.n ADH r~'pon .. , dilutional hyporuo. tremia moy drvdop, {b} This 'YJl<' ofhyponatr~mi . is rare, since hypovol~mi. would allO nimulate renal N.+ r~ention, 2, H,O-= group (H,O ""~ntion;> N.+ r~t~ntion) with or without ed~ma a, Edematous disorders {I} Congesti"" h~~n f.ilur~, hqJatic cirrhosis, . nd Mphrotic syndrome {2} Although th= conditions .re oft~n """",iated with normonatremi. {Ott th~ preceding sect, [V,B,2l , hyponatremia {typically mild} may also occur if th~ ratio of tbN.+ to tbH,O is decreased. b, Expanded ECF volum~ {but without «Iemo} {II Syndrom~ of inappropriate ADH "",,,,,ion (SIADH) {aJ This is chara.ct~riud by nonphysiologic rei..... of ADH in th~ prestnce of hYI""',molality and hypervolemia, Excess ADH with unr~strict«l H,O intak~ l~.d. to fiN.H,O ",""rption and dilutional hypon. tremia, R~nal N.+ excr~ion (perh. ps due to ANP) is incr~...d becou .. of activation of mlume receptors,'

508

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY (b) Nalfologic, pulmon.,y, and thyroid di""rd~n ar~ ow;oci>tffl with th~ 'rndrom~ of inappropri.te ADH ''"''retion, ••• '" many drug•. A f~ cast. h:IV~ oon reportffl in dome'tic marnn",],.'" {2}

fu~sivt

administration ofN.+_poot Huid.: Administration of 5 % dalro .. or 0.45 % ..lin. could lr.d to a dilucionaJ hyponatrrmi .. {3} Primary polydipsia moy cau.. hyponatremia but typically js • minor abnormality b.COUst funcrional kid"q' Gm nertl< Ih. oonsumal H,O."

3. Shifting ofH,O from lCF to ECF •. A markffl or ""n;sunt inc"",,,,,, in dfa::ti"" pla,ma o!JI\OJality (t.g., b«au.. of hyp<m«i to dre,..,... 1.6 mmoJIL for g1urosr con"",mrations < 400 mgldL .nd 4 mmoUL for glucose con""mmion, > 400 mgldL 1'J b. Conru ... m proe<SSeI {osmotic diu ... is and kffonuri.} :dso contribute to the Na+ loss .nd thu. hyponamm",. 4. ShiftingofNa+ from ECF to ICF a. AmI< dam"l:e to the ""II membrane of mu.de lib.rs :dlows Na+ to mo ... from a high con""m.. tion in ECF to a lower con"",mration in ICF." b. In thi, condition. th.", it a ronmrrem influx of H,O .nd fCo'+ and .ffiux of K+ .nd PO. to "'us< hypovolemia, hypoca.l""mi., hYJl<'rbl.m"" .nd hyp<rphosph>lemia. 5. Shift of N.+ from intr.v,,,cul.. to atrava.llCular '1"''''' a. When utin., typi",lIy. Na+ .nd ct poor fluid, i. in the alxlomin:d ",vity {u,."prritonfum}, diffi... ion of N.+ and Cl- from the plasm> to the l"'ritonnl fluid may ",u .. hyportat"'mia and hypochlo"'mia." b. Experiment:dly in dog" hyponar"'mia and hypochlo"'mia we", det«ted within 1- 2 d of the O"""t of utoperitoneum and wer. marked .ft.r 4 d." 6. K'" depletion cou,i"l: mifu in N.+ and H,O' (..e Eq. 9.ld) a. As d=ribed l. ter in thi, mapter {,~ Pou.!ium Con""mration, sect. V.B.2}, K' con b. Ion from the body duting a variety of gasrroimeuin:d .nd ",n:d dilOrd.rs. If these loss.. are not replacal by imake, the body will b.com. K'depleted. b. Th.,e K'" 10JSeS d«rease the atracdlul.. [K1, whim creat.,. gradi.m for K+ mo ... mem from cdb to the ECF. Bn::.u"" prol bind with intra""J]ular buffe...nd thu, do not .dd to the imra.cdlular o,molality. However, the K+ loss from the ""J], lowers the imra""J]ular o,mol:dity, .nd thut H,O mov.. from cd], to the ECF, thu, diluting the ECF [N.+]. 7. r.. udo_hyponamm", a. This may occur when the .erum [Na+] i, del
9/ MONOVALENT ELECTROLYTES AND OSMOLALITY

509

Excess H+ p
t [H'J

[H1

', -

"

t [K1 ·.

j

""-

[1<1 C~

PIa""", ',[H1 j(K'J ""

""- ,

"[H1

[1<1

[H1

C"'

[1<1

C"' Abiosis d"" \0

0"""" K'

loss

Fig. 9.2. H+ and I(' mili in :ocid·b..e disor<\en. • In an inorganic . cidosis. th< .. is :on >
• In." :oI.blow. [H'l in ECF is nducod. As H' shifts out of ""u, to . qui~br.lte co",",ntntiolll. K' mte" ",lis (10""" tb. pl .. m>: hypoblemi. OC<W""5). Th< iUustntion ,00'" tho :olhJotic It... b.ginning with H+ loss: tb. H' < due to othor p~. • Conv< duo to other proc<sses.

b. Loooratories may m~. !Ure .. rum or pl:lSJn" [Na+] by dirttt pot~ntiometry by using ion_sdttti"" d«trod.,. Such assays do no. requir~ .,mpl~ dilution. SO n =s solids in th~ pI"""", do not affttt .h.m. Th~ d«.rod .. det«. ch~ Na' "ctivity in th~ aquams pha.. only. POTASSIUM (K) CO NCENTRATION I.

Physiologic process<. A. S.rum [K'"] is n.... rly el :dt~r th . ..rum [K+] (Fig. 9.2) . l. An inorg:mic or min,,:d .cidoois (QluKin":! :md .hm K+ d""s no. nttd to l~.v. th.

.0

alJ.

510

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY b. Acid~mja =y promote the Joss of K' from cdl., but the K' i. excretal in urine with [h. organic ani"", (e.g., L-1a.cYle or AcAc) that at• • 150 ncr.tal during locric .cido";, and k.{~idosi., r.,~ivdy. 3. Treatment of a normoblemic acidotic sUte may COUK hypokalemia that reH,""cs tbK+ depletion in an animal. 4. Meubolic albJosi, may am .. mild hypokalemia. 5. Respiratory acidoses and alkal<>Se'l are not associaud with alt ..ed ,
denio!"." E. Plmna [K+] is r~uJ.ted through two /lUjo. proa.... s: {I} distribution ~ttn EC F and lCF, :md {2} r~nal excr~ion, L K+ di,tribution btt_. n ECF :md lCF a, Epin~phrin~ .nd in,ulin promot~ th~ upuk~ of K+ into ~lls through th~ .ction of a N, +_K+_ATPa .. pump, T hi, df"'t ofinmlin i, ind.~nd.nt of its actions on gluro", upta~, b, Hypubl~mia promotes th~ cdlular upuk~ of K+, wh"..... hypobl~mia promotes th. loss of lC'" &om ~ll" 2, Ikn,l acretion of lC'" a, Typicolly, K+ is resorbal btfor~ th. ,",nal tubular fluid r.aches th~ distal n.phron, Th~"fo", th. [lC'"] in tubular fluid ~ntering th~ dist :d nqJhron is n.ar uro, b, lC'" i, sn::retM prim>rily by th. princip:d ~ll, of th~ coll~cting tubules (Fig, 9,3), Aldost~ron. promotes this proa:ss by stimulating Na+_lC'" _ATP,... in th. bawlat_ u :d m.mbran~ ,nd o~ning lumin:d lC'" chann~ls, Hy~rk:d~mi. ,nd angiot~nsin II a" th. major ,timul:mt, of :ddoneron~ ,,,,,,,tion, Mr"lOOOrticotropic hormon~ (ACTH ) and hyponammia:d", ,timul.t~ :ddoneron~ rd~=, but the df"'t of hyponatr~mia is diminishM if th~ ~f&ctin blood volum~ i, adry flow, Th • • nh:mcal =mion pr~nnu th. h~rk:d.... mia that might bt cau..d by dehydration_inducal oliguria, 5, Plosma [K'"] i, .Iso influ~ncal by int~,tinal and mtanams process." abwrption of di~ary lC'" by th. intestin~, loss of lC'" vi. f.al, and 10... of lC'" via .wnt. [ I.

An:dytical oonc
9/ MONOVALENT ELECTROLYTES AND OSMOLALITY PerHubular fluid &

511

Ii I cell A~1r Ita

+----------::--

" . .--

AlP _______ •

----- .

,.

".

a-

Fig. ',I. ) . Actions of :ddoottor oompla (AJoo..Rq>tr) stimuht.. th. 'J'Ilth..is of :ddooteron<_indu=l prot';ns (AlP), whiclt rruy incloo. romp<>n. foUowing: I. No- is ""roped from th. aU to the p. , •..,rption of No· through . n open«! No+ cb:mnd. 2. K' is pumped into tl>. ""lJ from th. p. No+_K'_ATPu. pump mull> in . net ntiw cbarg. in tl>. tubul:u fluid (3 N.+ ,<SO""'" .00 <:. n. ,nention of W in th. tubular fluid (tho W that p""",ly Ie...-es and , ... nt<J"lI tho ptincipal ",lis) and thuo inc, ..... ren:d """,tion of H'. TI>. net """It of .Jdoot
2. Unit con"" rsion: mEqIL X I = mmolll.., and mgldL ... 3.9 = mmollL (SI unit, n~are;t 0.1 mmoIlL)' B. Sampl~ for [tc+] quantiution J. s"rum i, p .. f~rrni Th~ [K+] is nabl~ for month. if the sampl. does nOt dd!ydm~. 2. K, EDTA .hould not ~ usa! as an anticwgulam for t~"ing pl asma [K+] b.causr th~ :ontico:agulam'. K+ will ~ addal to th~ pl asm •• nd k m=rural.

c.

AssaY' I. 10n_.tI=iv~ d«trod. """'Y' are th~ most common. 2. Flam~ photomet~rs for m.-asuring [K+] we,., th~ gold stand..d but <=
III.

ar~

no

long~r

H YP'rkalem;" A. Thi, typically occurs wh~n th~ re nal n cretion of K+ d«rea= or K+ ,hift, from ICF to ECF. H Yp'rkaJ.mia m.y:also occur with incru!ed inu k. of K+ or trutm~nt with K+, .sp«ially if thu~ is r~nal compromist. If the :ocid_bo. .. statu. i. normal, th~n ..,rum [K1 tends to rdl= tblC'.

512

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

PerHubular fluid & I Aklost9fOOg

Aldo-Rcplr M

r.

.'---- ...

"'''', --c,-l,,- HCO, a-

a-

-----

Fig. 9.4. ~ .. ion of H- by typ< A in",rcaht«l ""J[. of tho di,,,,J nephron. AId"",,,,o,", en«,. th. typ< A int=.t.ted ",Us and binds to <=pOOl pro"'; .... n.. a1d05t< (Gf) to form HeO,-, whim it ucb:mg«l for a - in th. p<,<",tion. HCO,- production. rnd tit"" :dblosu.

0. ATP ... pump: O. =tnrup<>lt
B.

'"

countel Inn,!",ltel (rntipon .. ): rnd ... ...,i"" :ocicl..

Disord~n

and patho~n= (Tabl~ 9.5) l. Shifting of K' from lCF to ECF a. M~.. boJic illorganic acido ... causing acid~mia: iliiftiJ\i: of K' out of cells wh~n H+ moves in (Stt Fig. 9.2) b. Rhalxlomyolysis or oth
"""ute.

con k induced by ,m« or an ... h~tic ill dys'rophin-ddici~m co,," and illdud~ hyperkaJ~mic periodic par:olysi. in quart~r horse.." and "">libly hyperkaJ~mic p 5 mmoJlL but did incr ..... from 3.9 mmollL to 4.9 mmoUL ~fi~r n~rci,.).JO c. Strenuou. eurci.. may co...., hyptrkaJ.mi... HyperkoJ~mia i. cou.ed by K+ rd~= from .cri"" myocyt<:S, Th~ K+ rd..... might k rd.,ed to ion shift, COUsN by ~ d<ern.. ill nondiffusible imr:ocdlular ~niom ,h., occur a, phosphoc"'~till~, a highly di"""i.,ed a.cid. hydrolyzes to neutral cr~atin~. '" Also, myocyt~. m. y k {2}

CoII~nit>1 disord~",: Th~

dam~.

9/ MONOVALENT ELECTROLYTES AND OSMOLALITY

513

Table 9.5. Disca.... and conditions Ihal ca u.e hyperkalemia Shifting of K+ from ICF to ECF (no changt in lotallxxly K+) •Metabolic ~cidO!CS cau.s..! by :K:C\lmulation of inorganic acid, Rh~lxIomyolysi, or olh~r mmde donugt Acquired: sU~nuou, uerci .. , ..izUle;, .. Ienium deficiency Congtniral: hyp"rkalemic p"riodic paralysis in quancr ho .... and p"rh. !" dogs M ... ive: intraVllKlllar hemolysis in animal, wilh K' rich erythrocJ'us Other m ... ive: lisme necrosi, Afi~r uerci .. in hyporhyroid dog, Inc..asalrotallxxly K' Dec..asffi renal cxcmion of K' '!knal insufficiency or failure {primarily oliguric or muric} 'Urinary rracr obnrucrion or le~ into lxxIy H YP""ldo,reronism 'HYP""drenoconici,m (Addison'.) Angioten,in-ronvcning enzyme inhibilon Hyp"rr~ninemic hypoaldosuroni,m Trimerhoprim.inducffi K+ re{ention Inc,",asffi intake Admininmion of K+.rich fluid Other or unknown meduni,m ~~tal dnin:oge of chylou. thoracic ~ffu,ions Perilon~a1 effinions in caU • A l.tui",,[y oommon dio.... 01 condition N ot<: Also OOruid.1 pseudo.hypeltw.mi. (_ tM text)

d. Massive: inu."">cul,, hcmolysi, in mimals wirh K'.rich erythrocyte;: English 'pringtr 'Jl'Inid. with phosphofrucrokinaso: ddici~ncy have: hy!",rl.:alemia concur.. nt with their hemolytic epiwdcs which might involve: K+.rich young erythrocyte,," e, Massi"" rissu~ ncero.is (in lissu.. other Ihan mu.de or bIO<X!), which i, cau.s..! by Ihe rdea .. of r .rich ICF from d~ad cd!., i• ...,n wilh mmor necrosi" .nerial rhrombosi. with lissue ischemi., . nd wmerimcs just prior 10 dealh, f, Hy!",nonicily (c,g" cau.al by hyp"rglyco:mia in diabet .. mdlim,) leads 10 an osmoric shifr of H,O from 0::11, 10 ECF, thcr..by inc..asing Ihe intracdlular [K+] {bur norlotal K+ rontentj, K+ rhen .hift, down a ron""'ntrnlion gudient into ECF. Although rh~ K+ shifr deere .... the imrao::llular [K+], Ihe ne{ effecr on rhe pl"ma [K+] dep"nds on many other f:><:lo," {e,g" .. nalloss of K+ due to keronuria and osmoric diuresis in diabete; mdliru,}, .nd [hu. hy!",rkalemia may nor ~ pr=nt, g. Pl.,ma [K+] inere~.al, but only slightly cxo::alalrhe up!"'r ..fe .. n"", limir, wh~n dog, with cxptrim~ntally inducal hypothytoidi,m we,", uercistd (running for 5 min): Ihe inerea.. did not occur in eUlhyroid dogs, The hyperkalemia may have: i>ttn causal by . n ICF ro ECF shifr ofK+ relalallo ralucal Na+· K+· ATPa...criviry in sL:delai mu,d~," Hy!",rkalemi. is nor cxptc1al in nonexcrci...:l hypolhyroid dog.,

5"

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

h. P..... do.h~rkal~mi. {ll In vitro h~molysi, or J~~ of K+ ~"'" of ddaYM r~mo""J of strum or plasma from .rythrocytes (a) If th. [K+] in .rythrocytel is ~.ter than the [K+] in plum •. th.n th~ Joss of K+ &om ~rythrocyt"" moy caus. psn.tdo.hyperkaJ~mja. The ratio of ~rythrocyt< [K'] to pla,ma [K1 vari .. among ,~ies and .m0J\i: brttds of dog .. (b) This form of ps.udo_hyperbl.m;" our occur in horses," K1m~ dogs (i.~., J.pan ..., Akit","', Jap.nesr Shiba,_ and rudy others), .nd cmk s.rum or plasma may 1101 apJ><"U hemolyud d .. piu sub,tomi:.! K' J.:okagr from uythrocyte" {el H.moly.is is not ""J"'CtN to em", hy~rbJ.mia in c;>ts or other bretds of dog•. {2} Thrombocytosis" (a) K+ is ,de=<:! from platdets in the clottM blood "'mpl• . The rd.as.d K+ is pan of the [K+} u...:! to mablj,h .. rum .. f"en~ intervals. Without a thrombocytmis, .. rum [K+] j, about 0.3-0.5 mmoJIL ~a{" than pu,m. [K+] in sampl.. oolla:t«i from th~ sam~ blood draw." {b} Wh~n thrombocytosi. i. mark«i (> I.OOO.<XXlIJ.lL). more K+ is add«i to the ..,rum during clotting than nornully ocrurs and thu, th~ me
9/ MONOVALENT ELECTROLYTES AND OSMOLALITY

515

of th~ variabl~ duration, and v.riabl~ .mounts of urin~ in th~ Jl<'riton~al cavitie;. {b} Uriruuy tract obmuction may cau.., ~ similar hyp
Hypoaldost~roni,m

{a}

Hypoadr~nocorticism

{Addison'. di ..as.} hypoplasia da;= th~ production of aldosteron. and cortisol. (ii) Hypoaldost.ron~mi. da;""...s the .ctivity of N.*_K'_ATP ... pumps in the basolateral m~mbran .. of the principal cell. of collecting tubul ... Thi. I....d. to d.er .......! usorption of Na+ and th ..~fore da;r.....i movem.nt of K+ into the tubular ~pithdial cdl. for passive =t.rion to the tubular lumen. This proce.. i. funh~r inhibita! b«:..u.. th~ numkr of luminal K+ channeh da;re:.,.. with da;reasal aldost~rone. {b} Inhibiton of angiotensin-convening ~nzym~ might cau.. hyJl<'rkalemi<> bn::au .. of the devdopm~nt of hypoaldosteroni,m. Ho~r. if ",nal function i. adaJuat~. K+ i, "".. ta! throuy. aldost~rone_independ~nt pr"",,,,, •• and thus only mild hyp is p.... nt. but normonatremi . is maintaina! by the action. of increasnl angiot~lI!in II and da;=sed AN P.' Cd} Hyp. normocortisolemia. hypoaldosteron. mia. and increata! plasJrul r~nin activity." Th~ d~f~ct that Ia! to the hypoo.ldost~ronemia was not deurmined. {4} Trim",hoprim_induced K+ retention" (a) T rimethoprim (.. pecially when protonated in acidic urin.) blocks the luminal N. + crunnds of the collecting ducts and thus .m like . K'_.paring diuretic."'" With ra!uced Na+ ..sorption. th~ da;triell gr.dient that promotes K+ ""r~tion is reduced. {b} Elfa;" of trimethoprim would k ~nhanced if the flow m . in th~ tubul .. were da;reasnl. bn::au.. K+ KCre"tion depends on a K+ gradient m. intaina! by high urine flow rat~. that carry KCma! K* away. thu, increasing th~ intracdlular to lum~n K+ gr.di~nt. b. Inereasa! intake of K+: administration of K+_rich fluid." e:speci>lly if th~re i. renal compromi.. 3. Repe. ted drainage of rnylou. thoracic ~ffusions:"'" PathoC'"n~.i, of hyJl<'rkalemia i. not esrabli,ha! but may k rel . ted to concurrent hyponatremia. In th~ presence of hyponat"'mi<> .nd hypovol~mi<>. much of th~ filtrat~ Na+ is resorbed in th~ proxiJrulI tubul .. and :l.'lCending loop of H~nl •. If Na+ is not ddiveta! to the dist. l nephron. Ie .. N.+ is resorbed distally .nd thu.s less K+ secr~tion occurs. 4. P~ritoneal dlinions: Four cats with peritoneal df",ions h. d hyp
Bilat~",l adr~noconical

5"

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY The hyponat .. mi. W:lS coII!id.raj a dilution'! one t1ut remlted from H,0 retention C;>U.ffl by stimulotion of ADH ,.least and chint ""mu. by a d=easM df«tivr blood volume. concurr~m hyponatr~mja.

[v.

Normoblemi. in acidotic or :dblotic .nimah A. Normoblemia in an acidotic a!lim:.! l. Breaus. inorg:mic acidemia is exptct.d to incre;;o.., ..,rum [IC'], normoblem;" in the pr=n~ of inorganic acidemia mggest' an aninul h", a d«re=d tbK'. TheraJ><'ucic correction of the inorganic .cidemia may [ower the .. rum [K'] into the hypokalemic rangt. Disorders that may produ~ Ihis pathophysiologic staU indude ",nal failure and some cast. of diarrh= 2 A> mentional rarli" (_ Potassium Cona:ntr>tion, S<'CI. I.e), aninu/. with an organic metabolic acidosis may not be, hyp",kalrmic. Thi, j, ~u .. either the K+ shift imo edl, is acrompani.d by.n organic anion shift, or r~nal excmion of K+ is incr ....S«I to moinuin da:tronrutrnity .. organic anions ..~ excmw, B, Normobl~m", in an alblotic .nimal ], B.alUS< alkaJ~mia is exp«:tw 10 da:r~~'" .. rum [K+], normokal~m", in th~ pr=n~ of alkaJ~mia SIl(;g<'m an .nim.l has :m incr ....S«I tbK+, Th~ .. ptutic corra:tion of th~ alkaJ~mia moy rai", th~ ",rum [K+] into th~ h)'ptrkaJ~mic rang., 2 This combiruotion of lindings is not exptctw kcaus< disord ... th~t cous< m.tabolic alkaJosi. typically do not conrummly inc,,",as< tbK+,

V,

Hypobl~m",

A, This typically occurs wh~n th~r~ is • shili of K+ from ECF to ICF, • d=n=I di~ary int:lk~ of K+, or an incrulffl K+ 10'" ""' kidn""" alim~nury tract, or .kin, If th. acid· bas< sUlUs is normol, th.n th~ .. rum [K+] t~nds to r~H<'Ct tbK+, B, Di""rd~" and path~n= (Table 9,6} L ShiftingofK' from ECF to ICF a, M~abolic alblo.e it might ho"" da:r=d tbK+ ("'" Pot""ium Con""mration, sect, V,B.2), c, Exptrimental endotonmi. couses • hypoblem",,'~" Th. hypokal~m", nU)' drvc:lop kcaus<: .ndotoxin stimulot.. the N.+· K+·ATP.". of .
9/ MONOVALENT ELECTROLYTES AND OSMOLALITY

517

Table 9.6. Disca.... and conditions Ihal ca u.e hypokalemia Shifting of K+ from ECF to [CF (no changt in lotallxxly K+) Metabolic alblo,is wilh alblem", [nc",=d insulin activity (n pid ina ..... ) Endotoxem", D=... ,~ toul lxxIy K+ 'D,""",~ K+ intah: anoraia or other r...!Om for not ~ating [nc",=d aa' lion of K+ [ncre. std ren:.! lOS! '[ncreastly '[ncreastd :.!im~ntary loss: vomiting, di.rrh~a, scquesr ..lion, acess .. Iivalion [na... std cutaneou, lou: .wnling in horsts Olher or unknown mecluni.ms 'Hypokalemic ren.l failun:: in cat, Hypokalemic myopathy of Burme.. kinem • A lel. tively oommon dioe ... 01 condition

(2) The ,""rily of hypoblemia wiU k ~nhan=:l if other proc= >Ie leading to K+ loss or shifting to [CF. b, [na . .. ~ acrelion of K' ( I) [ncre=d renal loss (a) [ncre.std tubul .. How (il [nc"",,~ flow of Huid through Ihe coll,""ting tubules allows for inc",=d """"'tion of K' from the tubular ctll" Rapid flushing of K+ IIUintaim a low tubular [IC'], which aUows Ihe paui"" move· ment of K+ out of Ihe a:1Is. (ii ) Polyuric nates th>t promote hypokalemi. include glucosuri. , N. +.losing nephrop.thi .. (pydonephrili., tubular internili:'! nephrilis, and possibly hyperala:mic nephropathy), and diuretic u.. (loop diuretics and Ihi".idc), (b) [ncre. std n::nal acmion of aniom (i) Kelonuria: AcAc and BHB (hlon~ bodies) .'" aniom thai are not resorbal in th~ lubules, Th. ir negative charges add 10 Ihe drcrro. chemical gradi. nt Ihat promotes K+ acrelion, es",""ially when there i. oonrurn::nt stimul. tion of Na+ rC1orplion, Animal, with hloaci. dotic diaktes mdlitus trnd 10 k hypokal~mic; however, the nel K+ balona: i, the re",1t of multiple concurr~nt p~, (ii ) Lacmuria: Uclal~ is a poorly """,rbal by renal tubules, Thu., incr~=d renal aCrclion ofL.la.ctate {in lactic a.cidosi,} oblig.tes Ih~ 10.. of eotion, such a, Na+ :md K',

5"

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY (iii) Biearboll.tu,i., Wh~1I pl.. rn. [HCO,-] exettds th~ renal ",pacity to con"'rv~ HCO,- (reruoJ threshold) or if th~r~ i. a tubuJ:or acidosi" the HCO,- th.t rellUin. in the tubllJ., fluid obligates the renal exc.. tion of corion, such :as Na+ and K+. (e) Vomiting or ~ue.<SOCiatffl with adr~n:ol hyp<rplasia and not nU>j>lasia. 61 Th..~ cots had. high.normal to incr~~"",,, [.ldost~ron~l ooncurrem with hypokal~mi •• nd an inc......d :oIdost~roM to plasma "'nin acrivity mio. The cots we .. cofl!id~red to ru.ve primilry hyption, or acess salivation {al Vomiting m.y cau,.loss ofK'.rich fluid .nd r~ducM ime.ninal.bsorp. tion of K' .nd thus le~d to hypobl.mia. Also, ~nhancM renal excretion of K+ {due to ... pon.. to iner ....""'" aldosuron~ ~crivity and d=~a"",,, av.ilability of Cn may ~dd to a d«re;;o=' tbK+. (b) With di""h~"" loss of d«:trolytes and H,O may bt massive. H 2 0 imak~ may contribut~ • dilutional oompon.nt to th~ hypokaJ~mia. In addition, J.ck of .bsorption of dietary K+ may augment th~ severity of the tbK+ d.pletion and thus hypobl~mia. (c) K+.rich fluid may bt s"'luester.d in th~ intestines of .nimal, with ileus, e:o;proally in horstS. (d) Beause ... liv. is • K+.rich fluid, loss of J.rge volumes of ..liva (becau.. of choking, • beer-lI.d ... liv.ry gland, dysphagi<>, or ptyalism) in horses and cattle con produ~ hypoblemi .." Initially, th~ concut",m 10.. of HCO,- may produ~ • mild acidosis. If th~ .nimal, also "'rom. hypon.mmic, hypochloremic, and hypovolemic, thw ren:ol

9/ MONOVALENT ELECTROLYTES AND OSMOLALITY

519

com!"'n .. tions (con..,rv:nion of N.+ and HCO,- , .nd excretion of K+ and H+) may l",d to • hypokal~mic m...:.bolic alkalosis. {3} [ncr~=d cutaneous loss: _ •• ting in horsc:. (a) Equin~ _rat is rdatively K+ rich :II comp..ffi to plmn•. Thus, profu.., .weating can l~ad to K+ loss, tbK+ d."l~tion, :md hypokalemi •. '~" {b} [f the hon" is drinking. H,O intah would promou dilution of th. r~maining K+. c. Oth~r or unknown ma:ru.nisms {I} Hypokal~mic r~nal failut. in cats (a) Cats with prog....in .. nal dis""", th.t cau,. chroni" ",nal f..ilu", ..e pron~ to tblC' d~pl...:ion {hlliopmi4} .nd thus d~elop hypokal.mia. {b} Th. ",act J>'Ithog.nesis of th~ hypokal~mia of chroni" ",nal f.ilu", is unknown and may b. du. to muhipl. factors: inc",asrd .. nal K+ exc .. tion, incr",...! colonic K+ exCl"~tion, .nd d""",asrd di...: ary K+ int:ok~. lkaU,. cats main .. nal cona:ntrating ability much longtr in r~nal di= than oth~r domestic mammals, possibly th.ir distal tubul., .. main ... pomin to aldon.ron. during hypovolemia and thus retain Na+ and """'~t~ K+. In ""prrim.ntal conditions, a N.CI_restrictffi di...: did result in hypokal~mi •• nd inappropriat~ kaliure;is wh.n th~ glom.rul.. filtration rat. was rffiua:d.'"' {2} HypokaJ~mic myopathy in Burmese kin~ns""" (a) Th~ Burm.,.. kin.ns had hypokalemia, mu.de wukness, .nd high .. rum cr~atine kin..., .ctiviti.,. The myopathy ~ pr""ipitatffi by me .. or ",erc,s<. {b} Th. pathogrnesi. of th. hypokal.mia i. not enablishffi but may b. du. to • sudden shift of K+ from th. ECF to ICF. SODIUM TO POTASSIUM {N.+: K'} RATIO I.

B=.... "",.raJ phy.ioJogic proces... involv<: both N. + and IC', it', not .urpri'ing th at [Na+] and [IC'] may changt concur.. ndy in th~ sam•• nimal. Calculating a Na+:K+ ratio may .nhana: d...:""tion of d""trolyu disord~ ... A. Th~ Na+:1C' r.uio is obtainrd by dividing a m~asurrd .. rum or plasma [N.1 by. m.:IIurffi .. rum or plmna [K+] (both expf<:S.liom in mmollL or mEqIL). B. A low Na+: K+ ratio is wmmon and ",,!"'ct.d in hypoadr~noconicism. It has bttn writt~n that a N. +:K+ ratio < 27 (or 25 or 22) i. diagnostic of hypoad .. noconicism in dogs. Th. lowest ratios will b.: found wh.n hyponatr~mia .nd hyprrkal~mia ... concurr.nt, but • low ratio is not uniqu~ to hypoadrrnoconicism.

[I.

CaUst. of ad"" ... ...! N.+:K+ ratio:"''' A d""",=d N. +:K+ ratio i. not r"luirffi to det""t th~ following disord... but sugg.,ts pm.sibl~ .bnormaliti., in Na+ or K+ cona:ntratioDl: A. HJ'PO"dr~nocorticism (Addison·s distase) I. Hyponat .. mia is cau...! by in"",=d .. nalexcmion of N .+ {due to d~CI".=d aldosteron~} .nd m.ntion of H,O (du~ to incr",...! ADH =ndary to hypoconilOlemia). 2. Hyperkal.mia is cau...! by d""r",srd .. nal stCmion of K+ {du. to d~CI".a ...! aldosterone}.

".

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

B. Dimhtl (dogs. cats, and ho"",) l. Hyporuotr~mia is CUlM by inc",,~ intestinal Joss of Na+, perhaps aa:ompani. d by incrrasN H,O intake. Horso; with SNore di<>rrh"" ... mor. prone to low N. +:K+ rat;", than

:or. dogs and

cats.

2. Hyperkal.mia =y occur ~u .. of ""idemi. """",i. ta! w ith HCO,- 10... 3. When ,..,n with a whipworm {Trichuris vu/PU'} inf«tion, Ih. diwrd.r h", bttn call.d ps. udo--Addi50II', di ..=. N.ithor .d.. nooo{tical hypopl",ia nor hypooldo,{~ roni,m are pr=nt, and respon,;v. hyperndo,{uonism is np«tw. C. Renal f. ilure (dogs and cats) l. Hyponatremia is COU..M by d,""",~ ability of tubules 10 , ..orb N . +. 2. HyperkaJ.mi. is cau=i by d~.. ..,.d ability to srcre!. K+ (primarily in oliguric .tat.. ).

D. Urin.,y tract obnruction {doC' and cm} or uropuitonrum l. Mild hyponm. mia might ~ found during obstruction. If uroperitoneum i, pr... nt, N .+ may diffust into ptriton~~l fluid to c""~t~ hyponatr~mi .. 2. Hyptrhl~mia ao.uKtatM drainage of chylou. tffusions-would remove ECF Na' and thus t nhanct tht stverity of th~ hyponatrtmia. 2. Hyptrkaltmia =y result from ont or mort prOct>RS. including d«=std .. nal acrffion of K'" (ao.US«! by d«rt~K
Phy>iologic proctSS.. A. S<:rum [Cl-] is ntarty tquival~nt to ECF [Cl-] . which is influ~nc:«l by ECF conctntn· tio", of N. · and HCO,-. Th~ ..fore. romplet~ interpretation of .. rum [ct] rtquires knowledge of strum [N.+] and.t l~~n a consid~ration of an animal·, acid.bast statu •. B. Control of strum [Ct-] l. Ittn.l """"rption and sterttion of ct' a. About 75 % of filttrM ct- is """"rbod in tht proximal tubules down a conctnt ra· tion gradi~nt cr~attd by Na+ .nd H,O resorption .nd through a form.t ... Ct

9/ MONOVALENT ELECTROLYTES AND OSMOLALITY

521

«dung..r. Angiot~n!in II stimul.t~. the proxim:d rubul:u r.""rption of Na+. Cl-• • nd H,O. b. Cl- i, .ctivdy re,orbN in the thick """"nding limb of the loop of Henle vi<> a Na+. K+.2Cl- tran'porter in the lumin:d membrane. Th. rat•• limiting factor i. Clddivery to th. loop. This proa-.. i, blocka! by loop diuretic> {•. g., fi.uo""mide} .nd is lIimulata! by ADH. c. Cl- i, p,... ivdy resorbN in the dist:d nephron by an d.crrochemical gndi.nt establisha! by Na+ movement through Na+ channd •. Aldo,terone promo... Na+ r..orption in the di,t:d nq>hron. d. Na+ .nd ct resorption in the distal nephron al"" involves an :ddosterone.ind... pendent Na+.Cl- cotran'pomr. Thi. proc= vari.. directly with N. + ddive'Y to th~ distal n~phron. Thiuide diumics block thi, cotrampo"er . •. Type A intercahta! cdls of the dist:d n.phron secrete Cl- wh~n H+ i, secreta!. Th. p=" i, lIimul.red by .cidemia (Fig. 9.4). Conversely, wh.n th ... i, an .lhl.mia or HCO,- = , Cl- i. con!erva! .nd HCO,- i. secreta! by type B intercala..d cdh {Itt Fig. 9.8}. 2. Alimenury tract function! peruining to Cla. G..,tric {or aoomaul} muco,~ secretes HCI .., P"" of the dig."i ... proc=. In h",hhy anim:d., th. secmed ct i, resorbN .rt~r it p...., into the intmin:d tmet. b. s.c.. tion ofCl- requires the generation of HCO,- (set Fig. 9.5). II.

An:dytical conc
Gaslric parietal epilhalial cell

,,,-

'"~

,,,-

cr

cr

HCO..

~.

00,

oo,'~

''''

''''

......

,,,",w

Fig. '.I.S. G .. tric or abonuul >eeletion of H' rnd cr. H'" . nd HCO,-.", form<. H+ (from H,O) is .oecr.. od into th. IUm
Ie.... pt."". lCIl, and V' .... pWma [HCO,l . 0, ATP .... pump: ... d 0, cotranspo't
522

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

B.

Sampl~

for

let) qu.mitation

l. s"rum is pr.f.rred. [C[-] is S{abJ~ for momh. jf th~ .,mpl~ does not d~hydrau.

2. Plasm. may ~ u.sffi.

C. Prineipl.. of assay. l. lon_sda:ti"" dectrod. as",)" ar~ {h~ most common. Oth~r halid .. (e.g., bromide and iodide) will r.act with the d«trod. 10 gin a falsdy inc,..,....:! [C[-] . sometim., markally'o. Bromide may"" P""'fit when ",,{ousium bromide is u,a! as an anticonvulsant. Oth.r .nion. (•. g., [acrate and BH B) may reoCl to gin mildly incrraonl ",,[u..; th at is, the Cl- v:due is incrrastd . bout 3 mmo[IL by J.ctau .t 10 mmoliL and BH B at 16 mmollL {i-STAT litera tur.}. 2. Coulom.tridampJ.ced by ion •..,l.ctive .Ieee,,,,,. a=ys. III.

H ~rchJor.mia

A. T his cypirnlly occur.; when th.r. is hyptrnm emia but occasionally is found ooncurmllly with a d«",asal [H CO,-] (i.~., hyperchloremic m",.bolic ~cido5eS). Con""'ptually, ch:mgcs in [Cn ~r~ rdatM to .tt~mpts to maintain d=rical n~utrality. iI..'l [N.+] inc,..,. ... , so doe; [C n . As [HCO,-] d<"Crta'leS, [Cn inc,='leS. B. Disord~" and path~n= (Table 9.7)

Table 9.7. Di..,a..,. and conditions that ca use hyperchloremia H, O-ddicit group {with hy~,chlor~mia :md hypernatr~mi<>} [ nad,",!u.t~ H,O intah 'H ,O dq>,iv:nion Def<"Cti ... thirst r~spon'" (hypothal.mic d~f«t) Pu,"" H,O loss (without ad'"'!u.u H,O "",J.""m btt.. insipidu. ("",ntral or nq>hr~nic) H,O loss ;> N~+ :md Cl- lou R~nal: osmotic diur~,i, Alim~ntary: o,motic di ..,h..,., osmotic ..qu
m

9/ MONOVALENT ELECTROLYTES AND OSMOLALITY

523

l. H,O-ddicit group (hy""rchlo""mic and hJ'P"rn>l""mic disord~rs) : Ba::.us<: ct i. the

2,

3,

4,

5,

IV,

m.jor anion that hdps maintain d~ctrial nrutr>lity in the ECF, di""rdw; th . t aus<: hJ'P"rnatr~mic dffiydration :d"" t~nd to creat~ hy~rchlor~mi . (S<:<: Sodium Con""'n_ tr>tion, =:t, IILRI), Cl- -aa:s.! group (and usually concurrent Na+ a "",...) a, Inc" >sal intah of Cl- vi. or:d or N administr>tion of 5Olutiofll containing Clmay :dso le.d to hJ'P"rchlo""mia, b, Decreasal ""nal acretion ofNa+ {hJ'P"r:ddost~roni,m} (r>r~) {I } Ex~ivt aldosuron. promot~. a~ivt ""nal Cl- {and Na+} ret. ntion, {2} HJ'P"rchloremi. (.nd hy~rn.mmia) may <X'Cur ifH,O is romictal or ADH .ctivity is ralucal, Aldost~ron~ e"",,~ may prevent hJ'P"rchlo""mia {= Sodium Con""'ntr>tion, Sett, llLB,2b}, HJ'P"rchlor~mic me{abolic .cido';, a, Alimentary 10... ofHCO; {I} Vomiting or diarrhea th.t auses a 10... ofHCO;_rich inte;tinal se<:re{iOfll may l~.d to HCO,- dqJl ..ion and m..abolic xid""i., Cattle that do not ingest saliv:l. {becau", of.n ' 50phageal obstruction} become HCO,- d~pl~tal bea...., their saliv:l. i. rich in HCO;, {2} The HCO,- secmiofll are .ho Na+ rich but Cl- poor, Th,,~fore, the ECF l. ft ~hind become; Cl- rich, .nd thu. hy~rchlor~mia d~dops, b, Rc:nalloss of HCO,{I} In proximal """ al tubular acid""i., the ralucal ra:iamation of HCO,- may allow mor~ Cl- to ~ t=>rbal with N • ., . nd thus hJ'P"rchloremic me{abolic acidosis d~dops. {2} In dist:d ronal tubular acidosis, th~ imp.iral .bility to =:ret. W might k linkal to the failur. of aCt _HCO,- shurd~, thus imp.iring secretion of Cland ra:iamation of HCO,-, Respir>tory alkalosi. {chronic} a, As. com~nsatory change to prolongal hypocapnia and alkalemia, the renal retention of H+ is incre• ..d and thur th. renal consc:rvation of HCO,- is raluced, b, Th~ fall in HCO,- is Wlanced by.n inc""... in ct and oth~r .nions that ."" not id~ntifiw, " Dehydration of the sampl. (~.poration or sublimation): Exposure of .. rum or pla!ma to . ir may :dlow ~poration th. t auses hJ'P"rchloremia, This is especially true of air-conditioning systems that blow cool dry air ovtr the sampl. proassing or ana!ysi. areas, Sublimation of H,O from frozen sampl.. may au", hJ'P"rchloremia.

Normochloremi. : Recognizing the p" ",n"", of normochloremi . may k important when or decreased ",rum [HCO,-] is pr... nt, because it sugge;ts the pres<:na: of an incr.....d .nion gap (S<:<: th. Anion Gap section),

eith~r hy~rnatremia

V,

Hypochlo""mia A, This rypially occur.; when th~re is hyponmemia (and pseudo.hypochlor~mi. v..hen there is ps.udo_hypon.mmia) or .n inc""ased .. rum [HCO,-] (a! occurs with metabolic alkalos<:s). It may aloo occur in • me{.bolic .cidOlis becau.. Cl- excretion accomp.nie; the enhanced r~n:d acmion of H+ eith~r via NH.+ acre{ion (Fig. 9.6 ) or vi. type: A int~ralatal cdls (Fig. 9.4).

524

FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY

Table 9.8. Di..,a..,. and conditions thai ca u.c hypochloremia

Cl-dcficit group: lIet Cl loss> H,O loss {loss ofC] -<:omaining fluid followed by incr~ H,O inc"". } Concurrent loss ofNa+ (~ the N. +.ddici[ group in T.bl. 9.4) M",.bolic ..!1uI""", 'Lo.. or ~u.strat;on ofHCi: vomiting, di'placnl abom.mffi, pyloric obstruction, bovine hcmorrh3i:ic bowel .yndromc 'BoviM ",nat failure Furos.mide coxicmis

Th",z.id. diur"';cs M...:.bolic ~cido..,. with .n incrca.Yd anion gap 'Kelo:>cjdos;,

'ucric :ocidosi. Ingestion of foreign .ubnan"", (e.g., ethylene glycol) thaI C",,
Shjfting of H,0 from lCF to ECF (Itt the ,ame group in T.ble 9.4) Shifting of Cl- from imravaocula, to atrav:ascuJ.r fluid (.tt the sam. group in Table 9.4) • A r.t1t;ve[y common dis< ... or condition Noto: Poeudo_hypocblo=ni. auy b. cau..d by dispbcom
B.

Di""rd~n

alld

path~n=

(Table 9.S)

l. Cl--ddicit group {Cl- loss :> H,O loss}

a. B«>Iu.. Cl- i, th. major ,"io" that hdp' maintoill d«tri",l lleutrnity ill tho ECF. di""rd~n that "'u.. hyponatremic ddtydratioll (via gastroint.. till:d. urinary. or curanam, 10M) t.nd to:d", cr.... t. hypochloremia (Ott Sodium CoIla.III.. tion. ,«(. V.B.I ). b. Hypochloremia ill the ab...lla. of hyponatr.mia SIlggem the pr..ella. of m.to· bolic :dbJo,i, (with an illcreastd [HCO,-]) or.1I increased allioll gap (with," illcreased rolla.lllration of.1I anioll other thall ct or HCO,-) beam.. dectrirn lleutrality mun be m.illlailled. Interpretatioll of HCO,- (or tCO,) alld ,"io" gap value, should be sufficient to id.llli!y these colldition,. but ",lcuJ.tion' for rorr«ta! [Ct] and SID may be u,a! {Ott th~ =tioll 011 Strollg 1011 Differ.nce .lId Suw:on', Method of Acid.Base Analysi, ill Chapter IO}. {I} M~tobolic .lhJom (a) Loss or srqummion of HCl (•. g.• vomiting. di'plac«i .bomasum. other upper g"'troilllestillal tract obstructioll. gastric ",flux ill hor .... or bovill. h.morrhagic bowd .yndrom.): Loss of Cl-.rich secretiom l....d, to dq.letioll ofCl- ill th. ECF .lId thu.s hypochloremia. {b} Bovin~ rrnal failu",: Canl. with rrn:d imuflici.ncy t.nd to become alkalotic. The a.cid.b ... change" may ... uh from .bo/IUSal atony. which cr....tes a functiollal obstruction:md thm srquestration ofCt (Ott Bicarbonat. Cona.ntration. =to m.B.I ). Aho. ",nl. excrrU more K+ via rniv. during renal failure. Wh," sw:dlowed. tho K+ may limit the absorption of Cl-.1O (c) Furosemide: Thi, rompet .. for th. Cl-.bindillg site in th. N.+. K+.2Ct trallspomr of th. lumin:d m.mbran. of th. thick asco:ndillg limb of the loop of H.nl•• nd thus inhibits Cl- resorption. Exct:s.l rxcretioll of Cl-

9/ MONOVALENT ELECTROLYTES AND OSMOLALITY

2, 3, 4, 5,

(rd. tive to H,O) couses hypochloremia. Concumnt hypokalemic mffilbolic alkalosi, =yoccur (Itt Bicorbo""te Con""'ntration, =t,IILB,Lb), {d} Thiazide diuretics act in the dinal tubule by interfering with Clresorption in. N.+_K+ cotransporter, Because thi, pr",,",ss occurs in the coni",l nephron, it doo; not dimini,h medulJ.ry hypenonicity and thus the kidneys mainuin conantrating ability, Thus, when S(imul.rrd by ADH, H,O re;orption will cou.. hyporuotremia:md hypochloremia by dilution, {2} Meubolic .cidoses with inere""rd anion g'''' {al In ketoacidosis .nd lactic acidosis, there is .n inerrasrd filtration of untuorbabl •• nions (htone bodiu and L_lactate, respectively) from pl""ma, The", :mion! obligate the renal excretion of N.+ to m.inuin decrrical neutrality, Thu., I... N.+ i, """rbed in the proximal.nd colla;ting tubules, Because Cl- resorption in tho.., tubules depends on th. ela;trochemical gradient establiJted by Na+ resorption, the dimini,h.d Na+ resorption dimini,h .. the Cl- rerorption.' {b} Ingmion of a foreign subsun"" that gtnerates anions (',g" ethylene glycol): Pathogtnesi, of the hypochloremia p:orallds th.r found with ketoacidOJis :md lactic .cidOJis, b«aUst the filtration of unresorb.oble anion, obligat.. N.+ loss, As ct resorption de~nd, frequenrly on Na+ resorption, Na+ loss lrad, to Cl- loss, Other examples of exogenous anionic mbst:m"", .re listrd in the Anion G.p section, (c) If there is .dequate renal function, the reruol response: to an .cidemi. i, to iner.... the excretion of H+ by the following procc:..e. that incr..... the excretion ofH +:md Cl- :md the production of HCO,-, The ne{ effect i, to rrdu,,", the ..""rity of the acidemia (excretion of H+ . nd genemion of HCO,- for buffering) :md rrdu"" plasma [Ct], (i) Acidemia stimulates the type A int.rcalatrd cdl, {Fig, 9A}, Iii} Acidemia stimulates th. incrra"'! ",ruol excretion ofNH ' in both the proximal and coUa;ting tubular epithdial ""lI, (Fig, 9,6), Thi, m.ro..nism of acid excretion in the di,ul nephron may be impaired with .norexia, possibly to help maintain muscle mas.!' H,O-= group (SCI1ur 'pa"" (Itt Sodium Conantration, sect, V,B,5) Pseudo_hypochloremia: The same f. ctors (e,g" lipemia .nd hy~rproteinemia) that couse pseudo_hyponatremia will . 1'0 cou,. • pseudo_hypochloremia if the [Ct-] i, me. ,ured by indira;t potentiometry or coulometridam~rometric titration .... ys ( BICARBONATE (HCO,-) CONCENTRATION AND TOTAL CARBON DIOXIDE (tCO,) CONCENTRATION L 525 Physiologic process.. A, HCO,- in the body i, a =jor buffi:r th . t hdps maintain blood pH at physiologic concentration" HCO,- i, produced from H,0 . nd Co, in ""lI, that have corbonic '" FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY Proximal tubular epHhelial cell Peritubular fluid & pi asma glut""" 08- --------0 gUi!lmi....• - ,IX! ", '" , ( ) Peritubular fluid & pi asma 3 N~+ G/na'''K" ", ",0 . '"'7' '"' 2 H<- H2O a-btogluta",t..- ,"oo-~' ' '"' Collecting tubular epithelial cell '"' .- - 0- ( ) - ------------ glutam~\o' GMD~ Glomerular plasma NH, _ -.. -f---- -f gUiamioo- 2el- 2NH.+ C Z Na+ "" ( ) "",/ Tubular lumoo -----------. ----------. -~ NHJ -- a- "00.- co, =::C::-::::--"' IX! ", _ _ H20 _~ NH.+ Tubular Iuman ----------. -Fig. 9.6. &cr .. ion of H'" vi. NH:. • Proxim.J =01 ""lis: Acidemi. ,,,,,,I,,, in tho incro....:! upuh..,d <1ivity of g1utlmin ... (GI"",r) and £IUtl""'''' d.hydrog"mp: :and 0. ootnnspor«r or coun«, ''''''pom, (rntipo'«'). tulm~ opithe~al anhydr:as<: .rythrocytes. proximal renal tubular cdh (luminal and intr:ocdlular), parietal ""ll, of ,he cmric and abom . ..lepithdium. interalatffl cdl, {tal ""ll, of the collecting tubules .nt... ,he peritubulu fluid ,hroUJ:h a Cl-.HCO,- achang 9/ MONOVALENT ELECTROLYTES AND OSMOLALITY PerHubular fluid & pia ,m, 527 pl'Q)(imaltubular epHhelial cell Glomerular plasma Na+ H,0 HCO! -l-- j--r 3Na+ TubuJa,lumon '" ", ~ 3 HCO , ' carbonic ""h) "" " ( ...' 'ro,- <_"-{ H,CO:! H,0 CO, i:1 ",-------. - Fig. 9.7. ProD""" tubulu mns H,O) combi ... to form HCO,-. whiclt it trrntpotnmn.. H' ..l....d from the H,O is :ov:oibbl. for ==r 2. HCO; con bt =:med (when there i. a m~abolie alkaJosi.) through. CI-. HCO; aclung.. in type B int..cobted ""lis of the dis .. ) nephron (Fig. 9.8). [I. Analytical OOnctpU A. Tles IIUy bt obtained from serum sample•. 2. Th. [HCO;) in serum con bt estimated indirectly by m.a.uring the [tCo,). tCo, ,dleeu the to ..1 .mount of CO, g'" that con bt libtnted from .. rum. At • physi. ologic pH of 7.4, about 95 % of th. potential Co, g.. is in the form of H CO,-, about 5 % is in diuolved Co" and < I % is in corb.mino rompound •. Th.",fo"" the [tCo,) i. n...ly equal to the [H CO;). tcO, is also called rota/ mrlwo dioxw CDorm, {t<:CO,}. 3. Unit conv..,ion: mEqlL X I = mmollL {SI unit, n.arest I mmolfL}' B. Sampl. for [!-ICO;) quamitation J. Serum is preferred, but pl ..1IU may bt used. 2. Samples should h. vt minimal aposu", to air btfore and .ft.. ""rum is harv.sted {a few minute. i. acceptabl.}. Beao.lJ.5< the Peo" of air i. n<ar 0 mmHg. C0"D will diffu", from the sampl. to air. Th. lowering of the Peo" in the sampl. "puU. HCO; out of solution" (shifts the equilibrium toward CO, and away from HCO;, thus lowering the [H CO;] and [W) of the sample) (Eq. 9.2). Th • .,m......ction occurs in vivo during apir.otion (OIl mown in Fig. 10.l) to remove H+ from blood, but the 528 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY PerHubular fluid & plasma Type B intercalated cell • "<X" ---,,",;-- HCO,- ----Fig. 9.8. &a .. ion of Heo,- by type B interc:ob",d ""Us of th< distal ntu mpo't<' or ==t<'" ,,,,,,po,te, [HeO,-] dec",= i. not d.t«:cM in physiologic states ~l1St th.", is rd"'ivdy much mor. HCO,- than H+ (mmoUL '" nmoUL, respa;tivdy) .od (h~ HCO,- Ih . , is u.=I i. r'pl. ""d. H++ HCO,- ---) H,CO, ---) H,O + CO, ---) CO, Jon to air (9.2.) 3. Th. [tCO,] in und.rfiJled blood coU«:{ioll tube. (•.g., 3 mL of blood into a 10 mL tub.) jt I... than in filJ.d !Uk. after contact with .rythrocytes for .bout 20 min.71 Erythrocytes may contribute to 10Sl of CO, into underfiJled tubes· de~d sp''''' {which initially would I>. demid of CO,}. b.cau.. =bonic anhydr ... in eryduo_ eyres acederates the convusion of HCO,- to CO,. Even gtt.t.. loss of Co, occu'" with expom'" to air I>.fo.. or aft .. ",ntrifug>tion. C. Principl.. of ass.p (thr.., variations provided) l. Vitro. enzymatic tCo, a. In a ""'Y albline environment. ne.rly all Co, is in the form of HCO,-. HCO,r" ,,", with pho.ph~nolpyruv:u. to form oxaloa.ceur.. which it coupled to • r".ction that con""mes NAD H. The dis;;opp.-aran", of NAD H is m....""=' by rdla:tan", .]>.rated CO, diff....,. into a H CO,- solution with • pH dectrode. Changc:. in p H rd.u to the amount oflil>.raud CO,. 3. H iuchi .nd Olympu. analyze", a. H CO,- ",.ct. with phosph~nolpyruvau in the p .... na. of NAD H to produ", oxalo.a.t. u. PO ••• nd NAD+ in the p.... n". of Q1u lytic .nzym... The 9/ MONOVALENT ELECTROLYTES AND OSMOLALITY 529 Table 9.9, Disca .... and conditio"" that cau.e increasctory .cido';. 'Hypokal~mia &rondary to ~nduran"" ra"", or inusti""l disord~", in horses Shift of H+ from ECF to [CF due to hypokaJ~mia Administration of sodium bicarbonat~, lacrat~, citrate, or magnesium hydroxide 'Contraction .lkalosis: vomiting • A lel1tively common di..... 01 condition No"': Spurious ina.... m>y be co....d by incr<:o=[ .. rum be..", dehy:t), comumption ofNADH i, proportional to [HCO,-] and is m... surffl via spffirophotometry, b, LD . ctivity also converts NADH to NArY, Extrcm~ inc......." in serum LD . ctivity COIuscd by rhalxlomyolysis am result in f:dsely incr ... scd [H COn," [[I. [nern=! biauboruote (HCO,- ) con""ntration or total COIrbon dioxide (tCO,) con""ntration A, [ncr~a scd [HCO,-] is usually associatffl with. mCl.bolic :dkalo,is, ~ither primary or as compc:nmion for a r~spiratory acidosis, Mc .. bolic alkalo .., ar~ typically producffl by the loss of W from th~ ECF (which cou""s alkalemi.) (= Fig, 10, I), For ....ch H+ that i, lost, th~ .. i, an «[uimolar generation of HCO,- , B, Disord~rs and p:llhogc:neses (Table 9,9) l. loss of H+ from the body a, Gastric lou (vomiting, or physical or functional pyloric obstruction) ( I ) S«:rClion ofH+ by gastric mucosa gc:nemes HCO,- (Fig, 9,5), [f th. H+ is lost by vomiting, dninag< of stomach contents (ho", .. ), or sequestration proximal to th. intestin., it is not resorbed in the intestine, Thcrefor~, th~ gc:n~ratffl HCO,- is not used to buff~r th~ W (th>! would otherwi .. han been resorbed), and thus it .ccumulates in plasma, (2) Concurrent hypovol~mia .nd hypochloremia l~..d to incr~a=' renal con""rvation of HCO,-, Hypovolemia nimulates th~ RAS to cousc th~ tubular resorption of Na+ . nd ct, If the anim:d is Cl- deplClffl, resorption of N.+ without Cl- in the dinal nq.hron incr~ases th~ electrochemical gradi.nt, which promotes H+ o«n.lion and thus th. gc:neration of HCO,(Fig, 9,3), (3) Cattle in r~nal failure may d~nlop a metabolic alkalOlis, The pm,.iling thcory is that th. cotd~ d"",lop . bomas:d .tony that l....ds to HCl sequestra. tion, which gc:n~rates th~ :dkalosis, b, Rcn:d loss of H+ ( I) Loop of Henl~ diuretics: Furosemide and oth~r loop diumics (bum=nide and ctha.crynic xid) block th~ .ction of a N.+. K+.2Cl- tn"'potter, thus, the 530 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY r=>rption of Na+. K+, and ct by tho """"nding limb of tho loop of Honlo i, da:r ...saI. Tho ...uhing impairal H,O =orplion :md incr ...saI fluid flow in Iho dist:d nophron promo" H+ sa:retion by maintaining. favouble H+ gradiont. Also. hypovolomi.> and hypochloremi.> I...d 10 increastd sa:retion of H+ .nd thu, increaKd HCO,- ~neralion. In .ddition, incr ...saI ",nal ncretion of K' may I~"" to hypoblomi. thaI :dro promo", alkalO!is {Figs. 9.2 and 9.4}. {2} Thi.>z.id~ diu",i", inhibit ~ Na+.CI- cotranspo"'r in the disc:d nq>hron by competing for the CI-.binding site. As notal in the pra::eding po.rag.. ph. hypo",lemia :md hypochlo .. mia promo" the form.rion of meubolic alkalo,i •. {3} Secondary to respiratory :ocidosis: Chronic respiralory ~cidosi, allows time (3-5 d) for ",n:d compensation comining of inc.....M W sa:retion by tubular ""II, (proxim:d primarily) and Ihu. increa=! HCO,- gtnemion {Fig. 9. 7}. The inc",as.d H+ KCrttion i, probably stimular.d by Iho acidemia. {4} Hypokalemia stimulate. H+·K+·ATP.", in the disul nq>hron, thereby promoting K+ retention, H+ KCretion, .nd HCO,- gtn~ .. tion (Fig. 9.4). Hypokalemia may also promot~ alkalO!is by shifting of H+ into ""II! in nchangt for K+ (Fig. 9.2). {5} Aft~r endu.. n"" ra"". or during intminal di,ord ... (e.g .• colitis). horses can d""dop m~ubolic .Iblosis. Sometimes th= alkalo!es ..., explain.d .. being causal by contraction .Iblosis {Stt Bicarbonate Con""nt .. tion and Tom Carbon Dioxide Con""ntration, ""'t. 1lI.B.4} or g.. tric ~um .. tion of HCI; such ma:hani,ms may be prestion of HCO,- . olher mech.nisms nuy be in",lvaI. (a) Ext~nsivo sweating ""'y occur during ~ndu .. n"" .."",. Equine sw...1 is K+ rich and ct rich compo.r.d 10 pi ..""" so .w...ting mult. in hYp"rtonic loss of th= da:trolytes in addition to tho 1= of body H,O [hal m.y not be replacM by drinking. {b} W.rery diarrh~a dq>lm. ct and H,O. If tho ho"" i. not ...ting. l:ock of fttd intoke would r.du"" tbK+. {cl As nplain.d in th~ for.going """ion Sol.a :md in Fig. 9.4. th~ concur· rent su", of hypovolemi., hypochlo .. mi. , and hypokalemia increa", renal "",,,,,ion of H+ and thus inc ...... production of HCO,- . T h.." process<s cr ... le ~ metabolic :dblosis. 2. Shift of H+ from ECF to [CF due to hypobl.mia a. Alkalosis can I~ad to hypob l.mi.> , but hypoblemi . am .Iso rontribut. to an .Ikalosis. b. If the", i. tb K+ dq>lrtion, a f. ll in [K+] in th. EC F promo". th. mov~m.nt of K+ out of th~ ""II :md tho mo""m.nt of H+ into cdh. T he higher intra""lluiar [H'] .Iso promoles W sa:retion by ren:d tubuJ .. cdls and increa=! goneration of HCO,-. thu, promoting :dblemi .. 3. Administration of sodium bicarbonal~. organic anions thaI gtnerate HCO,- , or magnesium hydroxide in ruminants or hone. a. Ex=< .dministration of sodium bicarbonat~ (whiJo t",ating acid.mia) m.y incr.... s.rum [H CO,-]. 9/ MONOVALENT ELECTROLYTES AND OSMOLALITY 531 b. M",.boli,m of som~ org:mic anions {~.g .• l_lacrat~ :md cimll~} by h~patocyt~. I....l maintain th~ alkalosi. by d«reasing r~nal H ca,acr~tion. In rrspon .. to hypovol~mi • • • ldo,tuon~ promotes th~ rtnal ,« .. tion of H+ .nd ct by th~ int~rcal .ted edt. and concurr~nt production of HCa,{Fit:. 9.4}. c. The prinury pr~ that result in th~ 10.. ofCt (vomiting. 5njum.. tion. diu"'tics •• nd sw .... ting) nuy al,o ",u .. the 10.. of H+ and produ", HCa; . nd thu, contribut~ to m",.bolic alkalo,is. Wh~n ther~ i. a loss ofCl- and .ccumula_ tion of HCa;. then th~ ct _HCa; ""dun!:"r in th~ gastric p..iet>l ~pithdial cdh (Fit: 9.5) or th~ Type A intucalat«i cell, {Fig. 9.4} >re prob.bly invol=l in th~ patho!:"nesi. of th~ hypochlo"'mic m"'abolic alkalosi,. IV. Decru..d bicarbon.te (HCa; ) concent"'tion or total arbon dioxid~ {tCo,} concent"'tion A. D«rUstd [HCa;) i, usually a.ssociot«i with m~tabolic :ocidosi,. ~ith~r primbolic acidosi, (,"" Fig. 10.1 ). Th~ loss of HCa; {or other lxxIy buffers} rfflUct. th~ buffering apacity of the lxxIy and thus allow. H+ to accumulau. B. Disorder> and pathogtneses (T.bl~ 9.1O) I. G..nuation of ""(".<::'l.! H+: If m~tabolic p. thways J::<'n~m~ mffici~nt acid to a ceed th~ buff~ring ",pacity of blood. then H+ .ccumulates to crute .cid~mia. HCa,- i, d~pl",«i wh~n it is u...! to buff~r the gtnerat«i H+: th~r~for~. thest conditions may b. ",frrr«i to as titration "c;do.... a. Lactic :ocidosi, occurs when cdlular m",.bolism switch.. to .naerobic g1ycolysi •. Th~ rrsuhant incre • ..d degrad.uion of ATP "'u... a ce",in rde ... of H+ ("'" Lactat~ Conctnt"'tion. stet. lll.A.lb}.ln neonatal ",If diarrh..... ruminal.nd enteric fermentation of lactost may b. the sourc~ of D_loctat~ .nd H+.'" b. K~to:ocidosi. occurs wh~n th ..~ is ""ct.. in ~-oxidation of triglyceride, in h~patocyte,. of ctrrain compounds {~ .g .• ~thyl~n~ glycol} c",. t.. an .cidemia bt-caUst th~ ",raboli,m of the rompound !:"nerates .cid (5« T .ble 9. 12). 2. D«",...d r~nal excmion of H+: As with ace.. gtneration of acid. [H Ca;) drcr ......,. '" HCa; (.nd oth.. buffers) i, ustd to buff.. H+ that accumulates in pl"'ma. c. Ing~,tion 532 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY Table 9.10. Disca..es and condition. thai ca use decre .... ed serum [HCOJ - ) or [tcO,) (metabolic acido .... ) G~ncr.ltion of excess H+ 'ucric .cido";. ·~o.cidO!j, Ingestion of ~ruin rompound. (>« Tabl. 9.(2) D'""....«I renal ""cretion of H+ '!knaJ f~ilur. 'Urop",itoncum or urin.ry lract obnruction Di,ul .. nal tubular acidosis {cyp. I} Hypooldo,{croni.m lnn"ased HCO,- loss 'A!imcntory [<>SSt.: di:orrheoo, ...quc'tration, or vomiting of ""oCIutic KCrclions Iknalloss." proximal rerud tubul>r acidosis (typo 2) Dilutional ""idosj, (npid infusion of 5alin m.. to > a. Ronal f:lilu .. c.rates .n acidemia ~U'" of the kidnq" inability to excrcte the daily acid loo.d produ=:l by mcubolic p:nhw:oy>. With progressi"" ",nal di",=, las NH' is ""creta! ba:.u"" th ... are few.. functional nephrons to form NH". Abllornuliti~. ill HCO,- and PO, ncretioll may also playa role ill the devdopmem of acidemia. b. Urillary tract obstruction. also produCft all .cidemi. oo:..u", of impairffi urillary ncr~tioll of H+. c. With uro~ritolleum. H+ it excreta! by the kidlley. bur 1I0t &om the body. Also. urine is typically a HCO,- _poor fluid. so whell the H,O in urille i, resorbed by lymphatic ...,...,]s. it dilutes the plasllU [HCO,-j. d. Distal rellal tubular .cidosis (ty~ I) occu" wh~1I the H+ =",ioll by th~ distal tubules is d""reasal 00:..<= of tubular disea",. It may also occur with urillary tract orutructioll .lId hy~rkalemia. e. Hypoo.ldosterolli.m (as Sttll with hypoadrelloconici,mj IIUy promote acidem;" through multiple m""hani,m •. Hypoaldost~rollism is .lto calla! fJpt 4"",,1 ",bula, "ddmis. (I) Reduced H+ (alld K+) =retioll by prillcipal epithdial cdl, (,"" Fig. 9.3). (2) Reduced H+ ",cr 9/ MONOVALENT ELECTROLYTES AND OSMOLALITY 533 b. R..n:.! 1= {I} In proximal "".al tubular xidosi. {tJ'P' 2}, th,,~ is a d~f= in HCO,con..,rvation in th~ proxin... l tubul ... Th~ d~f= may bt du~ to abnormal N .+ r~rorption or to th~ pr=nu of a carbonic .nhyd",,~ inhibitor. Th~ d. f= =y bt p>n of ~ith~r an inh"itffi or xquirffi Fanconi', syndrom •. s..sid.. proxim:.! r~nal tubul...cidosis, oth" findings in Fanconi's syndrom~ indud~ r~nal glurosuri {2} Acquirffi proximal r~nal tubular .cido,is has bt.n ... n with muhipl. mydom. , hypocalu mi. , and • v. ri.-ty of drugs in palpl~. In dog" th. disord~r bttn ottn with hypocal",mi. {du. to hypop:muhyroidism and hypoviuminosis DJ, strq>to7.0tocin and mal~ic :ocid treatm~nu, and an onrd""" of amoxicillin."-OO Acquirffi proximal r~nal tubular .cido!is Won rq>Onffi in • mar., but th~ om,. was not d~t~rmin.d." 4. Dilutiorut! .cido!is m. y occur with rapid s:din~ infusion, which may dec",as. [HCO,-] by diluting ECF HCO,- . How.""r, .bsolut~ chang. caused by dilution is ""rectal to bt minor .nd thus cau.., • minor ch.IIV' in blood pH. S. In vitro loss of HCO,- from th~ .<;>mpl~ {ott Bicarbon. u Con",ntration &ct. II.B.2} C. Decr~a.sed [HCO,-] or [rCo,] with hy~rchlor~mia: HJ'P'rchlo .. mic m~ubolic .cidosis mongly mggests th~ prestn", of .. rut! tubular acidosis, ~ith~r th~ proximal or distal form. \. For proximal .. rut! tubular acidosis, proximal tubular di ...... rffiu= tubular secmion of H+ and decre;;ose; rons ..v.tion of HCO,-. As shown in Fig. 9. 7, proximal tubular HCO,- coruc:rvation normally I..ds to th. mov~m.nt of HCO,and Na+ ions from th~ cdls into th~ int~rstiti:.! fluid .nd blood. IfHCO,- ions.", not bting formffi in th~ proximal tubular ",Us btcat= of inhibition of carbonic anhyd .... , th~ Na+. K'.ATP ... pump cr.... t...n dectric:d gradi~nt that ~nhan= Clre;orption. Th. n~t result is decrea=! [HCO,-] , incr .......! [Cn, and ther~fo .. hJ'P'rchlo .. mic meubolic xidosis. 2. For dist:'! ren:.! tubul .. xidosis, disul tubul .. di ..a,. impair.; the secretion of W and thus d= ..",s th~ g<:n~ration of HCO,- by th. carbonic anhyd .... r~.ction. As shown in Fig. 9.4, decr~ased g~n~mion of HCO,- by th~ typt A int~rc:datffi .pithdial cd], dec",as .. th~ r~nal =mion of ct. Th. n~t r~sult is d=~...,d plas= [HCO,-J, inc",a.sed plasma [Cl-] , .nd th~refor~ hypc:rchlon::mic m.-t.bolic .cidosis. 3. Th~", i, confi"ion in th~ liuratur~ ..gardiJll: wh.n it i, appropriat~ to chara.ct~riI.e an .cidosi. as . .. rut! tubular acidosis. a. On~ vi= i. that th~ cJas..ification of r~nal tubular acid",is should bt limital to thOst disordm in which. ",n. l tubul .. dioord~r produces. hJ'P'rchlo .. mic m.-t . bolic acidosis without :m incr .......! :mion g. p .nd th~ .. i, no ti~nu that might h. ve: an incr.... ffi .nion gap ....., b. Th~se: differena::s =y bt considen::d rd . tivdy unimponant, but th~ lack of con!ist~n ru.. 534 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY {2} In r~nal failu"" the def.aiv~ ",nal acmion of H+ i, ~u", of too few filllction:d n~phrons. Th"" n~phrom that a.. still functional Jruly .till ~ abl~ to =m~ H+. Ikcau .. th~ ncr.tion of PO, :md oth.. aniom is abo impair.d, th ..e animal. may han .n inc",aS«! anion g.p. {3} Unfortunatdy, "'m~ .nimal, Jruly shift from th~ r~rictin group to the nonrestrictin group; th at i., acut~ proximal tubular di ..a", (e.g., toxicOli,) Jruly c",at. proximal ",nal tubul .. acidosi., but if th~ da~ i. KV~" enough, th~ di ...... may also damag<' enough nephrons that :m :mimal ent~'" a.cut~ ..nal failu",. ANION GAP I. o.,finitions A. Cation; an atom or mol«:ul~ with a po,itiv~ char~. Monov:dent eotion, ha"" on~ positive ella,!:", dival~nt eotions have two. B. M(a",,,d ca.iDn duv,,( {mC' }; th~ cha,!:" concentr.tion of th~ Jruljor monov:dent eotion, (Na+ and K+) whos. .. rum .ctiviti.. or concentratio", a", di=dy m=ut.d. ikcau"" th ... ions a", monov:d~nt and m~asur.d as fr« iom, their ion concentration. a", live charge, div:d~nt anions ha"" two, and trivalent aniom (•. g., PO...·) han thr..,. F. M(IlSIJ"d IIniDn ~harg( {mAl; the charge cone m. Anion gap = mC - rnK = ([N . +] II. + [K+]) - {[ct] + [HCO,-n (9.3.) Phpiologic proe<"" A. Strum i, always drctrieolly n.utral; that is, total positin charg.. 9/ MONOVALENT ELECTROLYTES AND OSMOLALITY 535 Table 9.1 1. Cation. and anion. of serum in healib (approIimate concentration. provided to .implify concept) Cations N,· K· "". /Mg'. W mmollL ion charll '"4.0 5.0 7.0 ,~, ,,, Anions Co HCO,Proteins Organic aniolll PO, SO, mmollL ion ch ..![ ,.0 "". 3.5' 7.5 LO '" Total To!>l • Mol.. con=>ttotio... of prot';", ..., not me.m«d t..c. .... of v.,; .. io", in moka.tJ .. ""'igbts of pro«i .... (Not.: U.ing the common anion g>p formulas. prot';", are th. m. jor compon«! . nion gap when th..,.. :oa:wnul... in plum •. i, from Na+ .nd K+ {the me' }, ~nd 134 of the 157 mmolfL of anion chatg<' is from Cland HCO,- (mK). B. Anion gap formula derivation (Eq. 9.4) A.. serum i, dectrieolly neutral, tC" = IA-. A.. tC' = mC' + uC' ~nd IA- = mK + uK, th.n mC'+ uC = mK + uK. A> mC' = ([N .+) + [K+j) . nd mK = [CJ-] + [HCO,-) , th.n ([Na+) + [K+)) + uC+ = ( [ct) + [H CO,-n + uK. R.~rr.lllging th. equ.tion, uK - uC* = ([Na+) + [K'j) - ([Cn + [H CO,- j). A.. anion C.P = uA- - uC', th.n .nion gap = ([N . 1 + [K*]) - ([Cn + [HCO,- j). (9.4.) C. Th. major purpo.. of eokulating .nion gaps is to id.ntify incr ... scd [uK) and th.",fo", an increaK in circulating anionic molecul.. (e.c. , L-lactat. and ketone Ixxiies) in stat.. of met. bolic acidosis {Fig. 9.9}. l. A> shown in Tabl. 9.1 I, uK ;> uC* in health; that iI, the mm of ch .. go; due to prot.ins, org.nic ions, PO" and SO, is cr ... ter than the sum of charges due to Jt:a"', fMC .... and H+. Also, the 'unmeasured" ~nion concentrations eon chan!:" mo", markedly than eon the "unm.asured" eotion roncentrations. 2. Clini",J]y signifieont chan!:", in the anion gap are usuaUy the result of chango; in uK. Major chang., in [fea"'] and [fM g"] are li~_thr ... t.ning. Only minor chanc., in [fCa" ] and [fMC"'] .rc ..en dinicolly, and thu. "'UK only minor chango; in amon g. p. 3. Most changes in . nion gap arc due to increased ronccntrations of the anions of organic acids. Th..., organic .cids ~ he endo~nous substances (e. c., L-larute and keton< Ixxiies), or they may be ~n.r:lled from eIO~nous subst:mc.. (•. g., ethylene glycol). 4. [nc",aSC's and dec ........ in protein roncentratioll!, especially albumin, eou"" mild incr ...... and deerea,." respcctivdy, in the .nion g.p. Most plasm. prot.ins ... in the uK group. FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY '" , I , ,. «'- -- -"-- ,. "," "",. .--" "'. ,. ~ "--- "- ~. ,,' <> --- ,,' ,,- <> ~, --- ~,. -"',,' 6 ---- ~. ~~ "--- - - -- ,,' -- .----- <> ~ "'. r:"c -- ,,' a- - -- a- 6 A B c D E F Fig. 9.9. Bll diagnms of :onion Il"P concJ>COon. n,. Ole' is d ... mwtly to cb. "i!'" from Ie.... IMg'+,:and rom< glooo~ru • ..1..",.. th. uK is d"" mostly.o mUG"" from po.. SO •• :ond .mom of org:mic . cids :md pro..,u", {Prl. Prott< m.;, ",btiv. contributions to .oul anionic charge. Catioru :md ani"", .fur contribu", to th. rnian g.p >t< ...i.hin the t:"'Y",haMJ am.,. [n th..., =mpLe.. tho Ole- coJ>Oivalent to d.. combined phJ"iologic concenm.'io", of mio", of org=ic , cids and pro",jn~ PO" ond SO, _ a [n 1 no,modt]o,.."ic mouboJic .Odos;' (th.enfOf< low [HOO,-l), tit. ina.osed rnion S"P "'1'",.. nOl imn...d macen.mio", of uK (organic .cid rniom, po•• SO" or other anions ofm. :ooids). Tb ... :ocidooes rruy b. orgrnic (• .g., u.<1ic 1< org.nic (e.g., betic :ocidosis) 00" inorg.nic (e.g .. ren:d f.oiJure ). D. [n. hypocb[o,.mic "",ubo~c :oJ.b[o,is (tl><",fo.. bigh [HCa,-n, tl>< :mion g>p is not inc,......! beawe tb. mm of ICTl and [HCO,l and tb. sum of IN.+I and 1K'1 b""" not cb1ll£ed. The.. :d1W"""" rruy <>«p><>tntion of H·..,d 0-. E. With conru,,,,nt bypoD1tremi.:md hypoch[oremi. , tl>< :mion g>p b .. not cbrng1dreoocorticiun. F. With bypoprot.inemu. tb. anion g.p i, decr<»< mm of IN.l + [K'l. These findings OCCIl' in bypoprot 5. With hy~rphosphaumia, .ach [ mgldl inc ...... in [PO,l is a..>ociatM with about a 0.6 mmol ion chugeJL inn..... in uA- and therefor< in anion gap {[ mgldL PO, = 0.323 mmollL PO,. at an ""erage n"!:,,ive valence of about 1.8}. Ill. Analytical concep" A. Unin: mmol ion chu~1L (same", mmoUL of ion or mEqlL of ion for monovalent iOn!, but not equivalent to ion cona.ntration for multival.nt iOn! like PO., fCa'+, and albumin) 9/ MONOVALENT ELECTROLYTES AND OSMOLALITY 537 B. A> with allY cakul>lrd valu~. th~ ,"ioll gap will be, only:as .ccurate :as the m~. !Ured value,. If IIttdrd. n&r to prior KCtions 011 the illdividuod dectrolyt ... e. The [HCO,-) uK IlIcrnK that cau.. hYp"ralbumillemia or redu~ the colI~ntr:ltioll of K:.'"' . lId fMC. B. Becau.. the anioll gap i. a calculat~d valu., the value may be errolleoUJ if th~ valu .. uK Table 9. 12. o;""UC5 and condition. that cause an inaeased anion gap Metabolic .cido... 'Loctic .cidosi" illc",....:! lactat. {eith.. l-1actat~ or D.1act.te} 'K<:toacido";" illcreased ketolle bodi.. {BHB or AcAc} 'Ren. l f. ilu",: illc",....:! PO., ,ulfate, or citr.te Massin rhabdomyolysis: prob.bly illcreased l.ctat~ . lId PO. llIgestioll of ~rtaill rompoulld. 'Ethylen. glycol {.ntifrtt"l.e}: illcr ...,.,j glycolate or oxalate Methallol poisolling {.ntifre=}: fortrulie Paraldehyde (sedatin or an.sthetic): illcr......d .~tat. and chloro.~tat. Mmlddtyd. poisonillg {.""il bait} Pellicillill {nry high dO.les} HYP'ralbumill~mia (millor chang~,) • A ,el1t;vely common di..... 0' condition Note: A .pwions inc, .... in :onion g>p lJUy oc umpt. hmd~ng. 538 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY Table 9.13. Di""a..es and condition. thai cause a decreased anion gap D'""...... in uA • Hypoolbumincffii myrJom. th. 1 is producing "'tionic proteins • A r.t1tively common dio.... or condition Noto: A .pmious doc", ... in :onion g"l' m.y 0Cp v.ru.. tit" may be d.cn...,J (..,. tho -I· V. DecrnK low,," the :mion gap :aids in Ih. imcrpm . lion of anion gap values thot .'" WRI or incrcasN; cru.t is. the .nion g.p v:duc would h:IVc bun g... lcr if the animo! had not had h}'ll'O'llbumin~mia. C. Di""rd~rs: Di~ .nd ronditions that da:rra .. th~ anion g. p ..~ pr.."mffi in T.bl.9.13. VI. Strong ion diff... na: (SID) A. Evalu>lion of SID ass • ....,' .cid_b= abnormaliti~ in th. comext of abnormal cona:n_ trations of ions (strong cations .nd urong anion.). SID roncqm a.. ,imilar to .nion gap roncqm but aho consid~r oth~r factors. B. Complet. nplanation of SID i, beyond th. sco~ of this book. but major asp«t •• '" pr... nud in Chapt"r 10. U.CTATE CONCENTRATION (L-U.CTATE AND D_U.CTATE) I. Phy>iologic process". A. Th~ glycolytic pathw:lY ..... robically conv.m glurosr into .n..gy (ATP) and gtn~rat~s pyruvat~. which may be conv.na:l imo L-lactat~ (Fig. 9.10). B. Th. major tissu. SOUfC< of L_bctat" is .Jcd<'t.1 mu",I•. L_bctat" diffu ... from th. muscl" fibe", to plasma and is yhn up by h. patocyt.... part of th. Cori cydr: glUCOl~ to l-lactat~ vi. ana.robic glycoly>i. in ~riph"ral tissues •• nd l-I.ct.r. to glue<>.!< via gluconeogtne.i, in h~patocyt ... H' p"tocyt., can also u.. L_lactar. for ATP production vi. the Kreb, cycl~. C. s..-c,u.. mammalian .rythrocyt~ lack mitochondria. glycolpis in erythrocyt.. produce L_IocY",. AI"". l _lacYt. from mu",l~ .m~", ~rythrocyt.. vi. thrtt m.thods: (I) diffusion .cross cdl membran ... (2) transporYtion via a monocarooxylate tran.poner. and (3) mnsporYtion via th~ inorganic anion-achangc: transport.. {band_3 pror.in)." Erythrocyt.. may .."", as. tr;m 'port.. ofL-lact.", .nd al"" a "I.ct.te .ink" that enhana:. L-Iactat. diffusion &om muscle to plasma (i. • .• mu",l. to plasma to erythrocyt..). 9/ MONOVALENT ELECTROLYTES AND OSMOLALITY gl""""" + 2 NA[)+ + 2 ADP + 2 po. POH ~ 2 pyruVBt aero~ ~2W 2 po. 2 ADP =::::::::::: 7 2 acetyl GoA 2 NADH + 2 W L • 539 ~2H+ 2 NAD'+ 2 GoA 2PO. 2AMP Krebs cyckI • co, + GlP + NADH + FADH, Net: +38 AlP Fig. 9.10. Produc .. of .. robic md """,robic glycoly>il. C. ubolism of ..cb gluros< molecuJ. remit. in two Prruv>t< moko.tl.•. ....., H' ions. :ond • net production of two ATP molecules. WMn tbe", is >.", oxyg.n.tion of ,,,,It. . oo:after Prruv>t< .n"'", mitocbondri.. pyron.. ~"'" the Kreb. citric ""id eye!. (trie>rbozylic ""id eye!.) with pyrun.. dohydrog..). Tn. pK.. of bctic:acid is 3.1. so nearly :ill of th.. bClat. produced ",m:Uns in ti>< :anionic form: very ~tde buffen H'" fOnn I>ctic :acid .. phy>i_ ologic pH v:ol..... k aplained in th.. text. tho ""idemia in I""tic :acidosis ",...ed by • ....,robic conditions come.! from th. .... of ATP (whim produas H1 fOr en '0 00<> _.. , ! HO = C OH, , 0"" , HO- C - H ,....,.. '"' O~c"-o- , O~c"'O00<> H- t - OH , , '"' , '"' • O'-"C .... CH, O'-"C .... CHJ ~""". """l""""lale ~,~ '"' IBciBle Osomo<s , '"' HO~l'"CH, " ~hydroxybotyrBle 1«o\onQ bodies Fig. 9.11. Q.,mical .true"'« of pyrun... bet .... :ond hto ... body mokcul<s. Kno..ing th.. m.mic.J composition of mokcuks c.., bci~ ..", .., und.ntmding of ti>< rebtionsbips of tboo< molerul<s in ",thologic " ..... n,. redoction of pyruY::l'" by LD in< rrujor fonn produced by aumm.Ji.., ""Jl~ . nd D·bctil'" is produced by bact .. i.. Of tbe th",. htone bodies. only A* ..,d """to... are truly ketones. A* can be converted to ~iti> D. Bacr~ria produ", D.Jacuu. and som. produ", L.J.crate. Th. "~reoi.om .. of L...Jactat~ (Fig. 9.11). D.locrate am :d.o k producnl in mamm:di.>n ",Us via the gIyoxaldsr pathway. which metaboJi= m~hylglyox:d produud durillg g1yroJ)"lis or vi.> con"""ion from a.ceton . ... III h. :dth. pl:.sm. colI",mrations of D.Ja.ctue are ""ry .m:dJ romparM to tho", of L...lactat~. FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY II. Analytical con""pu A. L_I""ut~ I. Common clinical :w~ for I""ute mU'lur~ th~ L_loctat~ produ""d by animal ""II •. L-loct.t~:w~ us< eith~r LD (s~rophotometric methods) or L_lacuu oxid.." (e.g .• i_STAT .nd NOVA). 2 L-loctat~ con""ntrations a", m=red by many blood gal analJ'Z"rs. but. depending on the aruolytical method. ~ith" whol~ blood or pwrruo is th~ prd'erred sample. In the NOVA instrum~nu. a plasma [L_l""tat"] is m.... ured in. whole blood sample by allowifll: only plasma to ~nt" the reaction chamber. B. D_loctau ron""'ntrations c;on b. meamred by high_performan"" liquid chromatography or by D_I""t>t~ dehydrog~n ... ~nzyrruotic methods. Th~se ..... Y" ar~ not commonly availoble in clinical l.boratories. C. Unit: mgldL X 0.112 = mmollL (SI unit) (Nou: Thi. i. a conversion for th~ .nalyt~ lactote. Th~ conversion factor for lactic .cid is 0.111.) D. Sampl~ for [L-lacute] I. Blood .an'pl.,. should be p~ or .nalyz.nl quiddy so that l_lacute produced by ~JYIhrocytes does not inc",... th" plasma [L-lactot,,]. If the sample c;onnot be p,""""ssed immediatdy. L_lactau production am be reduced by collecting blood into a tub. containing sodium fluoride .nd chiUing the blood until th. plasma is "'Parated &om th~ ~rythrocytes: fluoride inhibits phmphopyruvate hyd .. rase (.ynonym: ~nol ...) of the glycolytic p:uhw:oy. 2. Id..lIy. blood should be collected from fr..,_f1owing blood (without. tourniquet) '" that blood nagnation doe. not r<sult in incr~a.ffl L-lacuu production. 3. L-loct.t~ con""ntrations .'" also m=red in bovin" milk. as in indic;otor of ruminal xidosiJ. and in lxxIy c;ovity dKuions as roden"" of bact~rial infection (= Chapter (9). 4. Special coUection methods for D_lacuu .amples we", not found. E. SUMan"". that int~&'" with th~ m"asur~m"nt of [L_loctate] will v.ry with th~ method """. ~ [L-lactate] detetminal by the i-STAT L-lactau oxida.. method. but limilar interfen::no:: is not d=ribcd for the NOVA L-lactat" oxid... method. 2. Th~ pm.,n"" of fr.., hemoglobin and hemoglobin_b..ro oxygen c;orrie.. in th" sample will result in falsely low L_l""tat" con""ntlOlliom if measured by th" L-Iacute oxida.e assay. probably b.c.u"" th" peroxidase activity of h~m., romo= the H,o, produ""d by th~ L-lactat" oxid... r..ction.I. Bromide falsely Ill. de=a... IncreaKd pwrruo L_loctat., con""'ntrations (hypr,filctaum;Il) A. L_I""ut~ accumulate> in plasma wh"n its forrruotion ",,<:«ds the removal by tissues (primarily by h"l"tocytic uptak" and glomerular filtration). I. Th~ prirruory r"ason for inc",asffi L_lacute production is hypoxia. a. During anaerobic (hypoxic) conditions. caubolism of pyruvat~ switch.,. from .,ntry inro th" Krdu cycle to L-Iactote production (Fig. 9.(0). It should be noted that this process gen"rat.,. L_lacute and not lactic acid and that th., conversion of pyruVllU to L_loctat. resulu in th" use of H+ (thus increaKd pH). b. Concurrently. changes in other met.bolic I"thw:oY" r<sult in th~ .ccumulation of H+ in th., blood {acidemia)." In aerobic conditions and via the Kreb. cycl~. cdh comume H+ whil~ producing large amount. of ATP. However. in ana~robic conditions. th" n~ti"" "nergy nalll. c;ouses. net dq:rad.tion of ATP and thu! 9/ MONOVALENT ELECTROLYTES AND OSMOLALITY 541 Table 9. 14. Discases and condition. that cause hyperia<:tatemia (L-laaa lC) In.d ddivery ofO, to tissu .. 'Stagnam hypoxia: shock, blood vessd occlusion 'Demand hypoxia: ,mnuous enrcise, .trugglifll: during restraint Hyponmia: r<:Spintory di""rden Hemoglobic hypoxia: anemia, methemoglobinemia Inc..... production by metobolic p:uhw:lyt Grain overknd Defectin m.ubolic pathwaY" for auobic glyrolY"is Hypc:rammon~mia: ur ... toxicosi., ammoniated fo~ toxicosis PyruV:1IC ddtydrog<:n ... d. fici.ncy Other or unknown mecluni,ms Sepsi. Canine babesiosis linr di",,,,,, T r.msfiuion of stored blood or p acked . rythrocytes • A rel. tively common dioe ... or condition production of H+ (Fig, 9,10 ), Othu metobolic chang.,,; ..tso add 10 th • • ccumuluion ofH+ (~,g., .Itu ed lipid m.taboli,m), 2, Inc",ascd L-lactat. production m.y abo be due to d~fective metobolic pathw:lY" (Stt th. following OCCI, B,2,b), 3, Potentially, decr....d .. mowl of L-Iactate from plasma con contribute 10 hypc:rlactatemi a, but it is uncommon for this pr~" to occur a1on~, B, Di""rde.. and pathogc:n= (Table 9, 14) L Inad {2} Equine colic caused by g.mointcstinal disorde .. (' ,g" torsion, strangulation, and infarction): Th. = ive L-la.ctate production may be due to poor pc:rfil!ion of ti .....es, but absorbed endotoxin! may .ho stimul at~ L.lactat~ production, and intesti",,1 ba.cteria may contribut~ D. la.cme and L-Iactate, In on~ srudy involving 14 hone" with incr ... sed anion gaps, L-lactat~ concentntions were incr"""! much mo", than D. lactotc concentrations," G.::nerally, the severity of the hypc:rlactatemia i. innrsdy proponional to the prognosi. in colicky horses, " b, Demand hypoxia {I} Stn::nuou, enrc;": Excc"in mmcular activity incr~."," .naerobic glyrolY"is and thus incr....s L.I""tate production and rel..... from mUlcl~, " {2} Strugglifll: cou: In cx~rimental nudies of m e" . induccd hypc:rglya:mia, struggling asiOCiated with harness .. m.int for blood collection incr....d pl:uma L-la.ctate concentrations, The hy~rlactotemia w .. con!idered to be caused by incr..... d muscular activity." Similar chang.,,; in other animal, would be ..,pected with .imilar condition .. 542 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY c. Hypox~mja: Poor oxygenation of blood could produ,,", sufficient hypoxia to cou"" • hy~rlaCla{.mi .. but this ,itu.tion is not commonly rerogniud. d. H.moglobic hypoxi De~tjn mlnspo,r of 0, to {;ssu.. amid product hyp",J.Claumia, but ,hi •• im.tion is not commonly rNOgniud. 2. Inc",,=<:! production by mHabolic pathway. a. Grain overload (in ruminants. ho ..... and ponies): Excess intah of narch (gr:lin or other sour=) iner"aleS th. fOnrullion ofL-lacu t. and by rumen {ruminams} or hindgut {'"tions. b. Dd'"clin glycolytic p"thw.". (I) Hypuammonem;" (a) This may occur from ncessi"" production of ammonium k.g .• urea toxicosis or .mmoniatffi rorage toxico.is). &om various causes of hep.ric insufficiency. ~nd &om urea cycle def.a. {b} High [N H:] may interf..e with the Krd>s cycle .uch th.r the . erobic production of ATP is defective. Generation of ATP switch.. to an""robic glycolysis. which produ= L.-laaar.." ~ {2} Pyruvate dehydrogen= deficiency in Sus=< spaniel. and Clumber spaniels =ults in excess rorm>lion ofL_lactau. " '" Pyruvate ddtydrog<'n ..e catalyze. the con""rsion of pyru ..... te to Ad:.<JA, which then enters the Kreb. cycle. With pyruv.te ddtydrog<'n .... deficiency. the ronversion of pyruv. te to L.lactate by LD is increo...d. 3. Other or unknown mech.nism a. Sepsi" The mechanism of hyperlactatemi. {L-l. ct . te} is frequently conside=i to be cau...d by poor ti,.u. perfusion." but endotoxemia by itsdf h .. betn reported to cause hyperlactatemia in people." Also. experiments in dogs indicate that the use of L_laetate by liv.. and oth.. tissue; during "'P.i. i. d«reased." b. Canine babesiosis: Hyperlactatemi. {L.-lactate} oa:urs in some dogs with acute b.besimis. but the pathog.nesis of the hyperlactar.mia i. not established." It dO<:'! not .ppear to be related to the deg ... of .nemia and thus is not caused by hemoglobic hypoxia. Because of the roncurrent hypoglycemia. the hyperlactat ... mi . is likdy caused by .a:demed an""robic glycolysis that conmm.. glucose .nd produces L_l""tar.. This ronmmption of glucose is a f... tu .. of "'Psis_induced hypoglyctmia {= Glucose Concentration. =t. l.V.C.I.f. in Chapter 14}. c. Liv.. disease: s.v... li""r dis ..... may cau.. hyperlaetatomia (L.-lactato). but the patho&<,nesi. i. not established. Th. hyperlactatemia might be due to excessive l. ct ate production (hep. tic hypoxia or d«rea...d mitochondrial function) and not due to def«ti"" utilization. d. Tramfusion of norffi blood or packed erythnxyt.s: During sto"'!:". erythrocyte> continue glycolysis to produce L_l""tate. and thus L.-l. ct . t..... rich blood is . dminin ..ed IV. D_uet.,,, IV. Increased plasm. D_lactate oonctntr:uions A. [D_l. ctat.] may be inc",ased in animals with diabet .. mdlitus." l. D_l""tar. conctntratiom were great .. in can with ketotic diabetes mellitus (mean .. 340 IlmoJiL) than in cats with nonhtotic diabetes mdlitus (m ... n .. 140 IlmolIL) 9/ MONOVALENT ELECTROLYTES AND OSMOLALITY 543 or in h""lthy eots {m~~n " 25 J.lmollL}. D_lactate may have bttn formffi through a pathway involving ao:ton. or m~thylglyoxal. 2. Ev.n though th~ro W :l'l • signifiemt diff..~n'" in D_lactau oono:",r.otion. among the group'. th~ in"'~as< W:l'l < 1 mmollL and thus would not oontribut~ dinically to incr""sffi anion gap ""lu... Abo, th~ D_lactau oono:ntr.otions a", much Ie", than found in mammals with L-Iactat~ acidosis {~.g., 10-20 mmollL}. B. Ab..orption of D_lactat~ (produo:d by ~nt ..ic bact~ria) em inc",= [D-loctat~] {not m~",ural by routine lactau ....... Y" but may be oontributory to incr..... d anion gaps}. l. D_lactat~ i. a major contributor to total Oactat~] in neonatal calv.. with diarrh~a.''''·u •• 2. A aU with exocrin~ panc ....tic insuffici~ncy had metabolic acidosi. and a ooncut",nt incr"".. in pl.. ma [D_lactat~] . Th~ .uthors condudffi that th. unrommon maldi~ni"" di""rd~r causffi ent~ric production of D_lactau by b.ct~ria.'«l 3. [n milk_fffi calv.., f.ilur~ of esophag~.l groon rdlex aUows milk to ~nt~r th~ miculoruminal cavity (callffi ,""minal drinking) in which bact~rial f~rm.nt~tion of lacco", results in th. formation of D_Iactic .nd L-lactic acids. Aft.. th~ ion.< a", abwrbffi (probably in imenin~), a metabolic .cidosis with incrrasnl plm "" co"o:mrations ofD_lactat~ may develop. Th~ rapid meta bolism of L-Iactau prev~ms it &om aCCllmulating in the pl",ma.'" 4. [n a group of 50 colicky hor"" with mild lactic acidosis, non. had incrUK v. R~lotio,,"hip to th~ .nion g.p A. Both L-Iactau and D_Iactate will contribut~ to an .nion gap (5« th. Anion Gap section). Each mmollL ina"".. in th~ Oactau] can account for e.ch mmollL incr..... in th~ anion gap. B. [f th ..~ is an ina""sffi anion g.p in ""rum or pl •• ma and the [L_loctat~] is not increasffi .ufficiently to account for th~ .nion gap., magnitud~, the incrrasnl anion gap could be due to D_Iactate or oth.. unmeasurffi anions (Tabl~ 9.12) ~_HYDROXYBUTYRATE I. (BH BJ AN D ACETOACETATE {AcAc} CONCENTRATIO NS Phy>iologic process~. A. K<:!ogen.,is occurs in hq.atocytes through ...ries of ",actio,," that conv~rt AcCnA into BHB, AcAc, .nd ac..ron~; th~ two aniofl1 {BHB and AcAc} .nd ac..ron~ are collectively callffi km"'~ btJdif1, but BH B doe; not han th~ ch~mical structure of a k..cone {Fig. 9.11}. The amounts of k.k:. and BHB in hq.atocyt .. are in equilibrium. Th~ equilibrium shifts toward BHB wh~n NADH i. abundant in hq.atocyt .. (~.g., in di.b.t •• mellitus), wh~r""s the equilibrium shifts tow.rd AcAc wh.n NAD+ is 544 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY abundant. Th. pIC.. of ."",o.cttic acid is 3.6, and Ih. pK, of p..hydroxybutyric acid is 4.7. Th...fore. >I physiologic pH value!, n •• rly all of Ih • .., mol.cules a,. in Ih,;, anionic form •. B. K.tog<'n.,;, is promotro by glucagon .nd inhibit.d by in,ulin. e. R.nal .xcmion of BHB hdp! with Ih. acr~ion of H+ kau •• Ih. pK. of BHB is ".:" that of acidic min., and thw it binds mor. H+ at a min. pH of 5 dun at a pl..rn. pH of 7.4. Similarly, A£A<:. .id, in Ih. acre:;on of H+. n. Analytical oonctpu A. Unin (Not.: Con""",;on f""iors our for anions and not Ih. corresponding acids.) l. AcAc: mgldL X 99 = IlmoliL or mgldL X 0.099 = mmoJIL (SI unit) 2 BHB: mgldL X 97 = J.lmoiIL or mgfdL X 0.097 = mmollL (SI unit) B. """"'ys: Th.", "r Ih,.,. t~. of ass.)" for hlOM bodies. l. Spa::trophotOffi""ic quantit;ui"" .uays us< BHB d.hydrog.nast 10 m.a.u", .. rum [BHB] by catalyzing th~ convmion of BHB to kAc. In som~ :=OJ"', a high [kAc] IIUy int"f,,~ with th~ r~~ction to produ"," a fahdy low [BHB].''' Hemolysis can d""r ..... the mu.ured v:dues. 2 The common nitropru ... ide method. (Ketostix strip. Aerte Upt at - 20 'c.'" D. When ketone lxxIies .re detected in blood or serum, they should :dso be in urin •. However, when comparing r~sults, the type of .....y .nd it. aruolytic properties should be considerw. For exampl., the BHB might be inc..ased in .. rum and urine, but the urine tm for htones (AcAc) may not be po.iti"". HoW<"VC:r, typically the urin:ory excmion of both BHB and Ad.c i, .ufficient so that the nitropru<side method will provide a po.itive htone ",action. E. In theory, BHB cm be converted with H,o, to kAc so that nitropfUS.!ide method. con be u..w to detect BHB. How...", BHB concentr.ltion. must be;. 50 mmoJiL in urine or;' 100 mmoUL in ..rum (usifiJ: 30 % H,o,) to produce more than a Ira"", reaction. Such ron"'"ntr.ltion. a.. not clinicolly r.levant for .. rum sampl.., and if urine BHB concent.. tions were this g"'.t, A£Ac concentr.ltions would probably be gr ... t enough to be d.t""ted routindy (Itt Chemical Examiruotion of Urine, ..ct. V, in Chapter 8}."· III. Increa..d ketone body concentration A. An increased concentration of ketone bodies in blood is Itnuntmid. The clinicol disorder i, called kim,is. ~tosi, occurs primarily for two r...sons: l. Ex=ive ~xidation of fimy acids result. in the production of more AcCoA than con be u.w by the Krebs cycle. The excess AcCoA, <:specially with glucagon excess or inmlin deficiency, enters the ketogenic pathway to form kk, which C>II be ron_ ""ned to . ith" a=one or BHB. Thi. occurs during diabetes meUitu. and !Iarvation. 2 The AcCoA produced from oxidation of lipids cypicoUy combines with onloacetate as it enters th. Kreb. cycle, which ",. ntu;o(ly results in ATP production. When an 9/ MONOVALENT ELECTROLYTES AND OSMOLALITY 545 animal is in • n.g.tin .n..gy status. ox:dooa.tat. is us.,j for glucona>g 1.4 mmollL. 2. Th. diagnottic prop<:nies varign05lic spc:cificity of th. Ketonix .nd K.toTest methods w... good. Th. diagnostic .. nsitivity of the K.toch.d: "'"lIS oon.tid..a! inad~uat. for u.. . . cr..,ning t.,t for subclinical ketosis. OSMOlAUTI AND OSM O. GAP I. Ddinitions A. O""f)"'!if]". the conantmion of a solute ap..ssal in moles of solute p<:r kiu,gram of solvent (mol/kg). [n the clinical ....ssm.nt of body fluids. o,moWity is ap ..ssal :lS mmoU~ or mO.m/kg {the .uthor> p..f.. the simpl.. SI unit of mmoUkii. B. Omw"'rity: the conantration of. solute ap ..ssal in mol .. of solute pl compl.tdy dissociates into two ions p-' (Avogadro's number) identical particles of it (based on the number of particl.. in 0.012 ~ of arbon 12). On. mol. of a mbst.na wc:ighs its g",m mol",ular weight (e.g .• 1 mol. of gluC<»e w.ighs 180 g and I mol. of Na+ w.ighs 23 g). E. O""f)tk P"uurr. th. fora. r~uira! to count.. bal ana the force: of osmotic solnnt flow through a .. mip FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY Connecting Tubular Epithelium Capillary Wall Peritubular Fluid Tubular lumen Interstitial Fluid Blood Osmaality 1000 mmollkg H,D 19.300mmHg Osmolaity 400 mmoVkg H,O 7720mmHg Osmaality 299 mmollkg H,O Osmolality 300 mmoUkg H,O ",0 ",0 CoIoiMI Osmolic CoIIoidIII Osmolic ,~- Pressure ,~"' ,,~"' ",0 "'" ",0 j Osmolaity 500 mmoVkg H,O Total Osmotic Pressure Grad ient in Collecting Tubu les Colloidal Osmotic Pressure Gradient in Muscle Fig. 9.12. Scherrutic oomparison of osmotic pr ..... ro and colLoid:ol osmotic pre.",r< . • In the left d .... ing of the renal oolkcting tubul •• undor pbyisiologic conditions. the peritubul., fluid h .. • much gro.... osmoWity than the tubub, fluid. Osmotic PlOSSllro can he c:dcuht«l from osmoWily (•• cb I mmollkg of osmoWity i. equivalent to 19.3 mmHg of osmotic p, ... w . ). Tbereforo. rn osmolality of 1000 mmoUkg oqU>.t., to . n Olmotic p",,"we of 19.300 mmHg. rnd rn osmoblity of olOO mmoUkg cre . ... 7nO mmHg of osmotic pr...... ro. 1b. lug< di/Je,..,« in osmolality c...... . hrg. olmotic presrun gradi..,t (:dmon 12,000 mmHg in this "". mp!'). n.. p=.""" of the ,e!1ted Gibb,,·Donnrn oqui~b,rum. The colloidal osmoric presrur. gradi..,t (18 mmHg in this enmp!.) t..,ds to caw< the movement of H,O (and.m:oIJ .olu .... ) from in"'rltiti:ollluid to pwma. The movement of H,O out of 0' into the pi ...... is d,e"'rminod by the bydr.mlic pressur< grodient:oao<s the .. mip«t. V. in Chap"'r 19). Note: The osmotic pr ..... '. g..dient in the diltal nephron is more thrn 600 times the coUoidal osmotic p, ... w e in the capiihry bed. n.. p=we v.ru.. .,. provided to illust..t. the IJUgninxle of the di/Jeren=o he ...... n the 10<= of osmotic presrur. gradi..,ts :and colloidal osmotic presrur. gndients. I. TDnicity: th. iffrmw omlolality of •• olution; th .. is. that solut. conemall (I nm to I J.IDl ) to ~ ... d~d out by gravity. Molt oncotic pressur~ of plasma is mu.a! by plasma pro{~n, (.bout 80 % albumin and 20 % globulint ). Pan of th. rolJoidal osmotic pr...!Ur~ i. mributa! to mtion, (•. g.• N.-,) that are attracta! to th~ nq:ativdy charga! pro..in. (Donnan ~ui/jbriu", iffitt). 9/ MONOVALENT ELECTROLYTES AND OSMOLALITY Table 9. 15. Solutco that contribute frovided fO oin'filfy conceft) Solut~ N,> K> ct HCO,- UN Gluco.., PO, .c." Me" Prot~in fO ""'""" oomolarity (approximate conccnU'lltion. M=urw conc;.,mntioll Factor to oollven to mmoJIL Comributioll to osmolarit!: 146 mmollL 4 mmollL 107 mmollL 24 mmollL 20 mgldL 100 mgldL 4 mgldL 10 mgldL 1 mmollL 7 gldL " "" " >46 mmoUL ) 4 mmoUL 107 mmoUL 24 mmoUL 7 mmoUL } 5.5 mmoUL 1.3 mmoUL } 2.5 mmoUL 1.0 mmoUL < 1.0 mmoUL TOial -t- 2.8 .X 0.32 " X 0.25 " Varies with prot~ill 547 Contributioll to toul osmolarity ill Krum of h.. hhr :mimal 281 mmollL 94 % of tOial 12 mmollL 4 % of tOial 5 mmollL 2 % of tOial 5OJUt~ solut~ solut~ No significant oontributioll 299.3 mmoUL Mon nonprot~ill ..,lutes do nOi comribute to osmotic pr=ur~ ill capillari... btCOUK th~ copillari... are p"rm.. bl~ to H,O and th~ ,mall solutes. Chapt~r 7 oollu ins illforma_ tioll regarding th~ m... u",m.nt alld im~rp"'tation of colloidal osmOlic pram..". II. Phy.iologic proce.... A. M smw ill th~ foregoing d~finitions. oUIlolality is th~ collc;.,ntration of ",lutes i"" kilogram of solv~nt and d'p""d. 011 the numb.. of mol,""ul .. or iOlls in th~ ",lutioll. Th~ major oontributor.s to ..,rum osmolality alld th~ir rdati"" comributions a", liuw ill Tabl~9.15. B. Major COIlc;.,ptS of rdati"" oontributiollS of solutes to Krum oUIlolality I. N a+ is th~ major solut~ ill .. rum. About half of th~ solute {mol.., not mass} ill .. rum is actually Na+ iom. 2. Cl- runs a do .. sa:ond. B=UK Cl- f"' FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY b. If th~ df'""tiv~ rumolality is d=~a.M. ADH KC'""tion is dimini,hffi. and thus th,,~ is less H 2 0 ruorption by th~ coll«ting tubules. incr~ H,O lOll. and thll'l a corr«tional incr~ in ron""ntration of plasma solut... 6. Eff«:tivr osmolality (tonicity) a. Wh~n diff,,~nt con""ntrations of oolut~ ar~ on two sid.. of. membran~ that is H,O p"rm ... bl~. th~ solm.. ~stablish an osmol" gr:odient that promotes rumosis (movem~nt of H,O to diminish th~ gradi~nt). If the m~mbrane is imp ~ff'""tivr rumolality = m.asurw rumoblity = ,., m.asurw rumolality - [u,""a] (if u'"". in mmollL) [UN] (if UN in mgldL) (9.S.) e. Knowlalg~ of ~f&ctivr o.molality is "",ful in two clinical situation" (I) D~t~rmining the prestn"" of hYp"rosmol" .yndrome (a) Wh~n the eff,""tive plasma osmolality rapidly ri ... (::> 350 mmollkj: or [Na1 ::> 170 mmollL).lll H,O I...ves ""lb. including neuron •. Con..,· qu.ndy. the :miJrulI can be dep=sed. stuporous. or ~vrn die from the hyp"rosmol:u nat~. (b) How"",r. if the hyp"rosmolar nat~ develops slowly. th~n intracdlul>r production of org.nic mol«:tJ.I .. such as taurin~. glycin •• glutamin~. K1rbitol .nd ino.itol (so-callw idiog~nic osmol..) diminish .. the osmotic gradi.nt •• nd thutle .. H,O I• • ves the ceU. [fhypotonic fluids are then administ~rw to reduct the plasma osmolality. there am be a rapid influx ofH,O into neuron. and d.-vdopment of ctr~braled~ma. (2) D~t~rmining physiologic r.. pon... th.t are expectffi in azot.mic animals (a) [f the hyperosmol.lity is cau..d by inCl"~a.M ur... ronctntrationl only (i .•.•• ff«tivr osmolality i. not incr .....d). th,,~ should not br a nimulus to drink H,O or rd ....., ADH (unless hypovol.mia is prestnt). (b) [f the hyp"rosmolality is due to .ff«tivr K1lut .. and not ur .... the hyp"rosmolality is upected to stimul.te thirst and ADH rd ..... . Ill. Analytical conctpu for mmolality (sre Chapt~r 7 for colloidal osmotic pressure) A. Osm.. l. Osmom~try is based on phy.ioch~mical prop"nies {called ('tI/ligatiw propntin) that dep"nd on th~ number of particl.. in a solution. Tv..o coUig.tivr prop"ni~s are u..d in clinical osmometry: 9/ MONOVALENT ELECTROLYTES AND OSMOLALITY 549 a. Fruz.iIll:-point dqH=ion: A I mol/kg solution of. solut~ in H,O (I molal) wiU ha"" a !'n.tting point 1.86·C low~r than th.r of pur~ H,O. b. V.por pr~ssut. (or dew point) d.prossion: A 1 mol/kg solution of a ",lut~ in H,O (I molal) will ha"" a vapor prossu", 0.03 mmHg lower th.n th.r of pur~ H,O. 2. Frttring_point o.momrt~ ...", mor. rommon. mo .. prec~ •• nd mor~ acrura" than vapor pr=ur~ osmom~Urs. Vapor pressur~ osmom"'~'" do not m~a.ur~ th~ contri_ bution of volatil~ solutes (• . g. •• lcohols) to total osmol.lity. (Not~: Colloid osmom_ rt ... mrasu .. rolloidaJ osmotic p..ssu.. and not osmol.litj'". = th~ Colloidol Osmotic Pr~.. ur. section in Chapt~r 7.) 3. Osm. indicates th. total ron""'ntration of ",lut.. in th...rum but d"". not indicau which solutes "'. p.... nt. 4. Serum i. th. rrquiral sampl~ for m.asuring osmolality; th•• ddition of antiroagulants for pl.,m. samples would be: adding solut~ to th. plasm>. Th. st.bility of a .. rum osmolality will dq> B. Osm.. I. Breau.. th ... is a dirrct rorrdation bc:twttn molality .nd osmolality and th ... is • .dationship be:t~.n molality:md molarity. knowing th. millimol.r cono::ntrations of solutes .ruobl~. ut to ~stimat~ th. osmol.lity that is exprcud du~ to tho .. solut~ •. How .... r. this ronvr",ion i. not exact bc:causc: of thr.., facto,,: a. Osmolality .nd osmolarity of plasm. solutes ..~ not rqu . l bc:cau.. 1 L of plasma contains .bout 0.93 kg H,O .nd about 0.07 kg solid •. Most plasma ",lid. are prot~ins. b. Th. ron""'ntrations of som. pwma solutes "'. not m=ral. and thus th.ir contributions to total osmolality a.. not includ.d in calculatiom. c. Som. pl..ma solutes "'~ not compl~tdy dis.sociat.d (• .g.• small .mounts of Na+ and ct a.. pr~ .. nt in plasma as NaCl). 2. At least 14 formulas ha"" bc:t:n u...d to calcul.u an estimat~ (Osm..) of th. tru~ osmolality {Osm.} of .. rum.'" Each formula indudes m=rM valu~s for som~ of th. m.jor solutes contributing to osmol.lity •• nd constanH or f. cto", to estimau th~ rtfrcts of th. oth.. solut~ •. Th~ formul.. vaty bc:cau.. of th~ following: a. Diff~ .. nt invrstigators att~mptM to ~stimat~ tm. osmolality by using difT... nt solutes .nd convrf!ion f.ctors. b. M",hod variation. for som~ of th. an.lyt .. produc<: difT..~nt results. which rrqui", difT~"'nt math.matical manipulatiom to best estimat~ tm. osmolality. c. Th~ dissociation of ionic compounds may vary among sprci... d. Th. ron""",ion of ..rum O!molarity to ",mm osmolality i. impuf.-ct. 3. [d.ally. """h laboratory v..ould d.t~rmin~ • best_fit rquation to pr.dict O"n. for that laboratory'. =p. How""~r. most usc: on. of four rq""tions (Eq. 9.6.0-<1). [n.ll formul",. N.+ and K+ cono::ntmion. a", in mmoUL or mEq/L [f UN and gluco .. cono::ntrations ..~ in mgldL, th~n Osm< = 1.86 ([Na+]+[K' ]) + [UN] + [glucosc: 2.8 1 (9.6a.) 18 (90Gb.) '50 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY If UN and glums< con<'~ntn{iollS ar~ OSJll" = 1.86 (INa+] + [K+]) + Osm. = 2 [N.+] [ur~al in mmollL, {h~n + [glucost1 + [u,,",.j + [gluros.] (M c.) (9.6.1.) th~ oompon.nts of th~ «\uatiom? •. 1.86 X ([N .+j + [IC'] ) or 2 X [N .1: an estim. c. of tho osmolality due 10 th~ 4. Wh at."" major four dectrolyu,; that is, Ih~ sum of N • ., K', Cl- , .nd HCO,- oon~ntration' {I} Breau.... rum must rem:nn dectrically nnllrnt, incr~=d ooncrntr>tion! of omu anions will b. associ>tal with Jo~r [et] or [HCO,-) if cation OODctlltr>{;Ons .."",in con,Unt. Thus, th. apr~ssjon estiJrult., the o,molality due to d«trolyu. """" when me", is an inc..astd anion gap. {2} Th~ inclusion of K' tends to yidd a more a.ccur>{~ estim. c. of osmolality • .. p«ially if (he", j, hyponm.mi. and hyperkalemia. Howenr, ba:au.. ""rum [K+] cannot chang. much (± 2 mmollL) without ""ious mfflical constq...,nres, nujor chang." in ..,rum [K+] co...., only minor chang.. in tot:d .. rum ""molality, {3} OchUI appro.ch the concq>tu:d contributiom of the d~trolytes with the concept tha t ] mmol of NaCI contributes only about ],75 mmol becau.., N .Cl, like oth" plasma .<:dts, i. not completdy dissociated in plasma.' With th. assumption that plasma is 93 % H,O, the ounol:dity due to N .+ .al" i, about ],88 X pl asma [Na+]; ],88 = ],75 -t- 0,93, The osmolality due to other .<:du {i,e" K+, Ca"+, and Mg'+ salt,} is about 0,]2 X plasma [Na+] , T hus , 2 X plasma [N . +] estimates the 01molality due to the d~trolytes if the r:.. ios of N .+ to oth" d~trolytes are physiologic. {4} When comidering the calculated osmolality, it i, important to recognize th .. th. fi ... pm of th. formula (th at which contains Na+ or N .+ + K') repr .... .. nt. the contributiom of aU ions, not just Na+ or just N. · and )(,", b, [UN] -t- 2,8; conversion of [UN] from mgldL to mmollL of urea (M, of UN = 28, and the", at< ]0 dL in ] L) c, [UN] -t- 3; approximate conve"ion of [UN] from mgldL to mmoUL of urea d [Glucost] -t- ]8; conversion of [glUC<>5<] from mgldl to mmoUL (M, of gluco.., = ]80, and th"e a", ]0 dL in ] L) . , [GluC<>5<] -t- 20; approximate convenion of [gluco ..] from mgldL to mmollL 5, An Osm.. i. oflitcle v:du. by itself; it is .imply an estim... of th. osmolality d ue to commonly measurffl ",lute cona.ntrations, If a meaoured ",lure concentration (i,e" N .+, urea, or glUC<>5<) i. increa.. O .mo, gap = Osm,. - Osm.. a, (9,7,) r"Presents the total solut. concentration in th. sampl. , a.m, estimates the solute concentration by...., of some combination of Na+, K+, urea, and glucost concentrations, O""~ 9/ MONOVALENT ELECTROLYTES AND OSMOLALITY 551 -'" :Jtu_",-- "'" , "' =:!<:=:= ~= = Hea,- --------- ~ , , , "' • --" HCO,· ----~- <> --------- .. ~ --------- ". ---------- A B c "'. ,,' D E Fig. 9. U. Bar di>gr:uru of .. rum ounoWity oonc. OSJDO. g:q> ar< .. itbin the K"Y",haMti A. In he:dtby animals. n<:uly:ill nonprot';n ",Jutes:u. monov.t.nt electrolyte. except for urea:and glu<=< (gluc.). Th. oomo. g:q> should I>< n..r 0 if m appropria.. formub for 0"", i. uood. B. In a normodtJoremic m i. not incr.ued. bee..,.. tl>. 1.86 (Na- + K') iliouJd account for tbe sum of :ill cations :and mio",. 11="", electrical """tr:o~ty mutt I>. m.mu.in«i. inc ...... in th.e <x>n«n" .. ion of unm< ","(Dmpa· nied by .ith.er iner< ..... in rations. doer..... in oth.er .morn. or both. In my c.... the 1.86 (N a' + K') should account for tb. sum of :ill c.,i"", :and anio",. C. When hyperosmoh.lity is rawed by :u.otem.ia, the <>Imo. g:q> is not inaeued. beau .. botb tl>. Osm" :and the Os",,:u. inplies for hyper<><-mot.~ty cawed by byperglyc<mia or hypernatrem.i .. D. When byperosmoh.lity is cawed by an abnorrrW nonionic ",Iu.. (•. g.• mannitol). th.e osmo. g:q> is in"",,,,,",,. bea .... the ""ogenous wlu.. inc"' .... the Osm. but is not included in the Osm,. formul .. E. When hypooomob~ty i. coused by bypona~mia:and hypochlor<mia, the ",mo. Il"P is not changed. bee""", the Oun. :and tl>. Os"" :ore dec .....d by the = amount. ,,,,.a. b. If the formul. u=I for O.m, is optimized to match Q,m.. v:due•• the osmo. gap .. f..ence interval would be near uro. c. On. cannot reliably interpret osmo. gap values if a labo .. tory has not d~ .. mined the rdation,hip betw«n paired values for Osm, and Osm.. for values d~ .. mined by th., J.boratory •• lthough markal incr"".." m ay be readily apparent. [v. Ev:duation of .. rum o,molality and osmo. gap. The major conap.. are ,hown in Fig. 9.13. 5" FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY Table 9. 16. Disorden and condition. thar a u"" abnorn,a1 .crun. oomolality or incrcued o.mo. gap. o~ Osmo. gap Abnormal [solut~l Disord~rs. 'lncr~ WRI [ncr~a= in [ncre~ Incrrasffl oonctntration of a nonanionic oompound other than urra or g1UOO5t Indicatrs a lrue hrpoomrmia Stt ch~ Hyp =Iion 5« ch~ Azot~mia =tion in Chapt" 8 S« th~ Hyp<,g1yctmia stCtion in Chopt« 14 Mannitol infusion (IV) Radiographic contrail ruMia (IV) Ethanol, methanol, rthylene glycol Stt Sodium Conct ntration, stet. V Inc,,,,,snI WRI Na+, urea, or g1uoo.., oonctntr.ltion. conditio", • A rohtively COJIUDOn di ..... or oondition. No ..: A ' p",iow inc.r .... in ouno. S1p wiU oemr if th.r. is • psatdo_hypon>.tr..ru. (_ Conc V. Sodium Imupretation of osmolality data (Tabl. 9. 16) A. Strum hypUOOffiOWity .nd osmo. gap WRl: An incr~ O,m. indicate, the COnetn_ tration of on. or mo .. solutes i, incr~as..d. Sino. th~ ouno. gap is WRI. th ..e is • proponionat~ iner~....., in O.m, :md O,m •. B. cau.., an incr ..snl Osm, ..",In from inn~asaI cono.ntration, ofN.+, u... . or glucosr. then [he inn~"",d O'm. i, due to inc..= 9/ MONOVALENT ELECTROLYTES AND OSMOLALITY 3. Th~ 1.86 ([N. +] 553 + [K+]) .nd 2 x [Na+] ap .... ions would und"es{imat~ th~ total ion con~ntration if th,,~ w"e illc...,..., ill cation ron~ntratiom oth" thall [N . +] or [K+]. Howenr. incrrases in [fCa'+] or [/Mg""] of2- 3 mmollL would cr.... t~ pathologic ,tates but inc..ast the ",rum osmolality by no mo .. thall 4-6 mmollkg. (Ifl.~ illcrease ill cation clt..r-ge colI~ntration could k m>lmed by all incr."... ill anion mar-g' con~ntmion. or by a r.duction in [N . , and/or [K'"]. ) A mang<' in o,mo. g. p of 4-6 ullit, would typically k ron,id".d dinically i"'ignificant kau"" it could .. pr~ .. nt phy. iologic or aruolytical variation. 4. E""n though th~ mmo. gap will illcrease kau .. of an incr....s.d ron~ntration of a nonionic solute. the .. call k a concurr~nt illcrease ill .nion, to cre.,e an . nion gap.' Meth. nol call contribute to .n osmo. gap. alld it, met~bolite (format~) call contribut~ to an :mion gap. Ethylelle glycol can contribute to .n osmo. gap. alld it, metabolites (glycolat. or oX>lau) con contribute to an :mion gap. [II ketoacidosi •• a.ceton. might rontribut. to 0Sffi0. gap. but A£A£ alld BHB contribute to th~ anIon g.p. e. S.rum hypoosmoWity ~nd osmo. gap WRl: A deere~s.d O,m. indicat.. th~t the con"'ntntioll of on. or mo", solut •• i. drcreas.d. Ba:au.. th. o,mo. gap i. WRl. th~", i. a proponionat~ deer......, in 0",,, .nd Osm_ .nd thu. ther~ must k drc",asal con"'ntn tions of N .+. UN. or gluco ... As UN ~nd glurosr contribute about 7 ~nd 5 mmollkg. r~'pr<:tively. in health. and ~ markal drcr...... ill ~ither (~.g.. from 7 to 3 or 5 to 2) cau,e. ollly. minor deer......, in total osmolality. any dinically ,ignificant hypoo,molality will be cous.d by hyponat",mi .. D. [f th"e is • pseudo. hyponatrrmi. (and p ..... do.hypochloremia). th~ a.m. will be an accur>t. value kau .. lipid, or proteins do not affrcr th. con"'ntratioll of major o,mol.. in plasma H,O. Howenr. th. a.m, will k drcreas.d kau .. of the .puriou,ly low [N . +]. Th~",fo ... th. osmo. gap will k artif. ctually inc",asal. References I. R.... BD. Poo, TW. zoo l. elm;'''' P/,;Ii.1.p ofAa·~IJ.u. • ..J Ei«"..1,,. Di-J",. Sth «Iiti.on. N ... Yo,," M eG ....·HiIl. 1. Scott MG. H....d JW. l...G'Y'VA. 1?99. E1«troi,..., ...<1 blood , . .... 10, Bucti, Aob..ood ER. cdo. r .... T,...... -I091. l'hiJoddpt.i" WB s."""", ~ J. luodba-, GD. I,"",,,,,, c. R.duI'K~ G. 1986. No.. ",..intoo PT. G...... CEoGknom LE. ZOOJ. lDbac bo~p~al, i~ • MiDi""", .'iduu.."" ...ith hypodipoic h,~, . I Am Y.. Mod Auoc 223, 178}-1787. S. II.l lactic ocid.o .. i~ oh" p. R.. Y" s.; sq,217_ll». 8. D-akloon CWo ZOOJ. Paintball t=ioo, .. in co.. 5" FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY in...., It.... RI, w.-nt-,; MS. 1989.~ ..... to.print =>do< ,, [J. G."""",tn<>I 60581--wnin<mi. in • OOOD.«. J Podi." 86,862-867. 18. Ort!. SR. Ritt E. 1998. TI.. n'pbn>tic oyndrom.. N £n&I J Mod JJoS, 1202-1211. 19. Duu.IAj, Cob", ND, RuoIJWW. Bn.mbau~ GW. Sdunin. DG, K...,..i. BS. l?Sll. U,;nuy indi=oefJ..,,,,, .n." adminiottatioo of crptalloid ..,"'ti".", J Y" Int<m. Mod 7.1.1_246. 22. Wd.. DJ. G«>e R. Sonit!. CM n. Mn...", A. Lip"," A. 1991. HrP"""u<m.i. and ),yp"bJ....ia ouociated .... th idiopothic ~, "'I".u.......uy inducod thy\otl>o" MD. Con A1'. 2002. H""' ........... and h"""W....ia --.doted .. ith ,10.."".. phuland p'~ ..ncd dfuoioo, i" • cat. Can V .. J .(},610-<>1}. 27. B,oitodo... odt ED. R..ot C R. 1m. I_pd.", "",rtioo. of ""tidiourtk 10,,,",,,,.,, in , .J.o&. J Am V.. Mod Aoooc , "".V 175.181_ 186. 28. van Vond .. ,n IK. KDoi .... HS, Timm .. m .... _SI'.. o, [I'. '\l,i; SP. Rijnb..k A. 2O(I.j. V, ,,,,,, ... in " 'p""O< to ... """" otimolati.on in IS 1"""1: ...." ....... P"lywia and poIydipoi .. J V" Int<m. Mod 18,8()(hW6. 29. Hilli" TA. Abbo .. RD. [J. 1999. Hypo<>.",mia, &ah.atinc ..., «>n JM. C&,l"", GP.}on<. 51., 1998. ElectroIyt< di ..... baoc.. in fodo..ith......, ,habdomro!yoi •. J Vrt I""", Med Ih 17}-177. }I. Ri.th..doon DW, K..hn CWo 198J. U"'p"i-..u... in th, foal. J Am V.. Mod Jw.oc ISh267---l71. }2. So""",, CF. Bo"", KC. 19701. MrtaboIk <~. do, ,~ ~imon,o.lly ind.....,J '01'''''' of .... =in< win,,!" bladd... Am J V" R... J5'[08}-10Il8. lJ. P.. ", GO. 0"", JR. V.........d. CA. 1981. s........ p" ..... m """"""'...... in aci.d..ok _"' •. N.M",,,, E.. Kloin LV, R..""""r; H. lrttth M. 1!lSI. Mo.lipoant loypnin J..... D. Ndv>-Atothba<].." G. Ad-DamiJaoo M. 1998. L.tIuJ "' ....... , .t..bd.o...",t,..;'.-,doted..-it!. _ .. and p ""] """.... ~. in ....... d,..trophln-. V" 1'.... 01 J5, 117_ IU. l7. Spi .. SJ. C"loon GP. Holliday TA. Cacdin .. GH Ill, I'kb. JG. 1990. H1P"bkmk p<.i.odi< p"al,..;. in J.oo" •. J Am V .. ModA..,>< 197,1009-1017. }8. J..yk PF. 1982. Hyp<.bl.mk p«iodic panlpi. in, ""'" J Am A..im H_A"", 18 <977- 980. '9. W.......an K. S"in, .. WW. Caabwi R. Zhanc IT. 1997. MoIyoio «...,..[..,. p/oo.ph.ofrurtokinao< d,6d",KJ' in Enrli.k ",.inc" id" Sa-n. ""'" (198}-1986). J Am V.. Med Aoooc 191,j5l----459. 41. Sdo .. fun. IA. van &n.t MG. K.ooima HS. Vakloij CB, P.. "". ME.. Bo.. P, Rijnbnk A. &.n. ME. 2002. E...doo-ind.....,J hy"",bJ, .. ia in kypotloyroid Ban,,, ."a.. 9/ MONOVALENT ELECTROLYTES AND OSMOLALITY 555 <> ..."'" po" .. i.m «><>«n ...tioD. ) Vrt In«m Mod j,oI7- 51. <15. Gwm·M..... DA. R=l N . Simpoon KE.. Milo, EM. 2006. Eifoct of..npl, typ< • ...d timin, of ....y. <><> fdin< blood po"";"'" """",,d.dm SH. B.nto. S. 1996. Acutt lymplooblastk Jruk..nia. hyr=tktmia • ...d p"..,Job1P""bt..ni. in • doJ:.) Am V" Mod Jw.oc 208,151- 239. <17. Lobrtti RG. 19"98. Hyptn,..i ... ....k kypoddo"""'nitm in • doJ:. ) S Aft Vrt A",>< 690JJ.-l5. "S. RubiD SI. Tool ... L. H.lp .. ~. in th, .. bbit "",tical coIloc~n , """ p«fut...Ii ..." in k.",.., •. Am J PhpioJ H"" OK Pk,..ioI284HI028-HIO}4. 5... T <>i.d """"""" ...d doctrolyt< «><>«n"'''''''' ...d .......". <=t,,;,,, of doctrolyt<. in "" ..... w;do indoct<J "oJotonu ... J Vrt Inttm Mod 19,1lJ.-1l1. 55. AJ... A. 1m. H~ iD cato. Stm.in V" Mod S"'1: (SmallAnim.) 9,153- 157. 56. Fcklmao EC. Nd. on RW. 1996. Hypttodt,<><>OOtticit .. (C•• bin,·, "J"od",,,,,,). In, c.";,,, • .J h{i,., .nd &jnwJ"";.,,. 2..d odttiot>. 187_265. Pbiladdpbi .. WB Sawtd,n. 57. Flood SM. Randolph JF. Gd= ARM. Rd>aI K. 1m. Ptimuy bypa-aIdo.""",;"" in ..... """ I AmAt.i .. H",p Jw.oc J5,jll ...... l6. 58. ~,CE.. Robi ...... WF. Hort.bk CRR. I~j. !'.imaty . !dot"",,".m (COOn·. oy<>drom<) in • cae A ao< «PO" .od .tyi... '" 00".,..'';'" .tpt<". I Small Anim Pract ;M,19J.-107. 59. Aoh RA. Htn"t)" AM. T ..1= So 2005. Prim.'}" loypt..Jdoo,,"'ni .... in th. ac A .,.,;" of II .-.•. J Fdin, M«1 s..., 7,17J.-182. 60. Rijnk.k A. V_""'., G. K..oiotta HS. van "'" W ....kn RI. '""" Sluij. FJ. II"" J. Bot. P. Bo.. WH. 2001. Hypt..J.J-"""; ... in • cat .. ;d, m''' .... itod odt"""",tical 'woo",. Vrt Q 1J,JS-4J. 61. J• ....d.i S. Dj.j..di..i.'&.,,,.l..a ...... SC. K..oiotta HS. van Do_ AM. V....!.ou., G. vanSluijo FJ. Y>.n d,n I~ TSGA.\1. Bo.. WHo Rijn ....k A. 2005. !'.imaty kyp.nkl_o=ni.m. , .. odi ..... '" I""~ «nal ...d blood ""' .. "'" in cab ... tl. "",mal aod,...Ju,ced "..,J function. Am I V" Rt. 65.620_627. 6 ... J""" BR. G",I'I'y<>.(. 68. ll £""'''"''''''0 s. 1~51,1722-17JO. 70. W .... C. G.mpb<>«n"'''''''' in canOtt aod fdin, "" .... ...d I*>ocl. Am. I Vrt Rt, 58,.43-.47. Elf"". ~ 9 o ~ ~ ~ s ~ z u ~ z ~ ffi ~ > o ~ ~ w ~ Z ~ ili -IlS 'l "" , i' H] .'I . i,l H'! "ill:. U .. i l .t .~. ~~ • 'I · e .o j i -. ' -l ' j .0 ~ 11 t· i ',~..: 11 :::; rJ d u .~ J 1 . i"~ai -s ! J' 1 I ·1' .; - . j i~ ~ - i • l' -ll ~ ~... i > ~] , ~ ~ .' ! 1 ~'H':l< .;2ii ~i f.- i ~11 .' i ~ 1 ~- i !· j i§' - . ,! -" i6 -~ Cl. €, ~ I". 1 -, ,I l' I i < ' I' •~ Ii ~ 1 ~ I" , .' ~ 112 j h'. ~ !! " U 1 . ·.,~. " i." . ,,~;, • I,,! 'J j " I. ..r hq: : 1i. i·ti;t11'~"" h,·'ji·. .~ ."~ ~ ~~l! . 11 l . ,~, 1 I " j'!~ $ . ~ ~~ " ~ ~ t-if ~ ] t::i ~ J -lI ! 1 " 1.1 d Bt;;.;l i ~ .~ <.2 ,5~ i , ~n , i~" . . J,"; , '" i J' 0 , i' ~ :~j I~ ~~i 'i~ ~ ~c1 ~J J;;r .~~ ~ ' I~~~~ jlJ!~ 1.:, 8 ~j Q ~ ~~ ~ i . ~l :!~ 'i 8 j ~1 J .~ .. ~~~~ .: ~ .fl i~ ~~ ~~I " I " ! j Jil ~l'i I I·!·~ 01. H j' 0;1 ] :l!~~jj ~ "',,~~.]. ·~i;l ·i ·~...:e::::~....;",s. -= ~';~£"";~Jir~1 .S 'u 1 ·f .§l,~ ~ ~I 1. ,"~U'l H ld!~~li ~i.~ I p. ~il "i <"Jo ~~ 8 ~ts ~ .~; ~~~~"'11 j ~ :! ;::J ...;S ~ ~~t j t~~ '"?j' j >, ~ , ~ u;.~. i' 3"I F"., i ~~~lj ~ n I ur • .,"1., • " J '" o. '-'. !'. I.."!z, •• ~ "',. 1. ,',1< • "! •.,~!. ~~ r§~*~~~~ :Ht~tE~ ~~ ~i ~j~~j~t ~ ~~ ~~a~ lH ~~~ 'I~~~~~j~~J! 'I~i ~~~I ~~~tJ{i ~~III1~~~il .; ~~< ~":~~l ~ g ~~~ ~~~a! 11 h Ji2le~cS ~ ~1ai hcS::'o~ol!l] ~lf.i~i~j!!~~~J~~J~~~~'~ ilt·~fil~l~!j~¥i~~i .~~ ... .... '" .... ~ .... '" .... ,; .... '" .... "'-'ol . ,.,., -I , . ... , . , "'. . '" , . , oii"'; . ., ~. "" '" " '" ... '" '" '" ; :. '" if., "' '" . '" . . s:: g~~ t~j~~~:j~'i l~~J~~~!'i~ij~ t·b i~~~~1~~i~~il~~1J~~~ ~ ' ~~~j~~ ~ U !H]UIHm1u11H~di}uwhhiHHJhujJlffI~H m .-\ .... 9/ MONOVALENT ELECTROLYTES AND OSMOLALITY 557 100. 0 .... 10 00. Nappauib." to......boli< acid.ooi. io dianlook al_ I N.", l}lo212S-2[J1. IO! . Ewudo.k IB, N.yIoo 1M. z,,1Io GA. 2OOl. Anioo &"I' "",..Lo" • ..;t!o ..... m 0. ond DL.-Ioctat< «>D«n".rioo in dianlook _ oat aJv",. J Y.. 101.. 0 Mod 17,940-942. 101. Poch, RA. Cohn IA W~~lotadt .. DR. SIooh... GD, N. ..... 1M, Zdl<> GA, E..ad.v.k lB. Williamo DA. R.""" CG. O's. .... Dr. 2OOS. D_lactk ocidooi. a"'" PI. 2001 . Drt..mination of "'" acid_b." ...... in loO hon.. admitted ..;t!o oolie ............ D«m> .... 1m and M., 1999. C.O Y" I 41,70}-707. lOS. au';""""", M'\l . Edfd,d, IH, E"... JW. 1990. Pro"yl"" pyool iov rtioD ca •• ", D·lactk ocidooi •. Ub Im'''' 62,)1 4-118. 106. Sa 0-.....,., ,'" ".to A..oc n(}J6--J4o. 110, 0 .". IR. Ri, ....., B. Toy\<>. Al. P, ... GO. Cbond .. R. G • ..In<. LB. 1984. c... ....._bydro.,.bu.,..." ... This page intentionally left blank Chapter 10 BLOOD GASES, BLOOD pH , AND STRONG ION DIFFERENCE Def initions ................................................................ 561 Physiologic Processes ...................................................... 561 Analytical Concepts ........................................................ Acid·Base Abnormalities .................................................... I. Metabolic (Nonrespi ratory) Acidosis .................................. II. Respiratory Acidosis ................................................ III. Metabolic (Nonrespiratory) Alkalosis .................................. IV. Respiratory Alkalosis ............................................... V. Classif ication of Acid-Base Disorders .................................. Hypoxemia and Hypoxia .................................................... Strong Ion Difference (SID) and Stewart's Method of Acid-Base Analysis ........... 565 57 4 574 575 576 576 578 581 584 559 Table 10.1. Abbreviations and symbols in Ihis chapler [x] 2,3_DPG "'DO, A~, BF. BE", BF. CO Co." CO,lCOHgb C"bon'l~ EC' FO,Hgb H,(:O, Extracdlular fluid Fr~e ionized c:olcium Fnctional ronumralion of oxygen in inspirffi gas Fr•• ionized m:agnesium Fnction of oxyhemoglobin in 10lal hemoglobin C"bonic . cid H,O Wat~r H,PO.HCO,- Dihydrog~n H« Hgb H~malocril Ie,> FI~ IMg' HHgb HPO,>0,0 p.co, p.~ p.~ p.~ P~ pH Plo, P~ PO, Po,:>P~ P~ S.o, SID SIDa-.....-. SID,_ SID. So, Sp~ tCO, "SA WRI SOl Conumralion of x (x = an. lyl<) 2, 3-D iphos phoglyc;.,rate ArI~ri:ol..tva>l" oX)"g~n lension gradiem Sum or IOI3J of nonvolatil~ ~k acids Base exc;.,,,, in blood Base exc;.,,,, in exlrac;.,J]ular fluid Base exc;.,,,, in plasma C"bon monoxide G~u. arbon dioxid~ C"boxyh~moglobin phosphate Biarbo""l~ Hemoglobin D""xyh~moglobin (rffiuccd hemoglobin) Hydrog~n phosphate 0xy\:"n comem P.rtial pressure of carbon dioxid~ in .ncrial blood P.rtial pressure of :olva>lar arbon dioxide P.rtial pressure of oXJ'C"n in m~rial blood P.rtial pressure of :olva>lar oxygen Pmial pressure of carbon dioxid~ - log [W ] P.rtial pressure of inspired oxygen Pmial pressure of oxy\:"n Phosphal~ including PO.:>-, HPO,'-, or H,PO,Phosphate Pmial pressure of carbon dioxid~ in ""nous blood Pmial pressure of oxy\:"n in ""nous blood P~rc;.,m h~moglobin 5aruralion with oXJ'C"n in m~rial blood Sirong ion diff~ren"'" Sirong ion diff~ren"'" using rorr«lal chlorid~ conc;.,n1ralion True sirong ion diff..~nc;., Sirong ion diff~r~n"'" alcu!.tal using x slrong ions P~rc;.,m h~moglobin 5aruralion with oXJ'C"n P..c;.,m h~moglobin 5aruralion with oxygtn of mcrial blood by pul.. oximetry ("pO i, fOr "pul .." oxim<'lry) T olal carbon dioxid~ Unidemifial sirong anion Within .. f~ .. n"" inl~"",l 101 BLOOD GASES, BLOOD pH, AND STRONG ION DIFFERENCE 561 DEFINIT10NS [Hfinitions of imponant t~rm. in this mapt~r: Acwmill i. a d~r~a=! blood pH {inc",a=! [1-rlJ. A/Iu./tmill i. an incr~ • .ffl blood pH (d~",a=! [H1 ). Acidosis is a condition in which .cid~mia t~nds to occur. An .nimal truly not bt .cid~mic OO:allSt of comp<ns>ting m~l,.nimlS or btcaUst of the width of th~ .. fu~n"" int~rval. i. a condition in which alkaJ~mi . t~nd. to occur. An animal truly not bt alkalemic OO:aUst of comp<nsating m~hani.m' or btcallSt of th~ width of the ",f~ren"" interval. Hyp=:apnill i. n=s CO, in blood (inc",a=! Pco,). [t i. also called hypn-rarhill. HypDmpnill i. a ddiciency of CO, in blood (d~ ..a=! Peo,). It i. al", called hyplXarh;a. Hypoxmtill i. a deficiem:y of di.solved 0, in blood (d=~ased Po,). Hypoxia i•• deficien<:y of 0, r...ching the tissue>. cells. or organell.. of th~ body. Hypnvinlilalion i. an n=sive rat~ andlor dq>th of respi .. tion leading to abnormal loss of Co, from th~ blood OO:aUst of increa=! movement of new .ir to (and from) th~ ga.-nchange regions of the lung. B=1lst hyJ>OC'pnia can bt the consequ~n"" of hypUstS mloction in the 0, content of th~ blood. incr= in the CO, rontent of the bhxl. or both. btcallst of d==d movement of new air to (and from) the g.. -nm~ region. of the lung. Beca= h~nia am bt the ron .... quenct of hypo venti luion, ",m<:{imes hypercapnia :md hypoventilarion are romi A/M/~Iis PHYSIOLOGIC PROCESSES I. &veraJ respiratory and nonrespiratory processes hdp maintain [H+] at a minute, but nable, con""ntration {about 40 nmoUL}. Met.bolic processtS rontinually produ"" W, and it i. eith~r excreted {via kidnqo} or bound to buff"", {HCO,-, PO., ammonia, mlfates, Hgb, . nd other proteins, mch .. albumin}. Of the total buffering c>p>city in health, HCO,- contributes over 20 mmoUL, whe"", the nonbicarbonat~ buffers rontribute less than 10 mmoUL I ll. AI th~ buffers work together, chan~ in one buff"'ing ')"lum reH~t manC'. in the oth~rs. [n clinical medicine. the bicarbon. te buffering .y"~m is ustd to monitor control of [tr"] and therefor~ acid.base SCatu •. A. The relationship of [H+] , [H CO,-], and Peo, in normal blood at 37'C can bt expressrd with the Hend~rson. H ..,dbalch equ. tion or a nonlogarithmic version (Eq. 10.1.). The [H,cO,] is calculated from a meamred Pco, value and th~ ",lubility """Hicient of Co, in an aqueous solution {Eq. 10.lb}. pH = 6.1 + log [HCO - ] , [H,CO, ] P Dr [W ]= 24 X [H~~,] Pco, X 0.0307 = [H,CO,] [H1 in nmoUL; [HCO,-] rx [HCO,] in mmollL; Pco, in mmHg (IO.lao) ( 1O.lb.) 562 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY B. As Sttll ill the lIonJogarithmic equation, [H+) is clearly ,datM to th. "tio of Peo, to [HCO,-j. When the ratio illerea.." in an aninul, Ih. animal ~m .. acid.mic. When the "tio d«""aso; in .0 animal, the aniJrulJ OO:offiel albJemic. Ill. Pulmon:uy functions rd.t.d 10 blood C'~ .nd acid_b= sUtus A. Expiration of CO, J..ad, to th. dimination of fT« H+ (Fig. IO.IA). ._.= A. HeaHhyanimal Vi --------------------~ H+ + Hea,' f .~ ~ -Z H,eD, Z "- 'P' , ~ ""-*"'" - Vi + """'- '" ', H,O + co2jg) ~" __ ' ,~ B Metabolic acidosis H+ .~~ '- , -Z H,CO, -Z H,O + co2(g) 'm '. H+ from metabolism C Respiratory acidosis , "" ,, '>- " , ,"" ,,, "" ,, '>- , ".,..."."..- -- -.. , 0 f> W + Heo, " H2C03 ~H20 +C 02(g) ~ , " ,, " -',," -- , , "" , " I H+ from metabolism D. Metabolic alkalosis ----------------------------- 1,-, - - " ," ------------------"" ~ E Respiratory alkalosis f I w+ HCO, H,CO, _ H,eJ • C02(g) expIred ~-~ - - ~g )" " ,"" - ..,.'- '- , , """".. ,, "" ,, "c ~ 101 BLOOD GASES, BLOOD pH, AND STRONG ION DIFFERENCE 563 l. Bwm.. blood [H+] is ""I}' low romp.=! to [HCO,-] (mio .. 1:600,000), thi, p""""ss does not low~r [HCO,-] unless thu~ is ace .. i"" g~n~r;Uion of H+, 2, It may ~ hdpful to con!id~r the bicarbonate ,yu~m going through H,CO, (Eq, 10,20), but th~ .ctu:ol reaction catalyud by c;orbonic .nhyd...e invol""s the diswciation ofH,O, rdease of H +, .nd r....crion of hydroxide ion (OJ-t) with CO, to form HCO,- without. H,CO, int~rmediat~ {Eq, 1O,2bj, Th~ .. ""tion is =",rsible, H + + HCO,- <--> H,CO, <--> H,O + COl((} (IO.2a.) H+ + HCO,-' ....... .......... ,H,O+ C O ,{, ) ( 1O.2h.) B, Hyp<:""'ntilnion incr ....... api .. tion of CO, and undo to cau.., :olblemia. Hypoventi_ lation dec"".,.,. apimion ofCa, and tend. to cau...cidemi .. Two type.! of chemore_ ceptor.; control respi .. tion: l. Cent ... l ch~mor"""Pto" {in the buin stem} ;ore nimulated by incr .... sed [H1 in surrounding fluid, 2, P~ripheral chemoreceptoR (c;orotid .nd :oortic lxxIies) ;ore ,timulated primarily by hyponmi. but :olso by acidemia (incr .... sa! [H 1 ), C. Oxyg~nation of blood .nd pulmonal}' ""ntilotion l. Th~ bas.ic physiologic aspects of th~ intercru.n~ ofO, and CO, ;oro described in Fig, 10,2, Fig. IO. L Scbem. tic rep,,,,,,ntotion of tb. b.. ic 00l><'''P''' of tt.. bicarbonat. buffering 'f''''m in health md in . oo_b ... disorders, Re'pi ...ory disorde" inVOM removal of CO, from pulmon1lf .. terial (capiU.",) blood, M .. obo~c diso,de" caw< . bno,mal oonc sbaw th .. . 1"Il" ucetS of HCO,- is :ov:oibb&. to buff... 1-['", B. In. "",t:obo~c . cido,is, . cidosis OC<W"S 1><="", of on< of two batic pr<>" l. ExC<Sl H+ . ccumul . ... 1><=""" of inc,.»«l p,oduction of orgrnic ",ids, in=»«l H+ ",leas. from ATP ....g., 0' d=.»«l ,enal oxaetion of H+, TIt", 0= .. H+ d,ive< tb. e<Jw.tion to tb. right md tbw 1.1<\, to oonsumption of HCO,-, 2, Ex""", loss of HCO,- vi. tb. .~""'ntuy 01 u,inuy SJ"tem ,«!u"", tt.. buffering C1]>1city rnd alto.,., H' to """"mub te, C. In • =pi .. tory . cidosis, hypoventil.tion~ ... reduced o~p~~on, of CO~~ which J.. dt to "':' t P,eo, md an T [H'] (J- pH), Without co"'P" .... oon, lHCO,-] IS lnslgruficontly lnc«ased.nd «rrwns WRl, Given tUne, tbe kidn<Jl""';]] compem>" 10, .cidemi. rnd conserve HCO,-, D. In • metobolic alkalosis, alblosi, oerun 1><=.... of one of toro basic p~ Witbout oompe .... tion, Peo, .. moint WRL ], Ex""", H+ is lott vi. g.. tric 01 ,enall, md • J- [H1 (1' pH), Without compem>tion, [HCO,l ,..,will WRL -=,.. 564 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY Inspired Air Tracheal Air PIo,. 159 nmHg PIco, ....... 0 mmHg Po,. 149 mmHg P"o,-l00mmHg Alveolus Expired Air P..,o . 47 mmHg PAco, . 50 mmHg PEco, < 50 mmHg 0, Venous Blood P~·40mmHg -----------------r, , co, P.,co,. 45-50 mmHg Arterial Blood , P. o,_l00mmHg , ,, P. co, • 40 mmHg [H+) • 40 nmoIIL [Hea,l • 24 JTVL , , ,, , __________________________________________________ _ f ' Fig. 10.2. Scho"",tic dnwiog of tbo ""chong< of 0, . nd CO, .. the .Jv.olw-capiJIary jWK1ion. Pr=w< v.ru.. "'" indoxki, b .. . Po, (Plo,) of 159 mmHg. Witb tl>< oonoibution of PH,o (47 mmHg) in the warm mob .., tbo Po, drops to 149 mmHg [0.209(760 - 47}1. In the .molu., the PAo, it [""",. 100 mmHg, b.a""" of th. int<.ro>Il£" of 0, with tho blood ..,d b.a.,.., of th. inc","""" pA =. ('50 mmHg) from th.e blood. • Alv=1us ro blood: [n bealth. 0, quiddy dilfu"", from tbe alvoolus ro tb. c:opilhry blood (Po, ""or 40 mmHg) to giv< • Po, of 100 mmHg. whiclt "'p,... nts th.e p",smn: """,t«! by tho disso[ved 0, in ph"""- Tn. 0, also diffuses into erythrocyt .. aad bind> to Hgb (O,Hgb) to u,=t< th. oxygJ". Owing ..,.,tb.,i., m n,,,,, api",d Pco, (upnogr.opby) provides inform1tion to 1lS<SS CO, production, pulmonory gas ..cbang., and .Jimi""tion ofca, by thr ..... thrtic 2. For th~ .. to k adaJuat~ oxygenation of blood, seve",1 pJ"OCCSS<::'l must k funetio",,]. a. Th. inspirM .ir must rurve adaJU 101 BLOOD GASES, BLOOD pH, AND STRONG ION DIFFERENCE 565 from alv""li to blood; th~ diffusion co.flici~nt for CO, i. 20 tim .. th~ diffusion co.flici~nt for 0,. D. T "onsport of 0, to tiSllu" I. On"", 0, has diffust [v. R,nal functions relat..d to acid_I>...., balona. A. Th. kidn.". han hy rol .. in mainuining :lCid_b= !latus (Rt Chapt~n 8 .nd 9). B. Th~ir rruojor functiolll that ~ruin to .cid_b... lIatus in h~th arr to excrrt~ H+, eith~r dir«dyor by incorporation illlo .mmonium (NH:), dihydro\:"n phosphat~ (H,PO.-), or bisulfate (HSO,-j, and to con.."", HCO,- . C. Hormonal.nd oth ANALYT[CAL CO NCEPTS I. Blood gas instrumenl1 A. Blood gas instrum~lIIs m~a.lure the [W] , po" and Pco, in blood. I. Descriptions of m..sured analyt .. a. {H'] i, th~ fr.., H+ cona.ntration in blood. It r~H«ts th. n.t .fret:t of bodily proc= on blood [H+] .nd is rrport..d '" pH, th. unid= n~gatin log of [H+] . b. Po, (mm Hg) is the parti.l pr=ure ofo, in th~ blood. [t rrH«ts the amount of dissolval 0, but d""s not measure 0, associated with Hgb. [t is u.. fuJ in ...... ing pulmonary gas exchan~. c. Pco, (mmHg) i, th~ po.rtial prossure of CO, in blood. It rrfl~cu the amount of dissolval Co, in blood and is "",ful to ...... alv""lar nntilation. 2. Blood g.. analyurs ust Arterial Blood High So, Low [WI High PFK acIiviIy I"""",sing [2.3.DPGI 0, Tissue Blood Hig-.&.I [2,3-DPG) Pulmonary Blood HigOOsl [H+I Venous Blood LOW9St Hgb affinil;' fa" 01 Low So, High [W) LowPFKactiYiI;' L0W9st [2.3-DPG) Lowo.t[H+) Hig-.&.I Hgb ~If'nily for 02 Decroasing [2,3.DPG) Fig. iOJ. Oxyg" in metabolic path"l>}"l. • CO, (from "",ubo~c p>thw.ys) diffuse. into pl .. ID1 10d then into . rythrocytos. Vi. tbe carbonic :mbydn>< (Gf) reaction, CO, and H,O .,. ron",,""" to HooJ-:md H·. 1be HCO,- moves to pWm1 in uclung. for CI-. Most ofthe W is buffered by the d.eoxyg.ted Hgb. Th. 00, proth><,ed in ti...,., is cmied in blood in oro forms: (I) .. diuolved CO, with . P,.co, ... or 45- 50 mmHg, and (2) .. Hoo,in erythrocytos after re.cting with H,O in the plosen« of carbonic :mhydns<. • Erytbrocyte.! en .. r in peripbmJ .......... itb high 12,3·DPG], but th. JJXJ« acidic . nvironment (ina: • ...d 1H'l ) inhibiu pbospbofructokin ... (PFJ:) in the glycolytic p>tb""y, and tbw the .... of 2,3·DPG formation deere ..... Ery"-cyt" j~ pub,,,,,,,,,] w.-l • 0, difi'we. from aIveo~ to pulmonary phuru and into erythrocytes. Becawe erythrocyt .. have tbei. Ie ... 12,3-DPG] and Ie .. t [1-11 in th. pulmonary ~I~ th.ir Hgb molethwoy, and thw th.,.te of 2,3-DPG formation ina ....,.. '" 101 BLOOD GASES, BLOOD pH, AND STRONG ION DIFFERENCE 567 cona.ntmions (Na+, K+, ct, ur...., g1uco.." !Ca'+, and Hgb), :md {3} cakulat~ SN~",1 cona.ntr>tions or values (HCO,-, tcO" anion gap, So" BE,." BE", .nd hematocrit), B, Cakulatffl acid·ba.., .nd blood gas ""lues: Th~ following v:du .. can bt cakUlatffl from the m + [H,CO,] = [HCO,-] + (Peo, X 0,0307) (IO.).) 3, {HCO,-j ....... (in mmollL) i. also call~d stand"d bicarbonau rona.nt"'tion (SBC), a, [HCO,-j-... .. is th~ [HCO,-] in pluma wh~n fully oxygtn. tffl blood from a "normal" . nim. l is "8 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY c. Positive alld !Iegalive BE., values provide """mially the sam. infurmation .bout th. patient :as does th. BE.,.. Th. BE., ",Ju.. should app'''''ch the BE... :as an animal krom .. more anemic. 6. Buff" b.... {or total buffer b....j (in mmol/L) a. BUff" bas, is th. sum of "buff" ions" of blood, including HCO,- , Hgb, .nd pl.. m. proteins. h. Buffe, b.... is reI.tffl to ~ exa:s.s as foUoWJ: buffer b.... ~ = buff.r bo... ~ + bast ex=s. Buffer b=-." is the c;;o/cuJatffl sum of the "buff" iom" in a !"Ili.m', umpJ •. Buffer b>St_ .... is the npn:1ffl sum of th. "buffer iom" in a hrM to normal. 7. So, (in %) a. So, is th. amoum of oxyhemoglobin in blood apres~ as a P"=nt~ of ch. total amount of hemoglobin .bl. to bind 0,. (S« s~s. II [Puts. oximmy] and III [CO-oximeter] for method. of meamring th. p"=nu gd b. Th~ So, d~nd, prim>rily on th~ Po, as de'Cfi~d by the sigmoid 0,-Hgb dissociation cu"'" (Fig. lOA). At a Po, of 100 mmHg. th~ So, i, apectM to be about 97 %. Acidemia. increased erythrocyte [2.3·DPG ]. hYJl<'nhermi a, and hYJl<'rcub", ,hift th~ dissociation curve to the right (So, decreases for a given Po,). """ .,. .,. l"' , - .... - "':(------------------- ! Shift <:UV ! 2. ncm..sod (2.3--DPG) __________ ,...'______ ~--- LWw.'!lI'Jll:f~L __ _ , '" ",. i 3. ncm..sod 19"l"''''\u"9 -;---------r--·~~~ ---, , ,, ,; 7-------------1------------------, - o· ~--~--~--_+. !--_oc__c~--cc 20 40 60 80 100 120 Po, (mmHg) Fig. lOA. Oxyg 90 % .. long as t'" Po, ",mo.iru . ' > 60 mmH g (d .. b«lUfO'V). When Po, vol .... .,o > ]00 mmHg. tho So, .. ill ... neo.. 100 %. [ncn...d [H1 . incn...d orythro. wiU ,hift the o.trV< to the .. ight. If Po, " 'J" t< .ligbtly in H gb molKuks. [2.3-DPGI difforen=. and 0...... foe.o.... Accordingly. tho diffe .. ..,. hoca.... of diffe .. "",rago P" values (P,o, ..... n homogIobin is SO % ...",... od with oxygon .. pH 704. 37 'c. and oW mmHg P,co,) differ: oor>es " 25 mmH g. ",ttl. .. 26 mmH g. peopt. .. 27 mmH g. dogs .. 30 mmH g. rnd c. .. .. 34mmH g...... ..,= 101 BLOOD GASES, BLOOD pH, AND STRONG ION DIFFERENCE 569 c. Som~ blood ga> instrum~nts calculat~ th~ So, from m.... surffl pH, p.co" and P.o, v:olu .. by using fOrmul:as that an~mpt to corr«t for umpentur< var;"tiom ~nd a compla formula that an~mpu to describt th~ 0, dissoci~tion curve.' Th~ colcul.tal So, m.y bt ~rronn>us btcaus. of ,,",umffl normal 0, .ffinity for Hgb and """mffl normal [2,3_DPGj.' {I} If th~ pH i. constant. but th~ po, d«r ......., th.n So, d«reases. {2} If th~ po, is ronnant, but th~ pH deer ....... (acid.mia), then So, d«r= If Po, is COlmant, but pH incr..,...,. (alkalemia) , then So, mcr ......,.. 8. PAo, (in mmHg): alveolar 0, pre ...... re {sometim •• abbreviatffl :as A} a. PAO, is the pani:ol pre...... r< ofo, in the :olveolu air (g:as). Two fOrmuw incor_ por.oting Flo, .nd P,co, con be uK (IOAa.) Complne: PAo, = Plo, - p.co, (1 - Flo,(I - R)) R wh~re PAO, is p. nial pressur~ of :olveolar oxyg<:n, Plo, is th~ partial pr< .. ur~ of inspiral oxygen, P.co, is the partial pr< ...... re of Co, in art~ri:ol blood, R is the anrag~ mnabolic r~spi"'tory quoti~nt, and Flo, i. th. f",ction.l conc;.,ntration of oxygen in impiral .ir (gas). Anerial alveolar oxygen umion gradi.nt: AaDo, = PAo, - p.o, ( IDAb.) .~ · ,.1 I. oll)"g~n ",t.na .. veo"if (IOAc.) .. ",UO: afA =~o, -- t~n"on PAD' Inspirnl oxygen f"'ction "'tio: P.o, / Fl = p. o, Flo, (lOAd.) b. Th~ respiratol)' quotient (R) is th~ "'tio of th. ""lum. of Co, producnl to th~ volume ofo, consumffl by a body. In other words, R is th~ "'tio of th. ~mount of CO, diffu.', an :assumption tru.t may not bt tru. if th~r< i. a mismatch in vc:ntilation_pertusion. 9. AaDO, (in mmHg) i, the art~ri:ol :olveolar 0, t.nsion g",di~nt (Eq. lOAb). Th. value i. uK 57G FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY 10. alA (unitb'l is the arterial alvtivdy suble with ch.nges in Flo,. II. P.oJFI (unid=) i. the inspi=' 0, fraction ratio {Eq. lOAd}. The value is used '" an index of the delivery of 0, to alveoli and exchangt ofO, in lungs. 12. Oft (mUdL) i, the 0, content. The O,ct r."resents the total amount (volume) of 0, in a given blood ""lume (IOO mL). induding that dissolved in plasnu .nd that bound to hemoglobin. 13. P,. or Po, (0.5) {in mmHg}: The p.. is ddined as the P.o, ofa .ample when hemoglobin is 50 % ",turated with 0, at pH 704. 37°C. and 40 mmHg P~. C. Tem~ratur. cor=tion factors for blood pH and gas valu .. with v.ri.cions in lxxIy r.mpc:nture I. Panial presm= of di""l..-ed gases v.ty with the tempc:ratu .. of the solvent. For example. if 0, .nd CO, content .. main constant in • "'mple but tempc:ratu .. incr ........ the Po, and Peo, values incr."", (hot gas exp.nds and thu. exerts more p=su",). 2 Blood g'" instruments measu .. pH. Po" :md Peo, in a 3 7 °C chambe:r. If the patient', r.mpc:ratu .. is 40°C. then the in vivo valu.. will be: different from those determined by the instrument. Correction f:>eto", for inc..asing tempc:ratu= .'" in T.ble 10.2. For exantple. if the Peo, wer. 40 mntHg at 37°C. then the Peo, would be: 44 mmHg (40 + 10 % of 40 mmHg) at 39°C and 48 mmHg (40 + 20 % of 40 mntHg) at 41°C. 3. The", are differencu of opinion and unan.,...=' qu .. tions regarding the need for tempc:rature correction of blood gas. a. For example. lowering blood'. r.ntpc:rature {as in hypothernti.} will lower the Po" but the 0, content of blood will not change. In the hypotherntic sme. is thelow.. p.o, value d .. ired for the lower metabolic rate or should the p.o, be: incr......d by incr.a.ing the Flo,? b. If r.mpc:ratu .. _corrected values .'" used to ntonitor a p.tient. and if the ""ti.nt·s lxxIy tempc:mu", chang... will ch:mgts in pH. Po,. or Peo, be: c;>UsN by changes in body tentpc:rature or other f:>eto",? 4. If. loboratoty .. ports tempc:rature-corrected values. it mould also report the valu.. measured at 37 °C. If all in vitro valu.. are reported at 37°C regardless of the body r.mpc:rature. then changes in Po, and Peo, values will reflect changes in 0, .nd Co, content of blood. T able to.2. Correction o f hlood gas and J:!H valu .. for variations in bod! temperature 'C 'F ,H "40 105.8 104.0 102.2 10004 98.6 - 0.06 - 0.04 - 0.03 - 0.01 None 39 " 37 Not<: Cornelion ",eton ulruloted from .... bli,Md formuW'" P=, Po, +20 % + 15 % + 10 % + 33 % + 24 % + 15 % +' " +7" None None 101 11. BLOOD GASES, BLOOD pH, AND STRONG ION DIFFERENCE 571 Pul", oximetry A. An oxi",~," is ~ drvi~ cru.t measllfes .nerial So" denoud herr :as Spo,. During the 1980., the pul.. oximeur became an ~=pted instrument in humon malicine for monitoring Hgb oxygen.rion during .nesthesia :md is common in the monitoring of critical cue patienn. B. The pulse oximeur determines the Spo, by a spectrophotometric method.' l. Light at two wavdength. (e.g. , 660 .nd 940 nm) is p.....d through tissue that contain. "'terial blood (e.g. , the eor lobe or finger in people; the tongue, lip, rar, toe, or tail in dogs; and the tongue in horses}."'ln dogs, the ben agreement of Spo, with S,o, occurred with the lip and tongue pl.~ments. · 2. At the higher wavdength (e.g., 940 nm), oxyhemoglobin .bsorbs morr light than does deoxyhemoglobin (rMuced Hgb). At the lower wavelength (e.g., 660 nm), oxyhemoglobin abwrru less light th:m does deoxyhemoglobin. From the meamrM abrorban~ values, a ratio con be calculated that refleets the 5.0, (Eq. 10.5.). S.o, = [O,Hgb] X 100 [O,Hgb] + [HHgb] O,Hgb% [O,Hgb] X 100 [O,Hgb] + [H Hgb] + [COHgb] + [MetHgb] {10.5a.} {1Mb.} O,Hgh {oxyhemoglobin}, HHgb (reduced hemoglobin), COHgb {corboxyhemoglobin}, MetHgb (methemoglobin) 3. The interferen~ coused by other light-..bsorbing rubsunces in the tis,mes is removed by measuring the light while arterial blood pul"" through the tissue. In pulse oximetry, it is assumed that the changes in abrorban~ values occur when o,..rich blood puh.tes through the tissue. The puhe qualiry con be evaluated graphically. 4. COHgb .nd m..rhemoglobin al", absorb light .t the measurement wavelengths .nd thus interfere with measurrments of [O,Hgh] if they "'. present (,.. Hypoxemi<> :md Hypoxia, sret. 11.0) and give falsdy incrrased 5.0, and 0,Hgb% valueo {Eq. lO.5b} Ill. CO-oxim..rer value< A. A CO-oximeter determines the different types of Hgb by measuring the .bsorbance of sev.ral w.vdengths of light. Eoch ty~ of Hgb has ~ unique ab""rb.n~ spectrum, and the con~ntration of rach type is determined. The CO-oximeter con be used to identifY methemoglobinemia or CO poisoning. B. Calculated values (Eq. 10.6) [tHgh] = [O,Hgb] + [HHgb] + [COHgh] + [MetHgb] [O,Hgb] O,Hgb% = [ ] xlOO tHgb HHgb% = [HHgb] X 100 [tHgb] {10.6.} sn FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY [COHgb] x 100 COHgb% [tHgb] MetH gb% = so, = [M. tHgbJ [ tHgb [X IOO [O,Hgbj [O,H gb] + [HHgb] x 100 FO H b ) O,Hgb] ,g [tHgbj O,e! = 1.31 mL 0, X [{Hg b[(SO, - -) g Hgb 100 o,Hgb (oxyhemoglobin), HHgb (raluced hemoglobin), COHgb (Clrboxyh.moglobin), MetHgb (methemoglobin), Fo,Hgb (fraction of oxyhemoglobin in total hemoglobin), 0,ct (oxy~n com.ntl l. Total Hgb (tHgb) OOllctntr.u ion rq",sent' th. mrn of the hemoglobin types as del"mina! by . b,orbanct photometry. 2. The pe=nt"i:e'l of ram typo of Hgb ... cakulatffi nom th. ooll"",mralions e. [v. d.terminal by Wsorbanct photometry. 3. a,ec is also c;;o/CUJ.tM from the oximeuh meamrrioUl COllctmntion, ofh.moglobin_b=d 0, "",ius, bUI the j·STAT provid~d .ccurat~ ""lues with only low ron""ntratiofl! of th~ c"ri~rs" 2 Compar.d to result, obtain.d from an Or",l«tivt d«trod~ ...... y. th~ 0, a)flt~nt m~. !Ur.d by . n [nstrum~ntation Laboratories CO-oxirnff" diff~~ rnor~ than 20 % 1 h .ft~r infi"ion with . hernoglobin_bas.d 0, curier. Th~ dirr.r~n"'" w~'" great.. with th~ l"g~r d<=g< of th~ blood substitute.'· Sample for blood gas and pH anal,..,i, A. Hepariniud whol~ blood: 0.05- 0.1 ml of hq... in {l.OOO units/rnll p 10/ BLOOD GASES, BLOOD pH, AND STRONG ION DIFFERENCE 513 4. Blood may k co]J""tal in .ith.r glw or quality pla!lic syringes, though Po, may d""rra .. mo", quidly in glass syringes. To pr""ent expo>u", to air, th. n...dl. mun b. sralal with cork or rubb.r immal",tdy aft.. coJ]""tion. Air should not b. in the 'yring<' during or after the coU""tion of a .ample. 5. Optimally, a hep"rinittd blood sample .hould b. tened (poim-of_ca.. tming) or transported to the laboratory immediatdy:after coU""tion. If not possible, acceptable blood gas .nd pH mulu con b. obtained if the h""arinittd blood is immersed in an ice bath .nd analyzed within I h. Placement of the .. mple in • ",frigerator wiU not mol the .,mple as quidly; do not place a ,ample in a fr=..r, becou.. erythrocyte> will I,.... 6. Excessivt h"".rin in the coU""ting .yringr con r.. ult in erronww valu.. ; there should only b. enough h"""rin to oo;ot th. n...dle and. 3 mL syringe. Thi. can b. a.coompli,hed by drawing 0.5 mL hep"rin into. 3 mL syring<' to the 3 mL m ..k and then forcefully expdling it by wing 3 mL .ir in the syringe. Leaving h""arin in the hub of the .yringr creat.. minimal alteration> in blood gas v:dues but m.y cause significantly d=..sed [ft:a>+] and significantly increased [Cl-]. Lugrr .mounts :d", cau.. lower Peo" [HCO,-], [K+], and lactate concentration. and higher Po, {if b",.thing air} and [Na+]." B. Erroneow blood gas .nd pH v:du .. b.c.u .. of poor .. mple coll""tion or handlifIJ: l. Exposu", to .ir (induding .ir bubbl.. ) or acess h'p"rin in the ..."ple" a. The Po, .nd Peo, valu.. for arterial blood and room air .re different (fable 10.31. The Po, and Peo, ofh"".rin will b. the .,me as room air if hep"rin i. exposed to .ir. b. The Po, .nd Peo, of blood .nd air quidly equilibrate; th. blood Po, incr..... {unl... the p"tient is on 0, therapy} and blood Peo, d""rra .... The lou of CO, from blood cau... d""rra ... in the [HCO,-] .nd [H+] of the . ample. c. Net result: i pH, i Po" J.. Peo" :md J.. [HCO;] 2. Delay in sample anaIysi. or failu", to chill the sample adequatdy {but no .ir aposure} a. Aerobic metaboli,m of leukoo:yt.. and platdets cau.., d=....d Po,. Ba..d on ,tability ,tudies for equine blood p.o" an,,",l blood should b. an:dyzed within 10 min {if kept at room tem!"'rature} or within 2 h {if stored in ice bath}. Th. p.co, and pH we .. stable fOr up to I h at room tem!",ratu""" b. Glycolysi, in leukoo:yt.., erythrocytes, and platdet, causes increased W production and thus d""reased pH. c. Net result: J.. pH :md J.. Po, T able 1003. Differences in Po, and Peo, values (mmHg) between air and blo<> Po, .. 150 Pc~ c , So",", Muir rnd Hubbell.'" Art..ial blood Venow blood 574 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY ACID_BASE ABNOR.,\1AUTIES I. Mffabolic (non ... pimory) ~cidosis A. Mu"bolir IUid~,iJ is ~ p"thophy.iologic 'tal~ in which . nOIl""'piratory process "'-USN [he accumulation of H+ in blood :md a d..c..... in plasma or ..,rum [H CO,-J. B. Metabolic acidoses are typically prodUcffl by the ex". .. i:"neration of H + or a loS! of HCO,-. Th. loss of HCO,· {or other body buffen} rwu",," the buffering cap>city of the body and thus allows H+ to .ccumulot. {Fig. 10.1 B}. C. Disorders and patho~n= (S« Tabl. 9.10 and th. OUOOCi>lffl tat) l. Ex~ .. generation of H+: lactic acidosis (including rumen onrlo.d). kff"",cidmi., and ingtnion and meuboli,m of ""ruin compound. k.g., ""hyle"e glycol} 2 D«reased renal excmion of H +: reruoJ f:ailur" uroptritoneum, dj,uJ reruoJ tubul" acidosis (typ< I), and hypoaldosuroni,m 3. Inc",,=<:! Hea,- 10M: gastroimmin. llo!SeS {diarrhea or ..Iiva loss in ruminants}, "'nallo.... {proximal ..nal tubular acido,is, t}'J'<' 2} D. The metabolic .cido.." .", wm~im.s d ..sified into on. of five groups: l. TitratioNlI addo,ir. [HCOn deere .... bn::;ouse it is b.:ing used to buffer H+ that is either b.:ing produced excessively {e.g., lactic acido.is} or not b.:iIll: adequately excreted by kidn.ys {e.g., ",nal f.ilure}. TimltioNlI is som"",h.t of. misnomer bn::;o...., titrating i, the ch.minry labor>to!}' proa:.. of adding a r~nt to produce. given .ffect k.g., adding acid to d~.rmin. the concentration of. b...,}. Th. decr ... sed [HCO,-J is cmsed by buffering, not by titrating. HCO,- i. on. of several buff... in blood and i, used to asse.. alt.rations in the con""ntrations of buff.... 2. 5«rftOry fU"idq,ir. [HCO,-] decr.,..., because it is b.:ing lo,t from the body {e.g., excess salivary 10.. or proximal tubul ar acidosis} or not b.:ing produced {distal tubular acidosi,}. Th. hallmark of the.., .cido"," is the roncurrent hyperchlo .. mia. 5«rftOry i. an .ppropriate adjective wh.n HCO,- i. b.:ing secreted {e.g., in ..Iiv.}. However. in the context of th. tubular acidoses, UCTftOry is not.n appropriate adjective beca...., the decre=d [HCO,-] i. not caused by the secmion of HCO,into the tubular fluid. 3. Organk acidosis: Th. deer.... in [HCO,-] is linked to the accumulation of.n organic anion (' .g.,lactat., .""toacetat., ~.hydroxybutyr>te, or citrate). The organic anion may represent th. incr ... sed production of an organic .cid ('.g., .""toacetic acid) by metabolic pathWllY', but wmetim.. the organic .nion and the H+ a.. b.:ing produced by differ.nt pathWllY" {= the L.lact.t. section in Chapter 9}. 4. /lUJrganic ad,u"is: Th. dec ..... in [HCO,-J i. linked to the .ccumul.tion of.n inorg.nic anion {•. g., PO. or ,ulfat. }. Th. metabolic acidosis of renal fuilu .. is som.tim.. called an inorganic acidosis {due to t [PO.]}, but th ... also IIUy b.: incr ... sed concentrations of organic anions {'.g., citrat.}. 5. Di/r;tional ad,u,iis: [n the SID appro;;och to .cid.b.s.:: diwrd ... {... the Strong [on Diffe",nce section},. dilutitmlll adJolh occurs wh.n an exce.. of fIN H,O in plasma dilute! the electrol),!.. proportionately (•. g., decre .... both [Na+] and [CI-] by 20 %) . However, • 20 % deer.... in [Na+] (' .g., from ISO mmolfL to 120 mmollL) results in • gre;;oter absolut. decr= than the 20 % decr..... in [CI-] {e.g., from Ito mmoliL to 88 mmolfL}, .nd thus the SID dec=ses. In the traditional approach to acid.base disord."" the excess of free H2 0 also dilut .. the [HCO,-] and thus causes a metabolic .cidosis ('.g., from 24.0 mmolfL to 19.2 mmoIIL). 101 BLOOD GASES, BLOOD pH, AND STRONG ION DIFFERENCE 575 Some pmple ..ho comid" the hy!",rchlo"mie ..cidosis that is omS«! by the rapid infusion of ... line (IN.+] + [Cl-] = 154 mmoUL, but [HCO,-) = 0 mmollL) to ~ • dilutional ..cidosis, &oline is not fr.., H,O but does cont .. in ~ high .. !",r<:tory ",piratory alkalosis), 2, [n nomenal disorders, the kidneys iner ...... the sec.. tion of W (.... Figs, 9,3, 904, and 9,6), [I. Respiratory ..cidosi. A, /U'P;'"tory IldiUsis i... pathophysiologic sme in which .. respiratory di50rd.. caus •• th • ..ccumul ..tion of H+ in blood .. nd . n inc"... in blood Pea, valu .. (hy!",rcapnia), B, Respiratory ..cidos.. occur ~cau,. of alveolar hypoventil.tion and thus .n impaired excretion ofCa" Meubolism continue! to generate H+, but the .nimal connot "move it .dequately via the bicarbonate buffering system (Fig, [o,IC), C. Hypoxemia will . crompany (.nd typically pra::ede) hypercapnia if p. tienu .... b"ath_ . . mg room all, 0, Disorde" or conditions (T .. ble lOA) E. Physiologic compensation for respiratory . eidosis I, [n rnpon .. to . eidemi., kidneys will increase the secretion of H + and, in doing so, iner ...... the plasma [I-ICO,-) (com!"'nsatory metobolic alkalosis), 2, The renal ... pon .. takes .bout 2- 5 d to rai .. the blood pH during chronic hypercapni .. , ,~" Table 10.4, o;""""es and condition. that cau"" respiratory acid... i. (hypoventilation)" Inhibition or dy'!unction of meduU . ry rospiratory cent" 'Drugs: .nesth..rics, 5«lativt., .. nd narcotics Brain stem di ..... (tr.unu, infection, neoplasia, etc,) Alkalemia due to • metabolic alkalosis Inhibition or dY'runction of respiratory musdes (diaphragm, ch.. t wall): tick p. ralysis, tetonus, botulism, myasthenia gravis, hypokalemia, .nd mccinylcholine Up!"'r airway dysfunction: fa"ign Ixxiy, vomitus, and mechanical hypoventilation Impaired gas ""crunge ~t pulmonary capillari.. 'Pulmonary disease: inf=ion, allergy, edema, fibrosis, neoplasia, .. nd hyaline membrane di ....... in neono.tes 'Restrictive di ..ase: pneumothorax, pleural effusions, ~nd diaphragm .. tic herni .. V.>cular di50rde ..: right_to_left shunu (e,g" patent ductm arteriosu.) • A ,.t1tiY,) bocawe ol,...we«! g.. udu ng. in tt.. lung, wiU ~koly also result in 1. 1',0" but tt.. deer .... m . y not b. ... flici.nt for the diso,der to be dauified .. . byponmic di.oro.r (fabJ. 10.9), Not<: Owing their /i"t &... d.Y' of ~f<, neorutal fo.:o], and cal""" may haY< gr.~t<, reo, v::d"", tban . dult :animals,''''' 516 III. FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY Metabolic (nonr.,piratory) .lkalosi, A. Mu..bolir aika/OJis i. a pathophysiologic sme in which a non ... pir.uory proc= cou ..s the depletion of H+ from blood and an iner....., in .. rum .nd plasma [HCO,-j. B. Meubolic alkalo"," are typically produced by 10.. of H+ (renal =:mion or gastrid abom..al "",retion) .nd the resultant genention of HCO,- {Fig. 10.1 Dl. The loss of H+ and the concurrent production of HCO,- are illustrated in Fig. 9.4 (type A intercalated renal tubular ",II.) and Fig. 9.5 (g.mic p.rietal epithdial ",II). Note that the H+ secreted by the gastric muco .. is produ",d in the epitheli<>l cdl; it is not lost from plasma. C. Disorde" and pathogen= (S« Table 9.9 and the associ<>ted tat) l. loss of H+ from body a. Gastric lou: vomiting or pyloric obstruction (functional or mechanical) b. Renal loss {I} Loop or thiazide diuretics {2} Secondary to respiratory acidosi, {3} Hypokalemia 2. Shift of H+ from ECF to intucellular Huid beau.., of hypokalemia 3. Administration of sodium bicarbonate or organic anions m>l generate HCO,4. Contraction .lkalosis: loss of HCO,-_poor Huid. which caus.. hypovolemia .nd incr .... s.d pl"'ma [HCO,-j (see Chapter 9) D. Physiologic response l. [n nontellal disorders asro.ted with meubolic alkalosi •• the kidneys are apeeted to deer ...... the secretion of H+ {a"'ption: ... "pmo.doxical" aciduri<> [discussed in the following sect. 3]} .nd ron,."", less HCO,-. a. With incr .... s.d plmma [HCO,-j. the .mount of HCO,- th.t enters the filtrate mayaett inhibits the ",ntul n:.piratory chemoreceptors and thus couses hypoventi_ lation. Hypoventilation deer .... s•• the excretion ofCa, (thus elevating the blood Pc",) and incr ....... the [H'j (compematory respintol)' acid""i.). 3. ·P..radoxiral" adduria a. It is called JUl",tbxit"ll/ beau.. the .ciduria i. concurrent with an alkalosis. b. When.n animal h .. concurrent alkalosis. hypochloremia. and hypovolemia. the kidney. m.y produce acidic urine (S« Fig•. 9.3 and 9.4). {I} Hypovolemi<> stimul.t.. the resorption ofNa' and ct in the tubules through the actions of aldonerone and angiotensin II. {2} N.+ resorption i. not alwooys accomp:mied by CI- {beause ofCt depletion}. and thus .n deetrochemical gradient is established that promotes the secretion of H+ {thu. aciduri<>} .nd K+ (thu, contriburing to a roncurrent hypokalemi<». {3} The "",retion ofH+ by tubules also increases the generation of HCO,-. which adds to the severity of the metabolic alkalosis. c. It i•• ,""n most &«\uendy in cotde but also may b.: ,,",,n in other mammal •. [v. Respintol)' alkalosis A. fUipirllttn] Illkal~lii is. pathophy.iologic nate in which. respiratory disorder depletion of H+ in blood .nd a deere • .., in blood Pco, values (hyJ>OC'lpni.). COU'ieS 101 BLOOD GASES, BLOOD pH, AND STRONG ION DIFFERENCE 577 Table 10.5. Discases and condition. thai cause respiratory alkalosis (hyperventilation) Hypox~mia (nimulation of ~riph,,:d ch~mor=ptors) {s"" T.ble 1O.9} Pulmonary di ...... {stimulation of nociccptin r=pton} Stimulation of respiratory cent~r 'Metabolic .cid",is Sqlticcmia (Gum nq;ati"") H~t nrnke or f"""r Drugs: ... Iicy!'tes and aminophylline Cent..1 nrurologic di~ t.. um., neoplasia, in!L.mmation, ccrd>rovaocular .ccident, and hq.atic enccphalopathy M.rnaniGll hy~rventil.tion p.in or .nxiery • A ,el1tively common di..... 0' condition Note: Wh.n determined by the i-STAT. the Peo, can be Wscly dCCRucd by the p''''''''''' of thiopental in the blood umpt.. s,,~=: B. Respiratory a1ka1o= occur ba:.u", of :dnol .. h~rventilation: respirations remove CO, and thus W.t. rate fast" than H+ i. king made (Fig. 10.IE). e. Disorde.. or ronditions [fable 10.S} l. Hypoumia disorde ..: Peripheral chemorn:q>tors (carotid and .ortic bodies) stimulated by hypoumia initiat~ hy~m::ntilation th at causes the increased loss of Co, and thus loss of H+. (Stt Table 10.9 for di ..a= .nd ronditions that Gluse hypoxemi .. ) Pulmonary di ...... may result in poor oxygtnation of blood {hypoxemia}, which stimul.tes h~rventilation. Ba:aUst Co, diffuse. more ..... ily than 0" hypc:rv~ntil.tion may caUst exccs.s loss of Co, and thus a respiratory a1ka1osi,. If there is acut~ hypoxemi., loss of CO, may k som~ha t sdf_limiting ba:....., th~ resultant alkalemia inhibiu respiration. How""", with chronic hypoxemia .nd respiratory alkalosis, the remhing romp<:ns.tory m<"l.bolic a.cid",is reduces the degr... of alkalemia, .nd thu. hypoxemia rontinues to stimulate hyperventilation." 2. Stimulation of the respiratory center: Acid~mia and OIh" factors, induding CCntrai n~rvous system di"'.... , may .timu!'te the CCntrai respi .. tory CCnt" to cause: h~rventilation and thus • loss ofH+. 3. Mechanical nntilation: Anim.!' assisted by mechanical ventil ators may d""dop a1ka1~mia if there is exce.,i"" 1= of CO,. 4. Int"f..ent: When dct"mined by the i_STAT, th~ P=, am be faI",ly decreased by the p",.. nce of thiop<:ntal in the blood ,ample." D. Physiologic com~nsation l. In response to .Ikalemia, h~a1thy kidneys decrease: the secretion of H+ .nd, in doing so, reduce th. ronstrvation of HCO,- (romp<:nsatory m<"l.bolic .cidOJis). Also, m<"labolic a1kalOJi, nimulates ty~ B im"",lated a:lls to KCme HCO; {Ott Fig. 9.S}.'· 2. The renal r.. pon .. takcs about 2- 5 d to ..i", the [H+]. 5711 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY Table 10.6. Blood sas data in .in'fle acid_bue disorders ,H Di""rd" Respiratory acidosi, R~spiratory acidosi, with compenutOlY m<1aboJic alblmis Metabolic acidosis Metabolic acidosis with compenutory respir>tory alk.:dosis R~spiratory alblmis Respiratory alblosis with compematory mrtaboJic acidosi, Metabolic alkalosi. Metabolic alk.:dosi. with com~mato~ res ~i .. to~ . cido,i, V. • p.co, ; -L-WRI ; • WRl -L-WRI ; ; - WRl • •• ; WRl ; - WRl ; ~ HCO, l WRI ; •• WRI • ; ; Classificotion of acid_b...., di""rd~" A. Th~ major pm~rns for the simpl~ .cid_~ dilOrde" are listed in T.bl~ 10.6. l. Simpl~ acid_ba .. disord~ ... re tho .. in which th~ p"thophysiologic sUt~ i, Jimit~d to a primary disturban"" {~.g., lactic acidosis} . nd th~ apected compens;;otory proc..s {e,g" hyperventilation}, Figur~ 10,5 includ.. an algorithm for th~ classificotion of simpl~ .cid_~ disord~rs, or mOl~ simple .cid_b... di"'rd~rs arc pr..~nt (~,g., vomiting causing a m~ubolic alblosis, and ren al failure causing a mrtabolic acidosis), th,,~ is • m;xd IIciJ_bllH disQrdrr, Phy.ical aamination .nd laboratory finding, may contain clues that an .nimal has • mixed acid_bast disorder. Such cluel include the following: {I}.n unapectw blood pH (WRI or oppmiu chan~) for a ginn clinical stat~, {2} p.co, and [HCO;] changes are in opposite directions {e,g" .J,. [HCa;], and i P.co, would indicate both m", . bolic .nd respiratory acidoselj, and {3} lack of any apectw compens;;otion (Ott th~ nat S«tion), Additional information on mixed acid_ba .. disord ... is .vailabl~, 11 B, Expected rompematory ch.nges in acid_b",~ di""rde" I, Compensation for re'pi m ory .cidmis or alblo,i" If th,,~ is not r~nal di ....., the kidney, a.. expected to alt" th~ir acrrtionl of H+ and HC O; {also oth~r electrolytes} to compenut~ for the acidemia or alb lemia, 2, Compensation for mrtaboJic acidosi' or alblosis: If th~re is not renal di ..... and the acid_ba .. disturban"" i, not of renal origin, th.n th~ kidney. are apectw to alter th~ir acr",ions of H+ .nd HCa; .ccordingly to compen""t~ for th. acid.mia or .lkalemia, Th~ ... pir.otory system is also apected to alt~r its acr",ion of CO, to rompenlat~ . ppropriately for acidemi", or albl.mias, 3, Wh~n th~r~ is ~ith~r respiratory or m~ubolic compensation, th ... is never rompl"'. rompemation, Blood pH may r"'urn to th~ .. f,,~n"" interval, but it will nev~r return to th. normal pH for that animal ., long., the disord~r causing the acid_ bas~ baian"" remains, 4, How much can these 'ystem, rompensat~ if given time to do "'" On. aspect of th. interpretation of blood g•• data is attempting to d"'ermine wheth~r th. animal', 2, When two 101 BLOOD GASES, BLOOD pH, AND STRONG ION DIFFERENCE Wh~t is the blood pH? __-~::C:-"CC;::::::::-__ dPco, 579 d'IHCO,l Al< aI""," if . PCO, if t IHCO,l I I I "'spirB1Dry acicIosi. mGtabolic Itcidosis respiratory alkalosis mGtabohc f . IHCO,l if ~ t IHCO,l I ",spir~1Dry acicIosi. wilh comp&n'''1Dry "",labolic " lkaIo". ~ .Pco, I ~blosi . t Pro, I I I mo1abo1ic ..adosis with compensatory r"'piralory alkalO&is r".piratory alkalosis with comp90.~tory malabolic IOCidosis mGtabolic ~l 10.5. Algoritbmic "I'pro.m to d ... ification of ..imp!. (not mind) ocid·b... diwrde ... R.kr to Tables 10.7:tnd 10.8 for ""J>«1"tory r.. parnes. Fig. respiratory or metabolic .yst.m, han romp"nsatro apptopriatdy. [f the data indial.1< that an .nimal has not achi""'" up"cted comp"nsation, th.,. may indicate the following: a. The animal has not had time to romp"nsate. Thi, i, e'p""i.Jly true of "nal comp"nsation for .cul< respiratory di""rdw;. b. Th •• nim.l has mo .. than one di""rd" that is alt..ing the ocid.ba.. statm, and thi, disorder is preventing .n exp<eCal comp"nsation. 5. To atuntpt to deurmine up<eCal romp"nsatiom in dog., eXp"rimenul ""id.I,.,. disorders were er•• tro and chan~ we", monitoral. Th. finding' ITom """,ral ""p"riment' we .. summarized in a r~i= aniek " data we" extractal from that article for T abl. 10.7. The proposed purpose of the rorrection f.cton i, to deurmine wheth" the changes in [HeO,-] or P,co, "pr...,nt a phpiologic comp"nsation to a 'ingle .cid.bas<: dilOrd.r or wheth .. oth.. f.ctors a", .ffect:ing the [HeO,-] or p.co,. 6. A fomlUla for calculating an up""tro P,co, i. providal in Eq. 10.7. The up<eCal P.co, value is f"'<J.uendy calculatro ming av,,"C, ... lues for hralthy dog.: that i., average P,co" anrage [HeO,-], and .n anrag. romp"nsation f.ctor. Breau.. of the biologic diff".nce, in dog, .nd the analytical diffi:"nce, in blood gas methods, tho...verage values mayor may not be appropriate for • given patient. Som. J> 500 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY Table to.7. fupeacd oompcn.ation. for acid_hue disorden in dogs Expa;t«i [HeO, 1 dun!:" (mmollL)" For ~ach mmHg Acut~ i in p.co, in: respim ory .cidosi, « 24 h) Chronic respiratory acidosi, (> 3- 5 d) For ~.ch mmHg ,l. in p.co, in: Acut~ respimory alb/osi, Chronic respiratory alkaJosi. For ~ach mmoJIL ,l. in [Hea,-] in: M~t>bolic acidosi, For e;;om mmoJIL i in [Hea,-] in: M~ubolic fupffi"cd p.CO:! chan~ (mmHg)"' t 0.15 (O.04--O.20) i 0.35 (O.29--O.43) ,l. 0.25 (O.14--O.36) ,l. 0.55 (0.43--O.76) ,l. 0.7 {O. 5-1. I} i alkaJosi • 0.7 (0.5-1.3) • &peete:l motboo. :or. not pree;" .nough. to justifY "'porting them to tho n.".mt «nth but m:oy be prea.. ~ougb to jwtify reponing ooJ>C "",,,")_( p.CO, ) + ( inHCO,_ HCO,) (romr.:""""") (m p.C O, - in he:;dthy dog pati~nt in h~althy d og aelor X (10.7.) Ex:nnpl~: Pati~nt remit" pH = 7.146: p.=, = 36 mmHg: [HCO;] = 12 mmoUL Average: values for healthy dog (Table 1O.8): p.=, = 40 mmHg: [HCO;] = 21 mmoUL ' ')= (40 mmHg) + (12 mmol l L- 2 1 mmoI I L) x (0.7mmol mmHg ) (mp,,",' .=, lL = 34 mmHl( 7. The calculation of ""~ted compensatory chafll:es in blood gas d.r:. should be considered an e"im:ote. If the g<>:>l of the int~rp ... ation of the data i, to determin~ whether th~ :mim:ol has more th.n one disorder affecting the .cid_b= ".tus, sometim.. th~", are other v..ays of arriving at the conclusion. a. If a dog h as ketoaeidotic diabetes mellitus and i, vomiting, th~ dog very likdy has both. mer:.bolic .cidosis (am..d by htosis) and a me{.bolic .lblosis {due to vomiting}. b. If a dog has. pulmonary disord.r that is causing a respiratory acidosi, and also has <'Vidence of renal failure, th~ dog very likdy h .. both a re'pir:otory acid",;. and a m..abolic ""idosi,. 101 BLOOD GASES, BLOOD pH, AND STRONG ION DIFFERENCE 581 Table 10.8 Examples of CIpcctcd oon'pcn.ation cal culation for metabolic acido.i. in dogs; CIpcdcd p.co, calculated using &t. 10.7 Blood gas <\at> for a dog: pH = 7.146 (7.310--7.420) [HCO,-] in p.,i~nt (mmollL) Example A B "" " C [HCO,-] = 12 mmollL (17- 24) p.co, = 36 mmHg (35--45) [HCO,-] in health (mmollL) Compensation factor mmHgI (mmollL) p.co, in hrahh (mmHc) "" 0.7 40 40 35 74 U U fup« p.= {mmH&l 34 30 " mrasurffl and aprctrd I',co, (mmH&l , . 6 En mp\< A: ExpKteC< b.~ "",. mred and ap<'Cred P,co, val"", is 2 mmHg and tbm witbin the ± 3 guid.li"", n..rd'or<, the "",...,=1 p.co, of 36 mmHg rs c:dcuhtod wing :ovttIg< P.co" >VC< be~ me. mred and ..peeted P,co, val"", is 14 mmHg :md thw mum gr •• ..,r thrn the ± 3 guide~"., This nl ...... gg .." tbat the", b.. not ~ an ' ppropri • .., ""piratOJY compem.tion for the m 8, Th~ eseiJrultM compcll5atory factors in thi, sa::[ion a", for dogs and w... det~r. minM during exp HYPOXEMIA AND HYPOXIA L Expected Po, A, An ..ial blood is the only .=ptable sampl. for th~ ....... m~nt of oxyg~nation of blood by the respiratory ')"lum, Th. expected 1'.0, varies with the Flo" ch~ 0, cont.nt of the inbalffl ga' (Flo, in deeim:d fraction, P,o, in mmHg): (0,2, 95- 100), (0,3, ISO), lOA, 200), (0.5, 250), (0,8, 400), and {LO, 5(0)," B, For room air, the Flo, i, n.ar 0,2, .nd thu, a P,o, of90--100 mmHg i, exptors i, r~portM to occur wh.n P,o, is < 70 mmHg" or < 60 mmHg,'" 582 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY T able 10.9. Di""a..es and condition. thai cause hyponmia" D«r~~.ffl inhaled 0, coment: high altirud. or dosnl nntilation a'ta Inhibition or dysfunction of Ih. medulla,y =pimo.), ",nte, (1ft Table lOA) Inhibition or dysfimcrion of the r.spiratory mu,d .. (st. Tabl, lOA) Uppy dysfunction: foreign body. vomitus, or m.rn..ni",] hyponntilation 'Impaired!?, exchange: >I pulmonary capillaries (.... T able lOA) • A ,.tui",,[y common dis< ... or condition 'Di",,"".. th" em", =pin,OJY ocidoois (t P,co,) b.oc •.,.. of ...du=:! g.. ""dtmg< in tho lung. (T .bLe lOA) will Jilr;;"ly :Wo remlt in .L P,o,. but th. d.cn ... auy not b. mflici.." to be d .... /ied • byponmi •. Not ..: Compared to >dult Ito,..", neon",J foal! Mn Lo..." P,Ol. bigher P,co,. high .. [l-ICO,l . ond .lightly Lo..... pH val .... t-:. .... of uoo.r. C. With the incrcasffl u,", of point_of_cu. in,{rumenu that haY< 0, d~rode,. mo,," PoOl values ar. ~ing m=ured. In fr..,.Howillg venous blood in an animal with a P,o, ntlr 95 mmHg, th~ PA will b. n~~r 40 mmHg. If th~ .. mpl~ is OOll«tM from an occludM v~in, th~ p.o, will b. low~r. If collected from capillary blood, th~ po, should b. b.tWttIl th~ p.o, alld Pp, values. D. Blood gas d.u ill ar~ differelll from thost of adult aninu1s."'>< l. Compo.rM to th~ .dult horst. a foal at birth hal lower P,o, (< 40 mmHg) and higher P,co, values (> 50 mmHg) durillg iu first few hours. The P,o, incr~,.,es quickly to lIe~r adult values by I d of age, .lId th~ P.co, slowly changes to adult ""lues by 4-7 d of age. 2 Similar p>ttems ~r~...,11 ill netmatal calvt •. The p.o, approxhes ~dult value. by I d of~, . lId the P,co, appro.ches adult values by 2 d of ag~. 3. Plasm. [HCO,-] values are .lso g=ter initially, corresponding to th~ gre.ter p.co, p=sures. IIW""". n. Hypoxmtu. (d«r.-ased dissolval 0, ill blood; d«",ased p.o,) =y cause hyfH'Xu. (decr.-ased 0, ddivtry or utila.tioll ofo, by tissues), but the", ..~ other disorders in which thue i. hypoxia but 1I0t hypoxemia. The diff~relll types of hypoxia call b. divided illlo sev~1I groups." Hypoxemic and hypoxic disorders or oonditions .lId the apected laboratory data for hypox", disorders .'" lined in Tables 10.9 and 10.10. A. Atm~lph"ir hyfH'Xi/l; inhalatioll of atmosphere that has d«",ased 0, oolllelll {e.g., high altitud~, allesthetic problem, or ill an airtight box} I. The inhalation of atmmphe", that has d«r.-ased 0, colllelll COII5<::I a decru.led P..0" and thus less 0, diffuses to blood {,t. p.o,} . lId less 0, bind, to Hgb {,t. So,}. Th~ combinatioll of ,t. P,o, ~lId,t. So, wulu ill, d=eased .moulll ofO, in the blood {,t. O,ct}. 2. If the hypoxemia stimulat~s the periph~ral chemora:q>tors (corotid or .ortic bodie.j, illcreased respiratioIlS result in hyperv~lIIilatioll and thus ,t. p.co, (r~spiratory alkaJosi,) if th~ CO, is ""rually exhaled (ste tidal hypoxia ill the lIat sectioll). B. ndal hyfH'XUr. d=used 0, upuke bec.use of imp>ir~d respiratory ach.lI!:" (~.g. , r~spiratory obstructiOIl, or hypovelllilation coused by allmhetic ~IIIS) I. The impo.irM ddivery of .ir to th~ .lvtOli hal the laJlle eH"ttt on the oxygellatioll of blood as oonditiollS that couse atmospheric hypoxi. ; that is, ,t. P.o" ,t. So" .lId ,t. O"t. 101 BLOOD GASES, BLOOD pH, AND STRONG ION DIFFERENCE 583 Table to. l0. Blood gas r ... uiu CIp«led for each general type of hypoIia HypoIia p.~ Alm"'Eh~ric •• • Tidal Alvwl:.r •• •• • • Hemot:lobic Siagnant HinoloIic D~mand WRl WRI WRl WRl WRl WRI So. WRl WRl WRI WRl Spo. p. WRl WRI WRl WRl WRI WRl WRl 0,<, WRl WRl WRl WRl WRI WRl WRl p.~ vA WRl WRI WRl WRI WRl WRl WRl WRI WRl WRI WRl WRl f ""00, P,o,/FI WRl WRI WRl WRI WRl WRl f· f· P,co, WRl WRI WRl WRl WRl-t' WRI-J..' WRl-C' WRl WRI WRl WRl , If the h<mog1obic hypo";. j, c:m..d by :oncmi •. tbe So, wiU b. WRI. If tb. bemoglobic bypoxu is c:wsed by m<moglobin.mi. , tb. So, ...ill b. d.ec:r • ..ed. • If th. hJ'P"I"rrW. r.. ul", in th. .timul. tion of ""'pimion, hrI"'"",ntwtion """,Id =ult in decr... ed p,co, :ond • ""pir:otory .Ih losis. , If tbe,. j, inodeq ...t. ventilation, tbe,. would b. :on inOl.nod P,co, :ond • =pir:otory . cidoois. •• ," .' WRl " •• •• • • • 2. Th~ iml"'iral respiratory exch:onge causes imp.ired excmion of CO,. Thu., th~", m"}' ~ i P,co, :ond • r~.pir:olory .cidosis. C. A/"","'r hypoxill: decr""sal 0, uplah kau .. of da;",a. when blood is ptrfusing nonfunclional alvroli; that is, blood is avail.ble for gas exchID!:", but the gas exchang. doe! not occur. Thu" blood from thos. vc:.. rls is nOi oxygen ated (",mains as "v~nous" blood) :ond Ihrn mix~s with 0"Yg~natal art~r",l blood from oth~r pulmonary tissu~. D. HmtDglubk hypoxi8: da:reasrd 0, bound to Hgh {r.g., meth~moglobin~mia or co poisoning} or da:r~as<:nd~nt on blood [Hgb]. 2. If.n animal is .n~mic, th~ O,<:t will ~ da:r..as 584 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY 3. With methemoglobinemia, both f.nom (F.l<-) .nd furic {F~} heme mol,"",l.. no pr=nt. In this F. HiJtofI!Xic hypoxu. is d.f<'CIj"" 0, u.. by ti"ues ba::au.. of int.rf.rencr with metabolic pathways (e.g., some drugs or alcohol). Blood gas dau will not deta::t an abnormality. G. Dmllmd hypllXia is incrrasnl 0, demand by hy~rfilllCljoning cdls (• .g., hr~r{hyroid. i,m, pyrnia). Blood gas dau will not d.u·c t an abnormality. III. Spo, A. A dao=illg Spoz typically illdicates the d...dopment ofhyponmia but only when the P,,,, i. < 90 mmHg. With great.. Flo, such .. occurs duting allesth.sia, greot.. poO, valu...", up<eta!. Ba::.u.. of the dtap' of the oxyhemoglobin dissociation curv. {Fig. lOA}, fallillg S,o, ""lues may not ~ dffaota! until the P,o, is < 60 mmHg. which might ~ considerably I.... thall npecta! for high Flo, ""lues. B. Errollam. value,,' With illcr.asing collcentration, ofCOHgb {.. in CO poisonillg}, the Spo, rem.ina! .t:> 90 % ...ell whell O,Hgb p'rcentage daoreased to 30 %, bec.u.. the oximet.. does 1I0t diff... ntiate COHgb from O,Hgb. Similar but I.... """,re, f.l .. positivr interf.rence w .. S,""II with increasing con,entr>tions of methemoglobin. A> mown ill Eq. 10.5b, the O,Hgb p'rcellt>ge will dec ..... with illcr....ro collcentrations ofCOHgb or mffhemoglobin. C. III ho ...., the Spo, ulld .... timated the S.o, by.n average of 4.4 % whell S,O, was ~ 90 % and ov..estimated the S.o, by.n averat:e of 4.1 % whell S,O, was" 90 %.l1' The erro .. might ~ due to diff... nt prop'ni.. of .quine blood compared to humall blood. D. III dog, with S,O, of~ 70 % , the avrragt difference ~tw..,n Spo, and S,'" was about 3-4 % {e.g., Spo, of 87 % .nd S,'" of90 %}, dep'ndillg 011 applicatioll sit. (tollgue or tail) alld type of pro~ {ear or fillger}. The ...son for the diff..ellce is 1I0t known." As with the equine dot>, pul.. oximetry ulld .... timated the S,.o, when it v.as high .nd o""... timata! the S,O, whell it was low. STRONG ION DIFFERENCE (SID) AND STEWART'S METHOD OF ACID_BASE ANALYSIS I. Ev:duation of dectrolyte relationships led to all .hemate mffhod of assessing m=bolic (lloll... piratory) a.cid_b ... disturballce. (Stew:ln's quantit.tive .nalysis of a.cid_b ... chemistry) .nd the calculatioll of SID. A complete description of Stewart'. method .nd SID COllcepts is beyond the 5COp' of this book, but major asp«t. alld a compariso" with mo", traditiollal a=ment of acid_b .... disorders is presented. For a complff. allalysis of Stewart'. mffhod .nd SID. additiollal ..f..en= should ~ studied.""" III Stewart's method: A. Electrolyt. disturballCe! are approacha! ill the context of phy.iochemistry with consideratioll of equilibrium equations, con.. rvation of mass, alld balallce of charge. 101 585 BLOOD GASES, BLOOD pH, AND STRONG ION DIFFERENCE B. Nontraditional ddinitions of ""id, and balO; ar~ usM. C. All da::trolyu. in biologic fluids >r~ considorw to b. involval in acid.b..., WLon"" and Jrulintenan", of d'""tric;;d neutr:dity i, uf"""Iw (Eq. 10.8a). [N. +] + [K+] + [f<:::.'+] + [fM g'"'] + [W] + [NH:] = (1 0.8ao) [OW] + [Ct] + [HCO,-] + [CO,'-] + [Alb~] + [par] + [uSA] wh~re x .nd J rq>r=nt the variable charges on .lbumin .nd phospru.t .., r~'f"""Iivdy, and uSA includ.. 50.>-, lacute, act:l"""'t>t~ . p.hydroxybutyrate, othor acidic anion. of m~uboli,m, and uOg<'nou. anions. ([N. 1 + [K+] + [f<:::.'1 + [fMg'"']) - ([Ct] + [uSA]) = ( [OW] + [HCO;] + [CO,>-] + [Alb~] +[POr] ) - ([W ] + [NH.1 ) .trong cotions - strong anion. = w~ak aniom - wuk cotions [fSID,_ = "rong cotion. - 'trong anions Th~n 5[0_ = weak anions - w.... k cotions 5[0_ = ([OH"] + [H CO;] + [CO,>-] + [Alb~] + [PO.'""]) - ([W] + [NH.+]) ( lO.gb.) (1O.8c.) D. Analyees involvw in acid.~ bal.n", ar~ consid..al to b. e ith~r independ~nt or depend~nt variables. l. Th. independ~nt variabl...re those analye .. that are regulatrd or chang..! ind~pend~ndy of oth~rs: Peo" SID, and Arm (= th~ ddinitiom in th. nat "",,tion). 2. Th~ depend~nt variabl.. are thOR .nalye.. that chane<' if .nd only if on~ of th~ ind.pend~nt variabl.. chango;. The dependent variabl .. includ~ H+, OH-, HCO;, and tcO,. E. Chango; in [H1 ar~ prwicrw from changes in con"'ntrations of 'trong ions. Th. actual blood [H1 {i.e., pH} is not m.... surw. F. Albumin i, includw as a contributor to acid.~ balan",. whereas it it not. formal. ized part of the classic tradition:d .pproach. However, albumin cone<:ntratiom do .ffect anion gapt and a.. consid~red in base ua:u colcu!.tions. G. Hgb i, not includw a, • contributor to .cid.base balan,"", wh ..... it is • part of the c;;dculatw BE. valu~ th.t is common in blood gas data. n. Diff..ence, in definitions b.tw..,n th. two sysum' am le.d to confusion.' A. Definitions in the traditional .... ssm~nt of .cid.bas. di!Ord~rs by wing pH, Pco" and HCO,I. An "dd i. a .ubstance that am give off a H+ (a proton) at a given pH (•. g., hydro. chloric acid i. copable of giving off H+). 2. A b"u is a ,ub,una: that can bind a H+ (• .g., HCO,- i, copabl. of binding H+). 3. An "ddt"js i. a condition in which ""idem", {low.. pH and high .. [H+lJ t~nds to ~,. 4. An ,,{hllo,js is • condition in which :dkalemi. (higher pH and low.. [H+]) t~nds to ~,. B. Definitions in the St""",rt'. method I. 51rt1ng c",;onl or ffrrmg "ni~1U are thOR ions that a.. completdy dissoc"'trd in phy.iologic fluids. {Nou that the definition doo; not consid.. th~ ion, that ar. complaal with prot. in or nonprotein ions; for exampl~, JI\05t .. rum colcium ions FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY '0; ar~ compJexw with anions and ... not frtt or compJ",dy diMOCiatrd iom. Also, Na+ and Cl- ~hav~ as though rh c. Ill. to mah jt rno .. alkaline. b. Strong anions (C]-, sulfau, bctate, .""to.ct:{.te, ~.hydroxybutyrate, :md other acidic product. of metabolism): Strong anion. (e.g., ct) ... comiderffi acid. b=.Ust when addrd to ECF. and jf th". is not a baloncing shift of .. ,{rong ion {•. g., addition of Na+ or ",mova! of I. em e}, H + shift. into the ECF to m"". it more acidic. 2. An IId,u"u i. a condition in which th". is.n .,.ass of strong anions {"acid."} or • ddicit of strong cations ("b .... s"). [n th~ SID approach. an .cidOJis is pr=nt if SID is drcr""-sN. 3. An "lhllo,is is a condition in which thue i. an ac= of 'trong cations ("b.!eS") or • ddicit of 'trong anions {"acid."}. In th. SID approach •• n alkaJosi. i. pres.nt if SID is incr",,-sN. 4. Wrllk rlrttrolyw.", tho"" drctrolyt.. that ar~ in "Iuilibria in physiologic fluids. a. W",,-k cations: NH ' . H+, .nd som~ y.g1obulifll b. W",,-k anions: HCO,- . CO,>-. POl·. HPO.'"". H,PO,- . and prot~ins {mosdy .lbumin} 5. AroT i. th~ mm of th. nonvolatil~ w.-ak .cids (HA + A-). In pl"'ma. th .... ocrur '" nrious anionic forms of PO, and proteins (albumin . nd globulin.). 6. SID_ is th~ diff.r.n"" b.!w",n th. sum of .trong ion cona:ntr:llion.; that i •• th. diff... na: b.:tw.,.n the mm of strong cation conctntrations and th ••um of strong anion conctntrations. By ",arranging Eq. ]0.8a. it is shown that SID_ {i .•.• strong cations minus strong anion,} .ho t for intupreting cakulatw SID valu ... Thu,. SID_ could b.: ca.lcul. tw if th~ .nalyt~ con""ntrations in Eq. 10.& could b.: m.amra!. Using th~ .,m. concq>ts :as St.wart. loI Sin~r .nd H.ning,"': ddinw buff.. w,.,. which i. id.ntical to Stewart'. S[D.' SID S[D_ = (sum of strong cation.) ~ (sum of strong anions) {IO.9a.} = ([Na+) + [K+) + [fc.1+) + [[fMg"')) ~ ([ct) + [uSA)) uSA includ., SO ..... lactau •• a.toaa:tate. ~.hydroxybutyrat~. oth...cidic .nions of m.ubolism. and aogc:nou. anions. 101 BLOOD GASES, BLOOD pH, AND STRONG ION DIFFERENCE SID, = ([Na+] + [K+] ) - [Cl-] S[D. = ([Na+] + [K1 ) - ([Cn SID, = ([Na+] 587 ( 1Mb.) + [L-lact.1t~-]}( 1O.9c.) + [K+] + [ft:a>+]) - ([ct] + [L-laetate-]) (l0.9d.) (IMe.) S[Da-_w = [N . +] _ -.,J - [ct] ___ (10.9£.) [N.·] whm[CtJ __ =[ct J.-- x [Na'C and th~ "m.... n normal" cona:ntrations of Na+ .nd inurv>l. for th~ patient's ""lues, ct are for tb~ appropriat~ ref~r~na: B, SID, (Eq, 10,9b):" This equation estimates th~ SID by using thr..: strong ion cona:n· trations thot a", routindy m~asurffi in .. rum or plasllU; this i. prol>ably on~ of the more common clinical SID formulas, Nou th at it d~. not indud~ the cona:ntrations of ft:al+, /Mgl+, or uSA; thu., changes in those: an:'!yte cona:ntrations would cause: th. estimat~ of SID to be inXCllrate, C. SID4 (Eq, 10,9c):"J' This eqllation estimat.. th~ SID by u.+), Not~ th at it d~. not indud. the cona:ntrations offMg" or uSA other tban L.!aetate (such as D.laet.te); thu., changes in tho...nalyt. roncentration. would c;ousc: th~ estimate of SID to be imc<:urate, E, SID, (Eq, 10,ge):" Thit eqllation estillUt.. tbe SID by u. '88 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY coD""mntion .ppropri.t~ for th~ J.bontory method (the ... me as usn! for patient', [N.+]) should b. <=d. G. Othu SID formulas ha"" bttn propo...! that include some but nol of the strong iOnl." ... Abo, th"• •"" nol standardittd !lam.. for SID formula!, and thus SID information should be, jllt<rpm«l cue!ulJy. For "".mpl., some formulas contain [fCa'1 , [fMg'1 , or [L-l. cmel . but nol [. cti",,,,,e;m] or [1J·hydroxybutyrat.]. R.f... n"" interv:lJ, for estimated SID .... Ju .. and possiblo au",. for .bnormoJ .nimned SID value, vary d.~nding on th. analyt .. th.t ... or are not uK .n IY. Comp.rison of SID rormul,.. A. Th. rdation,hip bttw«n S[D_ and SID, is shown in Eq. 10.10. SID, is. good approximation of S[D.... if ooll""mrarions of ft:a'+, fMg"", and uSA :He unalJ. How"""., when th~"" i, a high [uSA] occurs in lacti" acidosi, or hto.cidosi" th~ SID, valu~ will be. c""~t~r than th~ SID_; chat i" ch~ SID, onrrnirruot .. th~ SID_, mrn '" Givtn Eq, 10,9a and 10,9b, ( 10.]0,) SID_ = SID, + ([ft:a'1 or SID, = + [fMC"]) - [uSA] SID_ - [fCa1+]_ [f},1C ] + [uSA] rdationship be.twttn S[D_ and th~ oth~r formulas u..d to estimat~ SID (Eq, ]O,9b----.,) varies with the analyt .. that a.. or ... not included in the formulas, It i, mo.. difficult to com!""" SIDa-____ with th~ oth.. SID formulas, How",,"r, th~ S[Da--.t would also ov.rellim.t~ the SID_ if th~ .. w~r. an inc=sed [uSA], C. Th~ ch:mCO' in SID valu .. that are ca.m by ch~nCO' in ",e:ok ion con"'ntr>tions can be >ttn by 'x.:Intining Eq, 10, Ila- f (Eq, ]0, Ila is th~ ... m. as Eq, 10,&), As shown in Eq, ]0, II~, chanCO' in SID, occur wh~n the .um of [HCO,-) , [Alb~), [POr), .nd [uSA) ch:mCO', L [f [Alb~) , [par), and [uSA) stay constant, crunges in SID, ..11= crung~' in [HCO,-) ; th.t is, t SID, ..1I«t, t [HCO,-) and thus a metabolic alkal",is, or J,. SID, rdl«ts J,. [HCO,-) and thus a metabolic acidosi" Ho~r, the rdation,hip betwttn SID, and [HCO.-J is less predict.bl~, or i, not present, wh~n th ..~ ... ch.nges in [Alb-), [Par), and [uSA], 2 Equation ]o,lle also mow> thot SID,_ depend, on two rruojor facto ..: [HCO,-] and [Aror], Thu., if PO, :md protein con",mratiofll do not change, chanCO' in [HCO,-] change the SID_, This is wh ..~ ch~ traditional . pproa<:h and SID approach to .cid_base balan"" hove commonality, a, Mmbolic alk.:do,i, = t [HCO,-] = t SID,_ b, MmboJic acid",is = J,. [HCO,-] = J,. SID_ 3, Anoth .. way to examin~ Eq, ]0, Ila .nd b i, in t~rm' of the buffering ca!"city of plasma, Th. d«trolyt .. in ch"", equatiofll th.t can buff~r H+ ."" HCO,-, par, and Allr, Thu., in Urnu of the ~ d«trolytes, an incr.... in SID_ indicates an incr.... in the plasm. buff.. con",mration (i,~" traditional metabolic alkalosis), Convtrsdy, • d«r.... in SID,_ indicat.. a d«""... in the plasm. buff.. con",mra_ tion (i,e" traditional metabolic .cid"'itl, B, Th~ 10/ BLOOD GASES, BLOOD pH, AND STRONG ION DIFFERENCE Givtn Eq. 10.8b. • nd 10.9•. , SID_ = ([OH'] + [HCO,-] + [CO,"'] + 589 ( 1O.11a.) [Alb~] + [PO.'""] ) - ([W] + [Nt·V]) B.cau", typical plasma concentrations ofOH', CO,..., W, and SID_ .. [HCO,-] + [Alb~] + [pO.'""] NH,:lr~ rdativtly small, + [pOr] .. [A.....], SID_ .. [HCO,-] + [A.o.] (lo.llb.) Giv~n [Alb~] (IO.Il c.) Givtn Eq. 10.10 .nd th.r typicol pl.. m. [f(;;,'1 and [fMg2+] He rdativtly small, SID_ .. SID, - [uSA] (Io. lld.) GivtnEq.IO.llcand 10.lld, SID, - [uSA] " [H CO,-] + [A.o.] or SID, .. [HCO,-] + [A._] + [uSA] (10.11e.) Colllidui~ Eq. 1O.9b. Eq. 10.1 Ie, and that anion gap = ([Na+] + [tc+)) - ([Cl-) + [HCO,-n, anion gap = SID, - [HCO,-) .. [A...] + [uSA] (10.11£) D. For tho", who . '" acrustomal to inurpmation of . nion g. ps (see Ch ' pter 9), Eq. 10.llf shows th~ rdationship bot",een anion gap .nd SID,. AI described in Chapter 9, the .nion g. p is mostly du~ to the ionic charge of proteins if there .'" not inc",a.o;ed concentrations of organic anion •. E. Th~ .nion gap calculation con .lso bo used to ",lcul. u a SID valu~ {Eq. 10.12c}. B.cause an incr~aKd . nion gap typicoJJy indi"'te. an increased concentration of anions other than HCO,- or ct, th~ incrn.., in .nion gap can bo used to estimate the [uSA]. This formulo would provide a mo", .ccurat~ estiJrulte of SID than does the SID, formula when there is a meubolic acidosis. However, the need to calcul at~. SID i. minimal if a d«",ased [HCO,-] h.. already bttn recognized. Given th~t SID_ = {sum of strong "'tions} - {.um of strong ~nions} SID_ = ([Na+] + [K+] + [f(;;,2+] + [fMg'+]) - ([Cl-] + [uSA]) wher~ uSA indudes SO,"' , l. ct. te, .cetoocetate, p..hydroxybutyrate, other acidic anion. of meubolism, and nogenous anions. ( 1O.12a.) And that an incrrased . nion g. p {AG} .. [uSA] (1O.1 2b.) (10.12c.) V. Interpretation of abnormal SID, values A. According to Suwan's method and definitions, changes in SID_ c",.te eitber an alkalosi. or an .cidosi•. ~ on the definition. and the calculation of SID" the major tYP'" of acid.b .... disord ... ~re lined in Table 10.11. Note that the apectal ch:mges in the d.".,ndent v:lriables {i.~., [H C0,-J and pH) :or. those apectal in the traditional appr".ch to . cid.b .... diJOrders; i [HCO,-] in met . bolic alkaloses, and ,l. [H CO,-] in metabolic a.cidoses. '00 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY Table 10.11. Cla.. i6Ci1oon of acid. base dioorocn from p.co" Aror , and SID fup«cro d.~nd.nt Chang.: in Cru.ng~ indTndent V:lf;.,bl. m .... sured analyte Cw..ificuion [!-leO, 1 pH ,l. P,co, i P,co, R.spiratory alkaJosi. WRl ; WRl " t Albumin' t PO, J. Albumin" HYP"ralbuminemic .cido";, HYP"rpho'ph . temic acidosis "" ; ""; ;,; i HYP"rruot .. mic or contraction ; ; ;,; ,l. ct,l. Na+ Hypochloremic . lbla,is ; ; Hypon>l""mic or dilutional i ct- HYP"rchloremic acidosis " Non.' Variable" Mec.bolic .cidmis :md i [uSA]' " "" ,l. p.co, ; ; ; SID/SID,' " "," ","1WRl " "It "",;..bl. chmg.: in Na+ R.spiratory acidosis Hypoolbuminemic . lblo,is .lblo,is'- acidosis" Metabolic .cido.is and i [uSA]' " • If byp<rprot< r-=gniud if it it kno.m th .. . ith.r • .L [H CO,l or :on t anion WOP (whim ind.ie>t.. t [uSA]) is p= 'II«:..,.. B, Ih,ffl on th~ ddinitions, ch' IIl:es in cona.ntr>tions of two ions m.y cou", a mixffl acid· b.... disord,,; for n:lmp[~, [ms of plasma H 2 0 could result in concurr~m hyper"". t .. mic alkalo,is and hyperalbuminemic acido,i., Th~ roncurrem alkaJosi, and acidosis in th. Suw:m method rai,es th. question of wh~th" th~r~ is noSIO",i, ,1O VI. AI ru:omm~ndffl by lOm~ authon, SID valu~s should ~ imerpretffl with routine blood gas valu .. (pH, Peo" and [HCO,- j) to d~t~mlin. whether th~ ani"",l has an acid. b"", problem and wh~her th. probl~m i, respiratory or nonrespir>tory (metabolic)," Jl If the probl~m is • nonrespiratory acid. ba", disturb:ma., th~n th. contributions of nonbiearbon. at~ dectrolyt .. con bt explorffl with Stewart's method, VII, Prior to th. propoord USf of the strong ion theory, an und~rst;",ding of:m :mimal'. acid. ba.. :md rlectrolyte disord~r was obtainrd through the interpret.tion of pH , Peo" [HCO,-] , BE" [N . 1 , [K'] , [Cn, :mion g' P, .nd albumin ron""'ntration (Itt th~ prrcrdiIll: 101 BLOOD GASES, BLOOD pH, AND STRONG ION DIFFERENCE 591 Acid_B... Abllormaliti.. =:tioll .lId Ch ' pt" 9}. Evalu.cioll of such illform>tioll h ... provid.d . lId wiU comillu, to provid••11 ulld.malldillg of all ,"imal', pathologic nar.. VIII. Tho.. who study SID theory alld its . pplicotioll star. that th. SID .pproach r"'luir.. 'J>'Ci..-spa;ific valu .. for ATQT; th.c is, the sum of the colI~m .. tio", of 1I0IlvoJ.til. buffers, .. rum or pl ... ma proteills, . lId PO•. " AI; st.ta! "'rli" (ill Ih. pra::.dillg sea. 1l.B.5) .lId ill Eq. IO.II e, pl ... ma Aror collsists primarily of th. variou. alliollic forms of PO. alld proteills {albumill and globulins}. A. Th.", h . "" bn.1I art.mpu to calrul. r. pl.. ma ATOT valu.. for h",lthy cotd.,"'" dogs!' cots," alld birds." Th. calcuJ.t.d valu .. (mnll ± sd) w... obtaill.d by u.illg ",ttlu from ollly to dog., 10 cou, 12 pigwll', alld 9 colv.. (or pool.d bovill•• lId oville pl ... ma) . For most of the dola, the variatiolls ill the calrulot.d pl ... ma ATOT valu .. .. p.... m the a pect.d variations ill pi.."", proteill alld PO. colI~mralions ill such samples from healthy , "imal •. B. Usillg Ih~ calruJ.ta! me.1I pl ... ma Aror valu...lId m... ural [HCO,-], all SID value COli b. calcttlal.d (Eq. IO.llc). However because ther.... biologic variatiolls ill Ihe ",rum COllctmratiollS of PO. and pror.iIlS, a mean Aror value may 1I0t b. . ppropriate for all heallhy allimals . lId will ddillitdy 1I0t b. . ppropri. te for :mim. ls thaI ha"" abllormal pror.ill .lId PO. collctmratiollS. Also because of the lock of "l:retmem ill ""'"y dillical assap, pl ... ma Aror values calculot.d for 011. set of assaY' may 1101 b. appropriar. for . 1I01her set of assays. Reference s .I""""',...... t. Hruod. JW. Soot< MG. 1?99. Pb,oOol< b..iJn b- ('- nc ioo dilFn"",,) .. """""" of • """ . ~ ..to'l' oci.d.h."., d~ kta Am.~ Saad J~Su.ppl IOn, Il}- 128. l. Jon« NL 19S7. BI..J G.un ",,,JAnJ.Bu. MryH' !.o. N"" Yodc n ...... M«Ii<.J . ... Satyn JW. CIoa"""" RL. Dolcini D'\l. 1989. M ... ,.,.d ,., akuJ".d o'Wa _ .. " .... in • popuJ..tion of pediauk int,,,,i,,, paci""" Rr.pi< Car< .l4J42-HI! . 5. Back<. SI. T,,,,,!,,,, KK. 1991. Pul" ~. la, Eh",mvI><. and p",,", plac.uo=, in k",lthr """,. J Am kim Ho.p Jw.oc Jl09- 14. 9. 1.1.. IS, Lui, F. Dri-.. B. T",, ~ Z RF. KuJlu R. «.,,, G. 2000. V.Iid. "'" of 0xn<" .. twa..... """.....,. """" io ......... ",,,. J<[ of k,modobin baoed orrvn ur .... (HBOC) infnoOoo. Clin Ub Sci [J,17l-179. 10. u. ... F. D..."", B. I..... IS . Rrro<- R. G......... RA. 2OOl. Yalidhy of . ""tia.t and mi=l VU. =woo ............ """ ............. io ......... b""" ..kOC' noodd af,,,, ...-'vi .. rion .. ;do ,....,.int: 00<>«>0 ... """ of b......p.bin.... oed _ atti ... An.nb Anal, 96,06-50. =, cc. p,...., Lo." H"I'P"" K. Rru..d. ML. Haakin. SC. 2005. ""'" m,n' of do. dfoc. of dilntioo of blood ..... pl<. ",.;tl. ...Ii.... k, pa.in "" blood "". dtxttolyt<. and Ioa.« """ ............. io <10" . Am I Y" Rr. 66.656-660. 12. Soott MG. H.....,J JW. t..G".. VA. 1999. Ekrtrolyt<. and blood P " " [0' Bur';' CA. Aah.""""[ E.R. .d •. r.... T__ , .. o/o;.,iu/ ~ }.d edition. 1056-1092 . Pkiladdl'bia, WB Sa.""" •. t}. Deuo. Ie. D.p. .... MP. lkn"""n AE. Wolf ST. M.. ~n D. 2004. Effect. of . pioV "",«rial and «ml"""""" and d",,"'" of "''''.V on do. atability of n-..n ;" 592 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY 16. R.... BD, P.... TW. 2001. CO";,.! ~ .fA.iJ-&s.,.,.,J E!.m.I,.. Dis, ....... Sd. edition. N ... Y",,1c McG"... HiIL 17. ;.sTAT 0,,,,,,,"'0. 2000. D.creaood reo, "',..It.....datod,.;th d.iop m...,.A._.... It., a...,I .. C lbom... 26. R.nn.y H.\l . Shu.... V. 2001 . S"..rno« and funclion of b"""rJobin. 10' Bnod.. E. Udrtm.ao MA. CoJk. BS, Kippo TI. s"li, ooI.o u. <>do. w,1ii.s"" H, ....."""" 6d. editioon, .4S- }5J, Nn. Y",h McG",.. _HiU. Il. Gootj<. P. Mo",. Y. Loc""';; E. 1994. J>..b. <>';m,"Y in bo ..... A "udr ...id. tb, .id ~f "" i••i.... blood ciKui,. In, p~ of tl., Fifth 10,.m.tioo.1 c...u< .. ofY"",io • ., An.. d. .... Am"j",n CoIkz< ofYrt S-...·. P.~ 91 ,1_ 16. .J(l. &h-. SW. 1990. An inuoduaioo to ..... "Ii: ;,.,n di/f, ........ Y" Oin N<>ac<>l. PD. 200}. C&kuiatio" ~f d.. total pl ...... <00«0""""" of ...,."..,j.cik ........k odd. and tl., dfmi... di._i,tioo co",""" of """",.bw, buff", in p1aun. /'0, ." in tl.. """" ;,.,,, .pp..... to acid-hu. b.laoc. io coto. Am / Y" R".6-(,IM7_ IOSI . J6. Conotabl, PD. 1997. A ';"'plilied """" ion modd foo odd·b... oq.ilibria, Applia.1ion to ..... ., pJ..na. J Appl S,....... -v. P~~8},297_}11. }7. Conotabl, PD, Stimplli HR. N ...... , H . B..d.a< dian ...... I Yrt I""m Med witbo., to 19,581 - SI!9. }B. Conotabl, PD. 2002. C okuJ.1ion of .. dabI" l. PD. 200}. C&kuiatioo ~f d.. total pl ...... of ...,."..,j.cik ........k odd. and tl., dfmi"" di._iotioo co",,""" of """",.bw. buff, .. in p1aun. /'0,." in tl.. "roo,;"'o . pp ..... to acid_bo" -v. CODC<"""""" b.laoc. io cm. Am / Y" R".6-(,IM7_ IOSI . 41. Stimplli H, T ayIo, M, McNi=II C. G&nc. Ay, C"... taI>k PD. 2006. E.p<~ = 29,1877_ 188 S. 4}. Cuobk. C. W""i.,.J;" M. Chba...o.T. Drt.y B. u ..d .. MP, ManiU, y, F..... A. G... cio P. 2OOol. H......,p.bin =wn .iJinity and «ztil.cio, foo"", of tl.. bIoood ""'J"I'" ...... p"" ift canin< • .,.J kli ... blood. Rto Y., Sci 77,s}-&l. 44. Cuobk. C. Di PN. 0...,."" T. Amory H . Marvilk y, Drt.y B, F. .... A. G...uo P. 2OOS. BIood-<>IJ"Z<" bindin& in •• altby Staoda.db.od bo ...... Y., I 169,251_256. Chapter 11 CALCIUM, PHOSPHORUS, MAGNESIUM, AND THEIR REGULATORY HORMONES Total Calcium (tea") Concentration ........................................... I. Physiologic Processes .............................................. II. Analytical Concepts of [tCa"'"] ........................................ III. Hypercalcemia ..................................................... IV. Hypocalcemi a ..................................................... 594 594 596 598 602 Free Calcium (lea"') Concentration ........................................... 610 I. Physiologic Processes .............................................. 610 II. Analytical Concepts of [fCa"'] ........................................ 610 III. A bno rm al Concent rations ........................................... 613 IV. Conceptual rel ationships of [tCa"'] and [lCa"'] .......................... Inorganic Phosphorus (Pi) Concentration ...................................... I. Physiolog ic Processes .............................................. II. Analytical Concepts of [Pi) ........................................... III. Hyperphosphatemi a .................. • • ............................ IV. Hypophosphatemia ................................................. Total Mag nesium (tMg"') Concentration ....................................... I. Physiologic Processes .............................................. II. Analytical Concepts of [t Mg"' l. ....................................... III. Hypermagnesemia ................................................. IV. Hypomagnesemia .................................................. Free Magnesium (fMg"') Concentration ........................................ Immunoreactive Parathyroid Hormone (iPTH) Concentration ...................... Parathyroid Hormone-Related Protein (PTHrp) Concentration ..................... Vitamin D Concentration .................................................... Calcitonin Concentration .................................................... Major Patterns fo r Calcium (Ca") and Inorganic Phosphorus (Pi) Disorderll ......... 614 615 615 617 618 619 62 1 62 1 622 622 623 625 626 628 628 630 63 1 593 594 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY Table 11.1. Abbreviations and symbols in {hi.. chapler [xl x con~lIIra{ion (x = . nalytc) 1,25_DHCC 24,25_DHCC 25_HCC Ca' + ECF I ,25_Dihydroxychol«akif~rol (ca/citriol) 24,25_Dihydroxychol=•.kifcrol 25_Hydroxychol<=lcif EDTA ,y 1Mg" GFR GH HHM ICF il'TH Mg" Pi PO, PTH PTHrp RlA SI IC,l+ IMg'+ WRl Calcium ExtrocdJul.r Auid Ethylcnali:nninHctraac;.,tic acid Frre ioniud calcium F,,",, ionized ffi"l:n ..ium Glomcrulor fihr>tion rate Growth hormone Humor'! hypuabmi. of malignancy Intrarellular fluid ImmunorraCI;", parathyroid hormone Magnesium Inorganic phosphorm Phosphate induding PO,>-, HPO.J.-, or HzPO.Parathyroid hormone P:l n lhyroid hormon~rda{«I protein lUdioimmuno ..... y Sys{~mc lmematioruol d'Unite. Total calcium T olal magnesium Within ..fcrena. interval TOTAL CALCIUM (tCa~ CONCENTRATION I. Phy.iologic P"""'''''' A. Strum or pI"""" c,'+ is distributM into thr~~ n"'jor fuctioDl. All GI'";n hd}jluid; is ;on;ud. but "'m~ is fTtt . nd 5Om~ is bound to anionic mol,""ul~•. l. c;.,n~ral conctpts a. fC~J<. {ofi:~n r~f~rrM to •• ;on;ud rlllcium}: About 50 % of [rCa>+) is jUI+. which is prestnt •• frtt ions in pI"""" H,O. Th~ [1Ca>+] i, th~ ponion of [rCa>+] th.t is hormon:dly r~latM .nd contributes to pathologic sUtes. b. Anion_bound c,>+ {I} Bound to anionic prot~ins: About 40-45 % of tC~J<. is bound to nq;>Iivdy ch ..gffi sites on prot~ins {80 % to :dbumin .nd 20 % to globulins}.' Sinct binding is charg. d.~nd.nt. changes in blood pH slightly :dter c,>+ binding . nd thus .+ ktwttn bound .nd frtt fractions. {2} Bound to nonprot~in anions: About 5- 10 % of tCa" is bound to citr>l". PO, . loct "'~' .nd oth... mall. diffusibl~ .nion5. 2 U.ing a filtration 'Y"l<m to "'parat~ prot~in_bound Ca>+ from oth~r c,>+ fractions and mrasuring [1Ca>+]. th~ distribution of Ca>+ fractions in 13 dogs anragal 56 % 111 CALCIUM, PHOS PH ORUS, MAGNESIUM AND REGULATORY HORMONES 595 Bones Parathyroid glands Blood · 1 ~Ca'+] • t [PTH] • t [1,25-OHCC) • "'" • 1 ]1,25-DHCC] Kidneys Intestine " ]ICa2'"] " ]Ca"/A-] · 1 ~C"'+] + t [PTH] Kidneys .. -- . . t IPO.J , . t [PTH] " " Mammary glands Fig. 11.1. IM1tio",hil" of calcium kinetics and tb. production of PTH and I ,25·DHCC. (No"': Ho,... kid ... )" lad I <x-hydroxyJu.: rnd thUll do not f i 11Ca'1:and i 11.25·DHCq. • Con"""ion of 25-HCC to 1.25·DHCC in kid ... }" is catalyud by I <X-hydrozyl.... n.. OCtMty of l<xhydroxyl ... i" promot«l by 1. 1ICa'1 :ond i PTH and inbibit«l by i 11Ca'1 :and i [pO.] . • Ca"" mobi~ution from bon<:and Ca"" .bsorption in intootiD<.,. promot«l by i 11.25·DHCq :ond i PTH. L..s cr+ mobili",tion and """''Ption ocrur if th.r~ i, 1. Il.25·DHCq or 1. PTH. • Urinuy Hcr .. ion ofC.'+ is enhrne K:~", 34 % prot~in bound, and 10 % bound to other anions.' In a group of 20 horses. th~ [1Ca'"'] ~nragM 51 % ofth. [tCa'"'].' B. M.jor factors that d.t~rmin~ .. rum [tCo'+] (Fig. 11.1) I. Agr: Young dog, (6-24 ",..k old) han .. rum [tC~l+] about 1- 2 mgldl gr~at~r than m . ture dogs." Young foals (I-GO d) have .. rum [tCo") sima~r to .dult v:du ...• Ki{{~n' {4--6 wk to 20-24 wk} hav~ ..,rum [tCo'"'] similar to adult valu~s.' Ag... rd.,ed data wer~ not found for c:dves. 2. Breau.... rum Co""' conctntr:ltion, in common ch~mistry profil~, rrpresrnt th~ [tCa"]. a drcrr= in .. rum prot~in conctntration {~'p«i..ny h)'l'O"ibumin.mi..} cau,.. a drcre= in th~ bound Cr .nd [hu. nt~r cau,. hypocalctmi... 3. Absorption ofCo'+ in int.. tin~ (mostly in a ..... m. but from duod~num [0 colon) a. In dog., cats •• nd cottle. intestin:d C~l+ .b50rption ~uir~. vitamin D [0 induct muros:d qJithdi:d ctn.ymhesi, of Co".binding prot~ins. PTH activiry augm~nt' ". FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY viumill D ""lions to inc' ...... c,'+ absorption, mostly by stimulating 1,25 _ DHCC production. b. B:.sa! on failu,," lIudi.,. in hon"., C,," absorption in {h~ equi". intesti". d.~nds I"", on viumin D and mo .. on Ih. amoum of di.tary Cal<-. ' Equi". kidn.". lack let-hydroxyl ..... (and thu.s do not COII""n 2S_HCC to 1,25-DHCC) but can product sm:.!l quantities of 24,25_DHCC.· 4. Resorption from or d.pmilion ofc:..>+ in bon,,'· a. PTH stimulates c,>+ pumps in the osuocytc m~mbran. ')"lum dUI promote Ca' + movr m. nt from bon. to bon. fluid to ECF. S«:ondarily, PTH induces 05{c/",u to contact bon. m>triI, or to mManct' stimulate osteoclasts to degrade bone by.nzymatic digtnion and acidifje>[ion." b. Vitamin D enh.Dces Co" r=>rption from bon. by promotiJll: ost.ooa,(ic 'Clivi!), and by .nhancing =pon.., to PTH." c. Calcitonin blocks ostroclastic ostwlysis through dir""t changes in ostroclasts and by reducing activation of os{rption through th~ form.rion of calbindin, a Ca .... binding prouin in th. dinal nqJhron. b. Angiot~nsin II nimulot .. th~ resorption ofN.+ in th. proximal tubul~, v", a Na*. Ca>+ ootr.mpon ,ysum. Ca'" i, concurr~ncly r~,orbed. Co.'"':md PO, inur>ction. I. Th~ [lei+] and [pO.] in pla,ma ... gr~at ~nough in h~althy animals th.t C.,{PO, ), complex.. would form if inhibitors "- ",,,.,,J ,.1,= c. [I. cru.t Analytical oonapl> of [iCa""'] A. Sampl~ I. s.:rum i, th~ pr.f.rred sampl. for [iCa'"']. HqJ"inized pl"'ma may ~ u,ed in "'m~ =p. 2 Blood .nti~lant' th.t bind Ca' + {EDTA, citrat~, .nd oxalat~} should not ~ u.!ed in samples for Ca'"' assaY'. 3. [n pwpl~, prolonged ... now occlusion during blood ooll""tion may inc ..... th. [tCa""'] by 0.5- 1.0 mg/dL I B. Common clinicl .... )"l"~ photometric assa,.. that mnsu .. [tCa"} (fIN + bound). I. o.C"50lphth.l~in assay: o . C ..solphthal~in r~.cts with Co'" to form a red complex. 11 / CALCIUM, PHOSPHORUS, MAGNESIUM AND REGULATORY HORMONES 597 111 dye colorim"'ric :w<>r: Bound Ca'+ is li~ralffl from anions and Ih~n Ca""' r~~CU wilh Arstruo7.0 III dy~ 10 produce a rolorffl compl~x. In som~ 'Y'ums, ""robic sampl~ handling may incr~ m.... surffl [rCa""'] by 004 mgldL wh~n Ih~ sampl~', pH incre;;o..,. kcauso of Ih~ loss of CO, (Ott Chopl .. (0). e. Unil ron""",ion: mgldL X 0.2495 = mmolfL, ~nd mEqfL X 0.5 = mmolfL (SI unil, n~ ..~'1 0.02 mmoUL)" D. Ik alUS<: a larg~ porlion of lCo'+ is prol~in bound bUI Ih~ body regulales Ih~ [fCi+], Ihe [rCa"'] may ~ d= .... sffl kca"", of hypoprolein~m", or h}'lX>"lbumin~m", when Ihe animal does not h :IV~' defect in regulaling Ih~ [fC~""'] . To eslim'l~ Ih~ dfecl of lower prol~in and :dbumin concemralions, correction or adjusling formulas h. "" bttn proposed. I. Canine adjusIM [ICa""'] considering :dbumin con",nlralion (Eq. Il.l a)' 2. Canin~ ~n:rz.o .djusted [rCa""'] = m~~IUr..d [ICol+] _ m.... surM [:dbumin] + 3.5 {± 1.3} (11.1a.) Example: If [ICa'1 = 8.0 mgldL &: [:dbumin] = 1.0 gldL; Canin~ .djusted [rCa""'] = 8.0 - 1.0 + 3.5 {± 1.3} = 10.5 ± 1.3 = 9.2 10 11.8 mgldL Im~rpreulion: If ch~ dog WlU not hYP""lbuminemic. its ,erum [ICa'1 would ~ from 9.2 10 11.8 mgldL in 95 % of conine sample•. Canin~ .djuslffl [rCa""'] = m~~IUr..d [ICo'+] - (004 X measurffl [TP]) + 3.3 {± I .6} ( 1I.Ib.) Example: If [ICa'+] = 8.0 mgldL &: [TP] = 4.0 gldL; Canin~ .djusI. d [rCa""'] = 8.0 - {OA X 4.0} + 3.3 (± 1.6) = 9. 7 ± 1.6 = 8.1 10 11.3 mgldL Im~rpreulion: If ch~ dog WlU not hypoprol~inemic, iu ..,rum [rCa"'] would ~ from 8.1 10 11.3 mgldL in 95 % of conin~ ,ampl~. ,ulhors do not includ~ Ih~ "± 1.3," which i, ~n .S!imale of Ih~ 95 % of Ih~ publi,hffl dal .. b. The formul. ,hould ~ USffl coutiously for Ihrtt r=ns: ( I) Ih~ ~djuslffl [rCa""'] is .1 besl .n eslimate; (2) Ihe formul. v..as g~ner:l!ffl u,ing conine rCa""' and albumin con",mralions dff .. minffl by one..,1 of assays, and .n id~mic:d formul. would probably not ~ oblainM if olher :assays w..e used: .nd (3) Ihe formuJ. does not ronsider Ihe v:uialions in Col<- binding 10 globulins. Ca1+ i, bound 10 albumin and som~ globulins. Th~ relalive amoum ofCa""' bound 10 :dbumin .nd globulins v.ries wilh diffe .. m albumin 10 globulin ralios; for <:Iampl., sampl.. wilh po.nhypoprol~inemia ""r.m Iho,. wilh hypoalbumin~mi •• nd hyp Som~ confid~n'" im~rval '98 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY individual. SlIU"'{ th.t rigid corr«tioll formulas may not ~ .ppropri.t~ • .,,~alJr in c;mk 4. [n a mro'p«t;ve .rudY'is of 1633 emine sampl.., it was concludffi [hot m= adju,tal [tc,l+] "'Iuation, do not rdiably pralict whffh... dog has .ith,r:m incrrasffi or da:r......d .<-]." This rrpon did not indicote Whffh" th ... was analytical ag,,",,mem ktWttn the method. uK th. basi, for th. ,.f,ten"" inurval, u,a! to cw.u!jr ... ulu as incr••K ~tw,""n m• ..,urffi and cakUlatffl values was not ~:duatffl. 5. It is =mm.ndffi that th. potent",] df«ts of dysproteinemi<> ~ consid"M when int.rpming [tea1<-]. If the formul,.. for :odjwting the ID<'llSur. d oonctntl Ill. H~rcal"'mia (Tabl~ 11.2) A, [ncr~• .al Ca'" mobili7..tion from bone or absorption in int~stine L [nn"asal PTH or PTHrp activity a, Prinury h~rparathyroidism''>-l' {I } P... thyroid adenomas or =cinomas stCret~ PTH th.t stimulates Ca'" r=>rption &om bone and incrra..,. Ca>+ absorption in the intmine, Renal excretion ofCa' + m.y ~ inc=std becau,. h~rcal"'mi. cau..,. an incrrasal filtered load (more filter<'d than can ~ resorbed even with incrrasal PTH activity) but not enough to prevent h~rcal"'mia. {2} Hypophosphatemia i. ex!"'Cta! because PTH is a potent phosphaturic agent, [f GFR i. decrras<'d, ther. may ~ normophosphatemia or hYl"'rphosphate_ mi. becau,. PTH cannot promote phosphaturia if PO, doe. not enter the tubular fluid. {3} The [iPTH] is exJl"Cled to ~ WRI to increased. Of 210 hyptrcalcrmic dogs with primary hyptrp:mnhyroidism, 73 % had an [iPTH] WRI .nd 27 % had an incrrasal [iPTH ]," In the pr=ncr ofhypercabmia, an appropriate phy.S"udo_hyperparathyroidism) {I} In some p.... ndocriM neoplamlS, a hypercalcrmic agtnt is produced by ",Us th at norm.Uy do not produce the "l:ent, Nearly all neoplasm. and e.!"'Cially carcinomas h.ve the potential to ~ I"r .. ndocrine, {2} Som. pa..endocrin. neopl",ms produ", PTH rp, PTHrp is • polYl"'ptid.; its first 13 N_terminal amino acids are very simil.. to thOR of th~ N_ terminal of PTH , It has bone osteodmic and re""l resorptive .ffects similar to PTH , Many normal crUs produce PTHrp, but very litd. of the PTHrp produced by healthy ",Us enters blood,'o PT H rp pIa". important rol .. in fetal development, but PT H governs syst.mic C.>+ homeostasi. after binh, {3} Neoplasms known to produ", PTH rp (Note: Only rome neoplasms of rach type ... as.IOCiata! with hYl"'rcalcrmi., and hyptrcal",mia without incr•• .al PTHrp doe. not exclude lymphoma or other neopl asms,) 11 1 CALCIUM, PHOSPHORUS, MAGNESIUM AND REGULATORY HORMONES 599 Table 11.2. Discascs and condition. Ihat cause hypercalcemia Inc....aI Ca>+ mobili7.;,lion from bon~ or absorplion in immin~ Incr ... sIion Endog<:nous vitamin D Granulomatow inlhmmalion: blastomycosis, hiS{opl~srnruis, cryptococcosi., pulmonary .ngiomo~ruis Neopla,m_associatal hyp+ Rc:ruol failure 'Hof!lel {common in both :K:UI~ and chronic} Dogs .nd cns {uncommon in acUie and chronic} Hypoad .. noconicism in dogs and cats (oth~r Ih~orie" incr~asal Ca>+ bound 10 prouin or cilrau) Thiazide diuretie& Increased prolein_bound Ca'+: hyp Hypothyroidism fju""nil~ onset} Rc:lainal fetus .nd cndom""rilis in a dog Idiopathic hyp<=la:mia in <:aU • A "1";,,,,ly rommon di..... or rondi,i"" Not<: n,. 1,G."1 in boalthy J"l"ng dog, « I }'J old, .. peci,jly in larg. br..,.u) may br up 2 mgldl higbor dun tho 1,G.'1 nf... """ in",rvals fOJ IJUru" dog>, '0 (a) In dogs, these neoplasm. include lymphomo {mu:dly T ctJJ},'~>I apocrine gl:md carcinoma," oth~r <:ar<:inomo. {pulmonary, n ..al, mammary, squamou, ctn, thyroid, and thymic}," and malignant melanomo," Lymphoma i, the most common neopl .. m associatal with hyptrcalctmia in dogs, followal by apocrine gland <:ar<:inomo, (b) In <:aU, th ... neopl"'ms includ~ pulmonary <:ar<:inomo, undiff~"'ntiatal e;neinoma, thyroid c;ncinoma, and lymphoma," {el In horse>, mydoma has been r~port~d to product PTHrp,17 {4} Documentation ofPTHrp involnmcm was limital by .vailability of valid ""Y" for many years, Other hyp 600 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY (a) [n dogs, thest n.opla,m, include epidermoid carcinom.'" and meta'tot;c adenocarcinoma." (b) In em, th.,.. lIcr PTH a=ys are usn! to det«! suppressal PTH production . ssoci.lpk incr~alffl PTHrp production by knign na>pl .. m, h •• bttn =gnized and h.. bttn caU.d humoral hyp,"Ilkmtill ojbmi[nllng.'" {2} Two dog> with schinosomi..i, cau,.d by Hrurobilharzkl IlmnicIl"" ru.d hy~=l""mi •• normopho.pru.t~mi •• incrrasnl [PTHrp]. and d=u• .d [iPTH].JO It WlL! conclud.d that th~ incr~'1ffl [PTHrp] v..as COUIffl by th~ ass<>pl ..ia. 2. Inc",-d vitamin D activity (hYP'rvitaminmjs Dj a. Ex=sive vitamin D a.ctivity couse; hy~rc;d""mia through multipl~ proc=. HYP'rpho1 abKlrption of PO,. (I ) It "imul.t.. formation ofC."_binding prot~im {i"dlbindinsl" in int<Stin<>1 mucosa 10 tru.t mo", dietary Ca>+ i, absorbed. (2) It .nru.n"", bon. resorption by making o,ta>lytic ""It. mor~ r~'pomive to ITH. (3) It d",rea.." renal aCre{ion ofCa'+ (~rhaps through incr~.=' calbindin 'ynth~,is). b. Sour"", of aogenou, vitamin D (I ) Ingestion of a rodenticide rontaining cholecalcif.rol (Quintox. Ramp:oge. or Rat_lk_Gon~). which i, conven.d to 25_HCC."" (2) Ingestion of ta.calcitol or cokipotriol by dogs"l-4J the latter (also known '" calcipotri.n~) king pr=nt in a topical ointment (Dovonex) tru.t i, u,.d for hutruln pwriasi' (3) Ingestion of plant. rontaining ~rgocalcif.rol {vitamin D,}~" including C~,"um diumum (d.y_blooming j . .... min.) in Florid." or S~"'num in Hawaii. Bruit. and Argentin.'· (4) Ex"",.i"" dietary intake of vitamin D ..."'.... including ingestion of mi.mr_ mul.tffi commercial ftt(j. c. Sour= of endo(;C'nou. vitamin D {I} Granulomatou, inflammation (a) Hyp 11 1 CALCIUM, PHOSPHORUS, MAGNESIUM AND REGULATORY HORMONES {b} Th~ 601 hypercal""mia is producM when nimulat":! histiocytes or macro_ phage. produ"" viumin D {1,25-DHCC}. {2} Neoplasm_associat":! hypervitaminOJis D: Inappropriately high vitamin D con""ntntions hav~ bttn rq><m..:! with amin~ apocrine gland ad~nocarci_ noma .nd lymphoma!' The neoplanic cdls may be producing vitamin D or stimulating vitamin D .ynthesis in other ""Us. 3. Neoplasia {h~mic or nonh~mic} in bone a. HYl"rcal""mic agenu other than PTH, PTHrp, or vitamin D have been '!.KICi.r..:! with malignanci .. in bone. Some dogs with lymphoma and myeloma ha"" localiud bone resorption and corresponding hYl"rcal""mia.'· The hYl"rcal""mic agents =y be working locally or 'Y't~mically and may include interl...,kin I, int~rl""kin 6, tumor growth factor, tumor necrosis factor, and prostaglandins.'·... b. The [Pi] m.y be within reference interval, or incr""s.d. B. Decreas.d urinary excmion of C." I. Ren.l inmfficiency or failure a. Ho ..... with ",me .cute or chronic renal di"a ~ {I} [n health, equin~ kidney> excme excess dieta,y C .... Renal diseases that decr""" GFR impair renal excretion ofCa'"' and thut aouse or contribute to hypercal""mia." lowering dietary Cal+ intake by switching from alfalfa hay to gmt hay aon r..:!uce or diminat~ the hypercol""mia, but the impairal GFR l"",im." Alfalfa hay aon cont.in 2- 10 tim .. the Ca'"' ront~nt of gr:w or mix..:! hay." {2} Hypophosphatemia =y be p...~nt. {3} Ho,"", with hYl"rcal""mic renal f. ilure have a dec",as.d [iPTH]." b. Dogs . nd aolS with acute or chronic ",n. l dise... {I} Occasional docs .nd aolS with acute renal f.ilur~ are hYl"rcal""mic. Hyper_ cal""mi. m.y be du~ to an incr...... d ron""ntration of C." bound to citr.t~ or PO,. Rai.in .nd gr. pe toxicoses frequently result in hyperaol""mic .cute ",n.l failu",.'" {2} Of dogs with chronic renal f.ilur~, 10- 15 % are report":! to be hypercal""mic, which 'Pl"'" to be primarily du~ to binding of Ca>+ to retain":! anion •. " However, mon docs with chronic r~nal f. ilure have a low_normal to mildly deer.....:! [tC."]. Binding ofCa'"' to retain":! anions may also yield normocal""mia {[rCa'"'] WRI} that mad". decr""" in [£CaU]. 2. HYI""'drenocortici.m {Addison', dis"",,} in dOf:s .nd aots a. About 30 % of dogs with hypoadrenocorticism are hypercal""mic.' .... Hypercal_ ""mia has also been described in car. with hypoadrenorortici.m."-'-' Th~ r""son for the hYl"rcal""mia is not esrabli,hed but prob.obly is at le.. t partially aoused by decre;;osed renal excretion and might be aoused by increas.d C.'"' binding to protein or citr.te. b. Renal Ca'+ excretion in adrenalectomized dOf:s is dec ..as.d by excessi"" tubular reKlrption ofC? ... The reason for enhancM tubular resorption is not esub_ lished but m.y inml"" angiot~nsin II or corti",l deficiency. {I} When dogs with hypo. drenocorticism become hypovolemic beaou"" of mmiting, diarrh~a , or impair..:! renal con""ntming ability, angiot~nsin II a.ctivity inc ....... Angiotensin [] promotes Na+ r..orption in proxim. l renal tubul .. via a N.+-C.'"' rotransport 'Y't~m." Thus, enhancM resorption of 602 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY No.' may :d,o promo{~ th~ proximal rubular r~50rption of Co.>+ .lId cau.. hy""rcal",mia. Concurrent h.mocon~mra{ion ;t.df =1 ,lightly inc......., the ..,rum [rCa"]. {2} Administration of glucocorticoid. dot. inc...... tOruli excretion ofC,>+. Thu., • ddici.my in cortisol may allow more C,,'+ to ~ resorbd. 3. RupturM urinary bladd.r in foals: Th... foal. =1 d~dop hypercabmia (.uthors' unpubJishal data), but most do not. When pr... m, tho hyptrcalctmia i. probably cau~ by tho re;orption of urinary eo'+ from th. pu;rone:d Clvity. 4. Thi".id. diuretics: Th... :oct in the dinal lIq.hron to promote hypernatruria and, =ndarily, volu"", d.pl~jon. Hypovolemia promor.. . nhancnl proxim:d tubular resorption of No.+ and, =ndarily, proximal tubul" ,<sorption ofC,>+." Thiazide:! abo promot< th. resorption of Ca ' + in Ih. diSl:d tubule •. " This form of hYP"rcole;.,mi. is rardy repona! in dom<:Stic aninuls but d~ occur in dogs. ~ C. Incr~'1ffl protein_bound Ca>+: In ""me case. of marka! hyperproteinemia :associata! with multiple myeloma, there is.n increase in nq;ativdy charged globulins th.t bind cotions including Ca>+. When K:." .uociate. with proteins, the [K:."] transiently dec,..,..,e. and the,.., i. compc:m.tory rdease ofPTH to incre-ase [1Ca>+]. The net result is inc,..,asaI [iCa"] and [1Ca"] WRI. D. Other or unknown mechanisnu l. Intravenous infusion of c,": This con be a mechanism if th~ ral< of administration ""ettd. th. rate of ..nat na.tion. 2. Hemocone;.,ntmion: If hYP"rcole;.,mia occurs, npeet it to be mild and :associated with increased protein_bound or small anion_bound c...>+. It may also be .dated to angiotensin ll ..... timulated transpon ofNa' .nd c...>+ in proximal tubul ... 3. Junnil ... onset hypothyroidism:" Thi. may be COUIffl by inaUKd int.. tinat.bsorp_ tion .nd dec,..,asaI renal ncr.tion of c...>+. 4. A maina! fetus .nd endometritis in • doC" 5. Idiopathic hypc:rcohmia: This wa. found in a group of colS that did not have rerocnizc:d hypercale;.,mic disorders. Som~ of the cats had calcium oxalat~ urolithiasis." IY. Hypocale;.,mia (T.ble 11.3) A. H)'I'O"lbuminemic hypocale;.,mia (hypoproteinemic hypocalcemia)' l. Hypocalcemia r.. ulu from. decmued concentration of nogativdy charged proteins and therefore of protein_bound c, ... The regulation of the [1Ca"'] would be adequal< in th... animals unbs then: is a concurr~nt defect in th~ regulation of the [1Ca"]. 2 It has b..:n called p"udo-hyptJ€ilktm;a becausr the [K:."] does not decrease and clinical.pwia, or inHamm.tion) .nd do not r"p')fld to hypocale;.,mi. or do not rd ...... adequal< quantiti .. of PTH to p=ent hypocale;.,mia. b. Hypocale;.,mia is couK+ load wh~n hypocale;.,mia i. presc:nt. 11 / CALCIUM, PHOSPHORUS, MAGNESIUM AND REGULATORY HORMONES 603 Table 11.3. Discases and condition. that cause hyp<><:'"1 cemia ·Hypoolbumin~mi. {hypoprotcin~mi.} Dec...asN PTH .ctivity Primary hypoparathyroidism {dam"l:ed parathyroid gl.nd} Psatdo_hypop:mnhyroidism {d,""""ascod PTH r~tor rc.ponsinness} Hypom"l:n=mi. {bovin~ grass t~uny} In. d'"'ll1ate Ca'" mobiliution nom bon~ or absorption in imcninc Hypoviuminosis D 'Chronic renal di~ or f.ilure in dog" cats, and ande Prot~in_Io>ing ~meropathy in dog, Dietary vitamin D ddici~ncy (rare) Vitamin D- rcaptor d.f'""t rickets (viumin D-Jq>le (in vivo or in vitro) T ~racycline . dminim. tion Co." d~p"'ition during fracture healing Oth~r or unknown m,""hanism • •Acute p.ncr .... titis in dog, and cats Urinary tr.ct obstruction Acut~ renal failure PhOJph .t~ en~mas Bliner btttl~ poisoning {camharidiasis} in horse; Myo!",thies: trampon tetany, ennional rh.bdomyolysi., malignam hypothcrmi.o, enduran",_type exercis< Acut~ tumor Iysi ••yndrome Rum~n o""rl""d (acut~ carbohydme ruminalengortrmem) • A ,el1tively common di..... 0' condition Note: n,. ItCa'1 m.y b. f.d .. ly «! if th. S1ffiple contains EDTA (wrong tuh. Of ronumiru_ cion of dot tuh.), A oonru,"mt fili< hyp<,luIemi. """"Id b. upe' c, The ..rum [iPTH ] will be: decr..,.,ed or low_normaL In th. pr=n'" ofhypocal_ ",mia and functional parathyroid gland., the [iPTH] should be: incr .... sN '" a physiologic r•• porue:, d Hyp"rphosph.t~mi.o is cxpected be:ams< of incr .... sN resorption of PO. by rell<01 tubules (bc:c.use of less PTH to promote pho.phaturia), and thus renal excretion of PO. is decreased, How~""r, renal cxcr~ion of PO. may incr~.,.lalCr bc:caus< hyp<rphosphatemia causes an increased filtered PO. load, 604 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY •. Hypupho,ph . temia enhances hypocalcemia by inhibiting renal formation of 1,2S_DHCC (excq>t in hor..,.). 2 P.. udo_hypop .... thyroidism a. In this r= disorder, PTH r=ptor.; or ""n.rTH rN:<-] d«",,",,,, kaUst of dirnini,h.d PTH .ff«ts on intesline, bon•.• nd kidnryo. b. The [Pi] may be incr..asN b=.Ust of inadrquate ",ill excretion of PO,. c. The [iPT H] should be inn.a=! b.=.l15t of th. hypocal",mia. 3. Hypom"1:n=mi. a. Hypomagnestmia co"""" by Mg>+ dq>letion may pradu,,", a function'! hypop>ra. thyroid suu and hypocal",mi .. Th. dy'!unction may be due to two proc....s: {I} Mg>+ 4'". {2} P.mhyroid cdh .=om. less PTH either ba::.Ust of dirninish«i cyclic adeno.ine monophosphate (cAMP) fonrullion (impairffladenyJ.te cyda.. ) or ~u .. of diminimffl com~iti"" inhibition ofC,,'+ binding to parathy_ roid cdls. In the p.... nce of hypom"J:n=mia. the .. may b. mo .. available binding sites for Ca>+ on the parathyroid cdls and thus a lesser [1Ca""'] is able to inhibit PTH ""retion." b. The.. m.roanisms may explain the hypocalcemi 111 CALCIUM, PHOSPHORUS, MAGNESIUM AND REGULATORY HORMONES Healthy stale IIC">+I IP~ 605 WRI WRI 11,25.IJHCq WRI liPTHI WRI Fig. 11.2. 5.<J... nti..l ....na during tb. developmthyroidism ",,,,.«1 by cmonic rm..l di..a.. in dogs. cats. :and atd •. Eventu..lly. rCa". Pi, rnd 1,15-DHCC oonc«l ]I ,25·DHCC] loads to .j. [/Co"] . nd perb.P' .j. [rCa'1 becawe of .j. int.nin.J c.." aboorprion :and .j. ct+ r..aption from bo .... • o.a.a>«l rl,25·DHCC] :also r.q.,ce:t tbe I .25·DHCC inhibition of PTH .ynthesit. and thWl PTH 'J'Iltbe.is inc"' ..... • D.cruo«l [fCa'1 cawe. t PTH prodllcrion,.j. c..lcitonin "'Ie....,..,d tl ....bydrozyI ... activity. • Increos«! PTH pro""""" viumin D-.d.pthyroidism is present. defa:live intestinal absorption of c:.'+ and defecti"" =pon ... in bone and kidney. cou.. hypocalcemia, a comp<::dcemia (3.9 mgldL), increased [iPTH], and increa=! [I ,25·DHCq." II wa. p.... nte-d bec:o"", of vomiting, diarrh .., muscle tremors, and mydri:.. i•. 3. Exocrine pancreatic insufficiency (dog.) a. Idiopathic !"Inere.tic a.cin .. atrophy, chronic !"Inereatiti., pancr.. tic neoplasia, or !"Incre.tic su~ry may lead to exocrine pancreatic insufficiency and thus co"", incomplffe dige;tion of di.ury lipid. and oth.r ingesta. ,,. FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY GFR1E~ f 25%I " Initial roo~1 dama9" Additional ".",,1 dama!!" Progrsss ·" I-~ (l.25-DHCC] (tCa'+] t I ·'" 1-- /-i--- s;",,,,,, Fig. II..,. ScM"",tiC path"l!" .... is of >«x>ooory rm:d byp<rpautbyroidi,m in dog •. cats. and CI,d• . am" "'p",.. nt ",f.re~ in",,,,:. " for GFR OJ . .ch arulyt< con""n,mion. • lniti:d renal .wn.1l": n,. "'quem" of . ""nts desaibed in Fig. 11.2 initially compo".."" for th. « of po. and inod.qu ... activ:otion of vitmn D. A ""' hom b. Di<"l'ry Ca'+ Jruly be, less available for intestinal absorption !>«:au.. of its binding to partially digoued lipid,. Abo, nuldigo .. ion of lipids Jruly redue< absorption of fat_soluble vit:omins, induding vitamin D. c. Dogs with exocrine pancr....tic insufficiency fr. 4. Prognancy, panuri.nt, or bctatioruol hypoca.lcomia a. p.nuri.nt hypocal""mi.> {milk f~.r} in cotd. {I} Hypoca.le<mi .... ulu from ""cess Ca" loss {vi.> milk or f.tal bone d~dop_ m.m} rdatin to im.. tinal absorption and bon. resorption. B="",,, most paretic cotd. hoY< an incr.oued [iPTH] .nd an inc..oued [I,2S-D HCC], ch. disorder involn • • n inad Di..ary facto" that promo.. me{;>bolic alkaJosi, Jruly da:reast th. targ<"l coli =pon .. to PTH.'" 11 / CALCIUM, PHOSPHORUS, MAGNESIUM AND REGULATORY HORMONES 607 {2} A d=~~.M [1Ca""'] l~.d. to clinical sign5 ofhypocal""mia {h~resthesi. and tetany ~arly, and p>resis to flaccid p:oralysis J.t~r} bet:.use of d~f«ti"" nrural transmissions and m...ct~ oontractions. {3} Hypophosph. umia may occur bet:.Ust of lactation, poor bon~ re50rption, and th~ phosphaturic . ction of PTH. b. Pu~~ral tetany or «lampsi. {bitches, mares, and ~'esl" {I} It is =ociattd with pffi< J..ct:uion :md corresponding pffi< los> of Ca" in milk. {2} An animal may ha"" ooncurr c. Preparturi~nt hypoc:d""mia in quttns'" ( I) Clinicol signs {:morm., dq,,=ion, .nd leth.rgyl ocrurrtd 3- 17 d prior to parturition. (2) Thrtt qUttlll had . d=~ .. td [tC~"]. Th~ [1Ca""'] WlL! d«",=d in th~ on~ cot in which it w;o. m~~lUrai. 5. Hyperca1citonism a. This is us,",lly cou...d by thyroid C-cdlneoplasm. (m~dullary thyroid <:;"cino_ mas), which .'" rmti""ly unoommon neoplasms reporttd mon often in old~r bulls. " Wh~n ow;oci. ttd with C-cdl neoplasm" hy~rcalcitonism umally couses only mild hypocal""mi. b«aUst of the following: ( I) Neoplasms a.. .'ittn in old~r animals, .nd their bones ar~ rdativdy ",fr.ctory to calcitonin {calcitonin does not inhibit PTH_stimulatal osteoclanic a.ctivity}. (2) l.ow..ing of the [K:~"'] cous.. a compens>tory inCfu"" in PTH production that attempts to m.intain a physiologic [1Ca1+]. b. Marhd hypocal""mi. may occur if a C-cd l neopl .. m d... roy> parathyroid gl.nds, which couses hypercalcitonism and ooncurrent primary hypoparathyroidism. c. Ex=- .dministration of salmon calcitonin {ustd to low~r the [tCa' 1 in hy~rvi_ tominosis D or HHM} may couse hypoc:d""mia. 6. Nutritional hypocal""mia (nutritional serondory h~rparathyroidism) a. Occurs with vitomin D-ddicient di~ts .nd wh~n diets a.. not b.lan""d for C~" .nd PO.; that is, the dietory Ca>+; PO. ratio is low~r than th~ d~sir..d ratio for th~ species. Th~ diet cous<::! th~ hypocal""mia, which stimulates parathyroid gland h~rpl .. ia. Imbal:mctd diets may include ex"" .. i"" PO. (comivord m... t diet) .ndlor. rdati"" or absolute ddici~ncy ofCa""'. The n~t df= it too much PO. in the body ~nd too litd~ Ca' +. b. A d«r~a.., in th~ [1Ca""'] stimulates parathyroid glands to couse .erondory h~rp:m.thyroidism. Th. incr ......d PTH rdeas. unds to kttp the [fCa"] near normal at the expense of the Ca>+ COnt~nt of bones, and thus oneomalacia m.y be p..sent, especially in young animals ( th~ bones of old~r animals ~re r~lativdy ..fractory to PTH _stimulattd osteoclastic activity). c. The dq;IN of hypocalctmi . depends on th~ ..""rity of dietary imbalan"" .nd th~ .VlIil. biliry of C~'" from bones (with Ca'"' being mor~ :IV.ilabl~ in young .nimal. initiaUy). d. Renal PO. nc"'tion is incr~=d b«ause of th~ phosphaturic actions of PTH .nd, in some co .... the high dietory intah of PO•. If th. r~nal PO. excretion is measurtd, on~ should expect inc.... td 24 h PO. cl ... ran"" and increastd fractional excretion of PO•. 608 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY D. E. F. G. 7. Ox,,!.!e toxicity"" a. Horses that ""I pJam. that haw high onlate content but low Co." cont.1It "b.mb I... diet;;uy Co.'+, and de ...]"p hypocalc;.,mi .. Such planu indude fuffd, pangola, SUIl";", kikuyu. rhub:ub. halogtton, gr• ....wood, and ."u!:SOb (OxtIlu fm-Ctlpr,uj. b. In ruminanu, Halogmm and curly dock (Rum(x criJPUI) =y cau.. hypocalc"ruia." c. Alfalfa h"" high Olulate content, bur ingtn;on dot. not cau.. hypocal",m", b=.Ust alfalfa abo has high Ca'+ conWU. Excess urill+. b. Jntrav~nous HCO,- infusion. in cats" {I} Both th~ [iCa""'] .nd Ih~ [fCa"] ar~ rq>Onffl to ~ da:rnstd in som~ cots ra;tiving intrav~nou. HCO,-. {2} Th~ p"thogtnesis of Ih~ hypocaletmia woos not upl.inffl, but pcts.!-ibl~ m«hani!ll" includ~ d=~• .ffl C." .. wrption in proximal tubul .. ~u,," ofCa""' oomplaing with Uet... HCO,-, alkal~mia resulling in d«r......d [fC.'+] , or increastd .. nal Na+ ucr~ion coming concur.. nt hy~rcakiuri .. c. M~.bolic alkalosis contributes to th. hypocaJetmia of parturi.nt hypocalctmia in cattl~ by rfflucing mgt! etll r~spon'" to PTH." 3. Furo.. mid~ Ir ... tm.nt: Furos.mid. dir«dy inhibits N .+ .nd ct r~sorplion in th. """'nding limb of Ih~ loop of H~nl~ .nd thu.s ..condarily inhibiu Ca>+ resorption in Ih. """'nding limb ba:;ou,," th~ passive resorplion ofCa" d.~nds on gradi~nu mablishffl by Na+ resorption.'~" T hu., wh~n furo .. mid~ is used to promol~ diur~sis, hypocaJetmia con d~dop. Thi. calciuric df«1 offuro..c:mid~ is u..d Ih.. a~utically to 1",. 1 hypc:rcalctmia. T hiazide diumics should not ~ u..d in such ca... ~u .. thry actually promote Ca>+ resorption in the di+ binding with dilfusibl. anions I. Ca>+ binds wilh EDTA . onlal~, ot cilrat~. given syst. mically or presc:nt in blood coll«tion .yst~m'. 2. T ~I",eycline is a C." _binding agent, :md its rapid intra",nous infusion may low~r Ih. [fC.1+], but th~ [tCa'·) ",mains WRI.'" Ca>+ d~posilion during fractu .. healing: S<:rum cokium conetntratiolll d«reasffl .tightly (about 0.6 mg/dL) during th. early stages of fractu", h~.ling in dogs.'" Th~ d«",as. may not ~ enough to co~ hypocaletmia by itsdf, but Ihe Ca>+ d~position during fraC!u", healing could contribut~ to hypocaJa::mia. Olh~r or unknown m.ch:misnu I. Acute paner..,.titi, (dogs and cou) a. Hypocala::mia may ~ p",.. nt in acul. fflematou, or h.motmagic panc""litis in dogs. HYpoc:!letmia i, common in cots with acul. p:mcrealitis, with a d«",ased 11 1 CALCIUM, PHOSPHORUS, MAGNESIUM AND REGULATORY HORMONES [IC~1<-] 2. 3. 4. 5. 609 be,ing mor~ common chan a d«",.w [rCa"]; • low [1Ca'+] was associ· .IM wilh • poor~r procnosi .... b. Th~ p.rhogen~ses of Ih~ hypoc:dctmia and d«......d [IC~"] ~'" nO! cl~~rly id.mifial and may involv. muhipl~ m.ro..nism •. {I} HistoricaUy, Ih~ hypoc:dctmia was anribuIM 10 Ih. binding ofC.'+ 10 fany acids in Ih. p..itonffi cavity (forming c.'+ so. ",); fmy . cids wu~ libe,ralM from n.crolic p'rilon~al fal b"cau.. of .ctions of p:mcrealic lip .... How.v~r, Ih..~ is linle .vid~nct 10 suppon Ih. ""p Ih""ry, and th~", is .vid~nct to ",furr il." {2} Orh.. Ih""ries wilh vari.bl~ suppon includ~ .bnormal hormon.! ..gu.1.lion of Ih. [1Ca"] {involving glucagon, calcilonin, or PTH}, ~mry of c.""' into edls b"cau.. of damagal m~mbranes, binding of ICa""' 10 pWJruI falty acid., and exlravas;olion of prol~in.bound C~'" b"caus< of increasM VlIKlllar p'rm ... biliry....." Uriruory lracl obslruclion" a. Mild 10 mod~ral~ hypocalctmi. may be, pr~s<m; Ihe [1Ca""'] i. d«",asal mor~ lhan Ih~ [rCr]. b. Th~ pathogenesis of Ihis hypoc:dc~mi. is not d ... rly und ..stood bm may be, rdalal 10 incr ...sal [Pi] .nd Ihe resuhant binding of 1Cal<. Also, Ihe renal mbuur damagt' lhal occurral during obstruclion Im}' incru.. Ih~ ren.! excretion ofCa" aft .. Ihe obslruction ha. been d ... ral. Acme r~nal f.ilur~ a. Hypoc:dcemia m. y occur in .nimals wilh .ctM renal failme, bm Ih~ call5t of Ihe hypocalctmia may be, eilh~r Ih~ renal f:>ilu", or Ih. concm",nt pathologic sUles." b. If Ih~ .ClM renal failme is prim.rily due 10 isch~mia, Ih~n p'rhap. a rapid-on...r hyp..phospharrmia l...ds to Ca>+ binding and hJ'll"'C'1ctmia, or Ihe amrr nq>hrosis raluces Ih. tubular resorplion ofCa'"'. c. Olh~r concur",nt p.rhologic sUI~. Ihal Jruly be, contribuling 10 Ih. hJ'll"'C'lctmia includ~ .cul~ p.ncr"'lilis, ~thylene glycol poisoning, and hypoalbumin.mia. Phosphale en~mas a. In r~por!al fdin. =', author.; propos.d that th~ hypocalctmi. d.vdoped b"caus< of hyp'rphosphal~mia." b. Hypuphosph.remia may product hypoca.lctmia Ihrough Ihr.., processes: {I} Acmdy, Ihe high PO. load drives the following lhogc:nesi. ofhypoca.lctmi. is not 610 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY und~rs{ood, but loss of pro{~in.bound Co.>+ into th~ intestinal Inet may contrjbut~."'" 6. Myopathies a. Hypoc:olc;.,mia may occur in a ..:ui~ty of «iujn~ ffiyop. thi .. k.g., tn ntpon t~any, enrtional rh . bdomyolysis, enduran""-tr~ enrci .., mon.min {oxirosis. postan.,{h~jc my",i!i" m aUl!ffl holSt syndrome, .typical myoglobinuria, malignant hypenhermi a, .nd .d,"ium myopathy) ......... '., h. The path0C"nesis of th. hypocalctffi", Jruly bt multifactorial (e.g., d=UKd intah, inn.asal renal Joss, incroasN mo",ment ofCa""' into damag«i <:tll., .nd profus. sweating). c. Swrating in hon". can crrat•• n alkalemia (hypochlo .. mic .Jk.:.Josisj that moy incr ...... c," binding to albumin and thus lower the [fCa'1 . Also, th.,e may bt di=t lOll of 0..'+ in 7. Acute tumor lysis syndrome a. PO, ,d.asal from [pM ""lis may bind Ca'+, and the c..>+.PO. romplnes deposit in {issues. b. PO, and lacm~ (producaj by hypoxic nroplmic cdl.) m"}' bind c,'+ in ",nal tubulu fluid and thu. inhibit tubulu resorption of C.l<. ,.. 8. Rumen overload (acut~ c.. bohydrat~ ruminal ~ngorgem~nt) a. Ruminant. cru.t ingest larg~ amount. of f~rm~ntable c.. bohyd.. t~ may d~dop .cut~ lactic xidosi., hypo""l~mia, '7.0umia, h}'Jl<'rpho.ph.t~mia , .nd hypocak" mi .. ,., b. Th~ p.thogen~.is of th~ hypoca.lctmia is not establi,htd but may .d. te to incrrasffi .....1 ucretion, btcau.. Ca2+ /lUy ~ bound to PO, or J.ctat~ in th~ tubular fluid. It may alo<> r~lat~ to d~position ofC.l<.PO, complu es in tissues. 9. H}'Jl<'radrrnorortici,m in dogs. a. H)1> """.t.'·' FREE CALCIUM (1Ca'; CO NC ENTRAT IO N I. Phy>iologic process~. (Ott Toul C,lcium Conctntration, Ket. I) II. Analytical conctpu of [fea'+] A. Tum. l. In dinical j:ugon and in /lUny vetorinary publicotionl, 1Ca2+ is oft~n calltd ionizrd cakium and is .bbr~i.ttd as iC., iC,'+, or ju.t C.l<. However, all calcium in lxxIy fluids is pr=nt in .n ioniztd sUt~ "ith~r as. f"", ion (i.e. , 1Ca"") or •• anion.bound ioniztd c,>+. Anion. cru.t bind Ca>+ includ~ prouin., citrau, .nd PO,. 2. In this book, those .bb"",iations ar~ u.td: calcium ion (Cr+), frtt calcium ion (1Ca"), and total calcium ion (tC.l<). Rc:f~rring to th~ unbound c," as fea'+ i. al", pr~~rrtd by other clinical ch~mists' and i•• nalogous to u.ing tT, and IT, for total and frtt thyroxin~, res~ivdy. B. Sampl~ l. B.cau.. of th~ pot~ntial changes in [1Ca1+] c;>u.td by pr~.nalytic;>1 variations, it is ~ .... ntial that pati~nt "'mpl.. and .. mples for d~t~rmination of r~f~ .. nct int~rval, ~ 11 1 CALCIUM, PHOSPHORUS, MAGNESIUM AND REGULATORY HORMONES 2. 3. 4. S. 611 coll«ta! . nd proce=d by using uniform .nd mingtnt crit~ri .. B=ust chang~. in [fC:."] may ~ rdata! to dirta'}' inuh and ponp.. ndial alb/osis, ... mples should ~ coll«ta! from dog. aft .. th~ dOl}! han bttn fana! ov~rnight."'" Similar changes would ~ a~tN in cats .nd horKS. ~pu bl~ ,.mpl.. includ~ ",rum, h~p",iniud whol~ blood, and hq,.,ini=:! pla!ma. HowrYer, s~al ronditions apply to .ll ... mples. For 300Curate ... ulH, blood ,.mpl.. mould ~ coll=a! .nd proa=d an""robically to ra!u~ changes in pH causa! by loss of CO, to air. Serum and pluma should ~ harvestN within I h of blood oolla:tion to ra!u,", potential errors CJ~ata! by l.ct",.. and H+ gtneration by blood ~lls. An ace .. amount of hq,.,in dlOuld k avoida!. Sample storagr a. [It:a''] is more nable in serum than in plasma or whole blood.'" HowrYer, silicone_separator tu~. should not ~ usa! beause the gel ronuins CT'".'" b. The [fC:.>+] did not change significantly in equine sera or hepariniud plasm. that was storN .naerobically for 4 d at room temperature.' Serum and plasma [It:a''] is repona! to ~ suble in glass tubes for I wk at 4 °C and for 6 wk >I - 20 "C.". c. [It:al<-] in heparinized whole blood i, reportN to ~ stable for 9 h . t 4-C. Samples should ~ prOct=d .n .. robically and quickly to avoid the effect of pH ch.nges on [fC:.>+]: raising the pH deer .... s.. [fC:.'+], whereas low..ing the pH incr ....... [fC:.'+] .' The [It:a>+] changes by . bout S % for ....ch 0.1 change in pH. Exposing ",ra to air by simply in""rting tu~, containing sera and .ir low~ra! the [fC:.l<-] (anrage 0.1 mmoUL lower) and raised th~ pH values (average, 0.35 higher).'" a. Albumin and other protein mola:ules h. ve neg. tively chargnl surface> that en . ble them to bind cations, including Cal<- and H+. Even though .n albumin molecule Ita. a mrplus of binding sites, changes in plasm. or strum pH alter the binding ofCa'"' to plasma or serum proteins. Thi. relationship i. illumatN in Fig.IIA. (I) A high .. pH incr .... se. the available negative-charge: site. on .lbumin and other proteins beau.., fewer H+ ions are presc:nt to bind them. This leads to incr .... sal protein_bound Ca" and thu.s da:rea.=l [It:a'1. Aerobic ... mple handling l....ds to e""pe of CO, to air, an incr...., in pH, .nd thu. a fahely low [It:a'']. (2) A lower pH indu~s the opposiu change. and thus increases [It:a'+] . Delaynl sample anal)"-i. will allow blood cell meubolic path,..,)" to produce H+. causing a falsely increasa! [fC:.'+] . b. Some ' ''.0)" . "ume a blood pH of7.4. Others normaliu: the fC:.2+ to pH 7.4 if the pH is .bnormal. If so, results should ~ reportN a! normalized or adjusta! [It:a'']. If a patient is either acidemic or alb/emic, a normaliud or adjustN [It:al<-] may be misl ....ding beau.. it does not represent the true [ft:.a>+] in th~ sample. Antico3J:ul. nu for whole blood and plasm. samples a. Hep",in is the only .ccepuble antico.guhnt for m........ ring [It:a'+] in blood or plasma ,.mpl.. , but heparin (a polpnion) does bind Ca>+. To minimize pr.. na_ l)'tical mor, the use of s~ial calcium_titrata! heparin tubes is rerommenda!. If the special tubes ar~ unavailable, then the heparin con~ntration should not exettd IS UlmL of blood"· 612 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY pH = 7.3 pH = 7.4 ProtoHn bound; 4 Ca>+ Free: 4 Ca'" pH = 7.5 Prol~ ., bound: 6 C,.:'<- Fr....:2 Ca2+ Fig. li A. Con""pru. l illwttotion ofth. d'fects of pl ..,ru Ol....um pH on tho [/Ca'1 . Th. dnwing iUustr>res th. effe.:. of the [l-I.] in plasma on the binding of Ca ' + to neg.tiy.,[y cb .~ pro",jn~ A similar efi'«1 oerun ..ith Mg'+. • At. pH of 704, thor. "'" Ca":ond H+ ions bound to pl .. m. or >t< four prot< . 1. m.n I... H· rnd roo", c."" bind to th. proteins. Tho binding ofC, "" to th. prot';ru decr ..... tbe [1Ca"]. In this illustmion .. itb • pH of 7.5. tho .amp!' a>nuins to.<> f= c.'. ions. ,.Ie... rH']. {I } Th~ [ft:~2+] may d«", ... from 5 % to 50 % by varying th~ amount of sodium h~parinate (rommonly callffi heparin) in the sampl~.lll In an a~riment. canine blood w>s rollected into .yringe; {to th~ I ml mark} that contained varying amounts ofh"J"lrin. The [fC.'+] in .ampl.. roll«tffi after heparin was forcibly apdled thr.. times ",..a5 ronsid ..ffi the most a<=m~ value. Th~ [ft:~J<.] d«reased by 0.2 mmoUl with about 0.1 mlof acess heparin (the amount in the .yrin~ dead 'pace ~nd needle). d«reased 0.5 mmolll with .bout 0.2 ml of excess heparin (heparin in the de~d 'pa.ce plus 0.1 ml in the syringe) , and dan,.,ed 0. 7 mmollL with about 0.35 ml of acess heparin (h~parin in the dead space plu, 0.25 ml in th~ syringe}.'" {2} Th~ CaL<- content in the calcium_titrated hep.rin .pproximates the exp«ted midpoint roncentration of fCa" in human plasnltffi heparin and Ca>+ in the pati~nt'. plasma, and thus appropriate ref..ence interval, must ~ availoble. {3} Th~ use of rinc heparinate was anempted as ~n anticoagulant, but th~ zinc interf..ed with th~ fC.2+ assay.''' b. Oth.. anticwgulant, that bind Ca>+ (EDTA, citme, and oxalat~) cannot ~ used in sampl.. for fCa>+ or tCa>+ =1'. 11 / CALCIUM, PHOSPHORUS, MAGNESIUM AND REGULATORY HORMONES 613 th . t th~ [1Ca""'] ~ reportffl with sp«i.l informatioll: typ< of sit< of oolla:tion, m<-] by using Ca><-_..l=in da:trod .. via pot~miom_ ~try. [f th~ illnrument also ronuim a pH da:tro r~mm~ndffl sampl~ , III. Abllormal oolla:ntration .. A. [1Cal+] i. tightly controllal by hormolles. Abllormaliti~s in [fe,"] illdicot< abnormal Ca""' r~gulatioll and may COUM dinical ';g'" or pathologic .vents. Although [tC.a""'] often p"rallds [fC,"] .lId i. a good sCI"ttlling t~st for di",rders of Ca'+ homeost ..is, [tC.a""'] does IIOt 1Ia:,,"sarily ",11= th. mor< "l""ant [1Ca""'] as illustralffl by the following conditio"" l. Hypocola:mia cou..d by hypoprot= in [tC. " ]. Ca'+ may ~ boulld to PO. or other , "io", 1I0t acretffl by th~ Urill" Y sY't<m." 4. [II. srudy inmlvillg 1633 sampl.. from dogs with chronic r~nal f.ilur~ or • variety of other disord~rs, th. sam. p<:rcem.gt of sampl.. h.d both illcreased [tCa"] and [fCa"] (19 %) and n<arly th~ "m~ p<:ra:mages of da:r<-] (27 % and 31 %, r~'pa;tivdy). Howo-v<:r, whell ollly th. dog, with chronic r~nal f.ilur< (n = 490) we,e colllid~'ffl, a h;gh~r p<:,cellUgt of dogs had incr~ased [tC.a""'] {22 %1 than inc,......i [fC,""'] (9 %), .lId .low<:r p<:rcemag. of dog, had da:reased [tCa'1 (19 % ) than da:"ased [fC,"] {36 % )." The dau from th~ ",tal failur. group suggest th . t . highe, p<:,a:ntag~ of [tC.><-] "'"a' th~ bound_Ca""' fraction (probably 1I0t prot~ill bound ) and th~ [tCa'+] does 1I0t ,dla:t changt' ill th~ [1Ca""'] ill collill~ chrollic ",tal milur •. 5. Hypc:nhyroid cot, may h,,,,,, d= II, 614 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY ""Jum. :> so % of th. i, .,{imatffl blood voJUmt in 3 h, Ih. a",,~ decr."", in [f<::.'+] w:os ".:" 0.3 mmoliL {1.2 mgldL).'" 8. [n hYJ>OC'l[""mic, 5- to l().d~Jd calves t .... t "'... acidotic btcau.. of dimh"", ch. [f<::.'+] w:as not decr• much:as Ih. [tCa'"'] prior to {'....Im. fit'. Both [tCr ] and [1Ca'"'] d=.:uffi .fm Huid th". py th.t .fflUad Ih..."",ily of acidosis.'" Th. .w:as rd.tionship, ktw«n blood pH and [fC~"l should ~ consid.ral wh.n acid.mic animal, ~'" .dmin;'{.fffl N.HCO, im,""v. nously, kaUst such th.",py can rapidly d<e,ra .. Ih. [fC."]."' B. Th. acid_b ... sucu, of.n anim:d can .ff= Ih. [ft:a>+] by Ih.tt processes: l. A> nplainal in Ih. all .a:t. II} dunging Ih. blood pH :of&ccs Ih. binding ofCa" to prot.ins and om., anions. Acid.mia ;ncreo.e; Ih. [1Ca""'] .nd alkaJ.mia d«r.~= Ih. [ft:a2+]. 2. Wh." {hu. is :m organic acidosis (~.g.. lactic acido.i, or kHoocido.i». th~ org:mic or a~toa~tat~} ill th~ pla.ma may billd Ca'+ to low~r th~ [Jt: .... ]. wh~",as collcur",nt .cid~mia would ",i.., th~ [fOr'+]. Also. th~ prese:n~ of th~ organic anion ill ",tal tubular fluid would d.-uu.., th~ tubular resorption of C ..... With these: muhipl~ f.ctors. th~ actual altuation in [Jt:a""'] may d~p"nd on th~ ~rity and duratioll of the organic acidosi,. 3. The blood pH :01'0 a!&cts pla,ma con~nt"'tions of PTH indq>tnd~ntly of [Jt:."]. The [iPTH ] illcreastd during ap"rimentaUy inducM mHabolic and mpir.otory acidoses if [Jt:a""'] Won kept COII!lant. 'lO Con""...,ly. the [iPTH] d«",astd during ""p"rimentalJy illduad metabolic .nd r~'piratory alkaloses if [fC:.'+] w:as kept const:mt. '" Thu,. ill clinical acid.base disorders. changes in [fC:.2+] could also be .d.ted to changes ill PTH. The altered [iPTH) in acid·ba,e disorders may refl«t its rol", in maint:lining .cid.base statu,. The PO, rdeastd from bolle act, :as a buff~r ill blood. and the PO, participate. in the ren:ol acre"tion of H+. Also. p""inemly increased PTH activity redu~. the r~nal acretion of HCO,-.ln iOIl IY. {~.g .• lactat~ Conceptual relationship-' of [tC."] .nd [Jt:a'1 . A. Thr"" major concepts must be r~memb",~d wh~1I imerpreting C.""' con""ntrations: l. The routille =)'1 of a ..,rum chemi,try profile me •• ure [tCo'1 . which include. thr"" Co'+ f"'ctions: fr"". proteill boulld •• nd bound to .nions other than proteins. 2. Th~ body r~culate' the [Jt:a""'] through th~ .ction, of "",~ral hormolle>. 3. s.:rum [Jt:r] v:olue. may be illaccume when blood i, not handled :maerobically. wh~n ",rum "",.ration from blood clot is delayed •• nd when ..,rum."'pa",tor tubes are u,ed. B. With these: COII~pt' ill mind. Fig. ll.5 illustr.ote, potenti:ol relationship" of the thIN Co.""' &actioll'. Major points th.t should be und.rstood include the following: l. An animal may han hYl""'.I~mia because: of hypoprot~inemia or hypoalbumin. om;". but th~ 'yst.m, that ..gulate [Jt:.-'+] are not d.f«tin {Fig. ll.5B}. 2. If .. rum prot~in con~nt"'tions ar~ WRI. then hypocahm;" typically indicates that tho [fC:.'+] i. d=eased {Fig. 11.5C}. 3. Mon hypercal~mic S>rllples ha"" all increased [Jt:.""] {Fig. ll50} 4. Normocal~m;" ill tho prese:n~ of hypoproteinemia mgge'lts:m incre>iffl [1Ca""') {Fig 11.5E}. 5. Th~ sevority of d«",asod [Jt:a'+] may 1I0t be evident from tho [tCa'+] if the COII~II· nations of the bound.Ca'+ fraction, .'" increasod {Fig. 11.5F} 111 CALCIUM, PHOS PH ORUS, MAGNESIUM AND REGULATORY HORMONES ~ 615 ". 13_ :g, 12_ E 11_ 710_ } ,. ~ 9_ J ItCa'+1 ReI. In!. IrCa"1 RO!. In!. ] ~ c , c..'·/k _ Fig. 11.5. Cono A. H.oJtlty oniaW: The [tC.'+] and ]/Go'.] .,. .. itbin resp«1i'" r.for.nco interv:o/,. B. Hypoprot';nemi. (bypooJbwninomi. ): Tb. bypocoJ=ni. i. c• .....! by . decr......! oonconttotion of prot';n_bound cr+. n,. ]RA'+] (the rogul.t«l roncontr.tion) .... itbin inrrt. ..mo< intervoJ. C. Primary hypop,"'byroidi,m, byp<>"it::lminwil D: n.. bypoc:dcemi. i. primarily caused by • d.cn...d [Ie....] boa .... of ';ther irude<Jwt< PTH or vitamin D activity. In thi.. .chom.tie =mp!'. the bound D. E. F. G. H. Co'+ rone 6. Th~ [ft:r+] is .ff=al by :dt~ral plasm. pH and th~ pres.llce of lIollprotrin :mion. in th~ plasma (Fig. 11.5G) 7. Excess h'l"'rin in a blood .. mpl~ am CUM a falsdyda;ro:asrd [1Ca""'] (Fig. I 1.5H). INORGANIC PHOSPHORUS {Pi} CONCENTRATION I. Phy>iologic process~. A. Pi rnsts ill diff~ ..nt form" deprllding on pH: H,PO. ..... H+ + H,PO.- ..... 2 H+ + HPO/· ..... 3 H+ + PO.'-. AI a pH of7.4, pralomirumt form, are H,PO.- alld HPO/- 616 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY Parathyroid glands amir.o __'Cl",~,_"-,,J_-. acid. ' + I [1,25-DHCC) I (rca"] + fl PTH] . t (PO, I Intestine +t (PTH) +1(1,25-DHCC] Kidneys + Bones PO, Blood + ,b + l [l,25-DHCC] ,..... --~, Cells '0, , :\' "Erythrocyte --'" :1 - , po;,. t di + t (PTH] Mammar glands Fig. 11.6. R.11tionshil" of PO, kin t [/Ct -] and i [1.25·DHOC]. • Conve";on of 15-HCC to 1,25_DHOC in kidneY' is catalyz«l by la-hydrozyl .... Th ••rtivity of [ahydroxyl ... is promot«l by 1. [/Ca'+] :and t PTH rnd inbibit<1ivity, • Insulin promot'" th. upt:th of PO, by ""lk Ho...... " ",J[ <4r=g."';U.uow PO, to =ap< from th. aIls and ~"', plum., • Dwing l:oc .. tion, • lug. >mount of PO, is ""creted vi. milk. • PO, p,,,,,,nt in plam" is mo,dy in to.<> forms (l-lPO,I- ...d H,PO,l, but th. "",.,wed phosphorus is ' =,.. in a 1:4 ratio, Unl= statM oth~rwi .. , all forms in thi, ch.pter ar< d..ignatM:as PO., About to % of Pi i, bound to cotionic prot~ins, 35 % is bound to nonprot~in cations, and 55 % is fr•• .' B, M.jor f:octors that d.. ermin~ .. rum [Pi] (Fig, I L6) L Iknal cl.~rance of PO, a, A major rout~ of PO, excretion is vi. kidneys in most mammals, so di"'rd~rs that cause decr .... sed GFR tend to ina ...... the ",rum [Pi], 11 / CALCIUM, PHOSPHORUS, MAGNESIUM AND REGULATORY HORMONES 2. 3. 4. 5. n. 617 b. PTH is • pot~nt phosph>!uric ~nt. In the pr=n~ of PTH, less of filt".d PO, is "K1rb..i in th~ distal tubul ... PTH ;octs throlli:h ~ cyclic . denmine monopholphat~ m~...,ng" syst~m to inhibit th~ cotrallSport of Na+ and PO,. Absorption of PO, in imestines a. If ~nim.h are nting, th~n th.y wiU typically oonsum. lu&" qu . miti .. of PO, th>! are .bsorb.d in im.scines. b. Absorption is mhan=l by 1,25_DHCC, either directly or through Co'"' complexes. Resorption from bon~: PTH stimulot.. osta>crt.. l"pid dfect} ~nd osta>dam {ddaya! effect} to rd ...", PO, from bon •. Becaus< of th~ pot~m phosph.turic action of PTH, th~ rd~= of PO, d",. not increa.. the [Pi] .. long .. "n.1 function it adeqUsphoryLnion n.«l PO, &om ECF. b. Insulin promow PO, .mry into "",lis by enhancing th~ movem~m of glucose imo "",lis. On"", in edls, gluoos<: i. phosphorylat.d to em~r glycolytic or glyrog.n symhesis pathways. Th. binding of fIN POl' to glurosr or oth~r organic mola::ul.. redu"",. the [CF con~ntntion of IT.., PO,>- .nd aUows mo" PO, to ~m~r "",J]s from th~ ECF. Animal ~ a. GH i. r~porta! to bt largely "'ponsibl., for th. im:r ...sed [Pi] in young. growing .nimals. GH increases the """I tubulor "sorption ofPO,.m b. s.rum [Pi] i. typically grnt~r in young, growing .nimal, than in th. adult animals u..d to mablish most "f,,~n"'" im~rvalsY [n pups (< 12 wk), .. rum [Pi] ref....:n"", imerval, w.".. about 5.7- IO.B mg/dL, wh~,.., .. . dult dog imervah w~re about 2.5- 5.5 mg/dL In kitt~'" {4-6 wk to 20-24 wk}, .. rum [Pi] "f".n"", intervah w". ~bout 5.0- 10.0 mgIdL, wh,,~ •• adult cot imervah w". about I.B-6.4 mg/dL A corresponding ~_rdated diff~"n"'" is that th., .. young ~nimals h~"" g=t Analytical con"",pu of [Pi] A. Sampk I. s"rum is the preferra! sample, but plasma may bt used. 2. [n vitro h~molysis or dd.ynl "moval of ... rum from. blood dot may iner.= .. rum [Pi] btoms< PO, is libtrat.d imo .. rum from ~rythrocytes (cytoplasmic PO, and membran., PO, rd ...ed from phospholipid.). B. AssaY' I. [n most oommon assaY', PO, ,..,~ct. with :nnmonium molybd.te to form a colored complex th.t is m...sured by photometry. 2. [n som~ systems, oonjugated bilirubin m. y im"f~", with th~ =y to produc< fal ..ly decr .....-d results rpSludo-h}poplmpm.mnu.},'>/< wh.r~.. , in oth~r =y s)"ltems, bilirubin has no .ffect or may produ"", faj ..ly incr.~..-d values. 3. [n sprctrophotometric . .. .". th. t us< the mongly acidic ammonium molybdat. ",~m, th~ ""&,,m con p=ipim~ immunoglobulin. to produc< a turbidity that 618 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY rn;ulcs in a f:d,dy incrnsN [Pi] by 1- 2 mmoUL r.. 3-6 mgldLI.''' This pos;t;"" int~rf~ .. nct has bttn Sttll in samp]" with monoclonal gammop. thi .. despit~ ... ~ modifianions mrn ., th~ addition of d<:{"'g~nt to pr~nnt prot~in pr~cipita{ion. C. Method, of «pressing coII"'ntnt;ons l. [Pi] is often r.f.,rffl to ., th. phosphorus ronctntration. More .ccuratdy, [Pi] js th. ""rum PO. reponffi as pho'phorm in mgldL It is not ,eporr..,j .. a PO, conctnlr:l_ tion in mgldL kcaust the .mount of •• ch form of PO. chang., with pH, .nd •• ch form has a diffuem ,dat;", mol«uJ.r mass (M,). Thi, problem it onrrome by rq>Oning phosphat. in SI units of mmollL. 2. Rehtion,hip bttWO'tIl [POJ and [Pi] a. I rumo] of H,PO.- weighs 97 mg. [n I mmnl ofH,PO.- th ... are 31 rug of P. Th...fo .. , a solution of 97 mgldL ofH,PO,- comains 31 mgldL of P. b. I mmol of HPO,J.- w~iChs 96 mg. In I mmol ofHPO,>- th~ .. are 31 mg ofP. TIm. fo .. , a solution of96 mgldL of HPO,'"" contains 31 mgldL ofP. c. B~u .. of th. equilibrium among th~ diff~"'nt PO, mola;ules at a pH of 7.4, I mmol of PO, a""rages .bout 96.2 mgldL D. Unit con""",ion for [Pi]: mVdL X 0.3229 = mmoliL (S I unit, n..,..est 0.05 mmoIIL)" [[I. H~rphosphat~mia rr.bl~ 1l.4} A. D«..~..ffl utinary PO, ac..-tion. l. Oisorde.. that d=rasr GFR {Itt p.. renal, rrnal, and postrenal :lWtrmias in Chapter 8}: Hyperphosph . tem;" oa:urs ba:ausc: PO, is not filtered oodeqlL>tdy from plmm. Tablc 11.4. Oi"""".. and condition. that cause hrperphosphatcmia D=rasrd urinary PO, excrrtion 'Oa;",asal GFR {S«: pmenat ... nal, .nd post",nal azotemw in Ch ' pter 8} Urinary bladder rupture or urin~ I~akagc: into tissues Deem=' [PTH] or activity (hypoparathyroidisnt) Acromrgaly Inc",ased PO, absorption from intestine Phosphat~ ~nrm. or in~ion of phosphatr urinary acidifier Inc",asal vitamin 0 (S«: Tablr 1l.2) lochrmic intestinall.. ions (may~ also mift from ICF to ECF) Dirt with ~ low Cal+: PO, ratio (rarc) Shift of PO. from ICF to ECF Myopathies: endurance: rid., in horses, aertional rh. bdomyolysis, malignant h~rth..mi. Acut~ tumor Iy,is syndromr Other or unknown mechanisms Hypc:nhyroidism in em uctic . cidoois Hypc:roodrenorortici.m in dogs • A rel. tively common dioe.... or condition Noto: n,. [Pi ] in gro.. ing ID1ffim:d.s m. y I>. up to 3 mgldl bigher tb1tl Pi ",!'.ron", intrrvili for .dul", of tl>. sprci<>. In vitro hemolpis or d.e1' J"'d r<mov.J of .. rum OJ plau= from blood .<1ffipL.s ..ill .JJo.. PO, from erythrocyto, to incr..... the IPi] in the .. rum OJ plam,.. Also" f.ohely incr. found in umpL.s with byprrbiJirubinrmi. or moooclon.J g:unmopathirs. 11 / CALCIUM, PHOSPHORUS, MAGNESIUM AND REGULATORY HORMONES 619 Th~ m"l:nitud~ unds to par. nd th. =ricy of .wt~mi a in dogs, can, and canl., but it Jruly not in hor~, 2, Uriruuy budder ruptu'" or I.abg. of urine into tissu." Hyperphosphoumia remits from decrnsed urimry excrHion of PO, from the body, 3, Dec",asal [iPTH] or "",tivity (primacy hypop.rathyroidism or pseudo_ hypoparathyroidi,m) {s.., Total Calcium Concentration, sect, [V,B}, 4, Acromegaly: GH increa= tubular PO, reKlrption,''' B, Incr~a.ed PO, absorption from intestine l, Phosphote en.m.,><.m or ingtnion of a phmphou uri""ry acidifier"" 2, [nc",ased vitamin D a, Hypervitaminmi. D in rumimnn: perhops inc",ased int.stinal absorption of PO, or incr~aK IILA2}, [nt~stinalle;io", r"'luiring int<Sti",,1 resection: Devitalizal inte;tinal muco .. allow. PO, to enter plarma (and peritoneal Huid), Also, shifting of PO, from ICF to ECF may ~ involwd, ,,, 4, Diets with low Co": PO, ratio C. Shift of PO, from [CF to ECF L Myopathie; (.ndurance rid~s in hor=, ex.rtional rhobdomyolysis, .nd malig""nt hyperth.rmia): "'I....., of PO, from dam~ musd~ fi~"'oo.-,·,,,,,· 2, Acute tumor Iy.is syndrome: rd .... of PO, from necrotic n""punic cdls'" D, Oth.. or unknown mechanism, L Hypc:nhyroidism in em: Thyroxin. may promote ost<'ClC!astic .ctivity to caUK rd •• .., ofC? .nd PO, from bon~, Hypc:nhyroid em may han hyperphosphot ... mi." dec=sN [ft:.l<-] , .nd an increaK 3, [V, Hypophosphaumia (Table ll5) A, Incr~.sffl urinary PO, excretion L Prolongc:d diumis: uss filtered PO, is re;orbed, 2, [nc",ased [iPTH] or [PTHrp] (T.bl. lU), 3, F. nconi syndrom~: Animals with Fannlni IJndrom( have " proximal tubular d.fect that deer ...... tubular re;orption of glUCOK, amino a,ids, and PO" Some people FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY '211 Table 11 .5. Di""a..es and condition. thai cause hypophosphatemia inc,",asM urinary PO. """..,ion Prolonged diuresis inc..=<:! PTH or PTHrp ""tivily {Itt Table I1.2J FaDoon; syndrome (dogs) D'""....«I intestinal PO, abwrption • Prolonged .norexia or PO. _ddicicnt diet PO,_binding "l:cn[. Hypovic.minos;s D Intestinal m.J.bsorption Shift of PO, from ECF 10 lCF Hypuinsulinism {ondogcnou. or exogenou.} Glu= inhts;on Respiratory alkalosj, Dd'r<:lin mobila.tion of PO, from bon. Postp.nuricm fU',sis (milk f~",) and ffiamp. d.a ..,,,d , .... lts. =Iricr Ih. {urn Flln('(Jni ryndromt to inherited or rongroilal form., wh.rns nth." indude a.cquiral di50rd~",. Th~ d=nlffi ",.orption of PO. in dogs with F.nconi syndrome IIUy callS< hypophosphat~mia and phosphaturia in 5Om~ dogs''"'''''' but not oth~ ...'" B. D«reo....d intestinal PO. ab50rption I. Prolongal morn", or a PO,..ddici~nt diet: less dietary PO, availabl~ for absorption 2. PO, _binding 'I:"nts: int.. f~"'n"" with PO, abKlrption 3. HypovitaminOlis D: .b",n"" of a promour of PO, .bsorption 4. Intestinal IIUlabsorption: dec ..a=' PO, absorption C. Shift of PO, from ECF to ICF l. Hyperinsulinism {endogenous or nogenous} a. Insulin promote. ~ntry of PO, into ""lis to phosphorylate the gluco .. that ente" the cdls. b. High inmlin activity could remit from imulin injections •• n insulin_=:ming neoplasm, or in response: to g1uoo .. infusion. 2. Gluoos<: infiuion (in hor...) lal to a hypophosphat~mia.''' The hypophosphatemia m.y be due to inc",a=' .. nal en;mion of PO. sn:ondary to solute diu ...is and also due to insulin_indue<:d movement of PO, from ECF to ICF KCOndary to g1uoo"" uptake. 3. Respiratory alkaJosil: Experimentally induced r.. piratory alkalosi. caused hypopho ... phatemia and inc",..,al mu.de g1ycolysi •.''' Phosphofructokin... is the rate_limiting enzyme in g1ycolysi., and alkalemia nimulat .. in activity. Thus, alkalemia promotes PO, movement into erythrocytes becau.. of the inere ..al phosphorylation during a=leratffl g1yoolpis. 11 1 CALCIUM, PHOSPHORUS, MAGNESIUM AND REGULATORY HORMONES D. 621 D~f«ti,.., mobilization of PO, from bone I. Postp:muri~m pare,i, (milk f~,..,r): Cow. cannot mobilize: suffici~m Ca'+ and PO, from bones to r~plo"" th~ c,'+ and PO, th~t ~'" lost in milk. 2. &hmpsia in bitch.s: the .. m~ m«hani,m a, milk f","~r E. Oth~r or unknown m«hani,m, I. Equin~ .. nal di, ...", (f.ilu .. ): Hypophosphat~mia may b.: 5ttn in "1I5<::I roncum:m h~rcal""mia. ,,. Howe,..,r, it i, not a oonsi,t~m finding and th~ p.thogen.,i, i, not known. 2. H.lothane .n.sth~,ia in hOlSe,,'" Mt~r prolongffi halothan~ anesth.,ia (12 h), ,ix horses h. d mild hYl"'rphosphaumia for the fi .. t hour, but then hypophOJph>t~mia devdopffl and I"'rsistal for 3-4 d. Th~ pathog<:n~,i, of th~ hypopho,phatemia wat not determined. F. P.. udo_hypopho'ph .t~mia: k noted in th~ .nalytical sc:aion (Inor-g.nic Pho'phorus Con""mration, ",ct. 11) the pr=n"" of ronjugated hYl"'rbilirubinemi. can ..",It in a f.lsely deer ...,ed [Pi]. TOTAL MAGNESIUM (tMg"") CONCENTRATION I. Phy,iologic process.. A. Serum or plasJrul magn .. ium i, di,tributed imo thr.., major fraction •. All Mg" in iNldy fluid; is ioniud, but some is &.., .nd 10m. i, bound to .nionic mol«ules. I. F,u magtwium ifMi'): About 55 % of tMgl+ i, IMgl+ present at fr.., ions in pl"'ma H,O. IMg'+ is the portion of tMg'+ that i, hormoruolly r.guh tal and oontribut.. to pathologic nat ... 2. Bound magn ..ium a. About 30 % of tMe i, bound to negativdy chargc:d ,it.. on prot~ins (albumin .nd globulins). b. About 15 % of tMg>+ is bound to anions such '" citrate and PO,. B. Mg>+ is loc;;otal in bon.. (about 60 %), in 10ft ti ....... (about 38 %), ~nd in th~ extracellular Huid. including blood (1 - 2 %). EJt""pt in C>ttl~, th. [tM g"] in erythro_ cytes is greater th. n in pla,m. (or ",rum). e. M.jor factors that det..min~ th....rum [tMg'1 I. Hypoproteinemia deer ...... the amount of bound Mg>+ and thu, may cau", hypo_ magnesemia (d«r..,...,d [tMg'+] ). 2. Ab,orption of Mg'+ in the gastrointestinal tract'O a. In ruminanu. Me is absorbed by the rum~n (and mayb.: int<::s{ine) in ~ process linkal to N.·_ K·_adenOJin~ cripho,phause. b. In monogastric .nim.l" Me absorption occurs in the di,t:d ,mall int<::s{in< :md colon ~nd i, enhanced by vitamin D . nd inhibited by high dietary c,'+ or PO,. 3. Excretion a. Mg'+ in feces may r~present unabsorbed dietary Mg" but also lost of endog<:nom Mg'+. b. Kidney> ( I) Me not bound to protein, pa"'" fr..,ly through tho glomerulor filtration barrier. Thu" a deerea.ed GFR can ralu"" r~nal acr.tion of Me and cau .. hypermagn ... mia. {2} If the .mount of Mg'+ ent~ring the proxiJrulI tubules excet:d, the tubular resorpti"" capacity, e],,~, Mg" i, ncr~ted. Osmotic diur.,i, .nd loop diuretics incr ...", ",ruol excretion ofMg2+. 622 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY {3} If Mg'+ is prestnt in bovi,,< urin., th.n th~ row is !lot exp«lffl to ~ hypomagn=mic b«:au.. Ih. pr=n~ ofMg'+ typically indicates th.t Ih. transport maximum ru.. oon exettdai Thus, finding Mg'"' ill urin~ indicates Ih., • cow doel not Ita"" hyponugn=mic tetany (gr:w trlany) unless cher~ i. a def«t ill Ih. r=>rption of Mg>'". c. Mammary gland during lactation: The ratio of milk [{Mg~ to .. rum [c.\1g'1 is about S. D. Hormonal regulation l. Antidiurffic hormone {arginine vasop .... in}. PTH, glucagon, calcitonin, and p. adrenergic agonist, can stimulate Mg" .b50rption in the cortical thick """'nding limb of the loop of He"l •. " 2 PTH all illueaK .. rum [eMg1<-] by illCIe",ing immiruol Mg'+ absorption and Me incrra,ing resorption in rrllal tubul .. and bon~.'" 3. Administration of 1,25_DHCC raluces the plaUIta [IMe], ~rhaJ>5 KCOlldarily to d<errasN I'TH .Clivity.'" 4. Thytoxin~ t~nds to dn:reast th~ pl:lSJIla [tMg"] by increasing Mg'"' exc.. tion in urin~ and ~ ,., 5. Aldost~ron~ promotes incr~aIM f=l and urinary Mg'+ excretion. Exptrim.nully. aldoneron~ infusion> dn:rras.d ruminal Mg'"' .bsorption.'" CHcreaset! .ldost~rone activity inc",_s the ..,rum [tMC1<-]. [I. Analytical conet~) should not be used in samples for [tMg'"'] assays. 3. Exeted into th~ .. rum from th~ .rythrocyres. In c;ml~, plum" and ~rythrocyte oone Ill. Hyperm"l:n • ..,mia (Table 11.6) A. D«r~"sed urinary exc"'tion: renal failur~ and other causes of dn:reas.d GFR B. Shift of Mg" from ICF to ECF: ExcqJt in canle, in vitro hemolysis. acti"" in vivo hemolysis, or th~ dday.d removal of ..,rum from a blood clot will allow erythrocyu MC'"' to be .dd.d to th. strum cono::ntr>tion. 11 / CALCIUM, PHOSPHORUS, MAGNESIUM AND REGULATORY HORMONES 623 Table 11 .6. Di""ascs or conditions Ihal cause hypcrn.agncoemia D,""rrasM uri""ry exc"lion '(k",,1 f. ilure and other GlUst. of d,""",astd GFR Shift of fMg'+ from ICF 10 ECF Active in vivo hemolysi. Inc .... M [PTH ] Milk fever Inae"M intestinal absorption of Mg" wilhout incrrasM PTH or PTHrp MgO, Mg(O H)" or .imilar :mlacids or calhartics (catde) MgSO, (horses) Olher mechanisms Excess intrannous infusion of Me • A 1.t. .i""ly common dis< ... 01 condition Not<: P.. udo·hyp<mugnn<m .. m.y 0C C. Increastd intestinal .bsorption of Mg'+ without inc,.astd PTH l. Ex~ .. 0",1 .dmininmion ofMgO (magne;ium oxide} :md Mg{OH), (m:ognesium hydroxide) to canle'" 2, Ex~ .. 0",1 .dmininr. tion ofMgSO. (magne;ium sulfate) 10 hones'" D, Other or unknown m,""h:mism, I, Milk fever: inc",astd PTH m"}' indu~ inae;;oKd renal resorplion of Mg'+, leading to hypcrmagnesemia.'" 2, A mn,ient increase in [cM g'1 :md [fMg"] occuu in Gltde po"parlUm, The ina....., may ~ rdalM to incroascd PTH . ctivity causing increasM ",nal r=>rption of Mgl<:md incre. Kd Mg" mobilization from bone,' '' 3, Ex~ .. intrannous infusion of Mg" 4, In people, Addison's discalc .nd hypothyroidi,m may c;m,st hypcrmagnesemia. Similar findings ha"" nOi bttn "'POrlM in domestic mammal., IV, Hypomagnesemi. (Table 1l.7) A, Hypoproteinemia: d,""",astd Me bound 10 protei", {d,""",astd [IMe]}, B, In.dequate ruminal or intestinal absorption of Mg"" l. Prolonged .norai. or poor feed intake (especially in l"",tating cotde): In one "udy that involved the de . th of 55 dairy rows, markM hypomagnesemia was pruent wilh clinical t"any {acq>1 in one row},'" For tC'lany to ~ ""OCi.tM with hypomagne",. mi., concurrent hypocal"",mia may ~ required, 2, Calv.. on a whole milk diC'l can dcvdop hypomagnesemia beaUst Ihe dietary Me requi .. ment of growing cal.",. is about 50 % gr....ler lhan the amount of Mg'"' in milk.'· 3, Grass ICtany in cotde'''''' a, The hypom:ognesemia i, considerM 10 ..suit from decreased ruminal ab""rption of Mg'"', but there .'" multiple thcori .. for the .bsorption defn:l, b, A lush grass diet is high in PO, .nd K+ content .nd low in Mg" and Na+, In rome lIudies, Ihe combination of high K'" and low Mg" was Ihe key factor th.t led to hypomagnesemia. Olh.r contributing faCIo" in ""me COWl! include high nilro~n fo~, increa!ed diC'lary org. nic anio"" and high aluminum fo~, 624 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY T able 11 .7. Di""a..es or conditions thai cause h ypomagnesemia • Hypoprol~ill~mia lllad«iuat~ rumiruol or intes{inal.b,orplion ofMgl+ • Prolongnl ano.aia or poor fN+ 'Osmotic dim • .i, 'K.rollu,;" Olh" or unknown mMan;,ms Bliml ked. poi'iOning in hOrK' Lacution tetany in Shed,md Jrulrc, • A rel. ti",[y common dioe ... 01 a",dition c. Th. roJes of "'do,lcronc in the proc= :or. nol cnablishM, but "'do,lcronc might ~ Ih. link ~tw""n K+ .nd Mg""'. Mg'" .bw'ption Jruly bt ralo=:! by factors ind.pend.1It of aldo,terone, such as the low N.': K+ ratio of rumen romcnu., t> d. Grass t"' :my is difficult 10 di>gllost postmon.m b«:.u"" of th. Jack of gross or micro,copic J..ions. A.ho, Ih. p"s[moncm pl:asm •• amples cannot bt u,rd for dct+ depletion. and the postmomm [tMg"] in tho .. fluids is mOf< S{abl~ than in pl"'m • . {I} If th~ .queous or vitreom fluids ~r~ to b. .n:dyz.al, they should b. antri_ fuge=y, a [tMg>+] <0.55 mmoUL ( 1.3 mgldL) was a.<s<> 10 mmoUL, th~n th"e ha. bttn too much post_ mon~m ch :m~ to consid~r the [tMg'+] a r.lia ble indicator of ant~mon~m ronantrations.'''' {3} Th~ [tMg'+] in aquams humor ~ S{abl~ up to 24 h postmonem in bovine '}'<S, :md, with on~ >=y, a [tM g'"'] < 0.25 mmoUL (0.6 mgldl) was associat«i with .nt~mortem hypomagn=mic t~tany.'''' 4. Ent~ric di ..~!eS a. Dogs with ~nt~ric di ....... may drvdop hypom:ogn~,.mia b.cau.. of increasal f.cal loss or concurf. e. Excess uri nary excr l".'" "l 11 / CALCIUM, PHOSPHORUS, MAGNESIUM AND REGULATORY HORMONES 625 D. Othu or unknown maohanism, l. Blister b.-trJe poisoning in hors.,,"'''' Th. pathogen.. is h.., not bttn documented, but possibly a lOll of protein.bound Me =y contribute. 2. Lactation reuny in Sh ... land mar .." 3. EndoroIemia in hor= au= hypom3l:ne",mia. Th. daor.,. .. is aused by the shifting of Mg""' into ""lis .nd might ~ medi ..ed by v:lSOp .... in or insulin.''' FREE MAGNES[UM (fMg""') CO NCENTRATION P"""""" {Itt Total M3l:n.. ium Con""ntration, Ket. n I. Physiologic [I. Analytical con""p" of [fMg'1 A. Turns: Most of th. sUrem.nU in the fea""' =cion also .pply to rerms used for Mg'+ concentntions. For coruistency, thes. abbrevi .. ion. are used: m>gnesium ion {Mg'"'}, fr.., magnesium ion (fMe), and toral magnesium ion {tMg'"'}. In clinical jargon .nd in =ny veterinary publiatiom, fMg'+ i. often called i~niud ma~'ium and it abbrev:i. ated .., iMg, iMg'"', or just Mg'"', and thm there is a pot.ntial for mitoommuniation. B. Sample: Mo. Ill. Abnormal con""ntrations. A. DOOrd." of tMg""' homeosu,is =y ~ disorders offMgl<- homeostasis or dOOrd." aff=ing .niom that bind /Mg""'. Mo .. studies a.. n=:l.d to esubli,h the pathologic coDSnju.n"". of altered [fMg',. B. A few published studies have: explored the potential ",lue of measuring [fMg""']."".'" At thi, tim., the many factors influ.ncing [/M g""] limit th. u..ful int.rp ..... ation of altered cono::ntntions. l. [n horses, both increased [fMe ] and daor.,.sed [/M g'+] wer. common prior to and after colic mrgery, but the reasons for the variations Wl.:re not d.... rmined." During exp"rimenul .ndotox.mia, both [tMe] .nd [fMg'+] Wl.:re daoreased. Th. changes occurred quickly and were thought to ~ due to movement of Mg"" nom plasma to cdl,.''' 2. Serum [/Mgl<-] re=in.d unchanged when ho .... Wl.:re fed a reduo::d.Mg'+ di ... even though there was daoreased renal Mg'"' exc ..... ion and daoreased Me cont.nt in muscl ... '>7 3. Both [tMe] and [fMg'+] in .. ra decre..,ed in ats fed Mg""..ddicient diets.'" 4. [n ats with nonketotic di . bet.. m.llirus or hlOtic dia~tes meUitus, the [/Mg'"'] w.., decreased more fr"'luendy th an the [tMe].'" Ho~r, dogs with di abetic kero.cidosi, rended to have a greatu [fMe] than did dogs with uncomplicat.d dia~tes mdliru. or healthy dogs: the reason for the greatu [fMg'1 was not '" FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY determina!. '" A> • group, Ih. [fMg'+] in the dog. with uncomplicatal di.btu. mdliru, was much more v.ri.ble (.ome low and some high) Ih:m th. [fMg"] in the h""'thy dogs. IMMUNOREACTNE PARATHYROID HORMONE (iPTH) CONCENTRATION I. Phpiologic process." A. PTH is. polypqltid. hormone (84 .mino acid.) produ=l by p.rathyroid gunds and inactivaud or dq;radffi by kidn~ .nd li~r. PTH is = ..Iffl by parathyroid gland, in respon .. to • danaMd [1Ca'"'] in blood, wh.rra, vil:omin D .nd an inc=std [ft:i'+] inhibit synthuis of PTH. l. PTH sa:,etion is priJrulrily mediated by £<::.'+ binding to th. par.uhyroid gland chief "",U calcium...,n,ing r~ptor {CaSRJ. This =tplor is linkal to a .tri.. of intra_ "",Uul" .igna/ing pathways th . t inhibit PTH sn:retion.'SJ,'" Mutations in the CaSR gtne <"'-II au .. increastd or d«r~ recq>lor activity and hypocalctffi", or hyp= in PTH. Alblo, .. {met;obolic and respiratory} inhibit PTH =<O,l>I B. Prim.ry target org.ns: bon~, inte:stin~, and kidn~y l. PTH promot~. incr .... 'M Ca>+ absorption by int~stin~ (in th~ pr~"'n"" of vitamin D) and incr.... snl ruorption in renal tubul~,. 2. PTH promot~. mobilization of C.'" .nd PO. from bon~ . 3. PTH promot~. ",nal PO, acretion by inhibiting resorption of PO, by tubules (. pot~nt pho,phaturic action). C. Th~ net df~ct of PTH activity is to inc",,,,,, plasma [ICa"] by Ca>+ mobiliution .nd d""",.. ~ plasma [Pi) by promoting phosphaturia. [I. Analytical oonctpu for iPTH assay. A. RIAs d.-vdopM for human PTH h:IV~ suflici~nt .p«i~, cross-r~.ctivity to b. valid fur domestic m:munab." l. iPTH v:dues g~n">IM by RIAl for intoct PTH or N_t .. min:d fragm~nt' rorrd>l~ wdl with a~M biologic PTH activity. Ba:aUst som~ immunoauays may r.... ct with preproparathyroid hormon~ . propa.. thyroid hormon~, intact PTH, or. PTH fragm~nt, th~ an:dyr~ of these .uays con rolJ""tivdy b. callM immunomutiw PTH (iPTH ). Only if th~ =y wa, 100 % .p«ific for intoct PTH v.ould [PTH) ~ual [iPTH). 2. iPTH ....,... t~nd to b.limitM to ~ndocrinology and larg<'r "&,,,nct labo..tori~•. 3. iPTH .u.,... do not d 11 / CALCIUM, PHOSPHORUS, MAGNESIUM AND REGULATORY HORMONES 627 ~Ust PTH is .""""ptibl~ to protaJlysis, the pr~fw.d 5ampl~ for PTH i. EDTA_ plasma that conuins a prouast inhibitor {.protinin}. If the sample is not analyzed on the day of collection. some investig.tor< write that plmna or .. rum without aprotinin i. au:q>uble if the .ample remaim frozen during .torage or transit.'" In • subility study at 21 'C and 37 'C, the .strum [iPTH] remain.d nabl. for 2 d with two prot"'" inhibito", {Pefabloc SC and 4_[2-..minOHhyl]_berrz.ene D. Iner~ Ill. .. rum or plasm. [iPTH ] (Table 11.8) A. Mon di",rde", that cause incrras.d serum or pla,ma [iPTH) are due to exces!i"" production of PTH by neoplastic par.thyroid cdt. or by hyperplastic p'r.lthyroid ceUs {>=>ndary to decrnsed [ft:.""]}. B. Howt""r, many dog> with hyperra1cemia due to primary hyperpamhyroidism do not have.n increas.d [iPTH). Insttld, the [iPTH) is WRJ but inappropriatdy high in relation to an incr~ [fC,>+), thus reHecting a defecti"" neg.ti"" fttdbad: on PTH secretion C. In one study, many dogs with hyperadrenocorticiun had unexplain.d iner.. ",s in plasma [iPTH) and concurrent normoca1cemia.'" IV. Ckcrea..d """m or plasma [iPTH ) (Table 11.9) Table ll .8 . DiseUC5 or conditions that cau.. increased fir T H) Increas.d PTH production by naJplastic cdl. NaJplastic pamhyroid gland (primary hyperpamhyroidism) Multipl~ endocrine neoplasia (type I or 2A) Increased PTH production by hyperplastic par.lthyroid glands (idiopathic or >=>ndary hyperparathyroidism) 'Chronic ren.! dis ..... Diet with a low Co... : PO, ratio Hypocalcemic di",rd ... (with a decreas.d [fC,1+)), other than hypoparathyroidism Pstudo_hypoparathyroidism {decrrased PTH receptor re.pomivene ..} Hyper.drenocortici.m in dogs • A ,.ll1;""ly rommon di..... or ronditi"" Table 11.9. Diseas.. or conditions that cause decn:ued fir T H] Decreased PTH production due to damaged or removed parathyroid glands (hypoparathyroidism) Decrrased PTH production due to inhibition Hyperviuminosis D Hypercalcemic di",rde .. {with an inc ...... d [ft:a'+]} except primary hyperparathyroidism Hypom:ognesemia due to Mg" de ple"lion 628 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY PARATHYROID HORMONE-RElATED PROTEIN (PTHrp) CONCENTRATION I. Phpiologic proa=. A. PTHrp promot~' C~l<- b:dance in th~ fttul and modulates cartilage. and bon~ d""Jopmenl. Aft~r birth, ch~ comrol of c,'+ balance 'wi{ch~. from PTHrp to PTH.2J B. PTHrp i, produced by many cells in f~ust. and .duJu, but plo,m. [PTHrp] j, vuy low in h~"'{hy .dult•. n. Analytical oonctpu A. Sample I. B.caus. of rapid prot~oJysj, of PTHrp, it has oon =mm.ndal that blood b. coll<'CIffl imo EDTA tubt. with .n added prot..,..., inhibitor {aprotinin or leu_ p"ptin}. Sqmat«i plasm. is .n:dyz.nl fresh or shipp«! froun to .. ftr~nce laboratories. '" 2. Som. laboratories, how"""., ""lues! ",rum 5ampJ.. bt shipp«! on ice. B. Assays designed to meamre human [PTHrp] (N wminal) are usa! for m.... suring conine [PTHrp]; how<'nr, the ,am.. """1 is not valid for equine [PTHrp]." 0"" human PTHrp """y at"" con bt Ulffl for m~asuring fdin. [PTHrp]." Ill. IncreaKd [PTHrp] in ",rum A. Som~ nwpl:.sms (espa;i:dly lymphomas .nd c;ncinomas) can produu ~nough PTHrp to cau .. an ina.... in ",rum [pTHrp]; th~ incr~'1ffl PTHrp .ctivity can muh in a hy~rcalcrmi •. Th~r~:dso a", ra", .. of inc",=<:! [PTHrp] in dogs with granulo_ m>lous di ..a=. B. Mor. information about th .... hypucalcrmic disord.n i, pres.nt«i in th. Total Calcium Concrntr>tion =tion of this chapt~r. ""m VITAMIN D CONCENTRATION I. Phy>iologic procesm A. Formation of vitamin D l. Chol.n~rol i. conw"N to 7-dehydrochol"'~rol. which i. conv.rtN by ultraviolff light to cholff>llcif.rol (vitamin D,). Vitamin D, can .1.0 bt of dillcif~roJ. 11 1 CALCIUM, PHOSPHORUS, MAGNESIUM AND REGULATORY HORMONES 629 6. PTH .ctivityand [C.l<-] aff«t th~ formation of 1,25_DHCC, but [Ca' +] apJ><"M'to b. th~ IIUjor factor controlling 1,25_DHCC rona:ntmions. On~ study indicotM that wh~n th~re is increastd PTH activity, low [tC.l<-] I.oos to .n incr~asaI 1,25DHCC ron""ntmion. wh~= high [tC,l+] lnds to a d= .......d 1,25_DHCC rona:ntration.' 6T ln th.t study, th~ [tCr] v.as m.... surM, but th~ alt~rM [tCal+] probably rdl«tM a chanc~ in th~ [£Ca'1 . B. Action! of vitamin D in dogs, cots, .nd cotd~. (Roles of viumin D in hones in rq:ulotion of [£Ca ' 1 appt.r to b. minor) .' I. Viumin D promo". imestinal upta~ ofC.l<- by nimulating the formnion of a Ca""'_binding pro"in (calbindin) in th~ mucosal q>ithdial ""Us. It al"" stimulates abKlrption of PO,. 2. Viumin D promot.. c,""' and PO, lib.ration from bon~ by seimuloting ostax:lastic a.ctivity. 3. Viumin D promot.. r~""rption of £Ca2+ by proximal .. nal tubules by stimulating th~ formation of colbindin. 4. Viumin D inhibits PTH synthesis in par.uhyroid glands by inhibiting tr:m.ICription of th. PTH mRNA 5. Net df«t of vitamin D: promotes hyptrcalumia 11. Amlytical oonupu A. Sampl~ l. S<:rum is th~ common .. mpl~ but plasma {with .ith~r heparin or EDTA} is 'CCq>t_ abl~. Cona:ntmiom of 1,2S_DHCC .nd 25_HCC are subl. for 3 d at 24 0c.'" S.mples should b. froun if an.lysi. i. dd»..-d. 2. Prior to mon assays, th. "'mpl. i. dq>rot~iniu:d or atractrd to f..., vitamin D meubolites from vitamin D-binding prot~ins (sprci/ic et-globulins :md albumin). Nearly all viumin D mol«ul~. are protein bound in ",rum. B. AssaY' I. The [1,25-DHCC] con b. m.... surrd by a radior<eq>tor asiaY.'" The assay uw;. viumin D r=:ptor from calf thymu •. The [1,2S-DHCC] am b. measurrd also by a RIA.'''''''' 2. [25-HCC] con b. m.... surM by R1As, though the :mtibody might oms_r ....ct with 1,25_DHCC.' It am also b. measurrd by. prot.in_bindiIll: ....y.'" 111. B=use of th~ limitrd availability of 1,25_DHCC .....Y" thue a.. f~ rq><>rts of .bnormal [1,25-DHCC] in domestic IIUmmals. Th. following lim ... ba...d on .. ponod or aprctM conuntr.uion! in various di,ord .... ' P05Sible r.latrd d~f«t. in I ,25_D HCC pathv.ays "" within parenthes ••. A. lncrea.od [ 1,25-DHCC] aptctrd or rrponrd l. GranulomatoUl di",...,: Macrophage:! IIUy produa: 1,25_DHCC. 2. PrillUry hyptrparathytoidism:'lO PTH promotes let_hydroxylase activity, which =ult< in incr.... rd 1,25_DHCC production. 3. lymphollU .nd other HHM di"'rd~n:'U" Either PTHrp promo"s let-hydroxylase: a.ctivity or neoplastic cd], produ"" 1,2S_DHCC. 4. Viumin 0 intoxicotion:"· 172 inae• ...d inuke. 5. Vitamin D-=ptor d.f«t richt<: vitamin D-dq> 6JO FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY 7. Chronic .. n:.! failur~ in hy~rcal""mjc dogs: This WlL! r~por{aI in 2 of 10 hy~rcal_ c;.,mic dog> diagno,al with chronic renal failure. whe,ra, 4 of 10 had d..:r~a=' [J ,25-DHCC]. Ikasoru for the incre>...d [1,25_DHCC] w,,~ not deurmin.d or propo."!.'''' B. ~r""=' [1,25-DHCC] upffial or rq><>md. l. (kn.] f. ilure, induding prouin_losing nephropathy: This may ~ CJUIM by 2. 3. 4. S, 6, 7, 8. 9, dre,.,....! functional renal riMue .nd th.",fore deere.=' production of 1,2S_DHCC, or by a lou of the vitamin D-binding protein .. '"'''' HYP",phosphatemi. not causal by incr•• =' vitamin D: Incr.......:! [PO.] inhibiu l a-hydroxyl"", .ctivity. which dao,,,,,,,,, 1,25_DHCC production. Hypom3i:n=mi.: This may cr..,• • pstudo_hypoparathyroidism, which dre,,,,,..,. I ,25-DHCC production, or hypomagno=mi. may impair PTH s«mion. Hypopuachyroidism: L... PTH activity c;m= l~<s l a _hydroxy\= activity, which d""rra= I ,2S-DHCC production, P.. udo_hypop.r.uhyroidism: uss ... porut to PTH caus.. l~ .. Iet-hydroxyla .. activity, which d""",ast. I ,2S_D HCC production, HHMJ<",. (This is p..h. !'" cau...! by hyp..cal"",mia, rMUc:ffl [iPTH ], hyp..kal~mic n~phropathy, or th~ pr=n"'" of an inhibitor of let_hydroxyl...,,) Prot~in_losing emeropathies in dogs'" (prob.obly d""",astd imestinal.bsorption), Viumin D-deficiem diet" (d""",astd im:ok~) Dog. with rubilin, amnionless, or megalin dysfunction and diminished r~nal tubular resorption of 2S_HCC and viumin D-binding protein had d""r~astd plasma cono:mmions o f 2S_H CC and I,2S_D HCC,'" CALC[T O N [N CO NC ENTRATIO N L Phy.iologic proo:.m A, Calcitonin i. a polypeptide hormone symhesized by thyroid C cdls that are pan of the APUD family of "",Us, (APUD i. the acronym for endocrine "",lls that have a role in amine pr""uf!lOr uptake, and d<earboxylation,) B, The regulation of .""retion is not thoroughly understood I, Calcitonin secretion is stimulatM by increa!M ron"",mrations of ICa2+ (major), IM g'+, et- .nd ~_adrenergic hormone., gamin, and cholecystokinin, 2, Calcitonin secretion is inhibitM by d""",astd [ICa'+] and increased somatostatin cono:mration, [I. Actions A, Calcitonin inhibits oneocbstic activity in bone, B, Calcitonin inhibits ren al rubut.r r=lrption of C." and PO., C, T he net effect of calcitonin activity is to decrease .. rum [tC."], [IC."], and [Pi] ilL Analytical cono:pl1 A, M ..ked differen",,, in amino acid s"'luen= OOWttn species may limit th~ cross-.pecies immunoreactivity in RIAs, B, RIAs for canin~ cokitonin ha"" been devdoped,"~'" [V, [ncm...! immunorexti"" calcitonin cono:mratiom A, MMuJlaty thyroid corcinoma'" B, Nonthyroid amo:r, especi ally from neural cren tissue 11 / CALCIUM, PHOSPHORUS, MAGNESIUM AND REGULATORY HORMONES 631 Table 11.10. EIp«led bonn one and mineral pallem. for major di,eases or conditions (without complications) Prim"y h y~rp'lf>th yro idi!JI\ Humonl hyp hyp [tCal.+] [Pi] [iPTH ] [PTHrp] [viumin D] t· .l.'-WR1 WRI- t, WRI WRI t 1 -L-WRI t WRI t t t WRI-1 1 1 1 WRI-t WRI-t WRl4 t WRI WRI-t 1 -L-WR1 -L-WR1 l' t WRI -L-WRI t WRI WRI WRI WRI WRI t WRI "-L-WR1 WRI WRI Prinwy hypopar:othyroidi,m WRI Vitamin D- r«:q>tor d~frct WRI t riches Milk f""r WRI-i WRI WRI-i 1 1 Prolongnl anomia WRI WRI WRI WRI 1 , i . ..,.,... the ",r.,"'J>C< int<'Caloemic ",nal £oj""" in bor>es, umi!'r d.u.....,. not found for oth ... .rumrn, I A q.... tion lJUlk (1) indi",,,,,, tlut th. apccted r",ult is no' mo"",, in most m:unrr",k Heal,by bor>es h."" very little vitlmin D, rnd one would "'pcct less in the prescn", of hYJ><'Cwmi., • MOl< common dun hyperc:de<mic ",rUJ biluJ< in moot domestic species ' Tbo apccted r.. ult is not Jrno.m in molt m:unrr",h, 00, in clinical """'. may r~p ....nt a rombination of factors; for aample , an animal with primary hyp<rparathyroidism may have hy~rcal""mi. bea .... of ex"" .. PTH a.ctivity, and hyp<rphmph. temi. beau.. of hy~rcal""mic nrphrosi. that produa References J, Eod... DB, R...k RK. tm, Min .. '! aod bon. ... ,taboIi.DL]'" lIurtio GO., A.b......J ER. cd •. T"", T_.J. ai""./ o"",u",.. 3..1 edition, 1}9l-t Sa.""'... of 632 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY 2. Sd"..c1 PA. C ..... DJ, &.ok. CL 1996. p,. J. t. S. 6. 7. 'l"""" Am) V... R... S7,26S-ZlI. YaR ..... K<>Ik JH, N.dudD .. RF. R.ful KR. 1lrooillI« D, W~T. 2002. H<pa<. EJ, Hdrtt RM, W,lkinoonj. H,,,... PRo 2OO}. ~,dat«l ,...ian.... in k""".~...d pl .. m. bi.och..nia.I "''' ,ooul .. in beo&ko and Ub....... "..;..... ... J Am V.. M«I A.oc 22J,) .j J6--1442. F""". CK. 1998. N ...... ,.l • ..,J p«ina .... dioco"". [n, R..d SM, R.y1y WM ...... E.j ..'_/~"""" }.{uIUin., 9}8970. PhiI. =,.. Sawod ..... 8. Sdo.ott He II. 1?98. C .... ..ic ,,0.1 fail"", [D' R« 9. 10. II. 12. lJ, I• . 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(P'Ilcl)-.-,lated P"''''''' PTH .• txl 1,1S- """thyroid t..,,,,,,,,,,,"",,1atcd protdn ronc ....;:th hwnonJ byp<< W'", *"ktOJ 5..""""•. =".rioo = oj I}. 28. N • ., LA. P.",alkAK. Lyman R. I~O. H~. ouociated"";:'" 111 CALCIUM, PHOSPHORUS, MAGNESIUM AND REGULATORY HORMONES 633 J.O. E..v lman RW. T yI", RD. Good RA. Day NK. 1985. H~ in ca .. ..ith fdin, _i= S(;'777- 781. ll. K1a~.o" JS. Bdl FW. H.rd", DW. HWh- 1%oIO}-IOS. 32. M ..."" DI. Pric< SM. s.a.. RM. Krook L 19~. Gaatric wciDoou...ru. p....d.obyp«pa". Th, bw..o ..1 k~ ofb.niu>""}" A nndy .pp...ciatcd ayod",,,,,. Am I Oin Path..! 10s,.!87_(92. }8. F..<\< IA 1994. lS(OH)..J.oIoalcikrol intozi.carion in """,. 1m Proc.cdint:> of th, 12tl. ACYIM F", ...... s... F....ci",o. 8l2--o> DD. 1982. ""u", v;'.min D, (",~f""") ",rioooi. in bo ..... C ... """" and "'p'.im.ntal ....tk •. J Am Y" Mod h - 18(U67--S7}. .. S. H...inp>o> DD. P,&" EH. 1981. ""u",,;tamin D, to";"'';' i~ .... ,," c... "1""" and """";m,mal modi .. of "', a>mp'''tiv< to.i.ci.,. of vitamin. D, and D,. I Am Y.. Med A"", 181,lJS&--I.J69 . .. 6. W .. o== RH . C&nadino RA. «rook LP. 1975. c."".", J,'"",._ A """".k plant";do US· dil.yd",~~if""" _ lj'" occi';.,.. BiocJ"m Biort.y. R.. Coonm"" 62,8S--9I. 47. Murll, E.. ay."" W. D, It..oo. P. van d.tn H. J Y" In'",n Med 80409-4lJ. S2. Sa>&<)' KCM. Prkt Gs, Yadtn SL 2000. Hyptocalcna.i. ;~ ca", A ,,,,,,.p«tiv< .tuody of 71 «n"'tio.. and oodium .ulfani.b.", dt"at>« hill-opacti", ....tu.ti.on of 4J (1991r-2002). J Yrt I"""" Med 19.66J--6N. DG. T.,...,.. do,. 634 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY 59. Sd"..c1 PA. C ..... DJ. 2003. Drt..n.inatioD of calcium &",tionation in 60. Pot ...... MA. Fd"",,,,, JM. 19l11. H~ • .-,ciotoro 42,,5"-"'65. 0........, CA. Poffnd.uv> EM, KIa.OD .. js. Joluu"", SD. Griffith DP. 1986. Eti.opo.""'_"io. dinia.l manif.... • tiono, and """'V"""" <>f =in< ~WD. o..iat, .... ~Ihi ..... V.. OJ,, N.. thAmSmaUkim P.act IMJ}-170. 65. G=o DS. r ....."'" ME. CJ.o DY, M .. ko.i" JE. 1985. J~'" bypothyroidiaa in • .J.o&. J Am V.. M«I Ju.oc 137,941i--95O. 66. Hirt RA. K"';.. l S, T dofalt M. 2000. s..",,,, kypaal=nia in • doc ..hl. • Med 1 ' '''I~26. 68. Oldl. .... .'lB. Rood, RK. Molloy cr, lipoon LG. 1984. Th., dfoco of _"ium on aIci .... inhibidoo of """yl." panthp<>i.d cyda",. Eod,oc.,;ooIou IIH88.J.-1890. 69. B.,k WW. Ki".".,1 Sf. W.-. MA. l ocbon MW. 2001. s..ro..duy k1P"l""'thp<>idi,m .nrib.t«l '0 loypom"U" .... mia in • "'-I; with protD<mtoatiooo io two dop wi ... protE568-H76. n. Sod",in.. CA. N.~ LA 2003. Yi ..",in D-.d.p< ....'o, .kkrn typ< 2 in • fo.,· """,do.-<>Id cat. I Am y" M«l A.ooc 22hJ37- 3J6. 74. H",", RI.. lOT ...... NA. Di.d ...."""'" "'''''''''....n in h~",ic ~ ..., "'.... Am I Y" Roo J(I,1587- 1597. 76. Golf IP. Ho,"' RL 2003. R..k of add-b." pb,oioiarion """" (cdamp.ia) in • Sbrtl",d pon, mat,. Au.! Y" I 47,40z-tv..i. 78. F..«"; AI. Hkhn.o MA. 1999. Pr.partwioo, kypoakm>i. in fo •• cato. I Am Y" M«l A.ooc 215,) 127_1129. 79. YOURJ: DM. Capm. CC. Block HE. 1971. Calcitonin Kcivity in..ltimobtand.ial noopl .. ",. fro". buJI •• Y" Patbol 8,19-27. 80. Fmu' CK. 1998. O1ootd, .. of colci.," m".bol.iom. In, Rud SM. B.yly WM • .do. bj. ;"'/."",J MuIid.,. I_ odidoo. 92j....9Joj. Ph.il.ddpki", WB Sawod .... 81. I""'" LF. 1999. HoJovtoo poiooninc; in ~ I Not To";", 8,J9s-toJ. 82. c"...,."ll WA. Whitlock RH. Sto., RC. T p.. DE. 1979. Edo.:rI=' dyoool. toxioooio in conk. Co""ll Y" 69,272-279. ...d cat. I Am kim Ho'l' A.ooc 8J. G .."", GF. TI...n MA. 1982. Edo.:rI=' w=1 (""'""'=) poi"";n, in .... 1s.l92-t97. 8t. G .."", GF. TI...n MA. H""" SA. G.."", RM. Hamu DW. 19I1-l. hdy dinioopatholo"" 6 .... in,. in in",";"'; ,th,"'" dy=!. Am I Y" Reo 45,2299-2303. 85. a..... DI. Lro .... d M. Mui. W HI. 1989. Eif=t of.....tium b.who..... in~ on ioniu0" ~,;., 46 co",. (199<>-1998). I Am Y" Mod A.ooc 219, 110j....1109. rr do, do,. oooe 111 CALCIUM, PHOSPHORUS, MAGNESIUM AND REGULATORY HORMONES 635 89. Apn.a1 N, Pitth .......ni CS. 1993. Acut. l"""""oo" A modti.,.. .... dio. . ... G..otro.o'''''*'V- loll j..-128. 90. W ......... Al. La. KH, N'pi .. 1W, !\OWni" po, D..cl. ..... y D. A.drod L 19I1 S. D..,.., ...... of ........ caIcium by i"", ..- l plaanu. f"", &'tty acid. io th, ..., A modoani..., fo. bypocaI=ni. io acuI< l"""""oo •. c.."""'Olnd.rico b."""", kypo<>k<mia and m<mb.",o-m«liat«! =<..;..., i"'..om .....tioo in ..,ft ci ...... M"&,,,,ium 7.91_ 102. 92. Droba .. Kj, Hu ~" D. 1997. Cooc.",.. rico of ionized ",kiom io pL-na from cam .. ith ",rthc.I obotruaioo. j Am V.. Mod An,>< 21 h ll92-139S. 9J. Vad,o SL lntrol of >CUt, , ..... fai)...., in 99 doc ~ j V .. ["'''0 M«I I b S8--64. 9' . Sdo. .. M . p, H ..... W, Wdkin. R. 1m. [atrop>i.e hn=PI•.,. pl"mnia, hypocok.mia and h~ io • cat . I Am kim Ho'l' A..oc n.J9.--.oII. 9';. Gucia_i.op<..jM. " - PI. Rwb jE. Zick"SC. Burma" .. H, F..,m"" L\1. 2001. p,....t.nc. and J""&D""tic importan« of k1l""""&""'"". iooiud aid ..... kvd di.~....!hod . of canth"idio toxicooi. io Am I Y.. Rc. so,l87- 191. 98. Hdm... RG. &\w .. d. WC. 1997. Clinicd f"""". of bIj .." """" poioo";", io "'loido. 70 C&O<>-&n< .... ,tiuod !.o,... j Am Y.. M«IA..oc 179.896-898. 101 . G.d,o F. G..d.", L SdI" G. WdI, M, Bon.....! ]aomio y, Gonin I..... D. N. Y.. p.thoI 3M 17- 123. 102. P..1iliu G. Y.n..., SI, Ma~ jM, C&d"", GP, j""", SL 1998. ElectooIyt< di.,..,baoc.. io fodo with ..... " ,lulxlom,.,lpia. I Y.. 10l<m M-Ouioti ..... o T . 1991. Preanalytial OaCS. c.,...,,,,,, """Oft. V......-.....,. FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY '" lIS. Bu .... PI, Elliott J. 19'96. Stud, of ak;"'" ho ....."". in (,Iio, hypw>mprtitioo. 1ua J V .. M.,....... «><>an"....... 117. 118. 119. 120. 121 . 122. 12J. 12 • . 12S. 126. 1l7. 128. 129. I.J.O. l}1 . l}1. l}J. l}'. l}S. 1}6. Il7. l}8. Il9. 140. 141 . 141. R... Sid S02-ISOS. E.. E.tq.. Je. lopn. [, R.. S, Garlia B, Rodri",,,.\1. 2001. Plaun. ioniUk of acido,i.-indu.ced j""" .. ", in aki.m 00 PTH oocmion in K "'" mrtaboli<...d ",. piu.tooy acidooi, in d" ~ Am J Ph,...,! ~ M,ta/, 286£780--£785. lorn I. Rodricu'" M, Fd.. okkl Aj, E_'!'" je. Ap.iJ .... T i.doc ..... iup.buIin. io du .. diif""m mttbod. fot i... tp..k pbotpbon... Ann aio Biodtom Z7(P, }),227- UI. I~"","o L'i. Cn.ttr SA. Randolpl. IF. &wo D . 1985. Eknolyt< ... .........Jitioo indoc" ....k pbotpb ... .......... io """ «tt. I Am Y.. Mod hooc Hr.', IJ.67- 1}68 . Aili", C Eo T yl" R. G...... « P. 1985. aioical. bOo.h....ical. acid-b.". and d ... "oIj .. .boo....... ;,;", in at> aft", h,pntoni< ..di"", pbotpbot< ''''''''' .dmini..ra"'~ . Am I Y.. Ro • • 6,98()....988. F..Jt... RJI I,. Ftuni oyndrom, .uod"od .. id. ,,0.1 d,. pb.ti. io "'" ..... "'" ,,,,,;,tL j Y" [ot<m. Mod 10,,12-419. IR. Btrtttd.w,.J, EB. 1976. Gluoo •...;... ooci.tod .. id. "",I tnbuIat dytfuncion in du«!laomji doJ: •. I Am Yrt Mod A.toc 16S,9}S-94}. Bo..It KC. j<>J"<' T . BI..".·Y_ B. G~JdtuutIAI. Cob", ND. RuoIJWW. &umbau ~ GW • .'id.min DG. K.t.,ti. BS. 19"9}. U,;~uy intJi=,ofho.... • ft<. ;"".vtDOUt adminiotn. ... ~ of ctpta!loid to!."''''. I Y" [ot<m. Mod 7.241_246. Bm.. b,., N. Ldbo.ki H. M.....,. SG. 1983. On...., mocb"';"" ofbn"'p~""'" d...;"1!: ..... , hypttvtnw •• tioo, E...in os, Willi .. N. 19"9}. BOod.,miaJ and b ", ~o.l cI.anu. foIIo...in& ptoIoo~ haIod..." .",<> """ N ... ium "txl Ma"".i"m .~ . d"" foriil> .1bIooi. do....,. '" Eo...,. 11 1 CALCIUM, PHOSPHORUS, MAGNESIUM AND REGULATORY HORMONES 637 1·0. McCoy MA. ZOO4. Hypom.p ''''''''''. and 0 ... data on .tt.oow kWDOUC ""P'ooi"", ronc<. It.. INJ7- 14S. 14 • . Ka...i TR. Woodb..,.,. AH. Moocom_Ka..ui I'.. 1990. Advn .. dJ«, ,,( .. ally odmini"",od map>< ~ "'" bydroridoo 00 rrum map><';"", "''''''''''....n and . d.d_..... bola<>« in ad"" cortl<. ) Am Y" Med A_ 196,nS-742 . I.(S. H,nninp., RW. Hon' J. 1m. Map><,; .... to';",';' in ""'" bona. J Am V" ModA..o< 2IhU_..8S. 1.(6. IUond IL. Kocabadi N. Spickicn UI'.. w....,,, M. 1995. n, ",<>«n . . .tion of ionhed .... ~.i"m in rrum durtnc; the pnipartupai.d ....,.",.,.,. and io..iut! aid .... and ""P'.. ium ron«ntn.';",,,, and f..;.", of ""P'ooium in ca .. wru. i.d ............ and do"troly<';"", "''''''''".... " in 122 doop...itJ. diab.", md~"", A ...... p«ili-c ...dy. J V.,I"«rn Med 18.612-(;17. in blood IS6. M."" FA. Boon GD. W"&,,, .. M.nn CC. Rubm. DS. 1998. [ODiud and ,otaI ""P'.. iwn f""", doop ..i... na ....!Iy ""'I"iud I""""';..! ",,,,hl •. I Am Y" Med h - 211,1l9S-1401 . IS7. s"""" AI. H. «ly J. KoI.o CWo T..ibio RE. Hioclodiif KW. SO[,... B. ZOO4. Validation of k,,( ... , calcium....... in, .ocq>to< in pu ....yroid r1 •."J pby';"J.v. Am I l'h,..iol Rcnal PIoy';"[ 286,FlOO5--F10 II. 160. R..m ......yl. 2006. Rocm.,""""",, in pb,...lot;ical aid ........ "'oootasio. Cli" o., m lob Mod « ,lJ7- Zl}. 161 . E.tcpo IC. Gaolia B. Goo PR. C&nto. T. R"d,;"", M . Apill, .. T oj,,,, E. 200}. Validation and cli..ical ~tilip of the I~ ACVL\l F.."",. s..nI,. W .. bin~n. 875. 16 • . E.tcpo IC. ~iIi.d ~""""'" in 1.00.... Eq..inc V,,) .w.476-tS I . 165. r • ."Jian MR. Mo'V" CH. CHI"", I'.. Sqrc GV. 1992. M<>. J ain [..,... 76.69S-7(l2. .,......k ""' ='"'''''' ronccn _"iwn- 638 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY Li..,,, D, Maooo RS. POO.lway of ",rub';"'; J,2S- 168. = 6«,1161_ 1166. 172. R..."bdb. WK. Kru ~ /,\1, Fi "C"ald SF. N~ RF. !1d [C), H=winhl HAW. 1991. Elucidation ~f tJ.. ""I.....c< of anin< (p.<»~o..in , A nooIoc..Ju bioJocia.I and prot';" cl.,miaJ ' pproa Chapter 12 ENZYMES Basic Principles in Clinical Enzymology.. .. . .......... . . . .... . ...... . . . ........ Alanine Transaminase (ALT) (Synonym Abbreviation: GPT) .... . ...... . . . ........ Aspartate Transaminase (AST) (Synonym Abbreviation: GOT) .................... Lactate Dehydrogenase (LD) (Also Abbreviated LDH) ............................ Iditol Dehydrogenase (lD) (Synonym Abbreviation: SDH)' ........................ Glutamate Dehydrogenase (GMD) (Also Abbreviated GDH, GLD, and GLDH) ...... .. Alkaline Phosphatase (ALP) ... .. ...... . ..... .. ............ .. ........ . ........ y-Glutamyltransferase (GGT) (Synonym Abbreviation: GGTP) ...... . .............. Creatine Kinase (CK) ....................................................... Amyl ase (AMS). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lipase (LPS) ................ . . . .... . . . ... .. ...... . ...... . ...... . . . ...... . . Other Serum Enzymes . ......... . .... . . . ... . ...... . . . . ........... . . . .. ...... 640 650 652 653 654 654 655 659 661 663 665 669 639 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY Table 12.I.Abb...,viatiom and symbols in Ihi.. chapler U,in~ ~glut>myltr.ms~r.IS~ (GGT:Cn)" [xl ALP ALT AMS AST activity 10 x rona:m"'lion (x = a""I)'I') AlkaJin~ phosphal"" AI:min~ Ir:m.. min ..... a.Amyl""" Aspanat~ tr:m.. minaK" ATT Ad~n""in~ Iriphosphal~ B·ALP C·ALP Bon~ CK cPLI EUSA ~U r-GIUlamyllransf~ra!~ GMD Glutamat~ dehydrog<'n"'~ GOT GPT Glutamat~ oxaloaa:lau I"'n.. mi"""'· lransaminast' Intestinal alkaJin~ phosphat.". Idilol d~hydrogtn= (rorbitol dehydrog<'n""") Liw, alblin~ phmphat""" Glutamal~ pyruvat~ Laclal~ d~hydrogtn= LPS Lip... NAD NADH NADPH P·5·P PU Niroliruomid~ SI TG TLI U URL WRI rona:ntr>tion alblin. phosph.t= Co'lirosl~roid·indua:d alkalin. phosphat= C,ealin. kin""" C.nin~ p:mc""lic lip""" immuno"'.ctivily Enzym~.linhd immunOlOr~m :way Fdin~ p:mc"'~tic lip""" immunorexlivily Glom~rula, filtralion r>t~ GFR GGT I·ALP ID L·ALP LD cr~~linin. .d.nin. dinud Rnlua:d niooliruomid~ .d~nin~ dinud phmphat~ 'n,. NC· IUBMB< 'tioJUl Union of Bioch<:mistJy:uxl MolKular Biology BASIC PRlNCIPLES IN CLINICAL ENZYMOLOGY prouins thai au alyz.<: ch~mical ,eaclions. Prol~ins lhat ha"" diff~r~m polypq>lidc nructu", bUI eaealyze Ihe same ch~mical ",.ction ar<: isDtnzym(S (or isozymcs). If Ih~ difftr~nt ~nzym~ mUCtu'" is creal«l by a pomr:molatioruol modificalion of an original gtn~ product, th~n Ih~ prol~i"" ar. is%mfl. Many enzym .. rcqui,~ nonp,ol to assin eaeal,..is, oon"",I· ing th.m from aJ>Omqmr1 (without oofaclor) 10 ""1omzJ"'~1 (wilh oof""lOr). Th... arc oft~n from Elfz}m(S ... 12 / ENZYMES 641 T able 12.2. Cellular or ti..ue .oura::o and half_lives of commo n serum cnzym ... EnzE~ Major m~nism. that lrad to inc",~ .. rum acrivi!r ALP Induction (I"l:estion) ALT Cdl dam"&,, AMS AST Cdl damag<' D~C""M ",nal de;oranct Cdl damag<' CK Cdl da""'l:" GGT Induction Cdl prolif~ration (Ingestion) Cdl damag<' Cdl damag<' Cdl damag<' GMD ID ill LPS Cdl damag<' D=e"M ",nal de;oranct Cdl ~rolif~ration • M.jor ""nub, .!OUr"", of pt..m. ~zym< , D ... from ov:oibblc but ~mi« Cdlulu sourct. of inc......M .. rum ~nzym~ activitr ·Hq.atocytes (L.-ALP) (C _ALP in dog.) 'Biliary ~pithdium (L.-ALP) o.t~oblam (B_ALP) M:munary q.ithdium • Hq.atocytes Skd~ul myocytes • P.ncr ... tic .cinu cdl, , Hq.atocytes 'Skd~ul myocyte. Cardi.c myocytes Erythrocytes 'Skd~t:d myocyt .. Cardiac myocytes Smooth musd~ myocytes (minor) 'Biliary epithdi:d cdb ' Hq.atocytes M:mlmary q.ithdium Hq.atocytes • Hq.atocytes • Hq.atocytes 'Skd~t:d myocyt .. Cardiac myocytes Erythrocytes • P.ncr ... tic .cinu cdl, linr neopla,m. Gamic mucosa .ctivity H:df_Iin" .. 3 d (conin~ L_ALP)'''; .. 6 min {cmin~ I_ALP)"o < 8 h ( ftJin~ L_ALP)m .. 2 min {fdin~ I-ALPl'''' 2- 3d (canind>O .. 5 h (conine)'" 7- 8 d (~quine) '''''u, < I d (conine)""" .. 2 h (equine) 'lt,'" < 2 h (conine) '" .. J d (equine) '" .. 14 h (oovind" .. 4 h (conine)'''' < 6 h (conine}"" .. 2 h (conine)'" wurces a vitamin sourct (e.g., P_S _P from vitamin B" and NAD from ni:ocin) but also may be ions (e.g., Ca'+, .nd Mg'*). I. Sou=, of .. rum ~nzym .. (Table 12.2 and Fig. 12.1 ) A. Serum ~nzym~. deocrikd in this chapter originate from cdl, (nog<'noUl to .. rum) .nd do not h.ve r«<>gniud function.< in blood. lkfo .. their rdea .. from th~ cdt., the ""rum enzymes may be in a cd!". cytopla'm, mitochondria, or membrane. St rum enzyme activity in healthy anim:d, i, rypirnlly .... umM to ..",It from physiologic process... 642 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY Liver Blood Pmooss.&. 01 .... grltdatioo am not known for most plasma .. nzymos. MltCI'tlphago>s probably involved. 8'" Liver AST LD ALP GGT CK GlAD AMS LPS ReMI GG' Fig. 12.1. Sourc... md rout", of,.mov:o/ of common....urn ~0ZJ'Dl<'. M ajor""""", of >erum ffi'J"D<' tbat contribut< to incr.,.>«I..,um a<1ivi,;'" ar. hep>tocyt .. (ALT. AST, GGT, GMD. 10. LD, . nd ALP). bj~ary epith.lial ""It. (ALP and GGl), .hle"J ond c:udi. e mwcl< /ib.rs (CK, ASf, LD, :md Ai 1), ost.oolans (ALp). :and pone,,,,tic acinar aUs (AM.'>..,d LPS). In vitro of AST.oo LD from ded rm:ymes rnd .nzym<-antipro..... ,.Ie... compkul. Tissu~_sp B. [I. activity incr~ase:s wh~n th~ ",I< an ~nzym~ ~nt~rs pl:asm. nettds th~ "'t~ of ~nzym~ inactivation or r~moval from pl"'ma.' [n C"nrr:d. th~", ar< fi"" m~:mi,ms by which ",rum ~nzym~ activity may incr~...,: (I) incre..,p<: from a "",II because of damage C;>Usffl by minor "",J] injury (=~rsible dam~) or when enzymes .r< reI ...sffl with cdl d~ath (necro,i.). [f intracdlular enzyme oon""'ntrations (cytosolic or mitochondrial) .r< gr ... t~r th. n Strum ~nzym~ 121 ENZYMES 643 ". 1V- ATP deplelion duo to hypoxia ... mitochoodrial toxins j E momb......... dalllllge C-'---7-I~ : E .E E ~: ";:'~ E EE EE E EE -if> E E E EE E Ee , E EE E E E EEE ~ " EE E E E E E E E '' @ E E F ~m": blobosoole mkMsod to :"I~ blood ... r-' _~ EEE ~'{:0 ~~. : :~:, - , ' ?,:,. ,' r' ~E Fig. 12.2. ReLe ... of cytoso~c ~zymes by bJ.bbing Of nK7OOU. Blebbing :and ...aos;' =y incr...... rum activity of ALT. AST. Ln. 10. GMD. CK. AMS. LPS. :and. to a mild deg~. ALP:and GGT. A nn.ty of imul", to cells auy em .. direc' n<;, or m<mbr .... bLebbing (irr.....ibJ. or , .... mbJ. damag<). [f ir~"ibLe ",U d:ur..gr OCCUJ'J. ",Uuhr rnzymrs or. "Le ... d with or withou, bLeb form .. ion. [f ",,,,,,,ibJ. dumgr OCCUJ'll. blebosomei form..,d lorer [Y"'. thus ",Leasing ,b.ir . nZJ'ID"'. E, "',;yrnr. a tracd lular con""ntrations. :md if th •• nzymes a.. activ. in plasma. th.n .nzym.. from cdb inc",= .nzym. activity in utracdlul" fluid .nd thu, plasma (or .. rum). 2. Enzym.. moy ~ rd.astnts binding to cytmkdrtal protrins. or. variety of ag.nt. (prot.:a=. Ii"" .... • tc.) that c:>u.. m.mbran. da~. Oner m.mbran. bld.s form. tho erll may: a. Rd.= bl. bosom.. ronuining cytoplasmic .nzym.. to pJ:.smo or lymph. wh ... th.,. ev.ntually ly.. and c:>U!lt inc",ast 644 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY o J Fig. 12..,. [ncn...d prodoction of ALP by induction. In h..Jdty animals (th. bft half of th. dn.. ing), ALP is m:ocbed to hep>'"'}''' """,br:u><s, mo", is Joe>tM on the ,,"ruJirular tit.., th. ,inusoid.J "",mbnn<. In .id: rnim.Js (tb. Iulf of th. d"...mg), drus' OJ m ... boJites (o.g .• biLe >«1 from th< .inusoidtJ membn .... S Fir' d. Enzym.. rd.~.ffl from neuron, moy not cross ,h. blood_brain barri., and thu. may not inn• ..., plasma .nzym. activity. 4. It is commonly.calM ,hat dam"l:' to ~lls "'lows enzymes to "Ink" from ~U, ~"'" of altered cd] membrane p"rmnbility. When damage is sufficient to ..!low an enzyme, a ["go prouin, to Jrak through a porou, m.mbr:Ifl<, then many oth.r intncdll1J., solutes {e.g., K+ .nd Co."} willl..ak out and ,h. cd] will di•. When thor. is .",,«sibl. cdl damage th.t inCf~""" .. rum ~nzym~ .ctivity, this "leab~ through hoI .." concq>t i, not applicoble unless th~ cdl. are cop"ble of quick rq>:Iir to limit th~ 10.. of ront~nU. B. [ncr~'!ffl ~nzym~ production by individual cdl. b=iu.e of induced .ynth~s.is l. This is th~ prim.,y mechanism for membrane enzymes and may ~. mechanism for mitochondrial .nd cytoplasmic enzymes. 2. buiucrion r~fe", to a nimulated incr~= in production of the enzyme protein via modifial transcription, tr:mdation, or othu processes. Endogenou •• ubnances (e.g., bile acids) or drug•• urn •• phenob.,bital, prednisolone. or prednisone m.y trigg~r induction. [ncr= in .. rum ALP activity r~SlIlt mostly &om induction (Fig. 12.3). C. Mor~ ~nzym~ produced by a ti!S\l~ b=iusr of ""U prolifer.tion, pmicularly for m~mb.. n~_'HOCiatal enzym .. l. NwpJ:..ia of the enzym~·s cdl of origin (e.g., increased ALP and B_ALP with o.tw ... rcoma, or increa!ffl LPS with pancreatic or extrapancre.tic nwplasia) 2. Hyperplasia of the enzyme'. ""II of origin (e.g., increa!ffl GGT with bile duct hYl"'rplasia, or increased B_ALP with bone growth or repair) 3. Convtrsdy, d~cr~.sed tissu~ mass may ~ associated with decreased .. rum enzyme activity or con""ntration (~.g., TU with p.ncreatic .trophy). D. Enzyme r~mov:ll from pI..,,,,, is decreased {enzyme has.n increased half_lif~}. l. Some enzym .. (e.g., A,\.lS and LPS) .,~ inactiv.ted or acr~tal by kidneys. Decreased r~nal blood flow l~.d. to d~cr~.sed ina.ctiv.tion of AMS .nd LPS. 121 ENZYMES 645 2. Some Deer.."""" . ctivity of most .nzym.. asse:s.sffl in routine ",rum ch.mi IV. M....... rem.nt of ",rum enzymes A. Activity ~r unit volume l. Enzymft are proteins th at cotalyze chemical ..actions. Routine a... Y' measu .. enzyme "crimI] by dec"'ting how fast a mbstrate i. consum«1 or how f"'t a product forms. a. Enzyme .... y malty: In the presence of acess substrate (S), the r~tion rate depends on the quantity of enzyme (E). More specifically, the reaction rate depends on th. rate of r~tion linm the enzym._substrate complex to the product {P} + enzyme: E + S ..... E_S ..... P + E. b. Nearly all serum enzyme =Y' are spectrophotometric. Th"}' may b. eith.. end_ point "''''Y' or kinetic "''''Y'. {I} End_poilU """YTo The reaction is stopped . t a specifi.d tim., and enzyme activity i. determin.d from the quantity of product form.d or the quantity of subnr.te UsM. {2} Kinnie "SMJr. Multipl. reading> are taken during a specifi«1 time period, and enzyme activity is det ..min.d by the ral< of the r....ction (or rate at v,hich product is being form«1). Most Cutrtnt enzym. assay. are kinetic assaY'. 2. Enzyme reaction! for common clinical .. rum .nzym........ Y' a. Figure 12.4 contains the initial ch.micol ..actiom cotalyud by the common serum .nzym... Th. initial reactions a.. umally coupl«1 to oth.. reactions th.t cou.. the formation or disappearance of a color«1 indicotor that con b. det"'t.d by photometry. b. Knowl~ of the enzyme r....ctions promot.. an understanding of enzyme nomenclaru .. and physiologic rol .. of enzymes. 3. Units used to express enzym. activity a. V = international unit = amount of enzyme that cotalyus conversion of I micromol. of substrate ~r minute under d.fined conditions (I U = I J.lmollmin). In this context, mbstrate indud... ubstances such as NADH or NADPH th" a.. also call«1 coenzymes or cosubm.t... Vnits are usually expressed '" UIL but occa FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY '" ALP compound-PO. . COIIl'OUOO + po. "'_I'"l.~+ ~I~nine Q.ko!ogIulanllll starch + H,0 "'-,:--;;: P'jNVBIo P-M' g~mat.. 7. --;,'"''''', ,,"''0-;c>' '" aSJlllrtalil~ :oo;;:':"",,,' Q_ko!ogIulanllll I'6.f' starch ""Smoots + unhy ",,&""""'111111 gUtamat" CK ~ ADP .........--, ...,;-... "'SlIt.... phosphat" y.glutamyl-p-oilroolnilide glycylgtyc;oo gllMmat" 7 GGr, p.nitroaniine --....,. y.glutarnyllIfycy\g Ivcina GMD~ 2-oxog1u["",lo NAD""""'-- .......,. NADH LPS _.fe ..._,..· + NH: d9Y<:eooe. + mooogly<:erides + \h'aIroI + latty adds Fig. 12.4. Initial ,..ctions in ....Y' fOf tho common c~nic:d .. rum enzymos. A...Y' .,< design«!.., that tbo ",,,,.limiting f.tctor is th. cauJytic :u:tivity of . .. rum ffizyn><. Methods of monitoring the m.mic:d ,.,.cnons ,bot "'" C1t.Jyud ~ tho enzym<s vary but typically inVOM .bsorption or ,ell.ct:u><,., pbotomt< drivt< or pyron'. to bel1'" mRNA, _ n g .. ribo,mdeic ocid: rnd NH:. aaunonium. c. Arbitrary ullits: Som~ ~nzym. uniu a.. namffl aft~r ~opl~ who d~dopal {h. a=ys. Eumples include Somogyi units (AMS), Signu_Frankd :md Karman unit, (ALT .nd AST assap), R05e_Byler units {LPS1, and King_Armslrong and Bodansky units (ALP). V.lues ap ..!Kd in .. bitmy uniu may b. much diffe .. nt from valu .. expressed in intern>tional units, because .. bitrary uniu may b. ddined by mark«ily different ......y conditions. It is difficult to impossible to mnwrt the arbitrary units to internation.l uniu acruratdy. Fortunatdy, moJl current methods haw been defined in international units {UJ. 4. The international unit does not normalize methods. Different assays may measure different amounU of enzyme activity in the same sample. a. Because of v:ui<>tions in some assay methods, enzyme .ctivities mrasu...d by two different assaY" may b. markedly different. Figure 1.5 ilJumate. the nwk..d differences that can b. found when one s.omple is analyzed by diff• ..,nt method •. Thus, aCetIrate interpretation of ... rum enzyme activity requires th at patient 121 ENZYMES 647 Table 12.3. Approximate changes in enzyme activities if Ihe .ame ,ample is analyzed al differenl assay lemperalures 25 ·C Common ~nzymes" 60-80 % Rd.tin ~nzym~ ~ctivily al 30·C 32'C "'''' 110-125 % 37"C 130-210 % • Including ALP. CK. lD. ID. ALT. and AST No..: Clunges :or<: ba>«l on 'eported oon...."ion net""'.'" In 2002. th. In .. m. Oorul Fffi<:r..ion of Oinic:d Cbem;,try publisMd S 37"C. v. lurs k oomp..ed wilh ,.f~"'n"c inurvals establi,hed for th~ assay u..ed 10 measur~ Ih~ p"lient', enzyme activity. b. Diff~rcna:' in assay m~hods cru.1 cau.. different m~.mred values include differ~n= in Ihe following: {I} Rc~ction. (forward .nd """"") . nd r~~nt oon""'ntralions {2} Subslrales (~spt'C",[[y AMS. LPS •• nd ALP a"ap) {3} pH of .....y r....ctions {esp«ially ALP =ys} {4} Incubalion or ,.. ction temp<mM ., IlgiL Th~se prolein rona:ntrationo air rd.livdy small when comp"rM 10 omer common serum prolein analytes such as albumin al 3 gldL or fibrinog~n al 0.3 gldL 3. Prot~ino d~ectal by immunologic ~~ for enzymrs m.y indude .ctin forms and inaCli"" forms or precursor forms. V. Enzyme nomenclalUr~ A. Th~ rccomm~ndalions of th~ Nomendatu", Commitrtt of the Int~rn'lion.l Union of Biochemistry .nd Molecular Biology.,. u..ed for naming enzymes.' Most enzymes wer~ n:mlM by Ihe r~~ction mbslme first (e.g. alanine and c,..linc) followed by on~ of six Iype' of r~.ction Ihe ~nzym~ C>lalyzes (e.g.. mlll,fer .mino group = lransamina .... lransf.. phosphate group = kin ....... nd oxidizc or r ... FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY numb" that d~=ibes iditol dd!ydro~n,...,). {h~ 'pacific r~'Cljon th~ ~nzrllY cotaly= (~.C., EC 1.1.1.14 for B. In ch~ convemioruol system, th ... w.., not a systeJrultic method of morning enzym ... Convention.l nam ..... still usrd by some laboratories and .uthors. C. Enzyme abb...vi.tion, .bbrm.tion, h", not oon .dopted. Abbreviation! in thi, {",,{book we", ''"'''ntllYndal by. group of chemisl, in 1975.' l. An international system for 2 For the ronnntional.ySlem, mall abbrevi VI. Sample p""""ssing, handling, and storage for common din;",] .. rum 90 % activity .. mains} in equiM .. rum lOr up to S h at 21 ·C, 24 h at 4 'C, and 48 h at - 30 'C; 73 % of ID activity w'" lo.!! by 24 h at 21 'C .nd 28 % was lost by 72 h at 4 ·C.' c. hoenzyme LO-5 i. heat labile, wher.."s LO_l is cold labil~. It is best to leave. sample for LO analy.i. at room temp""ru", and analJ'Z" it within 24 h. d. Some ALP isoforms ar~ h~t labile (B_ALPj , whew.. oth... are heat suble (I-ALP .nd C_ALP). Thaw.-d .. ra may have greater ALP activities than fresh .. ra. e. GGT activity in hep"riniud equine plam'" w'" suble for 1 mo at - 20°C'· C Sampl~ quality l. s"ra or plasma with hemolY'is may yidd errOnalllS results lOr thIN =sons. a. Enzym.. of erythrocyte. may b. .dd.-d to .. ra or plasma to inc=sr activities; lOr nample, incrras<"d AST, LO, .nd possibly ALT .ctivity. 121 ENZYMES 649 b. Chemical constituenu of erythrocyt.. Jruly participate in the enzyme :way; for a ample, gluco",-6_phospru.te, ATP, or ~denylate crcw. for CK activity. c. Hemoglobin 1m}' interr." with light transmission in spffirophotometric =rs .nd yidd fat .. iner.... es or dec",,,,,,", d'p"nding on :way design. 2. Sera with devated bilirubin (icwic ",ra): Bilirubin Jruly interfe.. with light transmission. 3. Sera with increasN visible lipoproteim (lip"mic ",ra) a. Lipid mola:u.les may interfe" with light transmission. b. Lipid mola:u.les cau.. unpredictable rnult. bea.... light transmission is hinder..! not only by numbe:r but .lso by.iu of mola:u.l... VII. Interpretation of increased ",rum enzyme activity A. Becau.. of the variatiom in enzyme activity measured by diffe .. nt assay., po.ti.nu' enzyme activities n..,d to be: comparrd to .ppropriate reference interv'!s. If inc....ed, the degr.., of inc",= i. um.!ly determined by dividing the patient·. enzyme value by the URL (the highm value of the ..f..en"" interval): for example, if a po.tient·, ALT activity = 500 UlL with ~ ..f... nce int.rval of 10- 50 U/L, then the ALT value is 10 tim .. th. URL (IO X URL). The ab",lute valu .. for enzyme activities may vary consid.rably betw",n assay methods, but the d~ of inc=... .bove the URL .mould be: n... rly the ",me. B. Th. magnitude of incr.....d .nzym. ~ctivity may limit the possible aplanations. For example, an ALT activity of 15 X URL is probably caused by hq.atocyte damage:, not "",e.. muscle damag•. Similarly, an ALP .ctivity of 10 X URL i. typically consid..ed too gr... t for B_ALP activity in ..:lult dogs, but the ALP activity could be: due to either l-ALP or C_AlP. C. eomider the half_life of the .. rum enzymes. For =omple, C K has • short.. half_life than AST. Thu., aft .. a single insult to mu.de, CK activity may .. turn to the ..fe .. nce int.rval sooner than AST ~ctivity. D. Intq;rate potential pathologic process." with other patient inforJrultion to form ideas or explanations for .n anim.!·, illness. VIII. Significan"" of incr.a..d .. rum enzyme activiti.. A. Serum .nzyme activiti.. a.. markers or indicators of pathologic proct.... (e.g., hepato_ cyte injury or chol.. tasis) ~nd not specific di..,a .... Many ty!"" of di ........ may cou.., common pathologic proces",•. B. For the cytoplasmic .nzymes (ALT, AMS, AST, CK, ID, ill, and LPS), the magnitude of inc ..... Jruly rdate to the .. verity of damag<:; that i., ,light damage may co .... values < 2 X URL, wher.."s ....." damage might cou.., values :> 50 X URL However, magni_ tude of inc",= d"". not differentiate reversible damag<: from ir ...... rsibl. dam'ge, or local damage from diff.... damage:. C. A mild increas. (e.g., 2 X URL) might not be: very important in on. animal bea.... other finding' cl.~rly indicote. definite diagnmis. However, in some cas<S, the ... m. ",rum enzyme activity might provide the only due of active di..,.... D. Becaus<: of the method of characterizing. patient'. enzyme activity (i .•. , comparing it to the URL), the magnitude of incr..".. is not .cruratdy d=ribc:d for most .nimal,. For =omple, if a dog's ALT activity prior to di ...... w;o. 20 UlL, and it """ to 200 U/L aft .. the on.. t of dis ...... then it rose to ten tim .. the basdin. valu•. Howev.., if the ALT ..ference interval was 20- 70 UIL, then the pati.nt'. 200 UIL value would .. pr..ent .n inc..... of only .bout 3 X URL .nd might be: consider..! a mild incr.""'. 6511 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY , , , I ,,, ,, I /Anirnal C I , ,, I I -,-----. , ,I ,,, I I I I AnimalS , " AnirnalA " - Tim& (days) Fig. 12.5. [ntorpre",tion of . ling\< mzym< :octivity. On the < URL (""'/b" un). FOI rninuJ A (•••), th. 3>< nJ ... ocrur~ p1tbologic p=>~ For rnimaJ C (_ _ ----1, the 3" nJ ... ocrur~ during. progr ... i", pathologic process. Tbus. tt.. dinic::tl signific"""" of th. .ing!e m<1S.U..".,,' of ffizym< :octivity may v:ory from mumJ to :animal. ~U'" prffii=.....lues ... Ulu:dly not av:ailobJ. for individual a!lim:.!., "'" must consider vari.tio[" among animals wh.n imorpreting .. rum E. Enzyme activity from a singl. sampling =y not rrfl«t the dynamic changes tru.t ffi")' '" occurring in a patient (Fig. 12.5). Sampling :.gain in 2- 3 d =y provide a tru.r i~ of the pathologic p,ocr....". ALA.NINE TRANSAMINASE {ALn {SYNONYM ABBREVIATION: GPn I. Phy>iologic process~" concqm, and facts: ALTis. cytopJ:.smi" ~nzym~ that catalyus a reaction th.t is involvffl in th~ d.... min . tion of :danin~ to form pyruv. te, which can ~nt~r the gluconwgene;is p:uhw:lY or th~ Krebs cycl~. r~nibl~ II. Tissue lOur"", of in"'~asal ",rum Ai T activity and ALT half_life " .listffl in Tabl~ 11.1. Ill. Analyti<:a1 concepn: Oth.r than variations in .....y t~m~ ... tures, the major f""tor aff~ing ",rum AI. T activity ~mong assay .yn~ms is the presc:ne<: or .bsence of the cof~ctor P_S_P. A. Convc:r.;ion of .poenzyme> to holoenzyme> with cofactor .ddition i. =mmendffl, although the ef&ct it vari. ble and often not of diagnostic rone<:ffi. B. In dog. and cats, th~ us< of p_S-p w>s a.sso 14,000 %} w:I' notal with the .ddition ofP_S_P."·1l C. Ckpending on the de-grtt of he moly, is .nd th~ =y, in vitro h~moly.i, might ",,"us< f~lsdy in",..,asod AiT activity. In one:way in which marked hemoly,is in"'~asal the 121 ENZYMES 651 Table 12.4. Disorder. or conditio,," tbat cause increased ALT a.;tirity H~patocyu dam"l:~ (dogs and c;m) 'Deg~n~"'tiv" hypoxia c;m~ by .n~mia or rongenion AnoJrullou.s: ponosyn~mic mUn! (typically mild) 'Metabolic: lipidosis, di. ktes mdlitm, fdin~ hwrthyroidi,m 'Nwplastic: lymphoJrul, m""astatic nwplasia, h~patocdlular carcinoma N uuitional: copp<:r toxicosis, h~mochroJrultosis Inflammatory 'Inf..ctious: lq>tmpirosis, histoplasmo1is, fdin~ infectious p<:rilOnitis, bact..ial chol.ngioh~patiti, 'Noninf=ious: chronic hq>atiti., cirrho.i. lnh..iud: ropp<:r S{orng~ di=, lysoKlmal SlO"'1:" di",aWl 'Toxic: "'roid h~p,"op,"hy, anesth""ic age:nts, t"""'eyeline, carprof~n, l '",uJrultic: hit by car Skd~tal mUKI~ damage: (mild rdati"" to CK changes) lnh..iud: canine mUKulodystrophy T",uJrultic: hit by car • A lell1ively common di..... 01 condition Note: Lim of sp ... ph~nob.orbital not compJ.t. but.,., provided to giY<: onmp\es, ALT from .. 30 UIL to " 60 U/L in «iuin~ ",ra, th~ int~rf~"'n"'" w.. consid~red to be: c;>...ed by both .peetral in!erfer~n"" and the addition of ALT from th~ ~rythrocytes," In . noth .. study using canin~ ",ra, Jrulrko-d h~mol)"is had no effect in som~ assay' and a moderau df= in oth~"" '4 IV, lncr~ A, B, C. D, ",rum ALT . ctivity (Tabl~ 12.4) Hepatocyu damage (",,,,,,,,ible or irr~er1ibl~) may occur bc:cau.. of a vari""y of insult. (inflammation, hypoxia, toxicants, trauma, etc,), Al T Jruly be: rd~ased from hq>. tocytes also during reparati"" "ages of liver di ... "" lnn~a ... in amin~ ",rum AL T activiry th.r ..~ associated with glucoconicoid th~",py Jruly be: caused by glucocorticoid_induced h~patopathy inst.,d of true ~nzym~ induc_ tion, Glucoconicoid. did not increasr ALT synthesis in cultured hq>atocytes," Canin~ ",rum AL T activiry may be inc",asaI in dog. t",atM with ph~nobarbitaL In a study involving 12 phenobarbital_tre>ted dogs, data from th~ histopathologic examina_ tion of li""r and ch~mical .nal)"is of li""r homogtnat .. indic;>ted that induction did not cau.. the incrra~ ALT .ctivity," Spont.n~ous mmde damage: typically is nO! associated with increased .. rum ALT activiry, but incrrased ALT activity d"". occur with "'m~ musd~ di...ases in docs .nd c;>ts, l. Young dogs with musruiar dystrophy may h. "" marknlly increased .. rum CK activity (::> 500 X URL) and have mild to moder.t~ incrra= in ",rum ALT activity « 7 X URLj,l7 11 Som~ dystrophic docs occasionally hav~ extr~m~ AL T incrra= (values 20 times a. gr~at a. those in nond)"trophic docs in the same rolony), 2, Dymophin-deficient c;>ts with acute rh alxlomyol)"is had m. rko-dly incrra~ C K activity (89- 2000 X URL ) and incr~a..d ALT activity {6-19 X URL)," 652 V. FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY Spm~. diff~ .. nct' A. In dogs :md cat<. AL T i. a major muhr of h~patocyu dam,,!:~, but in .. rum activity is also incr=td by """"~ mUid~ disra ... B. H~po.tocytes of ho=, and allll~ h:IV~ "" little ALT that it is not a u..ful marhr of h~po.tocyt~ dam~ in thn< s~i ... ASPARTATE TRANSAMINASE (AST) (SYNONYM ABBREVIATION: GOn I. Phy>iologic process~" oo""qm, :md facts: AST is a cytoplasmic and mitochondrial ~nzym~ th at catalyzes • r.ve",ibl~ rraction invalva! in the deamination of "'p.nau to form oxaloa=at~, which can ent~r the Kreb, cycle. II. Tissu~ III. Analytical oonctpn: Oth~r than v.riations in .....y t~m~ratures, variations in .. rum AST activity ar~ minimal :mlong asiay syst~m. if ... ult, are rq>Ontd in UIL. How"""r, as for AL T, assay m.thods including the cofactor P·5·P may gc:n.rat. diff.",nt results from th""" lacking P·5·P. IV. IncreaK ",urct' of in"'~asal .. rum AST activity and AST half.lif~ .'" li Table 12,5. Diso,dc" or oonditiom that cause increased ASJ and LD activity H.po.tocyte damage: 'Dogs and cats (s,"" hepatocyte damagt conditions listtd for incrrastd ALT in T.bl. 12.4) Hono; .nd catde 'Dege:n"ative: hypoxia cau.s.d by .n.mia, congestion. cholelithiasis 'Metabolic: lipidmi. or fat cow syndrome, di.be,to; mdlitus, equine hy~rlipidemia Neoplastic: lymphoma, metastatic neoplasia, hepatocellular carcinoma Inflammatory 'Infcctious: bact"ial hepo.titis (Ty=r', di ..a",), bacterial rnolmgiohepatiti., infectioUi necrotic h.,..titis, hepo.tic . b = Noninfectious: Theiler'. di ..=, chronic hepatitis. cirrhosis Toxic: iron toxicity, pyrrolizidine alkaloid-rontaining planu, aflatoxins, .hih clov" toxicity 'Skeletal or cardiac muscl. damagt (= mu",l. damagt conditions listtd for incrrastd C K in Tabl. 12.8) ru..... or condition Noto: lins of .p wiU.uow AST aad LO from orytbrocyte> to inc", ... ..,rum AST .nd LO :octivitios. • A "1"i",,ly common 121 ENZYMES V. 653 Spatocyt~ damal:" in dogs and cats. How.ver. it i. not as ti .. u~ specific a. ALT and thu, i, not a, valuabl~ di.gnostically. B=l.u.. canin~ AST is rq>Ortl AST might provid~ a be,tter indication of active htpatocyte dam~. lA.CTATE DEHYDROGENASE (LD) (ALSO ABBREVIATED LDH) I. Phytiologic process". conctpt ••• nd fact, A. LD i. a cytopla'mic .nzym. that catalyzt, • """,r.sibl. ",action that converts pyruvat~ to lacut. at th. ~nd of an .. robic glycoly.i •. B. LD ii"~1 a", tetramm of .ith.. hem subunits (H) or muscl. subunits (M ): LD_l = H HHH. LD_2 = HHHM. LD-3 = HHMM. LD_4 = HMMM •• nd LD-5 = MMMM. Id~ntification of i,;o.,nzymts that are causing th. iner ..... in total .. rum LD activity could inc",..., th~ diagnostic vatu. of LD activity. However. ilO [I. Ti",,~ wurer. of inc",.. 111. Analytical conerpu A. Variations in =y ttmptrature:s can alt.. LD activity. B. Som. =y' mtamre LD activity in a pyruvatt to lacu,. rtaction. wh..... oth... m.asur. LD .ctivity in a lactat~ to pyruvat~ rt.ction. Th. LD a.ctivity in th~ two typ.. of rt.ctions can vaty consid~",bly.'" ptrhaps bc,caust LD_l i. inhibit [V. Incr... c. dam~. By it..lf... rum LD activity i. a ""INning test for hq>atocyt~ or mu""l. dam'4:'. In a group of um. LD activity provides additional information that may hdp ""plain activities of mort tissu.... pecific .nzymes {i .•.• ALT.ID. and CK). D. Alttration, in LD i,oe:nzymt !"Itt.rn, in dog, with lymphom.:" Th~ ptrerntages ofLD iso<:nzym.. w<:'" dettrminal in four groups of dog. with lymphoma: Group A had no tr ... tm.nt. group B was utatal with st.roid>. group C was in rtmi ... ion. and group D had recurrtnt di......,. Th. dogs in group' A. B. and D had grtattr ptrctntages of LD_2 .nd LD_3 and low<:r p 654 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY IDITOL DEHYDROGENASE {ID} {SYNONYM ABBREVIATION: SDH} I. Phpiologic processt., OOIlcq.ts, and faa. A. ID i. a cytoplasmic ~nzym~ th at cotalyus fmeta... 10 sorbitol {or glucitol}. B. Th~ ~nabJi,hffi nam~ for th~ ~nzym~ ~ r""~,,ibl~ ..action involving conv~rsion of is iditol ddtydrogt'II"', but iditol is not a mbnnte or product in clinical assay•. The Tissue router, of inc""asal",rum ID . ctivity and ID half.life ",eli'IM in T:.bl. 12.2. Ill. Analytical oonctpu ,,= A. Other than vuiation. causn! by diff... in :way t.m~rarures, variations in ..,rum JD ""civily are minimal :nnong assay 'Y"ums if re,ults no rqm'tM in UIL. B. ID:ways "" nol consistently included in strum ch.mico[ profiJ.. , in pm bta.u.. th. enzyme'. stability j, a oon""rn for routin~ handling of mail_in sampl... [v. Incr~ .. rum ID activity A. This indicot.. hep:itocyte dam"i:" and Jruly bt rrv..,ible or irrrversible. focal or diffuse. It i, unusual to find [0 >etivity:> lOx URL. B. 10 activity is used primarily in horse. and cottle btc.u .. other common hepatic cytosolic enzyme. (AST and LO) ar~ not liver 'p«ific, and ALT i. not a ....,ful nurhr of hep:ltocyte damag~ in horse. and conle. e. [0 activity is some"tim.. u..d as a marku of hepatocyte dam"i:" in dogs and cats, but ALT =ys are more commonly available and ALT is mo .. stable. GLlITAMATE DEHYDROGENASE {GMD} {ALSO ABBREVIATED GDH, GLD, and GLDH} I. Phy.iologic process", COfl<'epts, and facts A. GMD is a mitochondrial enzyme' that cotalyze. a rrvef!ible r""'tion involving ronnr_ ,jon of glutamate to 2-oxoglutarate. [n people, ""ntrilobubr hep>tocytes have relltinly mor~ GMO activity than do the Jl<'riponal hepatocyt ••. B. GMD is not a rommon abbrrviation for this enzyme, but it is a r«:anunend«i abbreviation.' Two mo", common abbreviations ar~ GOH and GLDH. GOH i. also u...d as an abbreviation for gly",rat~ dehydrogenase and glu"",", dehydrog<'n ... . [I. Tissue Klur"". of increased ",rum GMD activity and GMD half_life (Table 12.2.): Serum GMD activity i. manly from hep"tocytes, but GMD is also found in many other tissues (~.g. , kidney, int..nine, and muscle). Howrv.. , damage to tho .. tissues doel not incrnse .. rum GMD activity, btc....., the enzyme d~. not ~nter plasma {~.g., kidnry and intestin~} or there is too littl~ in the tissues (e.g., mu.scl~ and salivary glands). III. Analytical con""pn: GMD assaY' han not h«n available in h«n widdy appli«i in other parts of the world. th~ United States but han 121 ENZYMES IV. 655 lna~ GMD activity A. This typically indicat.. h~patocyu dam"l:~ that may "" r~""nibl~ or irrevmibl~. focal or diffil5~. Disord~n or conditions {Iist«l in T.bl.. 12.4 and 125} that alU" hep.to_ cyt~ daroag<' may incru"" .. rum GMD activity. B. Ru«l on r~sults of li""r biopsi... th~ diagnostic .. nsitivity of ina~aMd GMD activity for diff~r~ntiating th~ p .... n"'" of li""r dis ...", from th~ ab.. n"" of liv.. di ..... in hor..,s was not as good .... that of GGT activity. but GMD had a ""mr p ALKAUNE PHOSPHATASE (ALP) I. Phy. II. Tissue rour",. of inc....«l .. rum ALP activity and ALP half_lif. (T .bl. 12.2) A. In dom~nic mommals. th~ .. appe>r to "" two g~n .. for th~ production of two i""n_ zymes: {I} I_ALP and {2} tissue-nonspecific ALP. Posttranuational modification of the nonspecific ALP cr~at .. diff~ ..nt isoforms of ALP: l...ALP of hepatocyt.. and biliaty .pithdium. and B_ALP of osttoblasts. I_ALP has not bttn shown to incrn", .. rum ALP activity.' B. C_ALP i, a uniqu~ canine ~nzym~ that is producM by h.patocyt~s wh~n ,timulat«l by conirost~roids. Chemically. its . mino acid sequ~n"'" i, the .arne as I_ALP. but it i. mor~ highly glyco.ylat«l. 656 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY III. An:dytical conctpu: Besides variation, in """y t~mp'ntures, variations in substrates :md pH Im}' cau .. marl.:«i diffor~nces in sorum ALP activity among =y sy.ums, A, In routine =ys, m~~mral ALP a.ctivity r~p ....,nt' total ALP ~ctivity ~nd typically includ.. activity from l_AlP and B_ALP (.bo CALP in dogs), Th~ rdativ~ rontribu_ tio", to total ALP activity vaty with ag~, B, In h~.hhy dogs, C_AlP contributes about 10--30 % of total ALP activity; it contributes mor~ in old~r dogs m..n in young~r dogs.'" Although often not routindy m""sur«i q>~ratdy, it i. hdpful to considor th~ diff~ .. nt isoforms of ALP wh~n intorpming ALP activity. C. Th~ rdatin contributiofll of th~ diff.r~nt ALP isoforms to total ALP .ctivity can be, d~wminM by ~nzym~ d.crrophoresi., sd""ti", inhibition, th~rmal subility, or ..,]""ti... pr""i pitat ion.' I 's-"" I. Affinity d""trophoresi. i. the connntional or stand.rd method but ..quires 'I""'ial "'Iuipment and i, tim~ and labor inl<nsi",."" L-ALP, CALP, .nd B_ALP am be, id~ntifi«i in c;onin~ .. rum. 2. ~isol~ .d""tivdy inhibi.. L_ALP .nd B_ALP activity, but CALP is rdativdy resistant. Mumr.m.nt of strum ALP activity with .nd without the ~ddition of Irvamisol. can hdp d"~rmin. wh",h.. mum of the strum ALP activity is du~ to CALP. 3. CALP it ..lativdy h""t nabl~ at 56'C :md 65 ·C, "'n~= L-ALP :md B_ALP~ .. h~ .. labil•. M~asur~m~nt of strum ALP .ctivity prior to and aft .. incubating strum in a h~."d waUr bath em hdp d~t..min~ wh"h~r mum of th~ .. rum ALP .ctivity is du~ to C_ALP. 4. B_ALP .nd C_ALP .'" stl.crivdy pr""ipitat«i by wh~at C"rm I""tin, wh~reas L-ALP i, not. ALP .ctivity be,for•• nd after th. pr""ipitation am be, usn! to ...imare B_ALP activity when combin«i with d",ormination of CALP a.ctivity by I"",misol. inhibition. [V. Incr.....d .. rum ALP .ctivity (T.ble 12.6) A. Cholmasi, {intrahepatic or p<>!th.patic} I. [nc",astd ALP activity 'Pp'au to be, primarily caUstd by iner•• """ production of L-ALP by hepatocytes .nd biliaty epithdium during obstructive mol.stasis. It is not known wh"hor ALP production is increastd in functional molestasis (..., Chapter 13). 2 M.ny di ...... l~ad to imp. ir«i bile flow, which leads to .n accumulation of bil~ acid, in hepatocyte.. Incr~..«i bile acid con""ntrations stimulal< L-ALP production, promol< accumulation ofL_ALP on sinusoidal hepatocyte membranes, .nd .. ~ linl.:«i to iner.. """ strum ALP .ctivity {Fig. 12.3).""'" Bile acids may promote L_ ALP rd..... from the hepatocyte membranes by promoting .ctivity of g1yco,y1phos_ phatidylinositol pho,pholipast D ...." 3. [n mol<::s{atic disorde .. , othor mbst..,,,,,s or facto .. might alto induct L-ALP production, or there might be d""",..«i biliaty excmion of ALP. In the absen"" of molesmi" there 'P!""'''' to be, too little ALP associat«i with hepatocytes and biliaty epithdium for hepatic necrosis to cau.. larg. inc ...... ' in .. rum ALP activity. B. Induction by drugs or hormones l. M.ny drugs h .... the potential to incr..... L-ALP .ctivity. Corticosteroid. {pr«ini_ son~ and pr«inisolon. }, phenobarbital, ..,d primidon •• '" rommonly .. ported to induct L-ALP production. The d'1:"" of induction typically d~nd. on do .. :md 12/ ENZYME S 657 Table 12.6. Disorder. or conditions that causc increased ALP activity (the i.ofonn produ ced i. in parentheses) Choles{asis, ~ith~r imrIDq>~tic or posmq>atic (L_ALP) ·Degcn~r.lli ... : necrosis or hepatocyt~ ,vidling tru.t l~",h to impaired bile How 'M~ybolic: lipidosi., diaktes mdlilUl, hy~radrenoronicism, choldithiasi. 'Neopla.tic: ba~ duct carcinoma, pancrutic cucinoma, lymphoma 'Inflammatory: ~riporyl hepatiti., cho!."'giti., choungioh~patiti., panc"'~titi. Toxic: pyrrolizidin~ alkaloid-amYining pum., alsik~ do... r toxicity, .porodesmin toxico.is Induction by drugs or honnon .. 'Ph~nobarbital, dil.min, primidon~ (L-ALP) 'Corticosteroid. (L-ALP and canin~ C_ALP): cndo~nou. or exo~nou. 'l1tyroxin~ (B-ALP): fdine h~rthyroidi.m Increased o.teoblastic activity (B_ALP): onoooarcoma, f.. ctur~ repair, riam Canine mammary neoplasm., knign and malignam B~nign f.mili.l h~rphosphata .. mia in Sikri:m hun:i .. • A rel. ti",ly oommon dioe ... 01 a",dition Not<: Lins of sp th< acliviti", up<'Ct«l in m a tw~ .rumah, Ing...oon 1IId .bsorption of colootnun by II duration of .dmininr.tion of m~ compound, It take; hours for hq>.tocyres to incrra.. L_ALP production (it may tak~ daY" for clinical ~id~n"" of incrrased production), and incrrasnl .. rum ALP activity may persi.t for 2--4 wk after .. mov:d of the drug,""'" a, In a study involving 12 ph~noWrbit:d_tr~~ted dogs, data &om th~ hi.top~thologic examination of Ii... r .nd ch~mical anal",i. ofJiv~r homog~nat~. indicoted that th~ incrrasnl ALP activity w;o ! not causal by induction," b, Data from hq>atic tissu~ homogenat .. analyzed aft~r 32 d of prednioon~ injections indicote that th~ n~roid tr~.tm~m incr....cd L_ALP production by inc=sing gene transcription in hepatocyt..," 2, C_ALP a, In conids, oortico.t~roid. {e,g" ~ndog~no"" coniool or exog~nou. prednisone or p.. dnisolon~} induce the production of a unique ALP iKlform of I_ALP called GALP {.ho called a_ALP for conicost~roid_induccd ALP, Sf_ALP for n~roid_ inducal ALP, and CAP for coniOO5t~roid .lk:din~ phosph.tase}, Th~ incr~ ..e in serum CALP activity begins about. w.. k after initiation of neroid th~rapy, Most experimental rndence "'ppom that coniOO5t~roid. up_"'{:uute a .pccific gen~ in canine hepatocyte. to produce thi. uniqu~ ~nzym~," b, Measuring CALP activity con be a ",r.. ning un for conine hy~radr~noconi_ ci.m bccau.. most dog. {83-100 %} with spomaneou. h~ ..drenocortici.m hOi"" inc",= 658 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY C. [ncr~a.ffl oneoblastic .ctivity l. Bon~ tesio", that co"",,, inn"asal os{ithdial erll, in m ammaty neopl:um, do h:IV~ ALP .ctivity." &rum ALP activity in dog, with oth~r neopl:u.ms wa. not ..... ssal. E. B.nign familial hyperphosphat=mia in Sib.rian huskies" I. Of 42 pups in eight rdatM litt~" of Siberian huskies. 17 pups had .. rum ALP activities .bout six tim .. the activities found in oth~r ~_matchM Sib.rian huokies. B_ALP wa, the i,oform cau.ing inc ...... d total ALP activity in.ll fi"" of th~ puppies for which isofornu wer~ a=ssal. 2. Th~ alUX of the incr .... xd ALP w:u not detuminM. &rum rona.ntration, of total mkium. inorganic pho,phoru •• and parathyroid hormon~ wtr~ not diff~'""nt &om tho .. of m atchM pups. F. Pregnant women IIUy have an incr .... xd .. rum ALP activity b.cau.. of incrnxd plaerntal ALP. ALP .ctivity did not incr.. ", in pregnant nur ..." Th~ incr~.'" in .. rum ALP during p'""gnancy in bitches i, too smaU to .ff~ ALP intuprffation." Plaerntal ALP m.y rontribut~ to .. rum ALP activity in th~ l at~_tum pregnancy of ailS." ru.vt h."" v. Speci.. and brttd diff~ren= A. Dogs I. ALP ha, high di3i:nostic sensitivity for dff~ing rnolestasis. ALP .ctivity may b. iner .... xd b.fore ict~ru, 'PP"'''. ALP valu~, may range &om < 2 X URL to;> 20 X URL 2. Inc,""asffl ALP activity inducal by rortioost~roid. result, from inducal synthelis of l-ALP and C_ALP. ALP values may rang<' from < 2 X URL to ;> 20 X URL" 3. Phenobarbital, primidone, and ph~nytoin are d~JCrib.d in cliniml studies:u ~ith~r inducing th~ .ynthesi. ofL-AlP or musing h~patic damage tru.t incr......,. .. rum LALP in dogs."' 4. Increasffl ALP activitYaiusal by incr~a ..d oneoblastic .ctivity in growing dog, (production of B-Alp) is typically mild {< 4 X URL)." 5. At l~ast rom~ Scotti,h t~rri~ .. have gr .... t~r .. rum ALP activities cru.n othu dog., and the diff~r.ner was g,""arer in dogs mort than 6 yr old. Part of th~ incrn!~ may be muxd by. high~r incid~nct in th. brttd of di, ....... that cau.. inc,""asffl ALP. Howrv~r, Scotti,h urriers without thes. di ...... had a m~.n ALP activity n.... r 121 ENZYMES 659 1350 UIL, wh~r~ .. 3i:~-matchal dog. without th~ di ... ",s h. d a m~an ALP n""r 230 UlL" B, Cats I, ALP h.. poor di3i:nonic smsitivity for dettcting chobrni" Cm typically a,", icwic befor~ ALP activity inc""' .... ALP valu .. may rang<' from < 2 X URL to:> 10 X URL 2, [n 12 (SO %) of 15 of cats with lipidosis, the ALP:GGT ratio wa. incr~..al {i,~" ALP activity increasffl mo,", than GGT activity)," Only 4 (10 %) of 39 of th~ caU with liv~r di ..ases oth~r than lipid""is (i,e" bil~ duct obmuction, cholangitis, cholangiohepatitis, na>pla,ia, hq.atic ntcrosis, and cirrhosi.) had incru,ffi ALP:GGT rati"" (m3i:nitud.. of chaIll:~ not r~portal), 3, It h.. be.::n rq><>rtal that from 43 % to 75 % ofh~rthyroid caU han incr~aMd .. rum ALP activiti.,., ALP activiti.,. typically are < 4 X URL In such em, the incr""offl ALP activity is cmoffl by L-ALP and B_ALP, wh ...... ALP activity in h~althy matu,", caU i, du~ to L_ALP, Some hypmhyroid em han incr~...d B_ALP activity in ..,rum without a concurrent incr~= in total ALP activity,·' C. Ho"",. I, ALP has poor diagnostic stnsitivity for dettcting cholm..is, HofS<S typically ar~ icteric before ALP activity incr,,=s, ALP values may ra~ from < 2 X URL to :> lOx URL 2, Th~", i, no evidence that glucocorticoid tr""tmenlS incr~... ..,rum L-ALP activity in ho ...., nor th . t ho"",. produ"" CALP," In two reportal = of steroid hq.atopa_ thy in ho ...., ALP activity was not m~amral,"'~ 3, [n horses with colic or int~stinal lesions, ALP activity may incr~ ... in the peritoneal fluid pr~dominantly beaus<: of inc",aso:d gnnulocytic ALP activity," but oth~r Physiologic P""'"'SS", concepts, . nd facts A, GGT i. associatal with ""ll membran.., It cauly= the transfc:r of gluumyl group. betwe<:n peptides and is inmlval in gluuthione r~.ctions, Many cdls have GGT activity, but biliary epithdial cdls, pancr""tic acinar cdls, and renal tubular q.ithdial ""lls . re classically consideral to h.n th~ g",.test . ctivity, [n som~ 'peei.,., mamm. ry glands .lso have high GGT activity, In these speeies, GGT is as.soci.tal with mammary glandular ~pithdial m~mbnn.. and milk m~mbnn .. in milk.~'" B, Colostrum of cow> h.. high GGT activity, and the GGT moltcul .. may be .b""bed from the calf intestine after colostral inuk~, PomuckJing calv.. may han GGT activities .. high .. 20 X URL (using adult ,",f~"'nce int~rvab) and up to 16 times the presuclding valu ..,",...", This physiologic ch ang<' can be uK '01 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY T able 12.7. Di..,rdc" or conditio,," Ibal cause increased GGT activity 'Cholmasj, {,.., choi.,t;"j, conditions lisrN for inc",asffi ALP in T ahle I 2.6} Bili ahih PhcnobarbiuJ, dilantin, primidonc Co'licosuroids: cndogcnou. or rxogcnou • • A ",t"ivoly common dis. ... or condition. Noto: lng,";on:and absorption of col<>ltnun by ....,...,n calves and PUP' m.y inc ....... rum GGT activity. [I. Tissue rourct, of inc",asal..,rum GGT activity:md GGT half.lif. "c iislw in Table 12.2. Ill. Analytical oonctpu A. s.rum GGT a.clivity vari .. minimally :nnong ......y .y&cms if result< arc .. porlM in VlL and ... ay tcmptntur. , "c the sarut. B. In .. srudy involving rat sampJ.. , hcr:uin was mown 10 nearly double Ih. GGT activity in an a=y u•.d r_g1utomyl.p_nitroanilid. as a substm~."" H~parin ~I.lO am caUst cru.1 reaction fluid, which v..ould int~rf~" with tnn!JIIission photometry.ll Th~" w turbidity in IY. th~ Incr<:a<ed strum GGT .ctivity (T.bl~ 12.7) A. Cholena,i, or biliaty h~rplasia l. Increased h~patic and plwIIIabli,hed." Disorders that cau", cholena,i, may:oloo indu,,", biliaty h~rpl.,ia and a resulum incre= in GGT activity. 2. &~rimenul data &om r>t studi.. indicate that incr~.sed .. rum GGT activity primarily dep"nds on the degr.., of hyp"rplasia of biliary epichdial ""lis .nd not on induction of GGT 'ynthesi', hepatocyte d:nn3.£<', or roolestasis." B. Associ . ted with drug, or hormones l. s"rum GGT ~ctivities tr'lI!i~ndy increastd to 2- 3 tim .. the basdine values from wttks 13 to 17 in dog. given ph~nobarbit:d for 27 wk. but mean .ctivity did not nettd "f"en"" imerval,!' 2. Induction lillIy not inc",,,,,, ",rum GGT activity in dogs king treated with gluco_ conicoid hormon .. , sin"" glucoconicoid, did not indu,,", GGT .ynchesi. in cultured hep.tocyt... " However, in prrdni.lOne_trnted dog., both hepatic .nd ",rum GGT activities increased, which suggested induction or the effeen of steroid hep.top.thy. " C. Hepatocyte dalilllgt in hor",,, EXp"rimem:ol data indicat~ that acute h~patocdlular n«rosi. lillIy cau.. mild inc",ast. « 4 X ba,din~) in GGT .ctivity.'" It is not known wh<'ther th~ incr~a!ed GGT rep", .. med hepatocyte n«rosi, or rd~"", ofGGT from ""II, b<"Cau.. of stCOndary roolest.,i •. 121 ENZYMES v. 661 Sptcies diff~ .. ner. A. Horst. I. Inc",_d GGT activity has beu~r diagnostic ..,nsitivity th~n do.. ALP for det«ting chol~st .. is or other bili:uy di"'rders in horus. In aptrim~nt;ol cholmasis. both GGT and ALP .ctivities inere • ....:!. but th~ GGT inc.... ~ (.. 7x) wa. great.. than th~ ALP inc.._ {.. 2xl." 2. Nwruual foals hav~ gre.t.. ..,rum GGT activity than do their mares. but not ba::;,...., ofGGT uptake in rolomum '" occurs in oth... p«i~s.n B. Cattl~ I. Inc",a.st<\ GGT activity is gen..ally ronsid~red to h. ve !>Hter diagnostic stnsitivity than does ALP for d WRI." VI. GGT activity in urine A. D .mag~ to renal epithdial erU, incre. """ the urinary excretion of .. nat GGT without inc",..ing th~ ",rum GGT activity. B. B«aUst th~ urinaty GGT activity dq> CREATINE KINASE (CK) I. Phy.iologic process... roncqJlS •• nd facts A. eK is a cytoplasmic ~nzyme that cotalyzes • r<"V~rsible r...ction involved in th~ transfer of phmph . te {PO,} from c",atin~_PO, to .d~nosin~ diphosph. tr (ADP) to form ATP. Cr~atin~ pho'phokinas~ (CPIC) is not an .ectptabl~ name for the ~nzyme. 662 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY B. CK is • dim~r. Th~ .. are four j"",ozymes th. t hove "",iabl. cdl dinributions: CK_1 dominates in br>in, CK_2 .nd CK.] in cardiac .nd skdeul muscle, .nd CK_Mr in mitochondria of many tissue •. C. Puppies and young doc' Ita"" gre>!" .. rum CK activity th,n adult docs. Comparal to adult dog. (> I yr old), the mnn CK activity WlU about 60 % ~a{" in the 6_ to 12_ ffio.oJd doC" 280 % g,ra,er in th. l _ to 6_mo-oJd puppies, and 410 % great" in puppie, less than I mo old." Also, th. me.n CK activity in small_brttd dog. « 10 kg) ~ about 70 % than activity in larg~breffi dog. (:> 25 kg}.'" Physiologic explanation, for th. "i:" and lxxIy.ju diff".nces w... not fuund. gr..,., KlU'''''. of inc",asffi ",rum CK activity and CK half_life are li,rN in T .bl. 12.2. [I. Tissue III. Analytical oonctpu A. Orher than v.riation, a1~ by differences in :way I.m~r>rures, .. rum CK activily ha, minimal varialion among ''''''y 'Y'1~ms if ..",h. a.. rq"Jn«i in UIL The an. lytical r:m!';O' of som~ comm"cial =ys are 100 narrow for "'m~ domenic mammal" and Ihu, .... fr"'lu~ndy may nm to b. dilUl«iro oblain numeric resuh, when CK .ctivily i, incr~..«i, B, In .. mples from healthy dogs, Ihe CK activily in strum sampl.. i, about 2,5 lim.. thaI of pla,ma .. mples," Th~ diff~ren"" may b. c;mstd by Ih~ rd..... of CK from platdcts during clolting," conine pl'ld~u h""c b..n ..""rt«i to contain CK .ctivity," Thi, diff..en"" m.y not b. clinically rdevant when the incrn.. in CK aClivity is moderate to mark«!, Incomplff~ r~mov:d of plaldm from plasma could lrad ro mildly inc......d CK .ctivity, C. Canin~ CK a.clivity in pla,ma WlI5 nabl. for I wk at - 4'C .nd for I mo .1-20 -c," IV, Inc=std .. rum CK aClivity (Tabl~ 12,S) A, A v.ri~ty of insults {pathologic and i.lrogtnic} may damage muscle fikrs .nd cou.. th~ rd...... ofCK from muscle fikrs, CK may k rde....d b«au.. of na:lOSi, or ..""nibl. ""II damage, Tahle 12,8. Diso,den or conditio"" Ihal cause increased C K activity Musel. ~ (mosdy .kdel:d, ocauionally cardiac, r>rdy unooth) ·D"t<'n~ ..ti",,: hypoxia coust<\ by ""enion or .. izu ... , """rtional rhabdomyolysis, .. ddl~ thrombus NcopJ.,tic: metastatic n"'pla,i. Nutrilional: vitamin E or ..tenium deficiency Inh"it«i: mUiculodystrophy, hyptrkalemic myopathy InHamm.lory: myosili' cou..d by N''''fMra, ToxopfMma, bact~ria or oth" ag<'nu: bovine endomffriti, Toxic: mon~n,in, castor kID, gossypol 'Traumatic: intramuscular inja:lions, hil by cor, ra:umb.ncy in hor..s .nd cold., seizures, ""enion • A •.J..ively common ru.. ... or condition Noto: lins of .ptcific ru..:.ses o. conditions:or< not compJ.« but:or. provi 12/ ENZYMES 663 l. Th~ m3l:nitud~ of inc"" ... con bt mild to nmm~ (< 2 X URL to ;> 100 X URL) and is som~wh>t proportional to th~ degJ'tt of mUld~ dam>gt. 2. From a .ingl~ insult (• .g .• recumbency or other traunu). there am bt vuy rapid inc= (hours) .nd a rapid dedine (hours to da,...) brcau", of the short C K half_life. B. Mild to mark~d inc=ses in serum CK .ctivity were .. ported in .no=tic cots with n ..oesoph3l:e. 1 tubt •.17 The exact p"thogtnesi. of the inc""ased activity is not known but prol>.obly involves muscle damag<'. Th. method of placing the tubts was not described. e. Hypothyroidism in dog> i. r~port~d to couse inc""ased C K activity. somr"lim•• nurked. but a pathophysiologic nplanation of the inc ....ed activity is not established." Wh .....s some hypothyroid docs ru.", inaeased ",rum C K activity. other hypothyroid dog> ha"" decreased ..,rum CK activity. D. [n vitro hemolysis may cou,~ faJ ..ly incr~.sed CK activity. Etythrocyr~. do not cont.in C K but do contain glumse-6.phosph. l<. adenylat. kin ... . and ATP. which may int.. f~ .. with th~ couplal ...ction. ofC K .uays. E. Injury to organs and tissu .. containing smooth muscle m.y muse incr ... sed ",rum C K activity but to. l.... r extem th. n with striated muscle dam~. Th... i, suflici~m C K in the bovine uterus that endometritis con couse inc..ased ..,rum C K .ctivity." Four oow> with moderate or s...... ~ndometritis had great.. CK values (.. 7 or IS tim .. as high. respectively) tru.n values in found in Ii", hralthy OOW>. The.. w .. a ooncur.. m. but rdatively minimal. ina ..... in AST activity. V. C K . ctivity in animals with neurologic di ..... se A. Brain .nd oth.. ti.mes of the e<mral nervous system oont.in high CK activity. Necro.i. or demyelination of neural tissu •• may cou .. increased CK activity in ctrebrospinal fluid samples. but n~ith .. is known to muse ina ...... in .. rum C K . ctivity. B. [ncr~.sed .. rum CK activity in domestic mammal, with neurologic di ... ... ppears to result from the rel ..... of muscle CK srrondary to oonvuhions or recumbtncy. AMYlASE (AMS] I. Phy.iologic process ... ooncq.ts. and facts A. AMS is. cytoplasmic enzyme that requir .. Ca'+ :md ct- .. oofactors and mtalyu. the hydroly.is of oompl.,. starch... B. Via dectropho ... is. four AMS is""nzymes (I- [V) ar~ classilied in dogs; itople and pigs h. ", salivary a_amylase. wh..~•• dogs. cots. ho"",s .•nd cotde do not. D. AMS activity in .. ra of he.lthy dogs includes p"nc... tic AMS. intenin. l AMS •• nd m. croamyla.. . ctivities. The Ii",r m. y also oontribut~. l. Compared to presurgical meamremenu. serum AMS activity in two of Ii", dog. did not d=....., by 7 d after pancreatectomy. In thJ'tt of Ii", dogs ... rum AMS .ctivity decr ......! by . bout 50 % . Th..~for~. in healthy dog>. serum AMS .ctivity m.y bt from p"nc"".tic and nonpanc ... tic ti.mes." 2. Up to 70 % of the plasma AMS in healthy dog> was of imestinal origin;" however. intestine is not consid..ed to bt a sourct of inc..ased .. rum AMS activity. In anoth~r group ofh ... lthy dog>. about 14 % of total .myl ... w .. bound to immuno_ globulins {macroamyla.. )." 664 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY E. Som~ jnv~,{icaton han found AM:S activity and mRNA for AMS in cmin~ liv~r."·'''''' 50 {h~ linr mij:ht rontribut< to w.dine .. rum AMS activity without 'loring appreci.bl. amou[I{, of th. [I. Tissue rourer, of inc",asaj ",rum AM:S activity and AMS half_life .", liSlffl in T .bl. 12.2. Ill. Analytical oonctpu A. Beside, variations in :way temptrarures, variations in sub,trates may c;m." marhd diff... nct. in .. rum AMS activity .mong ..say .yn~m •. B. Thu~ a.. thr« major tYP'" of :ways that m... u.. AMS activity. l. [n s;occharo!:"nic assaY' ('lU"clulrfJ_ m.an, "mgar' :md -gmy m..,.,., "produ~"). AMS Qlt;;o/y= th~ degr",htion of ,urch to produ~ glum .. :md malt"",. Th"", assaY' ar~ typiQllly ooupl.d with oth~r ..action! to m.... su'"" th~ glucos< producffl by th~ AMS ,""xtion. Canine .. rum oonuin! gluooamylase and maltase {'ynonym: 0._ gluoooidasel. Both may produ~ fahely increa=! AlvIS activity by ..acting with the malt"", prodUcffl by the AMS ...-..etion." 2. [n amylocla'tic a..... Y' (IImyU,_ m.... n' "narch" . nd -l"ftut me. ns "broke"). a dye bind, to the av. ilable starch. If th~re i, less bound dye: at th. ~nd of the ... ay. then there w >s mo .. AMS activity. Th... :ways were the p ... ferred amin~ AMS assay. kau .. gluooamylase did not interf.,"". but they a... not easily autom. ted. 3. In chromo!:"nic assaY'. AMS analyzes th. cl~a~ of. dye bound to a .ynthetic ,ub"rate. Some of th ... a,say, are accq>table fOr me. mring Qlnine AMS activity. whereas others are not." C. Spa:ific AMS isoenzyme> c:m be meamred. but their clinical value is limit.d. [v. Inc==! .. rum AMS . ctivity (fable 12.9) A. P:mc..atic acinar ~ll d~ l. Acut~ pancr....titi. i. th~ most oommon c;>use of damage to pancreatic acinar cdh. and it may =ult in the rd ...... of AMS ... pa:ially in dogs. The d . mage: m. y lead to Table 12.9. Dioorde" or conditiom Ihat cause increased AMS and LPS activities 'P. nc ..atic ""inar ~n damage:: inHammation. neapl ••i. (AMS . nd LPS) D«... =! renal cl....ranc. P.....nal di,ord.rs: dehydration. shock (AMS and LPS) ' Renal disorde,,: acute or chronic ...nal di, ....... {AMS .nd LPS} Po,crenal disord~rs: urinary tra.ct obstruction (AMS :md LPS) M""roamyl ... mi. (A,\tS) Other or unknown medt:mi,m Dexamethasone tr .... tm.nt (LPS) Pancreatic or hep.tic neo pla,ia (LPS) • A ,.t1tiv 121 ENZYMES 665 ""illar cdl lI«ro,i .. Th~ AMS may gain a= to blood vi<> nin' draining th~ paner"as and vi. lymphatic vesstb."ln a~rim~mal canin~ p.ner.,.titi" .. rum AMS .ctivity ~aks within 12--48 h .nd ~rsim for 8- 14 d,'" 2, Spn;ies variations a, ~rum AMS ""tiviti.. in dog. with acut~ pallcreatiti, rang.. from WRl to ""mmdy illcr......,j {> lOx URL}, b, In em with 'pontalla ",s paner~.titis, .. rum AMS .ctivities rang.. from WRl to mildly incr.,....! {< 3x URL},'· '-'" In a~rim~lIIal pancr~.titi. in six em, .. rum AMS activity did not illcr.... ~.'·, c. M~asur~melll of .. rum AMS activity has not """n u..ful for di"l:nosillg panc",_ atitis or other di ..as .. in hor... or ",nle. 3. P.ncr.,.tic lleoplasi<> call .lso I~:od to incr~...d .. rum AMS activity. B. D~<:r~...d renal inactivation or acretion I. In dogs, dillical and a perillYmal data indicat~ that the half_life of plasma AM:S ill== when renal blood flow or functional ",nal tissue d=eases.''''''' Exp (mean in=, 2.S-4.0 time; th~ baseline values). Clillically, the incr~...d AMS activity is apaxtd to ~ < 3x URi ill oonditiolls that cau.. d«reasal GFR without pancreatic acimr cdl da"",&". 2. Exactly how renal functions illfluen"" .. rum AMS .ctivity has not """n esubliohtd. a. V~ry littl~ to 110 AMS .ctivity is found in th~ urine of healthy dogs, ~rhaps ba::au.. AMS does not pas.! through the glom~rular filtration barri.. or """'u.~ it is illacrivattd or rtSOrbtd by rubular cdh aft .. it passes through the barri ... b. There i. a oorrdation between amylasuria and proteinuria, esp«ially glomerular proteinuria. Thus, it appear> ill health that the glomerubr filtration w.rier limiu th~ amoum of AMS that r.,.ches th~ tubular lum~lI, or ~rhaps • larg~ amount of luminal prot~in r~SI,]ting from glomerular di ..... ime&res with AMS inactivatioll by the tubular epithelial cdls."""''''' c. Ex~rimemal damage to proximal rellal tubular ails increased urinary loss of AMS. but rdatively not.., much •• urillary 10Sl ofLPS or lysozyme. Proximal tubular epithdial aIls ha"" some ",pability of inactivating or resorbing AMS if AMS d""s pas.! through the glomerular filtration barri... '" d. Formation of macroamyl... molecules ill blood also influences serum AMS .ctivity. Mllcrt111m}w, a oomplex ~tw"" AMS .nd all immunoglobulill or other prot~in, i. too large to pass through a healthy glomerul ar filtration w.ri~r .nd thus may han. longer half_life than nonoomplextd AMS. Macro. myl ... mia colllributes to the hyperamyl ... mia in ",me protein uric .nd :I7.Otemic dog.; how"""r, hyperamyl ... mi<> was still present in "'m~ .. ra .fter the macroamylaK molecul~s w..~ removtd. " 3. The imponanct of renal inactivation or acretion of AM:S by fdine kidlleY' h.., 1I0t """n ~subli,htd. Of 32 ",ts with rellal f.ilure, to had hyperamyl ... mi<> {slight to 3x URL}.'" C. Imestinal AMS h.., 1I0t """n .hoWII to incr • ..,e total .. rum AMS .ctivity. LIPASE {LPS} I. Phy>iologic process.., concepts, .nd facts A. I'IIncrrilfic LPS is a cytoplasmic enzyme that r~qui ... Ca'+, coli!""", alld bile sal" as cofactor>. It "'taly= th~ hydrolysis of triglycerid ... Th~ panc"'.tic LPS mol«ul~ '" FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY (M, = 48,OOO) and colipo... ar~ ,maU ~nough to p .... through ch~ glomuular fihmion barrj~r 10 be, inactiv:nal or acretal. B. &v,,:d [ip"= .r~ in Ih~ body, and ~ach has sptcific roJ .. in lipid metabolism. Mor< inform.tion .bout lipid m sinuooid., .nd hydroly= TG in low-d. mity lipoprotein mob;uJ ... 5. HomwnMms;tiw LPS in :adipocyt.. cot>lyu. Ih~ hydroly.is of stor«i TG .nd the libtmion of fatty acid.; it i, ,timulat~d by ~pinephrin~ and glucagon. 6. L}""Dmal aew IPS i, ~n intr:lcdlulor LPS that cotaly= th~ hydrolysi' of chol~u~rol ~u~ ... C. Compar«i to p... urgical m=urem~nu. ",rum LPS activity in four of fin dog. dec",as«i by 50-75 % by day 7 after panc=tectomy. Ho~r. LPS activity incr~=<:! by 40 % for on~ dog in the ... m~ uudy. O at> indiau«i that Ih~ p.ncr .... was a oontributor to .. rum lPS activity. but ther~ wer~ oth~r tissue source, in some dog.... Th~ nonpancreatic LPS in ",rum might"" gastric lPS or hepatic LPS. ar~ li,tM in T .bl~ 12.2. II. Tissue JOur"". of inc",=<:! ",rum LPS activity and LPS half_life III. Analytical oon""pu A. Clinical LPS =y methods h."" vari«i greatly. yidding di'pa""e results coused panly by v.ri ation, in analytical.pecificity for pancre;otic LPS. l. B.,ides variations in ....y temper.tures. variation> in substrates =y cause =rkM diff~ .. n"". in ",rum lPS activity .mong assay .yu~m'.'" 2. Exogenous oolipase i, ""Iuir«i for an ..... y to mumre total panc=tic LPS .ctivity. and roli~ i. not always includ«i in LPS assap. 3. Exogenous Ca>+ enhanea LPS activity. 4. [n some .ssays. heparin =y enh . n"" .. rum lipoprotein LPS .ctivity sufficiently to incr",.... rum LPS activity.'" and other lipa= Im}' bt d.tect«i. l l This is especially tru~ for :ways tmot use 1,2-diglJ'C"ride as a sub,trate. B. A newer ~nzymatic LPS :way mo. bttn purport«i to move C"'~ter analytical . pecificity for pancre.tic lPS and btu.. diagnostic .. nsitivity for p.ncreatitis than. 1.2- [V. IncreaKtion of publi,h«i data rq;arding serum LPS is romplicot«i by the wide variation in =y methods u.sffl. The following are general oonclusion" 121 ENZYMES 667 B. P. nc",. tic ""in.. cdl d"",~ l. Acut~ pancreatiti. i. th~ most common cou", of dam"l:" to pancr~~tic acin:or cdh and will ... uh in th~ rd..... ofLPS. esp«ially in dog .. Th~ dallU&" Jrulyor may not caus~ acinar cdl nectosis. Th~ LPS may gain a.c<:6S to blood vi. ",ins draining th~ panc",a.; and vi. lymphatic ",,,,,I.." 2. S~ies v~riations a. ~rum LPS :>etivity in dogs with ""ute p"ncreatitis ranges from WRI to extremdy incr lOx URi}. b. In .pomaneous fdin~ p:mcreatitis • ..,rum LPS activity ranges from WRl to mod~ratdy incr. tic neoplasia in dog" In Ii", of six casts. histoch~mi. cal . nd immunohistochemical finding. indicaud th. t p"ncr.atic . nd hepatic neopl .. ms we", potemial sourer. of mild to Jrulrked increaS«! ",rum lPS activity.'" Th~ ",me dogs did not hove significant incr.-ases in strum AMS ""tivity. F. As.lOci . ted with dexam",hason~ tr.atm.ms in docs'" I. Hyperlip".. mi. {< 2X URi} v..as found in 24 h~:dthy dogs by day 8 of m~tm~m with d=>mechason~ .ither at 2 mg/1q: or 0.2 mg/kg. In dog, with neurologic di",=. hyperlip"..,mia of incre~.ing ...... rity {mean value. 4.6x URi} ocrurr<'d by day 10 of treatmem (initial do .. at 2 mglkg and th.n r.ducal do ... ). Th~ number of dogs ''''''pled ""ri.d from two to eight on diff..em day•. 2. Microscopic ""idence of p"ncreatic d~ wa. not found. :md concurrem hyper. anlyt.",mia wa. not found. G. M ..k<'d hyperlip""'mia may ~ associat.d with wid .. pread steatitis cauS«! by LPS effects on adipo'" ti""e. P:mniculitis may ~ detect.d as firm ruocutaneous mass .. consisting of fat with a mild inflamm. tory compon~m.l2l 668 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY Table 12.10. Other serum enzymes Strum Dj,old" au.." Diagnonic value 5'·Nucl..,tidast Aldol"", t t Argin= i H'p"tobiliary di= .. Musd. fikl dom"&,, H'p"tocyu dam. g. Cholinestera..' ,l. Organophosphat[' toxicosis, inf<'Ctions, t i chronic liv t [ntestin:d mUC05a i i Nropl""i. (histiocytic) lsocitrate d.hydrogtn= Lom:yl :nnino~p{jda!~ Malt"", Muramidast (1y><>Zyme) MaJ.te d.hydrogtn= Omithinr carbamoyltr:m,f.""" T rrP"in-like immuno"",,,,,{iv;ly (TU) (Ott Chap!" 15) t t • PJ .. m. poeudocho~ ...."r... or butyryldtoJU>."onoe: not • Also ulW J.ucm. aminopopti.w. V. Pancreoo[ic Ii"".. immunoreactivity CPU} rumagt Hrp"locyu damagt HrpalocyU damagt Panc,,",. I;t;, or d«rr.,ffl GFR Exocrinr pancrr.tic insufficiency in dogs .nd cots .~cltoJi .... ton>< of thl myoneunl jUBCtion A. To impron the analytical .p«ificiry of LPS .....ys, immun~Y' w". drvdopffi by u.ing .n antibody .ptcific for mnin~ p.ncrO C. Cats I, [n upOntaneou, pancreatiti., wh~n a fPLI assay and a decision th=hold of 10 !AglL (",f~ren'" int.",:d, 2,0--6,8Ilg!L) wer~ u...i, the diagnostic .. mitivity of the [PLI] was 100 % for em with moderate to ..""'" pancreatiti. (.i>: em) .nd 54 % for caU with mild pancr.atiti, (eight mts),''' PU mrasur~m.nt' had mo", di:ognostic ",n,itivity th an did TU measur.menU or abdominal ultrasound. 12/ ENZYMES 669 OTHER SERUM ENZYMES M~ny ",rum ~nzym .. ru.v~ bttn ........d in an m~mpt to find btuor indialors of p.lhologic nale, involving hepatocyte, or olhor edl, (Table 12 .10). For a v .. i<'ty of re''''fl!. mO!t have failed to bt :as dinirnly v:..luable :as Iho .. ducribtd ... rlior in thi, chapw. Decre• ...d TLI has bren shown 10 bt valuable in di>gnO!ing exocrine pancr~ alic inmfficiency. Incre>sn1 TLI has bren found in acti"" p:mcrealiti. (Ott Cru.pt~r (5 ). R e fe re n ces t . Boyd JW. 198J. n.., "',d.....i..... "Iati.>, to u.a.,.... iD pI .. ma ''''1'''''' and ioo<.uym .. in di" ..... of • ..i"' .... Y.. aiD P.d.oI. 12,9--24 . 2. Gom GJ. H"", ... B, I...n..tn. JJ. 1990. 1'1...... """,bun. bl.b 1"0.",""'" • ..d "'P'''''' A roonmon f,,,,,,,, of t..p.ooIic and mirod.ondtiat du BioIo,,". N<>mnk. 5. Baron DN. Mo .. DW. Walk" !'G. Wi!k;""'" JH. 1975. R..-i..d Ii.< of .bb.",i.tiono 1"0......... of 'nzym" of di."",""" importan«. J Clin P.d.oI. 2M92-59}. 6. Co.otro-e-Silva 0 J•• F"n«> CFF. Picin.to MANC. Sou.z. MEJ. M-=_ SA. c.....; .. R. 1989. H,pui ...i..duc.d iDe .... ", in pl .. "" and "rum y.ri .......p ... nopo.. tx;. 1985. S"bili.,. and .""'V d.anoctni.tia of mzpn0" ~tio in ",,,. Can J y" R.. 7O,}I}-JI6. It. O ·NtiH SL. Fd.dm.o BF. 1989. H~ ... facto. iD dinicd d.....; •...,. and ~'matol.oc:7 of th, ~ Yrt Clin P.""'] 18,58-68. IS. Hodl.,. sr. HoIfm ... a WE. KoohJ ... d .. idt MS. s..-±i RK. Do""" JL 1990. mtc. of ~<>ido on aLWia, pbo. pk ...... aknint ....inotr.n,{,.."' . and pmnoa-dut.myIo ........ " in ooltwod door; k<pato 4},89--98 . 16. G. okin CL. Min" LM. M"'_nJS . Hoffm••m WE. 1IurtonSA. Gd .... HC. lhk SL. Mith JB. Slu.... DH. C.ibb AE. 2005. u... hi''''P''''''lou ...d li"" • ..d "rum al ...... ....;.-......... " and alblint p~ .. ", artiviti .. iD Muock N, .... I h I056-106t. 18. Yal J Y.. In"", Mod H-ID-I-iJ. 19. G.d,n F. Gudo", L. Stil" G. Wdl, M. &n..ndJ.omin Y. G<>nin. J..... D. Ntiv>.Aoodob.dot. G. ~ DamiJa..o M. 1998. l.ttlu..l p. Y.. P.d.oI. J5,1 17- llJ. 20. Flti.b.. GA. Wakim KG. 196J. n, fa" of 'nzym";" body Buld" An ~""ntal .. ".[,. I. Di..ppndi,;"m. J Ub Oin Mod 61,9/1-106. ' 70 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY 22. Mou DW, H,od,,",,,, AR. 1999. Oinial ''''J''''''''v. In, Bwcl. CA. AaIr.oood ER. .do. Ti, .. T,""'-" ./C/;"i..J a.. ......,. 3,d odhlo.., 617_ 721. Ph.iLaddphi.. WB Saw.d.... lJ. CU 449--452. lS. 26. 27. 28. 29. }O. }I. n. lJ, }•. AE. Smhl. KC, N...-.. JR. 2OO}. An ...!u...... of diapoootk data in com"..i"", .. tJ.. ",rult. of Ii"" biop ... , in m""", ,"""... Eq.in, Vrt J JS,SSt-- SS9. MOI.... N. Knf< W. 199.(. Dia~ .. t... of ~",dd.yd",,,,,, ... <1.tra K. Ball .. WW. KIck PA. Hacdonk MJ, Mdj... DK. 1997. A p.~&ic fuocti.on fo..Jblin< p~.. "" E..dotm.iD drto.ifia. ..... , Ub lnvat76,JI9--lZ7. Bak. ...... M, H.m."" E.:l.hans; L. I'ikuJ. S, ~ G, Radj""" j, B.~Pi.boIaJ. Bud..< R. ZOOJ. n.., roI" .f ....,..in. and o.lhlin, pbo.pb.- in mi".. alhation po<>«>O. Acta BiocJ.im Pol. 50,)019--1038. c.m.,. SA, Randolplo JF. M .. W ...... T, st."" M. 1991. EHect of ooIootnom in~nioD on , ,,,,,,,.-Pnamyltun.f..... and alb.Ii"" p/o<>"",,"" oc';';';'. in....,.,.".[ I"'P" Am I Y... R... 51,'9"9--50 • . s..,ocki RK. HoIfm ..... WE. H.""" R. Sd..rtr.. DI. 19"9J. Q..aotibc.""" of bon. .tb.lin, phoopb....., in ani"" .. rum. Y ... Oin r.th.>1 2;!' 17- lJ. H,.".k AM. HoIf"' ..... WE. Sanooki RK. Sd",dJ", DI. Do."" IL 19"9J. Q..aoti .. ti'" d"m.inotioon of 0<[...... • lkalin. p/o<>oplu.tao<;"'""""" in foo.l • ..t .duh .. """. I Yrt ]""'" Mod 7,20-14. Lank" IA. Hammon HM, Blum JW. 200 I. k~,;~," of p"""".pu .. mylt"'•.rn..... lkalin. p/o<>.plu.tao< and .. part,,<-<"'inottan.f.. ... in 0010"",,,,, ",ilk and bb,.[ plaama of cd..... fed /in. 0010. ....... at 0-2, 6-7. Ilr-ll and 24-25 h of... birtl.. I Y" Mod [AI 48,179-185. SyoLlinu. M, Takipdti M. y.....d. I, H .... imotoA. 1m. TI.. ~ = 59~j.-909. }5. M.J.aIIooph."'" i""",'Y""" in anin, .. rum. Y ... Oin r.thol20,51- 55. }6. SyoLlinu. M, Tili(>Kki M, Y.....d. I, H .... imotoA. 1m. Sq-atioa and q~nof <"'~toj,d.i..tuc.d. ...... , • ..t u..... •Ibfu.. p/o<>oplu.tao< i""""f""" in anin, rrum. I Y" Mod [AI " ' ,60J-610. l7. SyoLlinu. M, T ili~ M, y ....t. I, H .... imo'" A. 1m. TI.. =in< alb.Ii"" p/o<>"",,'''''' A ,...;"" of d., i..,.,.",...... in ..... "', ... t.lytia.1 mrthod • • <><1 d..;. dJa&oottic appli<.""". lpo I Y" R.. 46c}-II. }B. Kid...,. SA. lack"", ML 1988. Diau-tic ,.,J", of aIkalin. p/o<>oplu. .... i""",'1"" ''P''otioon by .ffinity doctrop/o<>", .. in .... ""'" c"" I Y.. Rr. 5;!, 106-))0. 19. I'uttlti H . RrkI..rt B, H.,....,... H . 19~9. lb, ........ ocU';';". of AP, , ........ -CT, GWH , GIT and CHE aft", rompl_ biliuy obou"";"" and choIcdocl.oavallittuJ. iD d" ",t. Oin CJ.im. kta 18 [,8 1-86. .. 0. .'iolt.. PF, Holfn.. nn WE. 19"95. Canin, "",~«>nnoid·ind......!.1bfu.. p/o<>.plu...... in ............. ooIubili=l by p/o<>.pl.olipar..u.ity in';"". Am I Pt.y.ioI. 269,c278-G286 . .. I . .'iolt.. PF, Holfn.. nn WE. 1999. Solubiliutioon of livn .thlin, p/o<>.pi. __ ito1_264. "J, .'iolt.. PF, Holfn.. nn WE. CJ..",b",. MD. Sd...1l'n DI. Kultknodomidt MS. 1994. H,pa"" total Ja.k".t.=y bik >oplu.tao<;"'=ym<" a oc=ni.., .,,' fo. krP""ad."""".. tkiun in doJ; •. I Am Y" Mod hooc ss, 2OM}4-Sl~ . .. 7. Eh.t..." N . D.rnd.l WS. Hoffmann WE. W'; ~d RM, Po... n BE. Wid. ..... SI. 1998. Pro"",otic ito""rtan« of .lkalin. p/o<>oplu.tao<.ai.n,. in ........ from doJ; • ...i!h app«> ;;; ~ ~ ~ N L.::Il . ~ i ,a ;z: · ~ ... j '" to' 0 , 'i Z .S ~ "I .! <, .~ JI ........ 8 < ~ i f-- 5 . • ] . ~ H H~. ~ . .' i ,,~j ! ~: ~ ~!~!nj ; j l .~ ~:::,g 1 ~~ .:'i ~ I : i. 1 .{ .1 , , ;. l ;=> t 1- OJ'!! .§ ~. ' ; ,:l' ~ ~ 11" ,s::.' " 1" 1! 1! 'u ~·'1; j~~ :;;.:; I ' "I] j ~~. ,> {{. q i6' ~sh Hi- ;j'.,.!, nI l,~ 8W . ~~ J~ ~~ . .~ t t ~ ~ j I! ,I," ii ;.••I ~i'j !~ s~ l:;;j 0 .. j~~ ~ j1'0 r' ~ ~ 1H I El ~h! i I , ~ ~';'o 1. li ~"'. ~! ,"! O. 11 ~ ~ i ' Ji~'o ~","I. i .31 ~~f.,!; <~ ~~ t- '~ 1~ ~f , 1! I~ P! ~ 1 ! it I.~ -q1! ,rJ~ ~ ~ ~ ~ t 1 i ~ I ~ l ~ ~ t¥ ~ ~ ~ i I .;: j :f I~..s j<~ ,~ ,.~ -<~,~ >, ~" ~~ l§i ~<1~ .!' 1 1 I < ~.... ,'j . ! , ' • , $ r 1 ," j ,< I ~ ~ I "'~;-Iis lie~ 1~ ~~ ~.,;~ td· F; ~ 1· 1,j .~ l ~ . a~ ~ ~ ~ ~ i(l .B !~'1 Jr. ~.i' ~ ~ '" ~ ~ ~ ~ ~ ...; ~ ~ "" I __ ~ ~ "..!; ~ ~ _N:t ~ ~ ~ ~ 0>2<0] ;:t"'f.-"..:lJ""c..o!ld:j'"'~"'.:!:t Q ~ d ~ ~ ~ ~ ~ ...; ~ ~ '""" '" ~ ~ ~ 0 ~ '" '" '"'" :> ~ Q Zf.-d~". ~Ci\S~ai-jj",=Cl .d!",d!~<..:.iI~~~ '" ~ ~ ~ ~ ~ ~~ ...; " ~c..ci!",,,, .. 5 .. JiI iLJ l~~jt ~ j~ l I~~~~ ;n~~ i ~ 15ij~ J ~p ~ ~!_ ~-:~~ <" !~.~ H~IZ ~ " ] d.,"! j., 1 · j"tJ·"l ~i 'j 1 ~ i '~I" i~O ',J, J';.~ , Ii I ~167j"'.i;"n''''J ; d 6i"ll· 'i~ ~1.~': ,,:> ~~ ] f~ :~ J ~lrj~~ ~ ~~J~i:: l §3 1 ~~.; ~~,g 1H-J.:J.1!....:, •• l l t_1 i~tl' I I] I ~tl'61Ii.~I~.,-~I'. ~ .i ~ ,i. I'J~ ..l18!· . :; BR~·5 :i1:i . j~~Udi ~": ;",,;;! .~ ::91u '~~~1 JJt!~~~_ j ·t~t~~i~~ ·f ~i11~iittI~~~J~~111~~ ~;Jf ~ ~~~~~~il "'t">~2.1 - : : . .; "~~~' J,-€~ .~ ;!l~!j~ .'E,~1!0~J·il" "~~j ~'" e .,;c_'~ .l' 0 ,-'< , .. )0 . , 1 · - FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY 7S. M.u.. PB, T abo.da J, Hoocood G, P.. ti.>""" BP. YaoSt.<>.AI'. 2006. Th, ,datioo",bip brt...... b"",~ indicct, oonom pmnta-p><>myI , , _.... na. CK. G...., FB. G...". DM, Frttman MJ. 1997. H""ci, ~pi<\oti . io ..... «tic., !acta,;,.,; Hoi"';" cattl., A "troop«ci", mod, of rrum bOoclo.miad ...notmalitioo, J Y" [0' .. 0 Mod I hlJl--2J7. 8')' Koma'ru y, lrol.. N. Tani,.... H, Kitau ... T . Yob .. H . [("i.., M . Ob"v.h H, T ... ".ki N, Ma.no K. MizocudU M . T.k<..cl.i Y. Tanip ... M, Nabm.", T . W......... H, M .. rup«""nl .. co... >«<>odio ~ to bi.ni •..,.. J V ... Mod [AI .j9»Ilz-.j86, 8... Go"",,](A, T",,,,..kl GH. Kmn..y MT. G ...... os, a.rJoom B. 1987. E..... 'i<m ofy~"tamyl "anop 86. Li""'- J. s.,,,,,,",,,,,,kl U. Hopfd C . W"II"..Jo.f" R. T"..-ltoId I. 198}. l'.tuym< activiti. . .. blood cd], .f",,,, .nidi.n. Y" RL< I" S,U 1-487. 89. Sat .... T . Fwl[ M . 200 ... =tin. kin... ...d "P'''- uni""" .... f..... iD "'"'" .. indica_. /'0, I«tom,on pi ..... activiti. • .f ...yl.", iooamyla"" ~pa'" and oyptiD_tikr immwoo., .. ci';ty in """. R.. Y" Sci Sb78-l!2. 91 . Stidk JE. Cad_ WW. Boon GD. 1980. [......, ...... in dittkaIIy ~ do, •. Am J Y... R... "bS06-S09. 92. c.., .... M. T<>pkG..0'l' KF. 1975. a.A..yIt... ..,oJ ~yLao. at .f th, op ",,,,tim.nt.lI, 121 ENZYMES 673 102. Hill Re. y"" W,oill, TJ. 1991. A<~", nocrothinc; pa<>«eRitio ...d KUI, oupp""oi", panaeo.titi. in ... , at. A ,,_tiv. .....ty of 40 "'"'" (1976--1989). I Y" ]n«>n Med 7,2j....JJ. 10J. Kite!.dl BE. S""""t-l. DR. CuJ[,a I. Hanold D. I~. OinicoJ ...d pa""'J..O< cbuop in "' .... im=tally i..d""od K~'" .,....,."oitio in.-. Am I Yrt Ro. C, 1170--117 J. 100. H.,.J"", EB. S-...b.ck DR. 1978. Elf",,, offuncti.ooal ~y"""" di •• pp'uanc< ."". of =in< ........ • mylu< ...d lipo ... Am I Y" Roo J9,1 }1t>-]J21. lOS. robin DI. 0 ........ CA. St.nn. IS, H.yd", DW. 19BJ. 5.""" ....yIu<...d~ ..... ...;..;,;.,. in do, • ...;do d."",k prim"l' • ....J faiIu= Am I Y" R.o "',(1J.i-..j 10. 106. lacob, RM. 19019. Rdati.ond.ip of win"'J' .....rIa .. ...;..;". ...d p""';nwt. in do, ~ Yrt Paohol 26,Jt9....JSO. 107. «<.ci.c lipo .. in ...;.,.", tiooo" <>i>taincd f""" ooio of K~'" p...".,.titio in doc~ Yrt Clin P.dooI }j,l9--4}. 112. G""" H. J...y RI, F.."j.icho" DS. 1968. A fw ..... cloaca-12. 120. S"in" 1M, FiDooio of klin< p."",,,.titio. I Y" ]0«>0 Med 18,807-81 S. 126. HoIfmaom WE. Do""" 11. 1977. Di.ppe",,,,, urn of intra'"'''''~'''' injected =in< olhlia, phoopb."o< i"",,'Y""" Am I Y" Ro. }.I!,ISH- IS56. 127. HoIfmaom WE. ~ WE. Do.n .. 11. tr77. s.. .... b.lf_lif, of ........ ""~..,. injatted;".-ina!...d b,p"';" .Ikalin. pIoooploa_ """"'Y"'''' in oh. cat. Am I y" Ro. }S,16J7.... 16J9. 128. H..n. r. 1997. E.quin< d"bdo...,..,ly';' .,....t...n<. ],,' It"bi""", NE. ed. C....... , n-.." ;" EfoWo. M.Jin_ 4, 11j....121. Philaddphi .. WB Saw,d, ... 129. Cudinrt GH. Li_n JF. F««llaod RA. 1967. Com.,...tiv. ;",..,rtip"'''' of ..... ", ",,,.Iin< pboopt.okiou." and ~tam~ to ....... ;".,.,,..;vioioo in oquia, pacalpic m,...p.binw'" Rn Y" Sci 8,219---226. 674 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY 130. Wakim KG. ~ GA. 196J. ne fa", of 'nzym" j., bod,. Iluld" An <>p<"'im,otal .-11' II. rn..pP''''''''' ,,"" of &lutunk--o~ ~ ["""'" ...iouo«>Do< R.. <;>CJ-477. Ill. Barton MH, Mo"';' DD. 1998. Di ..... of...., ~v... ]'" Reed SM. Bayly WM. 001 •. E~", 1"m-IM.Ji,,',.,.. 707738. PhiI., dairy 00'0" . R... V.. Sci Il,U,7_1b8. l}t. P"odknd RA. Kran." JW. 1970. U", of ",,,.m "''Y'''''' .. aid. '" di.~ . Adv Y" Sci Comp M«I Iq,61_ 10J. u s . IliU U, F.;..J,] R. H""" H, Moyn D . OhlUJ""'" in the «trac elm",'" Bi«/,m,Mtry of 0-..,,;' A.m. ..... hl odiboD. s... Diq<., ~ Il7. Siduaann L. Bon.,,, R. Bo .... CA. c..iotti F. Ck.c·R.naod P. F...... d G, F.. «,., CA. ""-Je. F<&nd. PF. Goll. Fj, H""h<J W . j. T""" PI, !<>31--<>.3 • . Chapter 13 LIVER FUNCTION Physiologic Functi ons of the Liver ................................... . . . . . . .. . Abnorm al Results of Routine Laboratory Tests Because of Hepati c Insufficiency or Disease .............................................................. Bili rubin Concentrati on ..................................................... I. Phys iologic Processes ...... .• ..•...... . •......... . •...... . •........ II. An alytical Concepts . . . ... . .••..• . . . . . . •• . . . . . .•• .•• ..... .••. . . . . . .. III . Hyperbilirubi nemia . . . ...... . • .. • ...... . • ....... • . . • ....... • . . . . . ... IV. Bilirubinuria ............... . •..•...... . •....... . ..•....... . ........ V. Icterus Index ............. . .• • .• • .. .. . • • • •.. ... • ..••. ... . . • • •. ..... Bile Acid (BA ) Concentration . . . . .... .. •• .••. . ... . • •. . .. . . •. . • •..... . • •. . . . .. . I. Physiologic Processes ................ . .. . ................ . • . . . . . . . . II. An alytica l Concepts . . . .............. . . .. . . ..... . .......... • . . . . . . .. III. Increased Bil e Acid (BA) Con cent ration in Serum or Pl asma ..... .. ....... IV. Bile Acid Chall enge Test fo r Dogs and Cats .................. .• • .. ..... V. Urine Bil e Acid to Creatin ine (BA: Crt) Rat ios ................. . • • .. . .... Ammonium (NH,+) Co ncentration in Plasma ...............••. • • ...... • •........ I. Physiologic Processes . . . . . . ... . . .. . . . . .. . . . . .. • •. • •. . . .. .• • . . . . . .. . II. A nalytica l Concepts . . .............. . . .. •...... .• . . •....... .. . . . . ... III . Hyperammonemia ................................ •....... . ........ IV. NH,+ Tolerance Test ...............................••. ... . . • • •. ..... V. Postprandia l Venous NH, + Tolerance Test for Dogs .....• •..... . • •. . . . .. . Dye-Exc retion Tests ... . . . . . . . ................ . .. . ......... • ...... • • . . . . . . . . 676 677 681 681 683 684 689 689 690 690 69 t 691 693 696 697 697 697 699 702 702 703 675 616 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY Table 13.1. Abbreviations and symbols in {hi.. chapler [xl ALP ALT AST "BA BA:Cn Conctntmion of x (x = Alkalin~ phosphat ... Alanin~ .n:dyt~) tr.ons;omiruu., Asp.nau tram.• min.", S. Bilirubin Bil. acid en Bil. acid to creatininr Conjug.trd bilirubin Dir,",,1 bilirubin Indira::! bilirubin Total bilirubin Unconjugat«i bilirubin Unconjugatffl bilirubin bound to "'humin (nonoovalrnt) Creatininr GGT GM D y-Gl utamylu'lnsf.", .. GJutamau d.hyd~'l<'" B, Bd Bi B, B" BuiAlb Heo,H,b ID LD NH, NH: SI T NFa UNSBA URL USBA USG ... WRl Bicuoonau Hrmoglobin Iditol drhydrogrn ... Lactalr dehydrogcnast Ammon"Ammonium Synemc Intrrn. tional d'Uni{~. Tumor n=o,is factor Cl Uri"e nOllsulfalffl bar acid limit U pp'r .. r".""" Urin. ,ulf. !ro bil. acid Ikfractometric urin •• p«ific gr:lVity Within ",r.rena. jru.rv:d PHYSIOLOGIC FUNCTIO NS OF T H E UYER The liver has many vital phY'iologic functions involving synthesis, ncretion, and storage, Wh~n a di.u", procn.! dam"1:'s cdh within a liver, ch.ng.s in hep.tic function may :oIt" the composi_ tion of body fluids, and the muhing . bnorm:oliti.. m.y be, detrctrd by laboratory assaY', L Functions inmlving body fuds A, Prot~in m.ubolism: Hep. tocyt., synthesiz<: mOS{ plasma prot~ns (ove:r lOOO prot~ins), including albumin and moJi globulins (aerpt immunoglobulins), MOS{ synthesis is tIL noW) ("from new"), eith.. from """111",1 (dietary) .mino acids or from none .. enti:ol amino acids made by hqJatocytes, B, Carbohydrate metabolism: HqJ. tocytes remove: most di.ury gluoosr from ponal blood and store glum'" as glycogen for em"gency nerd" Gluco", from hepatic glucona>gen_ esis is the major wurer of gluco", in th. blood of fasting animals, 13/ LIVER FUNCTION 677 C. Lipid m~uboli,m: Hq.atocyt.. mak. fmy .cids, triglycuides, chol~n~rol, and .polipo_ prouin' for lipoprot~in!, and .. urifY chol..wol for pho,pholipids, H~patocyt .. degrad~ chylomicrons from ponal and .ysumic blood and .. mon lipoprot~in remnants from .ystemic blood, 11. Storag~ functions: H~patocyte. no", including iron and cop~r, glyro~n, triglyc.ride, and a vari<'ty of d~ment', 111. Detoxification: Hep>locym modifY or degrade ~ndogenou. compound. {e,g" uric .cid, neroidal hormon~', poly~ptid~ hormones, .nd hemoglobin} and exo\:"nou, compounds {e,g" st~roidal hormon..} via a wid~ rangt of ch~mical "'.ction!, including conjug.tion!, oxidation., r.duction., and hydrolysi., Hq.atocyt.. a", a major siu of NH' fixation (i,e" NH' incorporation into ur~a or .mino ""id.), [V, Actions of mononucl~ar ph:ogocytic ,ysum: The liver filt~rs blood (.ynemic and poru!; apability =nd only to 'pl..,n) through the action, of Kupff~r "",U., Th. mauophagt. ",move: damaged "",J], {erythrocyt.., l.... kocyt.., and platdm}, inflammatory m~diaton, organi,m" and endotoxins &om the blood (.nd thu. also d<'toxili ..), V, Excrrtory function: H~patocyt .. changr compound, to H,O_lOlubl. fom" for ucr<'tion via the biliary ,ysum, urinary 'Y't~m, or int.. tin .., Hepatocytes dq;rad~ cholesterol to bil~ acid. for ucretion in bile, A, Bil~ i, fomlN by thr.., processes, [n • BA-d~p<:nd~nt pathwooy, thr actin on:recion of BA produ"",. an O1motic gradi~nt that incrt.... H,O excretion, In a BA_ind~p<:nd~nt pathwooy, active: tran'port of Na+, glutathionr, and HCO,- promot.. bile formation via N.+_ K·_ad~nosin~ 5'_tripho,phat.. r, Third, ,,"crecin promot .. bil~ formation by ,timulating HCO,- .nd ct- =rrtion into bil~,l B, Component' ofhrpatic bil~ includ~ H,O, BA (at l...sr 50 % of bile 1OIids), Be, lecithin, cholest~rol, fatty acid., dectrolyt .. (N.+, K+, Ca'+, ct, and HCO,-), .nd a vari<'ty of H,O_lOlublr w;;osu., [n th~ biliary ducts . nd gallbladd~r, BA_rich bile i, form.d by resorption of H,O .nd lOme rlettrolyt .., ABNOR.,\1AL RESULTS OF ROUTINE lABORATORY TESTS BECAUSE OF HEPATIC INSUFFICIENCY OR DISEASE I. Routin~ laboratory t.. t result. may suggest th~ pre",n"" of liver di....,., or d"'rt.s.d h~patic function (Tabl.. 13,2 and 13,3), How,"""r, individual r.....lt. are not .prcilic for hep.tic insufficiency or di ......-, that i., oth~r disorders may au .. the abnormaiiti.., [I. [t i. imporunt to ret:Ognizr th. diffrr~n"'" brtw..,n abnormaliti~, that indiat. hrpatocdlu_ lar di, ...... , biliary di ....., or hep.tobiliary di ..... , brcou"1Om~ pathologic process.. affett mostly hep. tocyte!, wh.",,,,, others aff~ct mostly biliary "",lI., Aho, it is important to ret:Ognizr dau that indicate h~patic insufficiency, A, Hrpatocdlular di ... .. may result from a wid. VlUi~ of pathologic ,Ut.. (i,e" d~g<:n_ rrative:, autoimmun~, .nomalou" mrtabolic, nutritional, neopl""tic, inflammatory, in~tiol1S, inhrrit.d, toxic, or traumatic), B, Biliary di ..... may al!O result from a varirty of insulu, It i. mmt often the ..",It of inflammatory, neoplastic, or toxic disord~n, Table 13.2. Complete blood count (CBC) and chemi.try t esl rcsulu (pathologic findings) thai SU88CSI or indicate hepatic disca.e or dysfunction Pathologic finding HTalic lesion sugge;ted Path0trn~.i, CBC mulu Acmthocytosis H~mangiosatroma Possibly vascular trauma Altered lipid composition of lipid metabolism def= of finding" crydltocyt~ m~mbrane An~mia An~mia HqJalili. (chronicl ,l. Functional mass" Codocytosis ,l. Functional mass Microcytosis ,l. Functional mass Ponooynemic ,hum Possibly,l. tran&rrin production and thus ,l. ddi""ry of iron to crydltocyt~ precunon ,l. Functional mass ,l. Functional mass Ponooynemic mum Cholcsl..is ,l. Be lranspott Cholcsl..is ,l. Utra production [nadequ<>te fixing of Nl-4+ into of inflammatory dis ..... Possibly,l. erythropoietin or abnormal prolein or amino acid met . bolism Altered lipid composition of erydltocyt~ m~mbrane Ch~minry assay r~sults D«r~ased U N Hyp H yp Cirrhosis HqJalopalhy ,l. Functional mass H )'I'O"ibumin~mi ,l. ,l. ,l. ,l. ,l. Hypoprot~inemia [nn~....dALT,ASf, lD, Ot GMD [nn~~..d ALP [nn~a..d GGT lipemia (!:fou) Functional Functional Functional Functional Functioruol mass mass mass mass mass 10, Cholcsl..is Bili.ry hyp<rplasia Cholcsl..is POMibly h~p"tocytr "'- ,l. Functional mass ur~a [nadequ<>tr biliary acrction of bilirubin i Production of chol~nerol ,l. Cl~ann~ oflipoprouins Hyp ~'" ,l. CI~ann~ of lipoprouins •s... mo", oompJ.tr ""pw.1tioJU in d .. pt.... for raclt particular arWytc, • [)"cr.....! function:d moss multo from 678 13/ LIVER FUNCTION 679 Table 13.3. Urinalysis, coagulation, fccal., and perironeal fluid tesr re.ult Pathologic finding Uriruolysi. results Ammonium (bi)urar~ cry,talluria" J.. Functioruol mass Ur .... cycle d~fb:r or Cholestasi, J.. Be tran'port J.. Functioruol mass Ur.II~ crystalluria" J.. Function:'! mass Bilirubinuria" Hyposth~nuria isosthenuria" of finding" InadaJuar~ fixing ofNH' into ur .... and J.. oonnrsion of uric .cid ro :.!lantoin InadaJuar~ bili"", naetion of bilirubin J.. Ikruol medull.ry tonicity du~ to decreased ur~a t N H: acrffion may inhibit oonccntrating mechani,m J.. Conversion of uric acid to :.!lantoin Co~.tion assay resuh, Prolongnl parti:'! thromboplastin tim~ or prothrombin tim~ Cholestasi, J.. Functioruol mass J.. Vitamin K-Jepend.nt oo:.gulation factors due to impaircd intestinal absorption ofviumin K J.. Cl .... rancc of inhibitors of roagulation f.ctors such '" fibrin or fibrinog~n degradation produm (fibrin fragm~n1' + fibrinogen &agm~n1') J.. Production of most ~lation factors Fa::.! a.m r~,ult, St.... torrh.... Cholestasi. Defrcli", lipid digestion because bil~ .cid, are not ddivtred to inlestin~ Peritoneal fluid analf'i, Transudat~ J.. Functioruol mass Cirrhosi, i N .+ .nd H,O ret~n1ion, J.. pl asma oncotic pressure, pon:.! h}'Jl<'fiCn,ion, J.. lymph drain:w: • &e mo,. complot. "'pw.1tioru in th. chap"''' for u cb pu,icub , m alyt., • Finding may 0C C. H~patic inluffirimcy i,. p:llhophysiologic S1at~ in which th. number of functioning hep.tocytes is markcdly reduced, Hepatic insuffici~ncy and hepatic failure are mu:.!ly considered synonyms, Di""rders th at cou"" hepatic in",ffici~ncy full into tWO groups: L Di""rd~" that destroy hepatocytes {hep.tocellular disc...,} may progress ,lowly, =ur epioodicolly, or involv~ rapid, ut~nsin necrosi., 600 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY 2 Porto.yn~mic ,hullts {cong~nital tho li,,« kcou .. of d=~ or . cquirall co"",,, ~ith~r hypopl ..i. or atrophy of nutri~nt' ",,,ching h~patocyu. from portal blood. h~. too f<'W functioning hq..tocyt .., but /lUny D. All .nimal. with hq..tic insuffici~ncy animal, with hq>. tocdlular di ....... do not han h.patic insuffici.ncy. Mon animal, with primary biliary di..,.", will d..vdop sn:ondary h'p"tocdluw di ... ",. M any '!Iim.!, with primary h.patocdJuJ:.r di ..... drvdop =ndary biliary dj,ordw;. Eith., h'p"tocdJul.r or biliary disn, .. =y cau.. hepatic inmffici.ncy. E. Laboratory res! ,.sults cru.{ indic;;otr hep.tic insufficiency do not cd] us which h.".tocd. lula, or h.patobiliary dist. ", tho animal has or wheth .. tho di ....... proc"", js ,."""ibl. or irr.""nibJ •. Ill. In dinical jargon, some pa>pl. ref., to hepatic enzyme """Y" {ALT, AST, ALP, GGT .• nd [D} '" Ii".. function t~m. How~ver. th .. ~ ~nzym~s >r~ not 50ldy h~patic in origin. incr~:asffl activiti .. of th= ~nzym~ do not dir~("dy indiat~ loss of .ny Jiv.. function • • nd Jiv~r function am ~ grrady rMUcM without incr~a.fflstrum ~nzym~ .ctiviti... To ~mphasiz< th~ last canctpt. would th~ strum activity of. hqJatic ~nzym~ inc",asr if on~ r~mov.-d an .nima]"s liver? [yo Ikcr~ functional hep. tic IIUS.'l A. Hepatic inmffici~n v. Chol.. usis A. Cholmasis is ddined in diff~rent ways. [n a dictionaty ddinition. rluJk,,,,,u it the ' stoppage or suppression ofba~ How."' Table 13.4. Enmp!cs of di.easeo and conditions that cause decreased functional hepatic rna .. (using the DAMNlT u ronym) .lHgenuati",: hypoxia au.al by anemia or congestion 'Anomaloul: pon0'Y't~mic ,hunt M~tabolic lipidosis. diabet~s mdJitm. hyperadr~noconicism Neoplastic: lymphoma. metastatic neoplasia InfLommatory lnf=iou.: leptospirmi •• hi 13/ LIVER FUNCTION 681 l. Th~ ''''ppagr component of th~ ddinition i, consistent with th~ ob"'rv:ltions of disundffi bile ducu (grossly) and bile plugs, lak ... or pigments (nticrosropic) th>l Oil< cau.s the osmotic df«ts of "",,,,.d BA largdy control bile volume, d«reas.d BA =<t~ .«tions. l. Bu is . product of heme degr. dation. wh",e .. BA i, a product of cholm.rol drgradation. Heme and cholesterol do not ili:ore phpiologic path"..ay•. 2. Bilirubin (Be and Bu) and BA ent~r hepatocytes through sinusoidal membranes but by diff",~m membrane transport systems. 3. Be and BA .re ""'r<'I.d &om hepatocyte> through cm. licular memb"III" but by diffe .. nt membrane tr.mpon sy"ems. 4. P.thologic .tote. that d:mlO{:<' hepatocyt~ membranes can impair both bilirubin and BA exc.. tion. Th... def«ts may k forth", link.d bn::au...ccumul. tion ofBA in hep. tocytes may cau .. hepatocyte damO{:<' that interf",.. with bilirubin metabolism. How""",. p.thologic states that sd«tivdy im",fere with t"lIIspon .ystems may imp.ir =<e'lion of one analyte but not the oth",. BILIRUBI N CO NCENTRATIO N 1. Phytiologic process." (Fig. 13.1) A. In plasrruo, th~ .. a.. thrtt fraction. of total bilirubin (BulAlb, 1k, .nd BII). Bu/Alb is dominant in the . bsen", of di .."",. Bu is constantly proouc.d from the degr. dation of heme from .. n=m erythrocytes .nd heme-containing proteins. In h... hh. Bu .nd Be are rapidly remov.d from pl.. ma by eith", livrr {Bu and Bc} or kidney> {Be}. The spontaneoll'l and covalent binding of bilirubin glucuronide isomers (Be) to albumin form, BII (Be-Alb), which, once £orm.d. h. , th. circulating half_life of albumin (.. 8--20 d)! Be and Bo con",mrations.re negligible in health. 682 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY -''''- ------------------------------------------ ~"'I ""'" "" I • , "" Be Hsp"tocyto • .{ ~ "" ..., • ~" '" ~ """"" ~ ~ ~ Ell Bilo ~ ~,~ , ~ ~ ,~ , • ~ I '''" ~. Int •• ti",,_ ,~ Fig. n .!. PhJ"iologic procosses in> o Wb.n Bu .,",," tho Ii"", ..,d its protein_perme:obJ. ";n"",i"', it .nt.... hep>tocyt .... itbout .tbumin and binds to Y.prot by. poci"" but fiociliuted process: binding proteins .nhrn"" tho proce>s by reducing tbe em"" of Bu b:oc.k to the ";nusoid1l pwm •. • Witbin Mp>t""Y""'. Bu is ronjug1ted with glucuronide (:wo g1u","" in bo,..,,) to form bilirubin monoglocoronide or bilirubin diglucuronide. roUecti""ly c.Jled Be. • Be is tnmported from hop>tocyt ... into ern.tiru~ (the f1t .. ~miting ''''p in bi~rubin excretion) by rn .... rgy..dependent tnrnpon 'J""'m for orgonic onions other than RAJ. • Be in bit. . nt .... the intestine ond is degnded to urobilinog ..... • If Be es<:1pe> Mp>tocyte, rnd . n"'" tbe blood. it ern p ... tluougb tho glomerulu filtf1tion bartier . nd be """",,,,d in the urine. Beau.. Alb does not p'" tbrough the glomerulu filtr:otion buMr of most rrucnmrn. Bu/Alb doel not en"'r .... utine in thoo. . nimrn. BuiAlb. Bu :woci. ted .. itb 1lbumin: 11.1,. mocropluge: Sb. ''''rrobilinog B. Sm:.!l :nnoums of bilirubin "'~ commonly found in th~ urine of h~:.!thy dogs. It h.., not oon •• ublished wh .. h.r th~ bilirubin is Be or BuiAlb. It i, frrqu.ndy >ssumed to be Be b«:.use Be is H,O .olubl. and passe. freely through the glom.rular filtration barri ... How",.. , sm:.!l amounts of :.!bumin ... :'!so commonly present in th. urin. of h •• lthy dogs, so some bilirubin may be in the form of Bu/Alb. 13/ LIVER FUNCTION 683 C. H ..ahhy horses hav.. gr.. at ...... rum [Bt] than oth ... dom.. nic anim:d" about 0.7- 2.0 mgldL in horses ""uus < 0.5 mgldL in othor aniJrulIs. 11. Analytical oonerpu A. Unit oon""rsion: mgldL)( 17.10 = J.lmollL {51 unit. n""rm 2 J.lmoIILl' B. Conv.mion:d s~rophotom.. tric m.. thod, {w", ch.. micol ous;oys} l. &ver:d bilirubin =ys .... u,al to mrasur .. :dl bilirubin fuction. (Bt) or only the non·Bu fractions (Bdl. Th.. cl ....ic bilirubin a"",y was the van d.. n B..rgh m.. thod. but it h .. bn:n r"Pla=! by oth ... ""'YS (... g.. M:dloy.Evdyn .nd J.. ndr .... ik.Grof m.. thods) for ....... ral d=de •. a. Bu ...acts .Jo~r than Be in most bilirubin ...xtio,,". so sub,uner. are addal to accd .. me it, participation in Bt :ways. When th .. xcd.. ram factors .... not presem. only the non·Bu fractions are mramr..d (calla! dim:t bUirubin ""ct'-on). b. From th.. m..asural [Bt] and [Bd]. [Bi] i, calcul.tal by subtraction: [Bt]- [Bd] = [Bi]. 2. If th.,... ..... ys a... u...d. laboratori"" Jruly ... pon th.. following roncrmrations that should ... preso:m th .. bilirubin fractions liSla! Wow. [Bo] i, not 'prcificolly m.... ural and cannot be calru!.ta! by th .... m.. thods. a. [Bt] (m........ ral) = [Be] + [Bii] + [Bu] b. [Bd] (m""sur..d) = [Be] + [B1l] c. [Bi] (colrulata!l = [Bt]- [Bd] = [Bu] C. Dry-ch .. mical method. u,ing thin.lay... rrag<:nt slid"" (Kodak or Vitro. instrum.. nts) l. Th.. asiaYS u... modifial bilirubin diazo .... ctions in thin.lay... rrag<:nt film' and a... d.. ignal to mrasur .. th .. following: a. [Bt] = [Bc] + [Bii] + [Bu] b. [Bu] c. [Be] 2. From those: musural roncrmrations. th .. foUowing a... calrulatal: a. [BO] = [Bt] - [Be] - [Bu] b. [Bd] = [Be] + [B1l] D. Procedural not .. l. Hgb im..rf..es with 37.0 .... ction. and produces falsrly low [Bt] with th .. M:dloy. Evc:lyn and J.. ndrassik.Grof m.. thod,. How"".... in th .. thin.layc: .... ~m slid.... Hgb f:dsdy incr",,= th. [Bt] and [Be] and faI ... ly dre ...a.= [Bu]. 2. Ultraviol", light degrad... bilirubin. In dir«t sunlight. [Bt] may d«t ..ase up to 50 % in I h. 3. Immunoglobulins may prreipita", .ft... th...ddition of acidic bilirubin ...agems. and thi, prreipiunt im ..rf..... with light trall!mission and co""", a f:dsdy inc ..... al [Bt]." Th.. im.. ncr occurs primarily wh .. n th..... is an incr""...d r-globulin ooncrntration {...g.. paraprot.. in.. mia}. A cl ... rly inc ...asal [Bt] (e.g .• :> 5 mgldl) con be "",ily rroognizal .. a fal... valu.. wh .. n th.... rum i, visu:dly not ictoric. In on .. co .... th .. [Bt] w;u "m""sural" to be :> 20 mgldl.., but th.. sampl.. wa, not ict... ic (ob ... rvation by S.L.S.). Minor im.. f..... nces. how"""r. may not be detreta! via this oomp.mon. 4. U,ing thin.layc:r did.. assays. th .. sum of [Be] and [Bu] m.y ""eNd th.. me .. u... d [Bt] {esprci:dly in hors..}. It h .. not bn:n mabli,hal wh",h .. th .. [Bt] is f:d,dy drer""...d or wh",h... the ,urn of [Bc] .nd [Bu] i, falsdy incrrasal. r..... 684 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY Table 13.5. Di""a..es and condition. thai cause hyperbilirubincoua lnc,",asal Bu production • Hemolytic disorders. "p«;ally atrav>SC\1]" hemolysis {= T .bl. 3. lOJ D«....«I Bu upuke by h.patocyw 'Fasting or anora"- (csp«ially in horses) 0«",,=<1 function'! mass {diffu.., hepatocdll1J.r di"" ... ; ~ the texl for cUffiplo} D«....«I Bu conjugation Dec..=<:! fUDClioruo/ mass {diffu..., hepatocelJuI" di""as.; Ott the texl for cUfilples} Dec..=d Be ncrctinn in Obstructive rnoiesrasi, 'H.patic cholmasis: lipidosis, diaktes mdlitu., ,toroid hepatopathy. lymphoma, hi ba. Ill. H~rbilirubin~mia (Tabl~ 13.5) A, Hyp",bilirubin~mia occurs wh~n the nt~ ofBu production a<:«te Bu so it does not accumul ate in plasma, b, Pathogenesis {I} An animal th at has a hemolytic ~n~mia may devdop ict~rus if th~ me of Bu formation ",,<:«lhogtn~ ..' of h~molytic disease, are d ..cribed in Ch.pt~r 3, c, R..mhs of serum bilirubin profile (T.ble ]J,G) {I} [Bt] may ~ mildly to markedly increased, {2} [nit;..lly, [Bu]» [Be], [fpersistent, then [Bu] nuppproxinute [Be], S«:ondary (e,g" hypoxic hepatocdlular degen~ration) or concurr~nt hepato_ biliary disease may complicate th~ patt~rn, 685 13/ LIVER FUNCTION Table 13.6. Bilirubin profil es in common pathophysiologic stat ... that cause icteru. , tt tt WRI-t tt tt t - tt WRI-i i -ii tt i -ii WRl- i i -ii F.. ting tt tt tt tt WRl-t WRI-t tt tt WRI- t WRI-t WRl- ii WRI- ii Obstructive Functional cholestuis ,od • The [Btl moy be mildly to madedly in==tivc int .. kr= ..-ith bi~rubin e. "'''Y'. {3} It has bttn written that bilirubin fr>ctiom do nOt help diff«rntiate hemo. lytic and hq.atobiliary icterus beausc [Be] is alwaY' gream th.n [BuJ.' Thi. rondusion .ppem to be supported by findings in a group of eight dogs that had roncurrent hemolytic .nemia and li""r disc...,.' but it is incon ,istent with the authors· experiena.. {4} [Bu] is often grr.ur than [Be] in animals with hemolytic .nemias. but it will not alWllYS be g,.ater. When [Bu] ;> [Be], it supportS the concept th at tho icterus is directly rdated to extravaKular hemoly.is. When dinirnl da'" indicate a hemolytic anemia .nd [Bu] < [Bc]. concurrent hq.atobili<>ry disease should be ronsidered. d. Oth.. expected laboratory findings with hemolytic icterus {I} Anemia, reCC'nerative if of sufficient duration, moderate to marked ","«ity {2} Hemoglobinuri<> and hemoglobinemia if the,. is mfficient intravaocular hemolysis {3} Bilirubinuria if sufficient Bc '''''pes from hrpatocyt .. or. with intravascular hemolysis, if thore is sufficient renaltubul .. home drgradation and bilirubin excretion into urine {4} [f pw.m. hopatic enzyme activities.re incre.=', hepatocyte dam,,!:, or cholest .. is rould be due to anemic hypoxia. 2. [nc..ased Bu production not :wociated with hemolysis a. Besid.. Bu from erythrocyte d.. truction. heme degradation also occur> with drnruction of erythr0C)1r precursors {ineffective erythropoiesi.} and degradation of other heme proteins (myoglobin, cyt:ochromes, .nd peroxid ....). b. By themselv.., these proce;ses arr not consid«ed to Gluse inc" ..ed [Bt]. HoW<"V«, they could rontribute to .n increased [Bu] if th«r .re oth« reawOl for increased [Bt]. D«reased Bu uptake by hq..tocytes l. Fasting hyperbilirubinemia a. DilOrders 6116 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY that are off fNSt (> 60 %) to conjugate bilirubin. " Fa'ting may make less gluco.. av.ilable in hq.atocytes for Bu conjugation. Administration of gluco.. to horst. with fasting hyp Ho",~, 13/ LIVER FUNCTION 687 if a di...,... i, ",using only d«r~a lffl functional mass and not obstructi"" clini",l ict~ru. i. not ap«tal. b. DiKlrd~," that Jruly "'u.. d«reaKd functional /IUS.I are listal in T .ble 13.4. 3. He=1ituy ddicienci .. in the hepatic upt:ok~ ofBu m.y Glu,e hyperbilirubin~mia and havt bttn recognized in Southdown 'heep" and Corriedale m'""p.17 D. D«r~...d Bu conjugation I. As melltionM in the pr«aling ,ection 2.a, • markal rMuction in functional mass would =1u"," Bu conjugation. Wh~n cytoplasmic r«tptor proteins.", "'lUratal, the uptake of Bu by hepatocyte. d«",as." . 2. Heralilary ddicienci .. of enzymes that "'talp conjugation reaction, occur in people and l.boratory .nimal, but h."" not bttn confirmal in our rommon domestic mammal .. A persinellt hyperbilirubinemia in an otherwise healthy Thoroughbral was prob.bly caused by a defective conjug.tion pathway. but the aact d~f«t v.as!lOt est.blishal." Th~ [Bt] rangal from 9.4 to 12.7 mgldL during. 2 yt evalu ation, .nd about 90 % of the bilirubin v.as Bu. E. D«re...d Bc acrecion I. Ob,"uctivt rholmam i. causal by a pathologic naU that obnruct, bile How through bile Glnaliculi or bile ducts (hepatic or ponhepatic). u,sions that impair bile How indude hep.tocellular .welling th.t romp"""'" canaliculi, periponal lesions that comp"'""' bile ducts, inf«tions and other proce.... that d~ bile ducts, .nd blocbge of bile ducts by ston.., parasit~" or neoplasm,. a. Pathogenesi, {Fig. 132} {I} [ncre...d [Bc] in .ynemic and thus sinusoidal blood ,alUm.. bilirubin r«tpto," on hepatocyus, .nd thu. Bu uplah is imp.iral. {2} With tim~, incnasnl [Be] le.ds to inc",...d [Bu], but th~ [Bc] is Hp«Ial to r~main ~ater than th~ [Bu]. b. R..,,,,lu of a .. rum bilirubin profile (Table 13.6) {I} Th~ [Bt] Jruly be mildly to extr~mdy inc",...d. {2} Typically, [Be] :> [Bu] {[Bd] :> [Bi]} ucq>t in horses .nd catd~. {a} Concur"'lIt hepatocellular di ..... m.y complicate panerns by d«r~..ing functional mass .nd the",fore d«r=ing uplah and ronjugation of Bu. {b} [n ho .... with rnolescasis, the [Bd] i. typiGllly < 30 % of [Bt]. The inc",...d [Bd] in horses mongly suggest' rnolest.,i,. {c} [n "'tde with rnolescasis, th~ [Bd] m.y be greater than or 1= th an the [Bi]. c. Other up«Ial l.bomory findings {I} Anemia Jruly or may not be present. If pre..IIt, it typically is nonr~gen~ rat i"". {2} [nc.....d .. rum activiti .. of enzym.. associatal with rnolestasi, {ALP and GGn (a) ALP and GGT .ctivities may be increased in dog. before icteru, devd0l". {b} ALP and GGT activities typiao.lly ar~ not inc",...d in cat, until there is Jrulrkal ob,tructi"" icteru •. (c) Biliary obstruction is uncommon in horse, and Glnle. When present, ALP and GGT activiti~, may be .ubst:mtially increasal. {3} s"rum activiti.. of hepatoceUular enzyme> (AL T, AST, LD, [D, and GMD) may be inctused bemuse ofhepatocyt~ damage. chol~nasis, th~n '88 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY Fig. n.l. Obstructin dtoLe.utic ict<..,.: Losions of biLe analiruli or biL. docts (hop1tic or posth.patic) inhibit biL. lIow. :and tit ... L.s" 01 no Be it =",,0<1 to tho in' .. ,; .... Be ~"'" .ynemic blood ~ • .,.. of (I) inc","""" perme .bj~ty of <arWicuhr tight junctio", ..,d lung< to th. '1''''' of 0;..., . nd th. omtral win. (2) Mp. tocyte n .,,<>cute (4) HyperchoJe,terol.m", OCCUn ill ""m~ rnoJestotic condition, ~nd COD bt nurkffi. but is not a ron,i'l.m finding (Itt Chapt~r 16). 2. F"nmmw{ clwkw,is {Hp.is-asStlCiIlud rhok,taiisj i, impair«i ncre{ion of Be from hq>.tocytes to analiculi in th~ abs.n~ ofbil;"ry obstruction. A function:d molesta_ si. w .. reponffl in dogs with &htrlmid roli and S",phykKtKcu, int"",~Jj"s inf«:lions cau,ing pneumon;". puitoniti •• urinary tr~ct infa:tion. ~ndocarditi,. and 50ft tiSllu~ inf«tion." Thi, 'YP'" of icteru, is mor~ commonly ~niud in ~ple and may ~ mo .. common in domestic nummah than h .. bttn r«ognized. a. Pathogenesi, {I} Inc",a=! [tumor n«rosi, factor alpha] (dira:tly or with ~ndotoxins) result, in d«"'''ffl BA tran'pon proteins in hepatocyte analicular m~mbrane>.lO.' Interleukin 6 aho int~~ ... with bile s:dl transpon. l l {2} It is not drar whethu the impaired Be o;c"'tion i. a diract defect or whethu it occu," on:ondary to canalicular membran~ dam~ ausa! by rerunffl BA. b. R..mhs of a serum bilirubin profile (Tabl~ 13.6) c. Other o;p«tffl laboratory findings {I} InHammatOlY changes in blood l.ukocyte> (~.g .• n.... trophilia. n.... tropen;... l.ft shift. toxic change. or lymphopen;"l {2} Other changes would depend on th~ dur>lion and the location of the ba.cterial inf«tion. 13/ LIVER FUNCTION 689 in th~ h<patocdlulu acretion of Be may em", a hyperbiliru_ and ha"" bttn =gniud in Corri«lal~ sh~. Th~ .b'""p :dso ho"" a d~fet:t in Bu upuke by hepatocyte•. " F. P~rsist~n"" of Bo in plasma l. Sin"" Bo h", the plasma h:df_life of :dbumin (8- 20 dl. an increastd [Btl may penist for more than 1- 2 wk aft .. correction of the pathologic defet:t in bilirubin acretion (e .g., surgical r~mo""l of a biliary tract obstruction). 2. Th~ penist~nt incr..,... in [BO] and thus persist~nt incr..,... in [Bt] i. not commonly rerogniud. [n most animal. with cholntatic icterus, the h:df_life of Bii may actually be .bon.. th an the tim~ required for the [Be] to det:re ... if there i• . H~='iury d~fici~ncies bin~mia [v. Bilirubinuria A. Excessi"" bilirubin formation (in hemolytic sum) or imp.ired h<patobiliary acretion of Be may iner.,.., plasma [Be]. Be easily p>JRS through the glomerular filtration barrier and is then acreted in th~ urin~. B. Bet:aUst of the low renal threshold for Be, bilirubinuria may be detected before hyperbilirubinemia is detected. [f hyperbilirubinemia (from Be) is prestnt, then bilirubinuria is apected. C. A> aplained in Chapt~r 8, • positi"" bilirubin r~action in • urin:dysis should be im~rpreted with kno,,{edg~ of USG ... especially in dog •. Moderatdy con""'ntrated urine {USG",= 1.025- l.040} of healthy dog. frequently give •• 1+ bilirubin r....ction; 2+ reactions occasionally occur with more conctntrated urine (USG",:> 1.(40). v. Icterus inda A. Definition: a valu~ that .. presents .n enimation of the ydlow di500loration of pl.. m. c;oused by hyperbilirubinemia B. Methods l. Potassium dichrom.te Jmthod" a. Icterus inda is d~termined by comparilll: th~ rolor of a patient·. pl"'ma to a set of stand"d 1Olution. containing potassium dichromate. The degr,"" of icrerus i. record«l in units from 0 to 100. Th~ plasma of healthy herbivo ... i. apect«l to be more ydlow becau .. c;orotenoid pigments nom plants may impart a ydlow discoloration. b. H~molyz.d or lipemic pla.ma am f:dsdy d '00 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY C. Clinical laboratories may subj<'CIivdy gr:od. ,h. =rity of ictmJ5 (mild, modem., or IlliIrk.d) by ....ssing the dec ... of ydlow to orange discoloration of pl.sma or ",rum. Strum [Bt] typically uett<\, 1.5 mgldl bc,for. visible icterus (in "" or mucom membnne,) is det=.d. Thi, d~nd. som~ha{ on the '1>«;'" and ,h. ob.. rvds ex~".nct. BILE ACID (BA) CONCENTRATION" I. Phf'iologic process •• (Fig. 13.3) A. [n h~.hh, the enterohq>. tic circulation of bile acid, i, highly efficient, and llrarly.ll bile .aIlS excretrd in bile .'" return«i to the livrr vi. intestinal .bsorption .nd ponal blood flow. Hepootocy\ll ..., \ PhJ"iologic procosses of bi!. . cids: ChoJ..tS'ibJ. f.o ... : (I) absorp,ion by the in'os'uw muCOlO :md en"""" in'o the portol blood. (2) deronjug>tion by .nteric b:octeru '0 • lORA and ,hen .bsorption by d.. in'ostinal muoou :md .ntnnc< in'o the portid bLood. or (3) degr.d. tion '0 . 2°RA (.i,Mr deoxycholic ""id or ~,bocbo~c acid). which may b.Y< two fat~r .. ion in d.. lft:es or ilioorption by 'M inteted by bopatocytos and renuned to ,he bi~ary Sf'tem '0 complete the .nterohepa,ic cirrubtion. In he:d,b. nearly all RA mokruLes "'" ..-ithin the .ntrrohepa,ic ciroJ.l.tion: wry few "'. in 'M SJl"temic blood. RA molecuLes that .>cap< the .ntrrohepa,ic cirrubtion can be cl< .. ed from 'M pluma vu glomerul:u filtration :md ..aeted in the urm.. I'SA. primary bile .ad: I'Bh. conjugated primary bil< acid: and 2'SA. ..-rondary biJ. .cid. Fig. !j.~. OJ chen<>o~c 13/ LIVER FUNCTION major solids in bil~ th at ~nUr th~ intellin~ .ft~r C.llbladd~r contracintestiruo1 absorption of BAs. th~ resulting high~r [SA] in port..! blood may .~.d th. li""r·s ability to extract BA... and thus thu", i, a postprandial incre= in [SA] in 'yn~mic blood. Typically. gallbl add .. contraction results from .ctions of chola:yno_ kinin th at is rd....d .ft~r ing~stion of a m~.l. Howev.r. gallbladd.. contraction moy occur at oth~r times. C. BA K<:r~tion from h~p"tocyt .. to coruo1iculi occurs through two P""""""; on. is N.+ d."..nd~nt and on. i, not. B. Ba~ 691 tion. 11. ,ahs ar~ th~ Aft~r Analytical conapu A. T~rms .nd units I. In rommon u.... th~ t~rm bi!r addJ r~f~ .. to a croup of cholm~rol..d.riv.d anionic acids .nd th~ir dis.lOci . ta! .nions. Th~ bil~ acid, indud. cholic acid. ch~nod..,X)"Cholic acid. d..,xycholic a.cid. and lithocholic acid. all of which a", 3a..hydroxylat.d bil~ acids. At • pH of 7.4. the dissociat.d anionic form. of th~ mol""ules dominau {•. g.• cholat~ and lithocholate}. Coll""tivdy. th~ anionic forms ar~ call.d bik 2. Unit conv.rsion' a. T oul bile a.cid; mgfL X 2.547 = ].tmoUL (51 unit. nearest 0.2 ].tmoUl) b. Cholic .cid; mgfL X 2.448 = ].tmollL (51 unit. n....est 0.2 J.lmoUL) c. Ch.nod..,xycholic acid; mg/L X 2.547 = ].tmollL {51 unit. Mar~st 0.2 ].tmollL} d. D..,xycholic acid; mglL X 2.547 = J.ImollL (51 unit. n.arest 0.2 ].tmoIIL) •. Lithocholic .cid; mgfL X 2.656 = ].tmoliL (51 unit. n.ar~st 0.2 J.lmoUL) f. O""",ionaUy. oth~r uniu are usa! (i .•.• mglmL and mmoIIL). Stt T.bl. 1.6 for th~ ronversion factors. B. AMays I. Th~ [BA] i. m~.. ura! in dinical laboratori~. by. spectrophotom~tric =y that utes 3a..hydroxyst..oid dehydrogenase in r~actions with primory and secondary ba~ a.cids or th~ir anions (eith~r conjugata! or da:onjugata!). Free Hgh in a hemolyud sample will co ..... a n~g,ui"" int~rfe"'nce in th~ .....y. Lipids in • lipemic sample cr~ .. e unacuptable errors according to th~ .... y's manufactur~r. 2. Th~ conantration of ronjugat.d bil~ acid. con be m... ura! by ~ith .. radioimmuno_ ...... Y' or ~nzym~ immuno ...... ys. The ........ Y' may be more a:onomical in ref~rence .am. laboratori~s. 3. Clinicolly•• bnormolities in total bile acid conantration .nd conjugat.d bile acid ron"",ntr.tion und to paralld each oth ... and thus .ith.. type of .....y con be ustd. 4. A IDEXX SNAP bile acid un is availabl~ to Kleen animal, for incr~aKd [BA]. 111. lncreaKd bil~ .cid (BA) con""'ntration in ... rum or plasma (Table 13.7) A. An incr....d [SA] con be coU.d hypn-rlu!kmia. but th~ t~rm is not commonly usa!. It should not be confus.d with hyperdtloremi. or hypercholest~rol~mia. B. Th~re are two m.jor pathologic processes that incr ..... serum [BA] in fasting dogs and cm; (I) d""r~...d BA d ... rana from portal blood and (2) d""reased biliary ncretion of BA (Fig. 13.4). Accordingly. increased ",rum [BA] supports th~ condusion that .n animal has d""r ... sa! h.patobiliary function. Increased ..,rum [BA] h", high diagnonic ",nsitivity for h.patobiliary dysfunction. How.""r. many primary and KCOndary li""r di ..ases con cou .. the dysfunction •• nd the dysfunctional state mayor may not be r~~rsible. Table 13.7. Discaoes and condition. Ihal ca""" an increased fasting (RAJ Ixcru.ffi BA ck=na from ponal blood 'D«Routd funaion~1 hepillic nws; diffuse he~locellubr disnse 'D«Routd pom! blood flow 10 liYet; congenilal and acquirffi ponosyslemic ihunu Decreoutd BA acrelion in bile Oi>:mucriYe choIescasi. 'Hcp"tic cholella,i,; lipidosis, di.bnes mdlitm, lIeraid he~lop:"hy, lymphoma, hislOplnmo,i., cytauxwollOl;., cirrhosis, cho l.ngitis, cbol:mg~I;I;S, periportal hqmilis. pyrrol iz.idine. alkaloid loxioo.is, lpo rodesmin tOlicosis. aI.ike dOYet (Trifo/ilim hybridum) toxicOIi. 'Poschqw-ic cholestasis; m olmgitis. bile ducr carcinoma, liver flukes, choldithia.i" choJ«}"titi., ~1K=titis, ~nc.e'llic carcinoma Funaional mol<::'luJis (Kf'Sil-mocialffi thol<::'lcasi,) 'A ..llti""ly common di ..... ot condioion Note: U .... of op«i6< .wordc:n .,.. ..mditioJU ~ not c.omplet. but _ provided Serum b;1<: acid con«ntntioru:= inc~ postprandiaUy. SA " SA to g;'" n ampi<s. ' SA i Fig. U.4. Pathologic P'-'" that inae- .. rum [M] (hyperdtol<:mial. T"" major I".hologk p'o<:uws c..... bypord>ol<:mia: , ~ MUD« &om m. portal blood: Th. d<&a may 0C0.l< ~ of ( I ) dcan-I up" "" of RA &om ,lw ";nuooidoJ blood bc<;au.. of. clea..sed fUnaionoJ Itepar" m.. o. bo:c:au.. (2) .~ is • .!tun•• , ~ bililU')' IlA _ion: Wh ......... is obouucuw choltswis Ul pon1OfYI .. mic BA. 692 ~ ..,...rgi.. ted '" Of functional choko ..." (4) •• ho m. .inuooidoJ bIoo ,h. 'n".ti".. 13/ LIVER FUNCTION 693 l. O,""",asal BA dtlran"" from portal blood a. Many pathologic stot.. (~.g .• inHammatory. m.rabolic. and dtin) involving hepatocyt~s callS< sufficient dallU&" to redu"" functional h'1"'tic IIUS.'l ~nough to impair BA de"an""."''''' b. Congtnital . nd acquired ponosy"emic ,hunts ~nabl. bile ..Its . b",rbed in the intenine to bypass the liver. =pt the enterohep>tic circulation. and ~nter the systemic blood."" c. Mild inc",a.-;.. in [BA] may occur when f=:l is withhdd from hors ..." The incr.... is prob.bly caused by. d=.....d hepatic dtlran"" rate of bile acids! 2. O,""",ased biliary BA excretion a. A variery of hep~tic and "",thepatic disorders can imp.ir bile flow and thus impair acretion of bile acids. Concurrent impairment of &: acretion is apected. but it m.y not be enough to cause hyperbilirubinemia. Similarly. when hepatic or ""nhepatic hyperbilirubinemia is p....,nt. increastd ",rum [BA] i. apected. b. Ouring obstructi"" choleswi •• th«~ is a down_regulation of caruolicular BA transport proteins. and hepatocyt.. may pump BA into sinu",idal blood insttld of into canaliculi. This process may aplain the "regur-gitotion" oon""pt of BA l.... ving hepatocyt ..." c. BAI are toxic to cdh. Thus. accumulotion of BAs in the liver may l... d to cdl dam"1:e .nd rel...... of BAs and other subst.n"". from canaliculi . nd hepatocytes. d. Cytokines {notobly tumor necrosis f. ctor alpha} have b«n shown to dec ...... BA transport proteins in hepatocyte canaliculor membranes .nd thus impair BA s,""retion.""" The /"<Sultont imp. irment in BA acretion is calledfonNional clnlmasiJ {see Bilirubin Coneentation. sect. III.E.2}. C. Mild incre. ses in ",rum [BA] occur in healthy :mimals after m....h. Postprandial inc",a.-;.. are also aaggerated in some hepatobili.ry disorders. Th... findings "e the basis of the bile acid challenge test (= stet. N). o. In hor .... increased serum [BA] was found in nearly all ho .... (36 of 38) with hepatic n,""rosi •• lipidosi •• neoplasia. or cirrhosis. In contran. only 2 of 78 sick ho .... without liv« dis,,..., had incr.,...J .. rum [BA] (",ferenee interval not stoted but upptr limit estimated from a graph to be 18IJmoUL}.>1' E. In cattle. the diagnostic sensitivity of .. rum [BA] varied .mong liver disorders: f.scioliasis {I 00 'Job. n = II I. biliary calculi (100 % . n = 2). hepatic . bscesses (53 % . n = 15). leptospirosis {71 'Job. n = 7}. and hepatic lipid""is (86 'Job. n = 36) {",feren"" interval not stoted but upptr limit estimated from a graph to be 40 J.lmolfL)."ln another rq>On. values for .. rum [BA] wtre incr.....d in cattle with hepatic lipidosis. hep>tic absc.:lRS.leptospirosis •• nd foucioliasi, with mean con""ntrations of 90--225 J.lmolfL at initial evaluation." IV. Bile ""id challengt ten for dogs and cats A. Principle: A 12 h f"'ting [BA] provid.. a baseline assessment of the .mount of BA that escapes the enterohepatic circulation .nd ente" the .y.temic blood. After ingestion of. standardized meal. g.Jlblodder contraction release. bile ..Its to the intmine. from which they are ab"'rbed :md then em« the ""nal blood. Thi, influx of endogtnous bile ""id, challenges the . bility of the hep>tocytes to keq> bile acid, within the emerohepatic circulation. 694 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY Table 13.8. Re:.u1u of the bUe add challenge l eU in dog" P~r<'<'n~ Grou~ H",lthy &v~re linr di",ast' Alilinr di",,,,,,' Porrosyslemic ,hun t" IU, liv "66 62 '"29 40 with fa,ting ~BA l :> 5 J.lmolfL '" 86.9 '" ,eo 17. S' P..""ntagt with 2_h postpr:mdial ~BAl :> [55 ~mol!L 2,' 78. 7 77.7 ,eo 12.5< • Illtd 15.5 )lmol/ll""'PnndWly) ..,.. d .. enni ... d .. II.. V:O/"" 2 .und.rd OOi.. ions . bow th.e mO 114/121 j.lmoUL 'Disorders indupWi . .. i.bou. Mp1tic involvemon•• di....."in. ted in'''V1.rulu coagubtion. brain """PI.. i.. bypo:odrenoco,ticism. glomerulon<"'tively. 'No.....= B. Procedure l. A fa,ting sample i, ooll""","d from th. dog or a t aft" • 12 h fan. Th.n, th. anim:.! i, ob.. rv.d while it ingest. food conuining prouin and fat (..g .• 2 t=poons of. amn.d food for tho.>< < 10 lb •• nd 2 tablespoons for lng. dog. ). A 2 h postpr:m di.l sample i. coll.a.d. 2. £ompl. h.mol,..i. mould b. avoid.d during blood ooll"'tion .nd procrssing b.ca""" Hgb int.n.r.. with the .p«:trophotom.uic BA .... ay. Postprandiallip<mi. ,hould b. avoid.d by first coll"'ting a fasting .. mpl. and th. n limiting th. amount of ing.stM c. rood. Publish.d d . u l. Dog'" crabl. 13.8) a. Fasting and postpr.mdial .. mples had high diagnonic .. mitivity for liv" disrasr .nd ponosyn.mic shunts. but the samples did not consist.mly diff... ntia .. th. typt or """,rity of th. di ...... Som. dog> with nonh. ""ti, di,.., .. IIUy have an incrrasrd fasting and postp.. ndi:.! [BA], but incr ...... are typically minor. b. Wh.n drci,ion thresholds of20 IlmoliL for fming sampl...nd 251lmoliL for postprandial sampl.. w... u..d. the diagnonic .prcificity of th. bar .cid oon~ntration approach.d 100 % and th. diagnostic .. ntitivity values w"•• bout 60 % and 75 % . resprctivdy (dna from a toru BA rITzymatic assay: diff... nt d",i,ion th ... hold, IIUy b. 1I• .dM for diff... nt assay.). 695 13/ LIVER FUNCTION Table 13.9. Re:.ulu of Ihe bile add challenge lesl in cats Pe=nt"i:~ Grou ~ H .... lthy All linr .Ii",... Porlosyst.mic shunt' Liv.. di ...", bUI nol ,hunt'" 1ll. liv.. di .."", nOi d.ucla!' 0 " N "" ~ BAl :> wilh f"ling 5 ~mollL 2.5" 73 "" lIS Pm:. nuge wilh 2.h poslpr:mdial ~BAl :> 10.0 ~mollL 7.' '""97 30.8' • The method of ....blidling ",r.,en« interv:ol ~ not rq>a05'O of compuison. the 2.5 % ..... mos that ,m"",,,,, v:ol.... hod . Gaussian distribution. Upp. porto.yJtemic ,bunt ca .. we", 321 140 I'moIIL < Diagn<>SeS (nwnoo of em InJ follow«l by th. m«lirn wtingipOltprondial lBAJ) indud«l I>.p>tie Jipid<>lB (n = 20: 281l42I'moUL). t..patic n 2. Cau" (Tabl. 13.9) a. Fa,ting and pmlp.. ndial [BA] valu., h. d high di"i:nOS{ic ",nsilivily for h'p. lobi. liary di",. ", wh.n Ih. res~lin URLs w... usa!, butlh. [BA] did nol diff.",n. liat. Ih. Iyp< or , ..... ity of Ih. diseases. b. Th~ diagnonic specificity for the ab..nce of liver di"",,,,, approacha! 100 % wh.n values:> 4 X URL and 2 X URL wn. considera! abnormal for fa'iing or postprandial sampl.., nspectivdy, but th~ di"i:n05tic .. nsitivily for hqJatobiliary di"", .. w .. only 49 % (fasting) .nd 8J % (ponpnndial) wh.n tho;.., d""ision threshold, ~'" usa! (data &om a lotol BA .nzymatic au.y). D. F.cto" oth.. than h. po.tobiliary .nd ponal.yst.m. that influence nsults J. F:lSting [BA] a. Spontoneou, contraction of Ih. gallbladder may inc",= fming [BA] unexpraa!ly. b. Fa,ting ""rum [BA] d'p"nds on Ih. ~nterohqJatic cyd~. for exampl •. intestinal di""" .. may impo.ir intestin.l abrorplion of BA .nd thu. lower. serum [BA]. 2. 2 h postprandial [BA] a. Th. pe:ik [BA] =y not occur 2 h postprandially beam", of varialio", in ga,tric .mptying, intminal tran,it lim., intestinal abwrplion. or other facto". b. Th. anim. l may nOi han .... t.n the food provid«!, or il =y ha"" a poor choltcystokinin respon"'. c. Th. gallbladder may han contract«! prior 10 fttding. 3. Th•• fo"'m~ntiona! faCiO" m.y OJntributc 10 th. rd ativdy common finding Ih at th. f"ling [BA] is g=ter lhan th. 2 h po,tprandi. l [BA]. Th~ lo~r [BA] in tho 2 h po'tprandial sample i. not of di"i:nostic valu •. ,,. FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY E. Condusions I. Th~ di.gnOSlic v:due of a ",rum [SA] is priJrulrily for detecting h'p"[obili.ry di..,.", or por,mySlemic shunt •. 2. Th. high .., ",rum [BA] oa;urs when th.r. i, a di ..... proces.l that enabl •• BA molecules to .oca~ enteroh.patic circuJ.tion. How~.r. many di",,,,,,,. or pathologic states cm am", the dysfunction. 3. Th. populnity of th. bile acid {esc {compa,«l to other lin. function tests, such as plasma [NH.1 or dye-ucmion (esI') is probably due to iu simplicity; th.t is, it d~ not ''''Iuir. sp<'Cial patient pr.pantion, sample coll=ion, or sample handling. V. Urine Bil. Acid to Cre.tinine (BA:Crt) R.:uio, A. Principl. I. As mown in Fig. 13.3 .nd 13.4, BA molecules that =1" th. enterohq.atic circulation are acret.d in the urin •. Whenon, there is d.f=i"" d •• ranceofBA from the porul blood or d=e=d biliary excretion of BA that lrads to BA entering the peripheral blood, the .. wiU be inne=d BA excretion via the kidney•. The BAconjugated with sulfate (mlf.ced bile a.cids) a.. mo .. w:lter soluble than nonsulfated BA 2 The .mount of BA excreted vi. urine should reflect plann" [BA] during the time the collected urine was formed. In the published studies, dogs "nd cats we .. not fasted prior to collection of the a""lyzal urine sampl...,. ... B. Pro<:ffiure I. The [BA] and the [Cn] are measured in • randomly collected urine 5ample. The urine concentrations of sulf.ted BA, nonsulfated BA, or sulfated plus nonmlfated BA a.. meamred using enzyJrultic colorimetric assays. 2. A ratio of th. ir concentratiom i, calculated. The reported ratio> represent" mixture of units; that is, ).lmol of BNmg ofCrt.- Unitle" ratios rould be calculated by ronverting the [Cn] of mglL to ).lmollL befo.. dividing the BA roncentration (also in ).lmoJlL) by th. [Cn]. 3. The rdative ease of a one_time rolJeaion of urine romparal to fasting and postpran_ dial blood rolJections makes this assay attractive fur :m=ing bile acids. C. UNSBA; Crt .nd (USBA + UNSBA) ;Cn have been mown to have diagnonic proper_ ti ... imilar to .. rum [BA] in dog. and aUS when ratios were compared to the gr....t.. t .. rum [BA] fuund in fasting or ponprandial samples {Tables 13. to and 13. II} I. With u.. of the ..teaed deci,ion thresholds in the population nudied, the ratios for canine urine had exceUent diagnostic specificity .nd po.itive predictive values fur T able 13.10. Result " Dog. Hralthy Hepatic disorders Om Hralthy Hep"tic disorders Not<: R..pon«l mj", '"" , 54 indud..d a call USBA;Cn ).lmoJlmg UNSBACn ).lmoUmg (USBA + UNSBA);Crt ).lmoUmg 0.Q....0.9 0.(}....42.6 0.Q....6.0 0.3-2377. 0 0.4-6.6 O.Q....23n .O 0.Q....1.9 0.Q....14A 0.7- 1.2 2.Q....12.9 0.2- 3.9 0.4-35. 7 multip~cation So""",: Tr";nOI . t al." and B.tkm.rn .. al.'"'"" bctOI of 100. 697 13/ LI VER FUNCTION T able 13.11 . Diagnostic f'ropcrtic. of BA.Cn ratio. in do!!" and cat Do,. BA (serum) USBA:Cn UNSBA:Crt {USBA + UNSBA}:Cn Diagnostic semitivity Diagnostic >p«ificity Positi"" prffiicti"" value Negative prffiicti"" value (.o) ,.o} (.o) (.o) "" 63 6i 67 96 (00 (00 (00 (00 (00 (00 Om " 10 " " " " BA (serum) 96 USBA:Cn 94 57 UN5BA:Crt 96 {USBA + UNSBA}:Cn 96 65 Not<: The r'l"'nrd rati", includrd a multip~""tion nero, of 100. The d.eciUon tb""boJd fo, SBA..,., tb. URL The <\rcision thresbolds fO, th. ntio v.ru.. ....,"" e:olrubted (me= + 2 sun<4rd "" "" '" '"'" " li""r disea,.., beause there we", no f.lse positivo •. How""",r, the decision th=holds yiddffl only f.ir di>.gnostic sensitivity v:du.. and poor nq:>tive prfflictivo values beause of frequent fal .. neg"i""", 2, With use of the sda:tffl deci,ion thresholds in the population !ludiffl, the ratios for fdine urine had ""ry good di>.gnostic specificity and positi"" prfflicti"" values beause there we .. few f:dse positivo., The decision th ... hold, yiddffl good di>.gnos. tic sensitivity values and fair negati"" prfflicti"" v:du .., 3, Prfflicti"" v:du .. =y vary comid.... bly with v:oriations in the prevalence of hepatic disea,..,. in populations of int...st, D, Esublishing the diagnostic value of the urine BA:Cn ratios will depend on the use of appropriate deci,ion thresholds and comp:"ing the remits with other methods of detecting livor disease, AMMONIUM (NI-4*) CONCENTRATION IN PLASMA L Phpiologic process<:. (Fig, 13,5 ) 11. An.lytical concepn A, Unin and terms L The an:dyte is commonly call ffl bu,1Jd ammonia (NH,), but the dominant form in plasma i",mmonium (NH/), At a pH of7.4 and a pK, of9,25, the NH/:NH, ... tio i, about 70, and thus the .. is rdatively little NH, in plasma, At body tempe .... {u", and pr=ur •• , NH, is a gas, Routine clinical = p use plasma and not whole blood as a "mple, 2, Unit convorsion' a, NH,: J,lgldl X 0,5871 = J,lmollL (SI unit, nearest 5 J,lmollL) b, NH / : JlgldL X 0.5543 = J,lmoJiL (51 unit, ncarest 5 J,lmoUL) '98 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY Fig. 1l.5. Phpjologic p"""'.... of ammonium. • Most NI-V is prod~ in in...,ines by digestion of di.tory pro«; ... or by th. m... boli,m of !>""teri•. So"", NH: is produ~ by the deamin1tion of..runo:ooids (in mrny ""lIs) :on ph ... (..p«ially in musd. libel'll). • Aft the .ynthesi, of ur.,. (in u... cycJ.), omino acid>. ond proteins. Uro. diffuses from bep. tocyte> to the sinusaid1l blood OJ the biL. corWiruJi, from ...rucb it m:oy be acrfl:od vi. th.e kidn.y> 0< d.. in, .. ti"", ''''p«1i""ly. Ur.. tho< rnt ... tb. in"",';... (from dio<, bil<, or bl<><>< n:d»orW os port of:m ..,terob.". tic cirrul1tion (:tIso ... Fig. 8.5). • Rerul """ecion of NH: moy occur by NH; passing through th. glomeruhr filt",ion barri.r:and oong """,.ted in the u.m.. NI-V is:olso fixed into UfO' in tho Mpatocyte> or into g1uumine (GIn) in tho «an tubulll cello. [n '''pons< to .cidemia, deomin1tion of GIn to g1u'1JJUt. (Glu) in the r.,w tubule. , ....1" in NI-V =''';00 (_ Fig. 9.6). • NH: it the mok B, """"'ys for NH' cona:ntntion L Methods a, Enzymatic method: NH, + a-htoglutor ..e + NADPH ---> glutollUte + NADP b, If an alkaJiniI.ing OV'nt is Usffl to con""n NH' to NH" NH, can ~ measurffl by. d}",. binding mr'lhod, c, Other ••up measure NH/ vi. colorimr'lric or ion.selective da::trode methods. 2 When [NH,l ""]u.. from two commercial analyze", were compar.d with value. gtnerat.d by the generally acupt.d wzymatic :way, ther< was good "!:r",,ment ~tw""n the Blood Ammonia Ch«ker and the enzymatic assay> but poor agr""ment ~tw""n the VetT~ and enzym .. i, .ssays. '" 13/ LIVER FUNCTION 699 C. Samples l. In h~althy fasting dogs, the [NH,J in artori:d .nd venous plasJrul s;omples w~re not s.ignificl.fitly diff,,~m, HOWO'Ver, in dog. with Ii""r di ...... , th~ .n~ri:d sampl.. had ~.t" [NH,J (S« Ammonium Con""m.. tion,..a, IlLA5}," 2, Th~ [NH,J in .. rum sampl.. i. gr~a t" th:m in pairM plasma umples.' 3, H~molrsi, couse; faJ", incr•• 1eS of [NH,J in som~ assay. kc.u", the h~m~ pigm~m im~rf~ ... with light tr.mmitun"" or b.cau .. erythrocytes contain NI-4+, " 4, S.mpl~ handling a, ld~:d: EDTA_ or heparin_:mticoagulatM blood is ddive:rM to a laboratory inun~iatdy aft.r coll=ion, Th. plasm. is harv.st~ immMi. tdy .ft~r arrival .nd roolM to 4 °C (in :m i", b.th), and chemic:d .nalysis is compl.tM within I h, All steps mould be complHM with miniJrulI exposure to .ir, b, The plasm. [NH:J is usu:dly consid~red to be suble for up to 4 h .t 4 °C and . dequ.tdy su bl. for 1- 2 d at - 20°c' e, SuooptiJrulI handling and instability probl.ms con l~ad to highly variable ..",Its," {I} Within bou .. of storage at room temp<:"tu"" the [NH:J will be up to 2- 3 tim~s th~ su ning valu~ b.cau .. of the g~n".tion of NH: from th~ d.gr.odation of labil~ prot~in. and .mino .cids (e,g" gluumin~) , {2} Delayffi collection of plasJrul from th~ blood enables NH: to be produ",d by ~rythrocytes .nd leukocyt .., thu. inc",asing the measurM [NH:J , {3} Delaynlanalysi. of plasma enables NH>'c) to ucop<: from plasma particularly if the sampl~ is not nopp<:rM or if:m ev.cu .t~ tube is incomplHdy fillM, Lms of NH>'c) will cou... d.cr .... in th. plasma [NH,J , ilL Hyper.ommon.mia (T.bl. n,12) A, Decreased d=. n", ofNH: from portal blood l. Di..a",s or conditions that .. duct the ",mm:d ofNH: from portal blood may ao.u .. hyp<:r.ommonemia kc.u", imestinal baa"ia produ", large quamitiu of NH" Two major type. of di",a= h. ve: this pathologic defect: a, Diffuse: h.patocellular di ...... that rMu", function:d m ... (e,g" cirrholi" necrosis, .nd lipidmis)" b, Ponosynemic .hunts, ~ith" congenit:d or acquired""'" 2, Dec",ased NH: d~ar:m'" am potentially be coused by congenit:d d~ficiencies that directly or indirectly involve th. ure. cycl~ {Fig, 13,6}, Th... disorde .. are rare, a, Argininosuccinate .ynthH.... d~fici~ncy has oon r~port~ in dogs. " b, A probabl~ case: of ornithin~ corb.moyltransf~ra .. {ornithin~ tr.onsao.rbamy1=J d.ficiency coused hyperammon.mia in a cot,'" c, Defecti"" mitochondrial transport of ornithine, • sUNUnct that i. transportM from cytosol to mitochondria in th. ur~. cycl~, probably COU.M hyperammon.... mia, hypc:rornithin.mia, and homocitrullinuria syndrome in Morgan fillies," d. Congtnital cystinuria is ao.USM by d~fective renal tubular r..orption of cystine; concur",ntly, the .. is d.fecti"" resorption of arginin~ and ornithin., A cystinuric cot had multiple episodes of hypc:rammon.mic ~n"1'halopathy during iu lif~," The amho .. condud~ that if di=ry 5Our= did not mainuin suffici.nt .mounts of arginine and ornithine for a function.l u"'. cycle (Fig, 13,6), then argininuria and ornithinuria couse Table 13. 12. DillellSa and ooAditioA' that "....e hrpenmmonWli. ' D ' D«1nK'd function..! nua: diffux hCl»toccliulu disnJI: ' D«1nK'd portal blood Row to ]j~r: oon~nital and xquired portos)'st~mic shunu Urn cyck mzylIIt ddicimcic. (oongeniul) Cystinuria ",ith ooncurunI arginuri a and ornithinuria (cat) Cob"Jamin ddic .....cy Incrca.ed NH.' production POSIpr;looiai Urn to~ic... js in aIde Siunuous acrcu.e (r;loehorm and dop) Urin.>ry infn:tion with uuasc.contoining ~eria and ooncurrent urMral oMuuaion (nlcstin,.1 disease in horw. Incr ...scd NH.· imakc NH.CI minim.ttion pcr o. or p"r r'""tum Ammoniated ro~ 10~icosis in can le Exposure to NH, from anhydrous NH , • A manvely common diotaH 0< condition Notr-li,u of tpeci6c: disordon or condino", ... no< complete but ,.,., p"""dood to gt... ."""f*o. A f..lldy incrnood [NH.1 m:o:y OCOlr if d.l.yed 'rWyUs of tt.. ampk reru.l .. in pro...",.. .. o. NH: produc""n by the blood «lb. Hepatocyte cytoplasm .... -.. ----.-._--- .. , " NH.'. HCO,- ~C,,,,~_. Omihne Mitochondrion " ", CylOSOl' • - •• ' . • _ Citruline,' A8pw~~~' lk.. Cyolo Argi ....... Argin.....:cinBta V'S. 13.6. n.. urea pwm. Tlw rola of ado""',.... triphoophate CATP) and phosphore ~ not . h"",",,• Carlwnoyl phoophatt 'Yntho",.., (CPS) tltal)'Uf the ",action in which NH;""" He0l "'" uled to produce athamoyI pboopn.te. Omimi .... arbamoyl .... n.fiotue (0Cl) c.al,..... the combination of corbamoyI pbosph.att oruI ornithine to produ.:J. citrul~ .... ArWninow up&r"'. 700 13/ LIVER FUNCTION 701 3. D,""",asal NH: cl~~r:ln"" can al"" occur with acquirM di""rdw; that cou.., • d~f~i"" u..... cycl~ (Fit:. 13.6). A col>:damin ddici~ncy {Stt Chapt" IS} may cou .. m~thylmalonic ~cid to ~ccumul~t~. which d,""r= the con""ntr>tions of N_aatyl g1utaJrult~. A d,""reasnl N_acrtyl gluum>le con""ntr:ltion couses d,""reasnl .ctivity of corbamoyl phosph at~ symh<'tase and thus less fixation of NH: into ur~ .. " 4. Th•• mmonium con""'ntratioIlS of Irim wolfhound puppi~s th. t wer~ 7- 8 wk old w~ .. 47- 115 J.1molfL comparM to 6-27 IlmoilL for :>dulu. The pathogenesis of th~ hyperammonemia in th • .., pups was not d.t~rmined. but Irish "..olfhounds do h"". a high inciden"", of ponosY"'temic shunts." 5. In 69 dogs with hepatic encephalopathy causM by ...."al linr di ....s.. {including 14 congtnital .nd 29 acquirM munts}. th~ [NH.1 in art~rial plas/rul . nr:agtd 1.5 tim~s the [NH<J of venom plasm •. The venom concrntr>tioIlS ~. be lower ba::.use of enhanud NH.t cl~...n"" from . mri. l blood by kidnq<. musd~. or other tissues. B=u.., of th~ diff,,~n"". some investig.tors recomm.nd that art~rial sampl.. mould be coll~M for m.asur~m~nt of pW/ruI [NH.+V· How~nr. coll~_ ing an .nerial sample i. not as .... sy as coll~ing • nnow sampl •. AI"". ba...d on th~ graphM providn! by the inveltigators. nearly all dog. that we", hyperammon._ mic basal on fastiIll: arterial samples ,..er~ . ho hyper:lmmonemic ba...d on falting venou. lamples. B. Inc......d NH: production I. Postprandial: Incr .......d NH: production during th. digtstion of. meal may cou.., t~mporary hyperammonemia. Thi. condition mould not be con.id~rM if the .nimal was .ppropri.tely fastn! prior to sampl~ roll~ion. 2. Ur .... toxicosi. in cotd" Hydrolysis of ur .... in th~ rum~n liber>t~. NH, that com_ bines with H+ to form NH'. This ""h. ruminal fluid mo", alkalin •. Wh~n th. rum~n pH i.;> 8.0. the NH,:NH.+ ratio mift. toward NH ,. which diHU .... from th. rum~n to plasm •• whe .. it combin~. with H+ to inc",as<: the plasma [NI-I.t]." 3. Strenuous enrcist: During ",,~rci ... two adenosine diphmphat. {ADP} molecules combin~ to form ad~nosin. monophosphate (AM P) and ad~nosine triphosphate {A TP} via the myokinase ",.ction of myocytes. The d•• mination of ad~nosin. monophmph at~ produces inosine monophosph. te and NH,. The NH, diffuses to blood and inc ....... th~ pWm. [NH.1 and pH." a. In gr~yhound •• the plasma [NI-I.t] incr~a...d from a mean of82 J.1molfL befo.. th. raa to a m.... n of256 J.1molfL immn!iatdy after the run." b. In quart~r ho ..... the plas"" [NH.+] incr .......d from • m~~n of 67 J.1mollL "'fo.. n"ciso: {t=dmiUj to • Jne;On of 137 IlmoilL immM;"tdy .ft~r n"c;"." In ~nother study. the me"" NH: con""ntr>tions befo... nd aft" ""erci.. ,.."e 37 .nd 113 IlmollL. respectivdy." 4. Urinary inf~ion with ur ....sr-<:Onuining bacteria and concurrent ur~thral obstruc_ tion: An azot~mic dog with suphylococc:d urimry tract inf,""tion .nd urethral calculi had a fasting [NH,] of2S8 J.1g1dL Th. hyperammon~m;" may h",,~ oon causM by inc ....... d NH' production in the urinary tract. d. cr.....d renal excretion due to obnruction •• nd l"'rhaps d,"""asal u..... cyd~ . ctivity due to . cido.is." 5. Intestinal distal. in horse" Hors .. with crnain intestinal dilOrders may have a markally increasnl pl.un. [NH'J and clinicol signs of rolic .nd encephalopathy without eviden"'" of h~patic diseast. An incr.......d plas"" [NH:J i. thought to be causM by incr..... d gen~ration of NH/ by .bnormal prolif~ration. of colonic ba.ct~ria and/or by inc ...... d intenin.l . blOrption of NH/. No singl~ type of 702 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY b3oC{~ria m~m c. h.., oon culm'ffl consistently in [hest cases. Di"l:lIo'is ''"'Iuire:! m~ .. ur~_ of blood [N H'J .'''''' Inn~a.ed NH: inuke or absorption l. N H,CI .dministration ~r os or vi. th~ colon (~ th. next sao!. IV, NH : Tol.r:mcc. Test) 2 Ammonia!ffl fol'3l:' toxicosjs in ",Iv. " A 30-d-oJd calf had . pl:um. [NH.1 that w:as oomid.ral increasal while nursing a cow that had inge,rM hay tr.atal with anhydroll'l N H,. ~ 3. Cattl. dying . ft.r uposur. to anhydrous NH, presumably would Ita"" incr • ..,al plasma [NH.1 . Canl. apnm.. occurs with the m. [iciou, rd......, of anhydrous NH, from 'to~ tanks. " IY. NH: tol.rID~ us{ A. Prinei"[,, By administration of NH,C] . ith., orally or rectally, a ch.JJ.n~ do .. of NH: is p,... ntal to the Ii"", via the ponal veins. Eith.. d«r.asffl h'!",!ic funcriollk [NH:J occurred before 30 min in some dogs .nd aft~r 30 min in others. " D. Interpreution of ",,"ults l. Fasting [N H.11 a. WRi; Th..e is no evidence of hepatic inmfficiency or a ponosY'umic ,hunt. The.. conditions rould be present, but the production of N H.+ is not ~nough to overwhdm th~ weakened 'Y'tem. Th..e is no evidence of. rare congenital ure. cyde enzyme deficiency. b. HYP"rammonemia; ~ Table 13.12 for potential pathogtn..... 2. Postchall~nge [NH:] a. WRi; Th..e i, no evid~nce ofhep. tic in.ufficiency, pono'J"I"emic ,hunt, or. rare congtnital ure. cycle enzyme deficiency. b. Increastd above r~f~rence interval; Th~ functional hepatic mass, porto.ynemic ,hunt, or both are det:!~astd. Rarely, • congenital or a.cquirtd ure. cycle enzyme deficiency is pr..~nt. E. Exampl~ r.. ults for d"!;s with pono'ystemic shunts are liSltd in Tabl~ 13.13. v. Postprandial venous NH: tolerance te,t for dogs" A. This procalure is simil. r to the N H.* tol ..ance t.. t (Ott th~ pret:eding set:!. IV) except dig.. ted food provides the NH: for th~ tol ..ance t.. ting. The major adv. ntagt over 13/ LIVER FUNCTION 703 Table 13.13. Ammonia tolerance le:§t res ult 10 H~althy dog, Dog 1° Dog 2' Dog 3' F:ming [NH:l fJ.t€IdL) 30 min [NH:l (llgldL) 88 ± 36' 155 ± 71 100 1000 370 1400 '60 R~sulu 10 h""hhy dog. Dog 4" Dog 5' Dog 6' for =y 2 'SO 56 ± 14 Fmi"i: [NH:l (J.tt!dl) 'SO 30 min [NH:l (Ilgldl) 76 ± 30 550 6SO 900 , Th< f.,ting [NH:J w>s WRI, rnd th ... h<potic function..,.. .uJlicient to hondl. th< NH, to.d during th< f.ning " ..., Th< rr=kod hyprr=on<mi. in the 30 min >1ffiplr is oonsi".nt .. i,b • pon",y"rmic sbunt Of h<potic inruflicimcy, • Tb. hsting bYJ>s .~ghdy inc .....d :md th"" no' "ro ..,'" of u .,., reported bee..,.. of the lock of rn.JyticoJ ogreernmt of th< two NI-I, ....Y' (no.. 'h< dilfe""t .... lues in 'h< he:dthy dog groupo). So",", M ry<:r et oJ." '" '" the NH: toleran"" test i, that th~ .dministration of NI-4CI solution is coII!id~ral .uessful, and some dog. llUy vomit th~ solution if it is administ~red orally. B. R...ics of ch~ procalur. I. Withhold food for 24 h and th~n f~al a commercial diet ronuining .bout 30 % protein to provid~ 33 kcallkg (.. 25 % of th ••stillUta! daily mcuboliLobl. en~rgy requirement). Thi, estim . .. i, . djunal for growing dog•. 2. Hqminized blood ;,; rolJectal prior to f.cding and .t 2 h int~rVllJ. (2, 4, 6, ~nd 8 h) after feeding. Blood ;,; chilla! during procusing, .nd th~ pl..,1IU [N H:l is m=ral immal;"tdy .ft.. pluma i. harvested. C. [n the publi,hed nudy, the postprandial [NH.1 did nOi aceed 16 J.ImollL in ... mpl .. from healthy dogs. Dog. with h~l"'toccllul .. di ..~se frrqu~ndy had a pr~prandial [NH,l and. postprandial [NH,l above 161lmolll without oignificmt change between preprandial and postprand;..l ""lues; the maximum cone<mratioll! occurred 6 h postprandially. Dogs with ponosySlCmic shunts had preprandi.l ammonium cone<mrations up to 250 Ilmolll (90th percentile n~~r 150 Ilmolll) and postprandi.l ammonium cone 704 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY Excrrtion of the;., dyes d.~nds on hepatic blood How. hq>.tocyt. upuh, conjugation {BSP but not leG}. ncret;on to the biliary sySlem, and .,u.roh.patic c;,cuJ.t;on (BSP but not leG). Impai'M acretion (causal by. d.f of the pathWllY) w;;u d.t Refe re nce s l. Tolman KG. R..j R. 1m. u.... fun.oti.oo. 1m Bwtio CA, A .......,.,./ ER. ...... T.- T,,,,"-,, oj'a",',,J a.. ..inry. 3,d edition. tlll-II77. Philaddl'bia, WB Sa.,.!, ... v. ... 2. 19M. .J', 10..... ,.,; M..JK.J ad;..."".. 27th cditioo. Philaddpbia> WB Saw.d, ... }. B..k PD. Nor'" C. 1m. Bilirubin mrtaboIi.n and th, b,.../itary hyp<>bilirubin<mi ... 2. H<patic upakr. bi.,.jiD" oon;., .no.., and =>rtion of bilinobin. &min ll".. Dio 14,JJI- J4J . ... "kDon-ch AF. !'alm. LA L.."« /J. Wu TW. 19/1.. . Drip.. ~f m .... malian biliprotdD ...d '''~'''' of bifu.bio ~ in vi", in...., at. I Oin 1m·", 74<76J-77fJ. 5. L...db.rr; GD. I"""", C. Rad..k"" G. 1986. N.,....,.J 0;,,,,,,, 8. R..tlruiun I. '"'" """ B""" WE. 1987. Bilirubin m".boliom io ~ I..patobilia.y ...d ha ..... lytk diocw.. Y" Q 9,zJ5-240. 9. ~ l.R. 199J. Equio, fa.tint:; hyp«bilirubin<mi •. Ad" Y" Sci Comp Mod Yl,115- 125. 10. MeSh"" BI. lumod'D IH. Y.m YE.. &jed ID. 198... Hypabili<.bio""'. in .... .-Io. Cao I Comp Mod 48,237- 240. ll. RM 1M. H",,;"'n RD. Collino RA. 1977. F..nn,; ...d "{oodln,; io .... , loctatinc dai.y =>0. 2. lb, ,oro,","!" ~f ~".. «ll "'octw, ...d fuoc""" foIlo"'ins; .....J.y fa ... I Comp P.thoI 87,25J-265. 12. Guy M1.. &w.o., DI. KrlkyLC. Almuao P. Bro.... I. I?SIS. l=no, in bob ",.Ical_...d i......cla';"o ...i .... lack of ",Jo.. .... intakr ...d birJ< ......., ,...... , kin .., ,,';vity. Am I Y" R.." S6,IS06-IS 12. ll. NoyIo, 1M. Kroofdd os,.Io/-9(1. I... Co....liwt CEo Kdky KC. Him<, IA. 1975. H~., ofbilinobin ",njup"" in.....,.al ... imal """""" Con.dJ Y" 65<;'0_99. 15. G"",...tl R. ,,",, ~dkio, LR. 1982. EH.ct of ~ odministn.';"D on equin, fanin,; kypabili<.bio..nia. Am I y" R.. ...HlOI-1!Ol. 16. Mia Mi. G"",...tl RR. Comdi", C E.. 1970. BiIirubi ... " C """""" ,nodi .. in nonn.l...d mutant SoutJ.do,.n oh..p ...i .... COOU";"I kypabili<.bio..nia. Pro< Soc fup Bioi Med lJ},955-9S9. 17. Mia Mi. G"",...tl RR. Comdi", C E.. 1970. Unronj.~ "od bilirubin '"''''1''''' in ""nnaJ...d mut",' Conioda[, ... "I'...i .... Dubin·I .... """" tyndrom,.!'roc Soc Ezp BioI Mod I}Stauiwo . ~pJ.oolu.ta" artivity. Am I Pk,..iol 267.G 1094--GIIOO. 23. I.in NC. 1986. 5,h,J"" Vd,';_., H""""Ioo. 4 .... oditioa. l'hiJ.dd.pItl .. U. &. F~,. 24. c",,,, SA. 199J. St. .... bit, .ci.d< iD rom""';"" ....... ] medici",. Y" Oin N....... Am Small Anim Ptact 23,625-657. '0 o., u, "....d.oI." 13/ LIVER FUNCTION 705 25. W ... HJ. 1996. 0inic.J .Dd paoboloOc.J ....di .. in bo ..... ,.;th b<patic di ...... Equi"" Y" ) l,!,146-156. 26. W ... HJ. 1991. &aJ.oti.on,,( tot.J ....... bOlo oci,d «noratio ... /"0. th, diagoooi. of l..paIDbiliaoy di.. . .. in ",ttl,. R.. Y .. Sci 1101 }3_ 140. Il. W ... HJ. 1989. &aJ.oti.on,,( tot.J pta ..... bOlo oci,d «>D«n'''rio... /"0. th, dia~. of btpatobilia.r di.. .. , in J.on. •. R.. Y .. Sci .06.264_270 . .l8. c...,,, SA. ManW...... T. st. ... MR. Wil",,,, E. 1991. fnJ .. tion of twd",·J.ouc I""'p,uodial uod ......bo., po"p.uodial ........ bit, oci,d. «>n«ntrationo fot di."",.;, oH,patobwuy di ..... in "",".) Am Y" Mod Aoooc 199,217_226. Z9. c...",SA. E..I. HN. )0""", SA 1995. ~,,( ....... bit, Kid. oo"" ...... tio ... fot di~. ,,(t..p..tobiliuy dioca" in cat>. I Am Y.. M,d A.ooc 207,1048_ 1054. J(I. B..onanoo A.\l . c..loon GP. Kanno..... B. 1988. CliDial uod di."",nk fea"",. ,,( .I"..., in • fooJ. I Am Y" ModAoooc 192,.J.Il7- J89. JI. HolTmann WE.. R.I." G. Ri"" oS. Do""" IL 1987. Alt, ..';",... in odccted ....... bOocbm>icd ",... titu", .. in "I.uid. aft.. induced b'patk halopatloy. R.. Y.. Sci Jl, 17_11. J9. Sdi,oon D. Hioab". K. 1957. lb, rom"' .... "" of .mmooi. in ..t.ol, blood. ,2-974. 40. Hitt ME. Ion .. BD. 1986. Effect. of.""'V "mpna'''''' uod timt on ",,"n, p ...... ........,..;. ronc......;. du, to • • ",,kd.""""o.k C. Rou S). QR. ](od,k, LI.. VItkb LK. Cupm"" KA. s.......n LL Pod,,",,,, LA 2006. H~ halopathy in ')''ti~.,,,, cat<. In, P«>«odinp"( th, 19"th ACYlM F~rum. D. ....... 794-795. 46. Batt,nby]A. Gi, .. V. Hall f). 2005. HYI""""""""" """";' in 1"""1: lti .......Iibo....d •. Y.. R..o 1).8,105- 107. 48. H.Jib.ortoo )C. Mo., ... 51!.. 1989. Noop«>ttio nitrovn-indocod""""";. tonoo.i • ...d """"",iatod food toxicitr ~. Y .. Qin Nootth Am Food""im. P]k d.a. ..".;.na d", to ",..d" f~.Am I Y" R.. .fl,218+-2186. po<""'....."" ......... R.."". ' 00 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY 52. Hall]A. Alkn TAo Fpathic k1f'<".mmoo.<>ni. and AW..im .. typ< II ,,,,,oc1'" in equid ... E.quin< V nam>i... oocl"od..ru. ''''' ["""" 1804l!S--489. 59. R..drui"" J. "'" <1m [o~ TSGA.\1. 1982. Roc .. l .... """' .. oo&..anc., _ io ...., .............. of port'! d«UIatioon io doc. with Iivn dioo. .., Reo V.. Sci lJ,21-- 1S. 60. w...... Me. Hin Re. G..ilford WG, Soott KC, )0""" GL. Bu ..,dt CD. 2001. rootpcan' DJ. Stromb«k DR. s.,.., EA. ZLnobk RD. Bu. .. DD. 1978. Ammo";. '.,[, Chapter 14 GLUCO SE, KETOAMINES, AND RELATED REGULATORY HORMONES Glucose Concentration in Serum, Plasma, or Whole Blood ....................... I. Physiologic Processes .............................................. II. Analytical Concepts ................................................ III. Hyperglycemia .................................................... IV. Hypoglycemia ..................................................... V. [Glucose] During Insulin Therapy ..................................... Ketoamines: Fructosamine and Glycated Hemoglobin ........................... I. Physiologic Processes .............................................. 708 708 709 713 719 722 723 723 II. Analytical Concepts ................................................ 724 III. Increased IFructosaminel and Increased Glycated Hemoglobin Percentage or Concentration ......................................... IV. Decreased IFructosaminel and Decreased Glycated Hemoglobin Percentage or Concentration ......................................... V. Correction of the IFructosaminel for Abnormal Protein Concentrations ..... Immunoreactive Insulin (lRI) Concentration in Serum or Plasma ................... I. Physiologic Processes .............................................. II. Analytical Concepts ................................................ III. Hyperinsulinemia .................................................. IV. Hypoinsulinemia ................................................... V. Immunoreactive Insulin to Glucose (lRI :G) Ratio ........................ Immunoreactive Glucagon (lRG) Concentration in Plasma ........................ I. Physiologic Processes .............................................. II. Analytical Concepts ................................................ III. Hyperglucagonemia Associated with Glucagonomas .................... 724 725 726 726 726 727 728 728 729 730 730 730 731 707 708 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY Table 14. I. Abb...,,,iatiom and symbols in {hi.. chapler [xl AcrH x coD""mralion (I = .n:dyt~) Adr~nocor{ioo{ropic hormone {ooniootropin} DM D;. I>.I.. mdlilus EDTA EthyJ.nali:omin~m""""lic GH lRI:G Growth hormone Glucagon_lih peptide F.ciliulin hao.. {ran'porle, Hcmotocr;t Hemoglobin [mmuno' N,F Sl Sodium fluorid. Syst~m. International d'Unites GLP GLUT Hcr H,' IRG IRI a.cid GLUCOSE CONCENTRATION IN SERUM, PLASMA, OR WHOLE BLOOD I. PhpioloCic process." (Fie. 14.1) r~Jaud .nd influcncnl by """"'fa! hormon ... For the hormones to influence mcc.bol;,m, th ... mun bt appropriate r«:q>lors and transport systems in the A. Blood [gluoo..,] is iarJ:ff cdh. l. Insulin activity lowe", blood [g1uoo..,j by promoting th. upuh, utili7..tion, or 'to~ of gluco .. by hepatocytes, myocytes, and .dipocyt.., Insulin i. not neffial for g1uoos< tr:msport into n~urom, lrukocytr., r rythrocytes , platd.t<, or hI'patocyt.., Insulin do.. influ.no: hI'patocytr glucaSl' uptah by .ltrring . ctivitirs of hrpatic rnzym .. that promoII' g1yroly.is or g1yoo!:"n synthesis, or by ralucing gluoonqenesi., 2, GluffiSt .ntry into most cdl. i. modulatal by a f.mily of protrim c;ollal f""ilitati"" hem", transport.rs (GLUT_l to GLUT_7), Imulin promotes glum'" .ntry into myocyte< .nd adipocyt.. via GLUT -4; insulin is not n..,dal for th. other carri.rs (r ,g" GLUT_2 in hrp.tocytes), 3, Glucagon :lCIivity incr='ll'::l blood [gluoo",] by nimul. ting g1uronrogrnrsis and glymt"noly.is, 4, Catecholaminr activity alt." blood [glucosr] by"".raJ mech. nisms",l a. An u,-..drrnrrgic stimulus of p.ncr.atic p _cdl. drcr ..... imulin rd .... and thus rroua. g1uoo.., utilization by hrpatocyt .. , myocytrs, .nd adipocytes.' b. A ~_adr.nrrgic stimulm of paner....tic ~-crll. inerr. ... insulin rd....... H owever, u,-..drrnrrgic rrcrptors prroominat. on pancr....tic ~-cdl., so catecholamines prima rily inhibit in.ulin Jrcrrtion. c. A ~,_adr.nrrgic nimulus ofhrp. tocytes incr='ll'::l glycot"noly>i •. ' d. An u,-..drrnrrgic stimulus of th. pituitary gland inc ......... th. rd • ..,., ofG H _ .. le..ing hormon. , which thrn causes G H rd......,.' 14 1 GLUCOSE, KETOAMINES, AND RELATED REGULATORY HORMONES 709 5. GH {som>lotropill} activity illc",,,,,,, blood [g1uco ..] by reducillg g1uro.. upta~ by myocytes alld adipocyte,. 6. Cortisol activity illcreases blood [g1uco",] by ,timulotillg g1ucollw!:"lIesi, alld creatillg a state of illsulill ",.istallce {Ott Gluco", Collcetratioll ill Serum, =t. IIl.B}. B. III mOllogamic allimal" f"'tillg blood [glurost] i. m.imaillffl by g1ucolleogellesi, by millg product, of prot.ill :md lipid G1taboli,m. III rumill. ms, propiollate from rumell fermemation is used for h"".tic g1uconw!:"nesi,. In horu" colonic propiollate comrib_ utes to glucolI""!:"lIe"; •. " C. F.. ting normoglycemia repre",m, • balallce ~tw""" the .ctiollS of illsulin (promotillg 'to'"l:e .nd utilization of fuds) :md g1Ucagoll {promoting mobilizatioll of fuel,}. Wh.n the", i, an imb.lallce alld fuel mobilizatioll domiruot .. , th.n the .nirruol will ~ hyp"rglycemic and potemially g1ucomric; g1uromri Analytical roncepn A. Term, . nd units l. As aploined in th. followillg stCIion C, th. [g1uro",] ill whole blood rruoy 1I0t be equal to it, collcemration in plasrruo or ",rum horvestffl &om the same blood. Assumillg ,imilar rom""t times with blood cdl., [glurost] ill .. rum alld pl .. rruo will be nearly equal to each other. 2. Ullit con"""ioll: mgldL X 0.05551 = mmollL {SI ullit, 1I..... t 0.1 mmoULY B. Sample for [g1urost] l. For most clilliGlllabomory m.-tho,h, .. rum i. recommendffl . nd plasma (especi. lly h"".nnized) am be U,ffl. Serum . nd pla,ma should ~ removffl from cdls within I h of blood rollection becau.. glycolysi, cominues ill blood cdl. in vitro, thus lowerillg [g1uro",]. a. If pl .. rruo or .. rum ru.. romact with ceU, at room tempe"tu", prior to cemrifu_ gation of blood, [glurost] typically d.er.,. ... about 5- 10 % per hour; rruorkffl leukocytosis .nd erythrocytosi, will .co::lerate the process. The me of g1uro", colllumptioll call be deer..""" by placing the blood sample ill • rool ellviroll_ mem, but clot formation alld comractioll :ore al,o reduced. b. Serum "",... tor rubes {rube. with gold or rrdlblack stopp""} comaill . n . ctivator {silica} that ellhallces clot formation alld gd that enabl", ...ier "'p""tioll of. blood clot .nd ",rum. After cemrifug. tion, th...rum [glurost] rerruoillS ,tabl. in a refrigtntor for at l.,.,t 48 h if the gel b.rrier i, imact. u The gd b.rrier m.y brrak dowlI ill tran,it. 2. Special coUectioll tube. romailli"l: NaF (tube, with grey stopP"") call be Usffl to block g1ycoly"; •. However, they:ore 1I0t routinely used in clinical medicill'. a. r compla.. with Mg", which is. cof.ctor for phmphopyruv.te hyd .. ta .. {synonym: ellolo ..} ill the glycolytic p"thw;oy. The NaF tube, may also rom. in .miroagulam, (oxal. t. or EDTA). r will also inhibit g1uco .. oxidase activity {and other ellzyme,}, .nd thus NaF pwm. should 1I0t ~ u..d ill glucose assap th.t ..... g1urost oxida ... b. Pl .. ma [glucose] dec"'a8ffl by 5- 10 % duri"l: the first hour .fter blood rollection. imo NaF rubes.'· The decr.,. .. may =uh from the osmotic movemem of H,O from erythrocytes to pl •• ma when the blood i. mixed with the hYp"rosmotic ",It. .1., ........... 0'__' ~ • t oorlM t_ Kld"oy Adf9flal I Fig. 14. 1. Phpjologic &non th1t inlluenu blood [gl""""'l. • Imostinr: Di....y carl>ohyd ...... (OIOI OR b,""'" dmm to JDOOOUcrharidrl (including gluro .. ) th .. "'" aboort:>od in .h. ""all in.... int. f..".. which they "".... porral blood and rho .. blood if not 'r"""''' ........«1 bj- hq>a~ ,ole, • Ponc..u: Ins .. Un..,d gluocagon Of" ...1oru«I from p....,..,acic fkdIs and a-cdl~ , .. poa;"dy.lnsulin orcmion" nim .. l.trd by ina.,.sed blood «><>«n' .. 'iono of gluooo<, GH, glucagon, or WI><> ocids. G1uagon _ ...ion is "imul...d by i~ blood <»natocytes .. pn>t«I by inc~ glucagon, cortiooI, 01 inca"'" .tarcn.. 710 f.y,u.c. 14 1 GLUCOSE, KETOAMINES, AND RELATED REGULATORY HORMONES 711 {NaF and potassium oxalat~}. Oth~r data indimu th . t it tak .. about I h for th~ P- to inhibit glycolysis compl~tdy, but th~n [glucost] r~mai"" S{abl~ for.t l~an 3 d." Th~ .miglycolytic dfa:t of P- d~nd. on its conuntration in th~ blood .ampk" c. Erythrocyte. are prone to lysi. wh~n exposffl to NaF . nd potassium oxalat~, .. ~ially if th~ .. i. not . n optimal .mount of blood drawn into th~ ~vacuat.d blood tul>.. Th~ rel ..... of .rythrocyt~ cont.nts (~.g., Hgb, inorganic phosphot.., ~nd K1 rruoy l. used for m... suring blood [g1uco..]. ~ral are d..ignal for u"" by JlO'pl~ who a.. monitoring th~ control of th.ir dial>.tic l1at... Th~ instrumenu typically will provid~ useful resulu if th~ unit is ol"'tating corr«:tly, if the sample i. colla:tal prol"'rly, and if manuf.cturer', dir«tion. ar~ follow.d pr«i.dy. 4. [Glu=] in arterial or mpillary blood i. gr ... t~r than in nnou. blood b.a"", I"'ripheral tissue! mnrum~ glUCOst. Th~ diffe",nu in normogl"""mic and normoin_ SlIlin~mic .tat.. is probably < to mgldL C. Wh . t i. m... su=i: whol~ blood or pl:asrruo [glu=]? l. Wh~n .n assay u ... whol~ blood for a sample, it i. important to know wheth~r th~ result r~pr=nt. a whole blood [glucost]' a pla.ma [g1ucose:]. or. calculatal pla.ma [g1uco..]. Som~ instrumenu m~amre and report whole blood [glurosr]. Other inmum~nts m~.. u",. whol~ blood [glum.~] .nd calculat~ a pl:asma [glum ..]. Of th ..~, some . ..mm. a normal Hct valu~, and thll'l the calculatal value will not I>. corr«t in an~mic or ~rythrocytotic sampl... " Som~ whole blood =Y' me.. ure molality and not molarity, .nd thus variations in th~ H,O content of blood (~.g., du~ to di.pla""m~nt of H,O by protein. or lipid.) will influen"" m~.. ural [glum..]. '"'' Th~ .. han btt:n ..nral reports of comparison. of [glUcost] m.:asu=i by differ~nt blood g1uw.. instrument. or :assays. Som~ of th= nudi...'" difficult to int~rpret b.aUst the types of [g1uco..] (i.~., blood, pl:asma, or strum) w~re not sp«ifial and diff~ren= due to differ~nt Hct valu.. w~ .. not consid~r.d. 2. Whole blood vc:rsu. pl:asma or se:rum [glumse] a. Glucose i. uniformly dinribut~d in H,O component. of whol~ blood (glucose: frec:ly diffu ... I>.twec:n pl"'ma and ~rythrocytes), but the .. are . bout 71 mL of Fig. 14. 1. «m,;nwa o Muod.: Gluros< upuk. by myocyte< i.s promot«l by inru~n tltrough 'f'<"ific insulin uceptolJ :on<:yt"'. where .. GH , gluc:ogon. rnd epinephrin. promo'" glycogenoJy.i.s to provid. glucos< fo, glyroly>i.s. o Mipoo< ti"",,: Insu~n promotes tit. upt:tke of glllCOll< by :odipocyt .. : GH ,«lucos gluooo< up ..!". o Kidn.y: If tit. ",nn tIt,,,,hold fo, tubular , .. orption of glucose i.s acnd«l. tit." hyp«glyc<mic gluOOllwi. wiU dov 712 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY H,O P'" ]00 mL of .rythrocytes and . bout 93 mL of H,O P'" 100 mL of pl..,na. Thu., th. H,O com.m of whoJ. blood vui .. with its Het. B=u"" Ih... is leu H,O in ''Yduocyt,. than in plmIla, th. [gluem. ] in .rythrocyu. is .bout 76 % of that in plasma, and th".for. whot. blood [gluco..,] ffi PIasm. [g1 urost [ ,;;b~lood "'~[.~"Oro::'~'~I" =,CCWh~o~I' (1.0 (O.OO24xHccin%)) (14.1.) b. The diff".nces thot may occur brtwttn who]. blood [gluro ..] and pw.m. or ,,,urn [gluco..,] ... lined in TabJ. 14.2. Wh"n ~r. :m.mi. it presSt1 will bt n•• rly njuaJ. D. Method, of m.asuring [glucost] l. Photometric assay" Mosr cu,.. m m.thods ...., g1uC<>St oxida.. (~fic .nzym. for gluro..,), g1UCOst d.hydro~n,..., or hexokin ... (mlym. for . 11 hexoses) for th. initi:'! rtlction. In oth~r =y', g/uco"" (~nd oth~r monosacdt ..ides) r~xts with o_toluidin~ or gluco .. ...dUct. r~~nt' such as copptr or f~rricpnide. Color changes can b. drt""t~d by .ptctrophotomrtrr or rdlectan~ photom~try. Liptmia, hemoly_ ,i., and ict..m am interfer~ with the light transmi"ion, but the degr"" of int~&r_ enct varies .mong ..."'}". Isopropyl :.!cohol cm produ~ a positive interferen~ with ~nt pad mrthod. (e.g. , Demostix) when blood is conuminatffi with isopropyl :.!cohol during blood collection; the re,wting [gluco ..] am b. grtltly incr.,...,j {cru.ng~ from 100 mgldL to nearly 300 mgldL)." 2. Nonphotom~tric =ys; Th.,.. modifications of th~ glucose oxid= mffhod, have an advantage ov.. photometric =ys in that the", are few .. chemical and photomffric interferen~ •. In th~ glucose oxi"" .. r~action, 0, is consumed .nd hydrogen p Table 14.2. Calculated plasma [glucosc] in a blood .ample with given whole blood concentration and Ha value If whole blood [glucose] {mgldL} is; 50 '00 400 ,eo C.lcul.ated pl"'ma [glucose] {mgldLJ i.; If Het i,; "56 '''' 45% 60% " '" '" '" '" '"467 '" '" 935 InCf~~"" above whol~ blood [glucose] (%) , "" No ..: Concmtr:otions of plasm. glueos< ....... caLcuh"d by using Eq. 14.1 :ond ... wrung v.n.bl .. in ''''''pJ. h:ondJing, md in vitro gIycoly>is "...,'" not p..,.." •. Agr<=>namtr1tions d.p mow 14 1 GLUCOSE, KETOAMINES, AND RELATED REGULATORY HORMONES 713 faJ..., d~cru= in [gluco",,] con bt marknl in .amples from pati~nts potassium bromid~ for neurologic dis"""". E. Anif.cnill hypogly""m", from ""Hular consumption l. Extreme leukocytosis: Som~ la>k~mw .nd inHammatory sUtes (= Chapter 2) han bttn rq><>md to cau,. hypoglJ'C"mia btc.u .. of incr..,...d glycolysis. O~p"nding on ,.mpl~ handling, th~ hypogly""m", could bt pani:dly cou..d by in vitro glycolysis. Incr""..d glu= consumption may occur btcause of increased numbtrs of leukocytes or btc.u"" of incr~aKkocytes {e.g., m:dignant blast cd], or after stimul.rion by granulocyte colony- stimulating f.ctor)." 2. Erythrocyt<>lis (,.m~ concq'" as with .,"reme leukocytosis): A la>kocyt~ need. more gluco,. than an ~rythrocyu, but typically th~re.re .bout 1000 times as many erythrocytes •• leukocyt ... Markal rubricytosis would .1.0 prob.bly cause increase-d gluco,", consumption." F- or bromid~; ~iviIll: Ill. HYP"rgly""m", A. As in summarized in th~ preceding Physiologic Proa:s.ses section. pl"'ma [glucose] is influencnl by many factors, so it should not bt surprising that ther•• re many couse. of hyp"rglJ'C"mia. I. Th~ ddinitions, diagnostic criteri., .nd clas..ifications of DM uK ami.). (2) Th. np"n commin... recommendal th., th~ dassificotions of "insulin_ dep"ndent" and "non-insulin-d.".nd~nt" OM bt droppnl btcau,. of th~ confusion ~n~rat.d by th~ir u,.. 2. A proposed cl...ification syst.m for conine DM" is simi!." to the classifications of pathologic hyp"rglJ'C"mia in Tabl~ 14.4. It consim of two major group": (I) inmlin-d.fici.ncy diabetes (IDD) includes those disorders th.r couse a progr=in loss of P-cdls (e.g., type: 1 DM, and p:lflcre. titis), .nd (2) insulin_r..isran"" diabetes (lRD) includes tho"" disorders in which there is insulin antagonism by hormones (e.g., tYP" 2 DM and .ndocrin~ DM). 3. For th~ p.rhologic hyperglJ'C"mic disord~", "",~ra.l other abnorm:d laboratory finding. may bt p....,m .nd can bt uKmlin~ p"rc<:ntage (, ... the Ketoamines: Fructosamine and Glycotal Hemoglobin section) 714 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY Table 14.3. Criteria for the diagnosis of diabetes nldlir"" in people l. Symptom, of diabetes plus casual p[",ma [g1ucO!~l ;" 200 mVdL ( Il.l mmollL). " UUU4! is dcfin~d •• any tim. of day without regard to lime .in"" last m....l. The dassic symptoms of diaktes indude polyuri.>, polydipsia. and unaplain.d weight loss. 0, 2. Fasting pluma [g1UC05C] ;" 126 mgldL (7.0 mmollL).' roiling is dcfinal '" no caloric imah for at least 8 h. 0, 3. 2. h pl"'ma [g1uco",,] ;" 200 mgldL (Il.l mmoUL) during an onl glucost loterance I""'.' Th. tesl .hould k I"'rform«l as deocribW by [he World H""lch Organization, using a glucosc lo.d rontaining an ajuivalcm of 75 g anhydrous g1UOOllt dj,solnd in waCcr. In the .b.. n", of unequivoal hyp with acul< mcubolic d 200 mgl 250 mg/ 150 mg/« tot.nn", ""'" mel oomp~c> tio<'"'i.ttent hyp (• .g., ,.tinof"tby or ",orin dis. ...). Until.imihr " .. dies "'" pe cri,.,ion in c~nic.t medici .... s,,~"'t: WHO Study Group'" mel Govin " B. Dioord~f1 and condition. (T.bl. 14.4) I. Physiologic hYP"rgl)Umi. a. Ponprandial hYP"rglyc;.,mia: Glucos< .bsorbal aft~r corbohydralr (starch) dig.nion inc,.a.st. glurost ~ntry into blood. Also, amino acids absorbal .ft~r prol~in digestion stimulat. Ih. rdeas. of glucagon which thrn promotes hYP"r. glyumia via glurona>grnesis. [GI=) in a monoganric .nimal .hould return to f.. ting values within 4 h. b. Excitem. nt or frighl: C.I.molamines (rpinqJhrin~ .nd nor~pin.phrin.) slimulat. glycogrnolyli. ( ~,.adr~nrrgic respon .. ) and promot~ GH rd..... (u, ..d,.nrrgic ..spon.. ).' GH Ihm interfrr.. with gluen.. uptak. by myocytrs .nd adipocytes. An u,.. dr~nrrgic stimulus of p.ncrralic p-cdls drcrrase:s imulin rd.asr and th ..s ..duc<" gluen.. utilizalion by h~patocyt .., myocyl~s, .nd adipocytes. Aloo, .pin~phrin~ may stimulat. ACTH ""r'lion, which would promol. hYP"rrorli. sol~mia .nd a.. ociatal hYP"rglycrmic dfrct •. " Th. magnitud. of th. hyprrglycr. mi. v ..ies among .prei.. and is probably g.. alest in cots (pr.k val .... n... 300 mgldL)." c. Suroid.a.\.SOCiatrd hyprrglyc;.,mia: Gluooconiroids prod ..crd by "stres.st<\" p:lli.nts stimul al. gl ..coneog.ne.i. and u ..l. a state of insulin =isl. nc;., by drcrr•• ing tho numbe:r or dnci.ncr of glue:<»< mrmbran~ lransport~f1 (GLlJr.4) and indirrcdy by incrrasing glucagon .nd fatty.acid ronuntration •. "'>< How""... th~ n.. mbe:r of imulin r""'plo" in t>rg~1 c;.,ll mrmbranes may .ctually be: incrra.std bc:coUst gl .. ooconiooids stimulat. th.ir formation." d. Dieslrus: Progestrron~ rdrastd from Ihr enrpus lutrum promotrJ ..I..... of GH, which drc ....= .. plak. of gluen.. by myocytrs .nd adipocytel." [n dogs, this 14 1 GLUCOSE, KETOAMINES, AND RELATED REGULATORY HORMONES 715 Table 14.4. Discascs and condition. that cause hyperglycemia 'Physiologic hyptory imulin secretory r.. pofl5C: 'P.n",~atic insular amyloid""is (major form in em) Obesity Other sp«ific ty~ of OM 'Pancreatic OM: p>ncre.titis, p.ncreatic arcinoma 'Endocrine {nonp>ncreaticj OM: acromegaly, glucagonoma, hy~rad .. noconicism, hyp<rpituil>rism, hyp GH i. produced by dUCIai epithelial cdls of mammary glands and not by th. pituitary gland. Il 2, P.rhologic hypthogencsis i. uncommon,It Invenig>lo" have shown that some diabetic dog> ha"" .nti-IJ-a:ll antibodies," b, Ty~ 2 OM {inmlin resinan", with inadequat~ rompcns>tory insulin secretory re.pofl5C:}; previoudy callM non--insulin-d.".nd~nt OM, typ< 11 OM, or .dult_ on.. t OM {I} Thi. JOrm of OM i. mon common in c;ots .nd i. c;ousffl by d. fca. in inmlin secretion .nd poninsulin =cptor defecl. in target cd]', th~ two major crit~ria for ty~ 2 OM,'" 716 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY {2} [n em, ""id~n"'" indicau. that .mylin (amyloid polyp (pancreatic amyloid",;') .nd dam"V', p-cdls. Also, amylin moy ffiffli<>U th~ insulin ""i'YD"" in target ""JIs." {3} P~njsmu hyp mu. 14 1 GLUCOSE, KETOAMINES, AND RELATED REGULATORY HORMONES 717 that h:od illc=std [fructos;omilleJ, the hypothyroid dogs w..e 1I0t hy~rglJ=mic." The collcurrent findillg ofhypothyroidi,m ~nd hy~rglJ=mia /ruIy, ill some cases, be. bc.cau .. of. gc:netic p=iispositioll to both thyroid di",ase alld OM ...... {g} Pha>dtromocytoma: [II olle study, .bout IS % of dogs with pha>dtro_ mocyto/rul. had mild to moderar. hy~rglJ=mia.47 Cat..:holamill" (q>illephrille alld 1I0tdtromo_ cytO/rul .timular. glycogtnolysi, alld promor. GH rd..... (>« Glu= Conettll colled diabetic dermatopathy, n=olytic migmory erythema, and SIl~rficial n..:rolytic d.. matitis. The pathogtne;is of the hyptrglJ=mia is not e:sublished but /ruIy illvol"" illsulin ruiJlallct, glucagon. GH. or alterations ill amillo acid, fatty acid, or rille meuboli,m. It ap~ar:s ,hat the di.betic .Ute devd0p' after th. O"""t of hepati, dist.... , but it has not }"'t i>ttll esublished if th. li""r di",.... caustS the diabe.tic state. (3) Drug_induced OM: ~"istem hyptrglJ=mia (2 or more dap) associated with u .. of. drug (a) Sr.roid. (glucocorticoids) (Stt GluC0.!e Cona:mratioll in Serum, ""'. 1I1.B.l.c) (b) Thytoid hormones: Studies ill cats.uggest that hyptnhyroidism creates a nate of ill.ulill res.inalle<, but the mecluni.m is 1I0t knowlI."'" [n • nudy in which thyroxin. was gi""" to create ex~rim.ntal hy~rthyroid_ illl' ill dogs, data indiaued that the glucose_induced hy~rglyctmia was prolollged bc.caUst of defective insulill secrotion." (c) Megtmol acet;;ote: A:J a "'roid, it promous glucolleogenesis; •• a pro!:",till, it stimulate; rd ..... ofGH. Both mManism. probably occur in dog., but perh ap' ollly the latter occurs ill cats." {4} inf..:tious DM (a) Cattle illfected with BYD viru. can develop OM that appears to result from dam~ to p-a:lb.>407 (b) Sep!is: An early respon .. to elldotoxomia i. insulill ... isune< .nd resulunt hy~rglya:mia. Hypoglyctmia /ruIy develop lat.r. Th.", at< "".raJ hormo",,] .nd rellular respoll"" ill "'Psi. that alt.. glu= metabolism.'" Ikcaust animal. with DM are comide=i more susc 718 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY g1ucon«>g~n .. is . nd rwuc<s gluco .. utilization by tissu ...... If th . t is th~ m«h.ni,m, hYJl<'rglyc;.,mi. would dq>.tocyt.., .nd thus hYJl<'rglyc;.,mia would ~ morr likdy wh~n th~ hy~rammon~mia i. COusM by di!Ord~r.s oth~r than h~patic insuffici~ncy {e.g., e~ss NH; production in intestin~, utea toxicosis, and ammonia for:agt toxicosis}. {6} G..netic DM (a) An inheritw form of DM occurred in h ..honds. Th~ onsH of the disord~r was frequ~ntly ~fo", 6 mo of:agt and was causM by ~-all hypoplasia." (b) A probable nmil;..l imulin_de~ndent DM was reponed in a group of adult Samoyed dogs. Th~ pathog~n ..is of th~ diaktic ,tote was not esublished."' (c) vruin breeds of dogs {e.g., Aillkan malamut~, Fini,h .pin, miniatu", lChnauur, miniature poodl~, and English. spring~r spanid} ha"" a higher incidenc;., of DM, but • gt'netic p:llhogt'n..i. is not establi.m~d in tho .. brffds.lO {7} Anti_insulin antibodies: Dogs with anti_imulin antibodies priot to insulin t",.tments have bttn rrponed, but without case details." ';'; {Note: D;..betic dogs matw with bovine imulin may d~nlop anti_insulin .ntibodi... }" 3. Pharmarologic or toxicologic hYJl<'rglyc;.,mia: hyperglycem;" :woc;"ted with occ;o_ sional or sporadic oodminigenu may produc;., a disorder th . t fulfill. diagnostic Cfit~ria for DM: I t t Tabl~ 14.3) a. Oralot intrannous gluOO5t {dextro..} ~nte" pl.sma faster than it is utilized, stoted, or ncret~d. b. Swoids {g1ucoconicoids} (.... GluOO5t Concentmion in ~rum, sect. 1l1.B.l.c) c. Megestrol acetot~: As. st~roid, it promot.. g1ucon«>g~n ..is: as. progtstin, it stimulat.. rd~= ofGH. Both mechanisms probably occur in dogs, but ~rh aps only th~ latt~r in cots." d. Ketomine stimulate. rd..... of ~pinephrin~, which promotes glycogenoly.i., reduced uptake and utilization of gluco.., and therefor~ hYJl<'rglyc;.,mia." e. Glucagon ant3J:Onize:s inmlin activity by stimulating gluconeogtnesi. and inhibiting glucose utilization and stO"'C'" f. Thyroxine: In a lIudy in which thyroxin~ was ginn to cr ... t~ nperim~ntal hypenhyroidism in dog•. data indicotal that the duration of the hyperglyctm;" .ft~r intravenou. glUOO5t infusion was prolong~d ba:ause of defectin insulin s«rrtion." g. Ethylen~ glycol" may inhibit glycolysis and th~ Kr~bs cycle, and thus may indirectly stimulat~ gluconeog~n .. is in "starved" c;.,Us. h. Xy1a:r.in~ .nd deromidin~' bind to u,-..drenergic receptors on ~_ceIls, ... ulting in the inhibition of insulin rd..... and thu. rwuction in glUOO5t utilization by hepatocyt~s, myocyte!, and adipocytes. I. Propranolol and other ~_adren~rgic block", inhibit insulin rd..... from ~_ceIls." Additionally, propranolol may creat~ a ,tate of insulin r~,istant:e ." J. Insulin (Somogyi ~ffi.ct): In r.. ponse to excess inj«tal imulin, an animal develops hypoglyctmia that lIimulates the rd""", of glucagon, ~pinq>hrin~, conisol, and GH. Together, the .. hormones promote hy~rglyc;.,mia. In a normal 14 1 GLUCOSE, KETOAMINES, AND RELATED REGULATORY HORMONES 719 .nimal, th~ hYP'rg/y,,",mia i, minimi=:! by insulin reI~ .., In a diabe,tic, insulin rdeast dot. not comp'flSau, :md thm the animal d~ops hyperglycemi<:es,Us,71 [V, Hypoglycemia A, Hypoglycemic disord~rs .r~ couKSt utiliz>tion by tissues, decrrased gluco.. production, or both, B, A diagnostician should always be, alm to th~ po .. ibility of a mild to e,"",me .purious hypoglJ=mia cr ... ted by th~ following: (I) in vitro glycolysis in leukocytes, ~rythro_ eyres, p!'tdets, :md polSibly bact~ria (Ott Glu""",, Concentmion in ~rum, sect, ILE) or {2} th~ bromid~ intuf..~nce in gluco .. oxida", a.. ay., C. Di""rd~ .. and pathogeneses (Ta bl~ 14,5) L Pathologic hypoglycc:mitocytes, myocytes, :md adipocytes .nd decr~ased gluC<>St production by hepatocytes, Inmlinomas may cous<: p'rsistent or sporadic hypoglyc~_ miadr~nocortici,m: Wh.n hypoglJ=mia is pre"'nt in this disorder, it i. probably beca<= of th~ hypoconi""l~mia (thus, decr~.sed gluconeogtnesis :md incrused insulin .. nsitivity in urgtt cdls)." 71ll FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY Table 14.5. Di""a..es and condition. (hal cause hypoglycemia Pathologic hypogly",mi. lncr""sal inru[in sn;re{ion 'Pane'''''t;'' P-cdl !I Growth hormon~ drficit ncy HypopilUiuri,m (da:reastd cortisol and GH) D""rasnl gluconeogenesis 'Hq.atic insufficiency/failure: a.cquiral, congenital 'Hypoad.. nororlici,m (d=.aM Da:,raoed gI)'COgffiOly>is Glycogen noJ'3l:' dis"" ... (ra .. ) lncr""sal glum.., utiliz.alion 'uctalioruo/ hypoglyctm", {'pomanams bovine ketosis} Exmioruol hypoglycrm", (hunting dogs, e"do!>ncr horst.) Oth.r pathologic hypoglyc. mial with uncrrc.in or unknown Non-~-all D path~neses rrognancy hypoglfC"mia Malonic aciduria in Malt~ .. dog. Pharm3oCOlogic or toxicologi, hypogl)Umi. In""lin Sulfonylur~~ compound. (glipiI.ide, glyburid.) Ethanol • A rel1tively common di...... or condition Noto: o.l.yo;f rn:ol";' of blood samples or f.oilwe to ",moY< .. rum or pw.n.. from blood ""u. appropri.tely .. ill r=>lt in f:olody low 19luIDll {2} GH ddici~ncy: R«iuc..d GH activity promou, in"'~:as«I insulin .. n,itivity and thu, h., th~ pot~ntial to cau.. hypoglyc.mia, but ..pom of hypogly"". m;" in lllimals with a GH ddici~ncy we", not found. {3} Hypopiruitari,m: Hypogl)Umia could b. cau..d by d",,,,:as«I """,",ion of GH or ACTH (and thu, 1<:Sl coni501 production), but hypogl)Umia i, not a common problem in animal, with hypopituitari,m. c. D=.as.d glucon.ngen..i, {I} HqJatic insufficiency: With mark.d .. duction in functional hepatic mass bea<= of congenital or acquir.d di~, the", :ore too few hepatocyt .. to maintain fasting normogly""m;". Oth~r <"Viden"" of h~patic di ..~ .. or 14 1 GLUCOSE, KETOAM INES, AND RELATED REGULATORY HORMONES 121 inmfficiency i, npttt":! (e.g., incr.~,ffi h'1'atic enzyme .ctivities, hypoalbu_ min.mia, da:reas.d [u",~ nitrog:p:p«t":! to b. marketUy und."""ight or em.ciat.d. d. D=ra,..:! glyoo~noly.is: Cong.nital ddici.ncie, of .nzymes nttd.d for glycoge_ nolysis may contribute to hypoglJ'C'mi •• nd accumulation of glycog..n in cd], {glycog), th.", is a huge n""d for gluoo"" in mammary glands. The cow> will become hypoglycrmic if gluconeog.nesis cannot m""t demand. ~osi, develop.! =ndarily b«au.. of enh..nc.d fatty"""cid allabolimt." {2} Enrtional hypoglyumia: Huntifii: dog. and .ndurancr horses may become h)'P'll;lycemic b«ame glycoly.is consumes gluOOst f.. ter th:m it i, .. plac.d by .ith.. glycog.nolysi, or gluconeogend.nt factor.; tend to c;ou"" hypmmatory ... pons< con,ume mo .. glUc:05e. Concurr.ntly, hepatic production of gluoo.. is r.duc.d b.G1u,. of acidosis and d=e~s.d delivery of p=ursors for gluooneog..n .. is." {b} (f th ... ar. num.rous org.nisms, they might contribute to the hypoglycemia." 722 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY {3} Pr.gmncy hypogl)Umi:a: A htotic hypoglyctmia occu'" in latt pr.gnancy in dogs. Th. p.thoctne,is of tho disoN" is not .,ublishffl." (4) M:olonic aciduria in Malt... dogs: Dogs with (hi. meubolic disoN •• hod nurkal hypoglyumia, but th. sp<>cific ,ozyrnat;" d.f= w:as not .,ublish«l. Th. dogs did nol han malonyJ~nzym. A (CoA) d=rboxyl= ddi_ ci.ney .... Da:,rasffi gluconwg.n .. is probably rontributrd to lh. hypoglyami.>, kcau .. incrrasN [nulonyl_CoA] inhibits pyruvatt cuboxyla.., :m aic animal may c;m,, , hypoglycrmia b«:au.. of th. = utilization of g1UOOR .nd deere= v. [Glucost] during insulin therapy A. S"i:d blood [glucose] curve l. Dac. from this procedu« nuy ~ u..ful for initial r~gulation of a dia~tic nate, for su.ptcterial blood [glurosc:] curv~ is created by measuring blood [glucose] in the following .. mples from hospic.liud patienu that a« f«l .nd exerci!«l as similarly as JlOS'ible to th~ir normal routin~:" a. An 8 a.m ... mple, jun ~for<: morning insulin injection b. S,mpl.. collect 145 mgldl.nd the 8 a.m . • nd 8 p.m... mpl .. had [gluco>e];> 180 mgldl. b. Do not chang<: the in.ulin d05t if the mdir [glu<:<>u] is bc:cw,""n 90 and 145 mgldl and the 8 a.m. and 8 p.m... mple, had [glUcost] ;> 180 mgldl. c. Decreas. th. insulin do .. if nadir [glurose] is < 90 mgldl or if either th~ 8 a.m. or th~ 8 p.m... mple had [glucou] < 180 mgldL 4. Guiddines for cats ar~ similar but with t«.ter decision th«sholds (~.g., nadir values ~cw,""n 100 and 150 mgldl). " 5. Because th,,~ can ~ marked variation in th~ .. rial blood [gluco ..] curn, from on~ day to the nat, especially during hospit:dization, a decision to :dter an insulin dose should comider other clinical information." 14 1 GLUCOSE, KETOAMINES, AND RELATED REGULATORY HORMONES '" Probl~m' that moy b. r"""ala! by .. rial blood [gluro .. ] curves includ~ th~ following: a. Insufficient insulin do .. : [f so, incr~= th~ do ... b. Too short an inmlin df'""t: If so, us. longc:r_.eting insulin or givr q12h. c. Ovrrlap of insulin df'""t b.cw.,.,n inmlin doses: If so, use .hon~r_acting inmlin. d. Th~ Somogyi eff'""t {= Gluro.. Cona:ntration in s.:rum, =to 1II.B.3.j}: If JO, ra!ua: the insulin d"" and rq,..,1t the curv. in I wk. B. Unexptcra! hypt~ might al", b. du~ to • transient disorder that d,""rrasa! in ..vrrityor resolvrd itsdf. 6. KETOAMI N ES: FRUCTOSAM[NEAND GLYCATED HEMOGLOB[ N (Hgb) I. Phy.siologic proces... A. Wh~n glu= ruets non~nzymatically with. protein'. amino groul", mo.dy at lysiM sites, a Schiffba.. is forma! that th~n oonvrrts to a mor~ stobie kffoamin~ adduct. l. G.:nually ~aking. fructo ... min .. are glyca.ta! prot~in •. [n dinicol cheminry, frurtoklminr refe" to kffoamin .. that are forma! by th~ posmanshtional ir~ .. ibl~ nonenzymati, linking of g1uCO!~ to albumin or oth~r pla.ma prot~ins (mosdy [gG); glyca.ta! albumin .c:rounts for about 80 % ofhumon .. rum krtoamines." Th~ corbon backbone of these ketoamin~s is id~nticol to frueto .. (h~na:: th~ name "fructo..mine"J . The half_lif~ of fruetosamine mol,""ul .. i. gc:nerally SI atal to b. ne.. 2- 3 wk, but th~ half_life vari .. among spa;ies and am b. alt~ra! during pathologic stat .... 2. Glycata! (g1yrosy1atall Hgb is a ketoamin. formal by the nonenzymatic addition of gluro.. to Hgb. Th~ half_Iif~ of glycata! Hgb i. gc:n~rally nat~d to b. 2- 3 mo but varies with the circulating lif. sp"n of eryrhrocyt.. (~.g., dog> .. 100 d, cots .. 70 d, and cattl. .. ISO d). Hemoglobin A" is th~ major subset ofhumon g1yca.tal Hgb, rq>r=nting glyca.tion of th~ .mino terminus of the P_globin chain. B. Th. formation of kffo.min .. in blood is po.itivdy oorrdata! with th. magnitude and duration ofhyp<:nds on the dq;rada_ tion or 10.. of the p..~nt mol,""ules (e.g., albumin or Hgb J. T ransi~nt hyp 724 II. FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY An:'!ytical conctpu A. [Fructosamin~] l. In th~ nitroblue t~J"37.0lium ......y. &uct<mlmin~ act. as a .. dueing "l:~nt in an :'!kaJin~ malium to ~n~r;Ue form=n~. which it d~"'tal .~rophotom~rically. Nonspecific raluction occurs within th~ first to min. so measur~ments :ue tahn after that tim~. However. other ralueing >gents in .. rum could cause fal .. _positive remits. In rontr>Sl. th~ fructosyl lY'in~ oxid= =y is conlid~rffl to ~ specifie for fructosyllysine. a .pecific glycatal amino aeid. a. In a romparison study. th~ fructosamin~ ronctntrations in conin~ .... d~er_ minal by th~ nitroblue tetrarolium . ....y wer~ .bout 3-4 times thou d~t~rminal by th~ ~nzyJrultic =y {about 100 J.l moUL}.'·' This study rai.es • conctrn about the validity of the [fructOiamin~] .. ponal from th~ nitroblu~ utrazolium assay. b. Modificotionl of th~ nitroblu~ tetrarolium :assay to ",mo,,", actions of rfflucing sUNtan= other th . n fructosamin~ rqrortally ..suited in [fructosamin~] of . bout 10 % that of the origin:'! nitroblue tetrarolium .ssays. '«l 2. Wh~n mu.ural by .pectrophotometric ......Y'. hemolyzed and icteric .amples can cau", e"on""us remits. B. Glycatal Hgb conctnt ration! or per""ntage'l l. Glyaual Hgb has bttn m~mral by chroIlliltographic. immunoturbidimetric, and chemicol ..... ys. ~ch with its own limiutions. Some ...... ys w~ .. d.. ignal to specifically det«1 hUIlliln hemoglobin A,cO wh~",... oth~", {e.g.. chemicol methods} det"'t multiple forms of glycatal Hgb. 2. Results.", um:.!ly reportal as th~ per""nt"l:~ of tot:'! Hgb th. t is glyaual. 3. If lipemi<> is p.... nt. the a.... y should ~ rompletal after washing the erythrocytes; lipemia wiU f. hely inc",...., perctnug.. in some .... ys.' ... 4. Th~", w;o. good agr..,ment ~tw..,n • rolorimetric =y .nd • chrom. tographic method with canine blood.'" 5. An immunoturbidim~rie manuf. ctural for m~a.!uring hUIlliln [glycotal Hgb]. has bttn u=i to m=ur~ canine [glycotal Hgb] ...·'.. .t.U)'. Ill. Inc==! [fructosamine] and incr~ glyaual hemoglobin perctntage or con""mration (Table 14.6) A. Di.~tes mellitus: Inc",= Table 14.6. Discascs and condition. that increa.e [ketoamine) Fruaosamine Glycatal hemoglobin • Hyperglya:mi<> (persistent)' Hypothyroidism • A rel1ti""ly common dioe .... or condition , s... T. bI. t 4.4 for causes of hyp 'Hypergly""mia (pe"inem) 14 1 GLUCOSE, KETOAMINES, AND RELATED REGULATORY HORMONES 125 Table 14.7. Di""ases and condition. Ihat decrease [Iretoamine] Fruelos;omine Glycatffl hemoglobin • Hypogly""mia (persist.m)' Hypoprol.in.mia Hyp • Hypoglycrmi. (persistem) An.mia (5« Ihe texl for vari.bl..) c. IV. normoproteinemic hypothyroid dogs, 9 of II had:m incrrascd [fruetosamin.] (nitroblu. tC"tra7.0lium =y), and the con""mrations d«reasffl after initiation of thyroid hormone supplem.mation." 2. B=d on studies in people. the increased [fruetosamin.] is the result of an in",easal albumin half_lif. {rcductd protein turnover).''' Hyperproteinemia l. In theory, hyperprotein.m;" could calJ.5t increased [fructosan,in.]. H"""cver, vari. tions in the duration of the hyperprotein.mia and vari.tions in albumin to globulin ratiO! result in v. riabl. fructoumine ron""mratiollS in hyperprot.in.mic sampl.. ."~"· 2. In one srudy, oorr«tffl [fructos;omine] in hyperproteinemic .nimal, did not improve the diagnostic valu. of [fructoumine]."' Decr.aKd [fructoumine] and decreased glycatffl h.moglobin per""mage or con"",mration (T.ble 14.7) A. D«ru=! [fructoumine] l. lnsulinollU in dogs: Persistem hypoglycemia caU'ffl by an inappropriate rd ..... of inmlin canl.ad to . decr•• sed .. rum [fructoumineJ.'''·ll' 2. Hypoproteinemia and hypoolbuminem;" a. Normoglyccmic dogs with hypoolbuminem;" had d«re.sffl fructo .. min. con""mrations. '''.1'' b. About 67 % of normoglycrmic cats with hypoprot.inemia had d«r..... d serum [fmetosamine}."· The [fructoumin.} was better rorr.latffl with [total protein} th.n with [.Ibumin}. 3. D«,..,asal [frucl05amine} h", i>ttn .lSOCi. tffl with azotemia .nd hYl"'rlipidemi. in normogly""mic dogs.'" However, the report did not describe whether the ' 7.0t.mic or hyperlipid.mic dogs h.d dysprot.in.mias. 4. Cau with hypcnhyroidism ha"" .ignificantly lower .. rum fructoumine concentr._ tions than healthy cats.''''''' Similar findings arc found in people with thyrotoIirosis. In B. D«reasal glycatffl Hgb perccmage or ooncrmration l. lnsulinoma: Persistem hypoglycemia caUsffl by an inappropriate ...I.... of inmlin can lcad to • d« ...... d glycatffl Hgb percrm.C" or con""mration.'07.l 00 2. Anemia: If [erythrocyte} is srable, then the dcgrtt of glyauffl Hgb formation will primarily dcpc:nd on blood [gluco..} during the lifr span of the erythrocyte .. a. If there i. an .cute .nemia (e.g. , bccaust of hemorrhage or h.molysi.) and. subsequ.m rcC"ner.tive "'porue:, th.n the glycatcd Hgb oon""mration and per""mage will be rcductd bccau.. young erythrocytes h. "" Ial glycat.d Hgb .nd blood [Hgb} i. d«,..,asal. '" FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY b. If th~r~ j, anemia of any origin and th •• nim.l has not bttn hYl"'rgl)Umic, then the [g1ycotal Hgb] will b. da:rrasnl ~"'" the blood [Hgb] j, da:,easM. ,.. w". c. Th. glye.tM Hgb p<,o:ntagc:s in dogs with acul< and chronic anemia, sima" to th"", in dog. with hypogly""mias cau,aI by !Joan """pla,i.. '" V. Correction of the [fructa.amine] for abnormal protein coII""mrations A. Ba::;,Ust dy.proteinemw affect [fructosamine] ind'P"nd.ncly of blood [g1uco..] •• om. author.; h.vt r«:ommend«i COrfflO{;On formul .. to adjust for the dy.proteinemi. {Eq. 14.2}. Th. formulas suggeuboli,m of albumin is d«""a=!, 50 it, half_life is incr~~.ffl. Thi. ",suIts in g... t~r glyation p"r mole of albumin. thus romewh.t counuracting th~ decruK in fructo.<_ amine caustd by hypoalbumin~mia.UJ Consajuendy. the rorrdation ~tw..,n .lbumin and &uctosamine concentrations is poor to moderate. '''":"C[,·:lb:":m:iT"el~ro:':h:~:I~'h:rCd:o"g", observed [fructosamine] x -..m:"' observnl [albumin] (14.2a.) . . 3.3g/ dL corrrcted canme [fructoumm~] = 180 IlmoljL X = 297 Ilmol/L 2.0g/ dL Corrrcted Exampl~ amin~ [fructoumin~] = 1 . . . median [total prot~in for he~hhy em Corrrcted fdme [fructos;omme] = observed [fructo,amme I X [ ' [ observed toul protem (14.2b.) B. A canine corrrction formula using ",rum [albumin] WlU r«:<>mmended ~atu .. of. gr~ater rorrdation coefficient (r) for .. rum [albumin] (r = 0.79) thm for [toul prouin] (r = 0.54}."6 HowrYer. in one ,tudy involving diabetic .nd nondi.~tic dog•. UK of the cor=tnl con"",ntrations did not mbsuntiaUy inc",= the diagnostic ",nsitivity or diagnostic specificity of the [fructos;;omine] (nitroblue tetr:l7.Olium .ssay) for DM.'" C. A fdine corra:tion formula using .. rum [total prouin] was r«:<>mm~nded beau"" of. gr~.ur rorrdation coefficient for .. rum [total protein] {r = 0.681 than for [albumin].'" Beau.. there was very poor correlation with ..rum [albumin] , the authors mggested that a greater proportion of the glyaued proteins we .. globulins. IMMUNOREACTIVE INSULIN (IRI) CONCENTRATION IN SERUM OR PlASMA I. Phy>iologic proc:=e. A. Insulin i•• polyp. Th~ ~mino .cid sajuen"",s of i",ulin molecules of dogs and pigs .'" id~ntical .nd have one amino .cid difference from hUflliln insulin. Equin~ i",ulin Ita. one amino acid differen"", &om porcine insulin. Fdine and bovin~ insulin molaoul.. ar~ similar but have minor differ~n""'s &om the molecu.les of canine, porcine, and human i",ulin.""2< 14 1 GLUCOSE, KETOAMINES, AND RELATED REGULATORY HORMONES 127 B. Physiologic stimuli for insulin KCretion include incre.w oonctntratio", of g1Uoost, xylitol, amino ""ids, .nd several hormone. (g1ucogon, gastrin, ,ecrotin, pancreozymin. gastrointeniruo1 pol~ptide, ~nd ~_adrenergic hormone. ). C. lnhibito," of inmlin secretion include somatostatin, a._adrenergic agonim, and ~_ adrenergic antagonists." D. Preproinsulin i. m. de by ribo,omes of p. ncreatic ~_cdls . nd is quid:ly cleaved to proinsulin that is stored in KCmory gr.nules of the Golgi oomplex. Insulin is formed after clea\'al:e enzymes (some C~>+ regulated) break ptide, . nd .plit p An. lytical oonctpu A. Terms .nd units I. lRI i, the preferred term for immuno=y me"urements of .. rum or plosm.. inmlin for two rea.mns: (I) measurements may indude proinsulin, and (2) meamrements of inmlin .re in immunoreactive units, not biologic . ctivity uniu of injectable insulin. 2. Units: 1lUlmL = mUlL; J.IU1mL X 7.175 = pmollL; and J.IgfL X 172.2 = pmollL {SI unit, ne~rest 5 pmollLl' B. M.aY' I. Mon measurements oflRI .re m. de using oommerci:d radioimmun""~ kiu and therefore .mi_insulin antibody reag..nts. Chemilumin=nt .....Y' .nd enzyme_ linked immuno"'r~nt .....Y' (ELlSAs) .re also av.ilable. 2. The .mibody in oommerci:d =ys llUy h. ve: bttn developed to react with porcine or hum. n in",din. There is sufficient cross-immunoreactivity th at oommercial .....Y' have: bttn validated for conine in.ulin. Commerci:d assay. llUy not ~ valid for fdine insulin.''' 3. Wide ranges of [IRI] h. ve: bttn reponed for conine, fdine, :md hUllUn """pies a.......d with oommercial inmlin .....Y' .... "~ll. The variation may pmi:dly ~ due to differenct. in nandaro or calibrator ",IUlion., bUi unaccq>table v.riation pgtnts. 4. 5 728 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY Table 14.8. Di""a..es and condition. thai cause hyperimulincmia inc,",asM insulin production and rd~= 'Functional pmc •..,.tic ~-all """pi",", {imulinom.j Xylitol [oxleo,i. Hyp th~ tat) • A ,ol1tiv.[y common dio.... or condition Noto: Anti·insulin an,ibodios may prod""" . positin jllterf...,,,,,,, in som< >SS>JI'. Ill. HYP'rinsulin~mi. ,.1.= A. The major reason for measuring [IRI] is to document the inappropriate of insulin from nroplastic P-<:dl.; t .... t is, too much inmlin ,d""snl for the miJrulI'. plasm. or ",rum [gIUoost]. B. Di""rdc" and pa(h~n= (Table 14.8) l. inc,,",asffl insulin production :md rd •• ", a. Functional pallcreatic ~-all na>pl..i. (insulinom.): NeopJ:.stic p.cdh may comismuly or .pondically product inmlin, which cau..,. hypoglycemia kc.UM of cnhancnl g1yrolysis, rMu=! g1uoon""&<,nesi,, .nd inn.=<:! glUoost uptake by myocytes alld adipocytes. b. HypugI~mia 1I0t c;;o.....d by d<er .... sn1 in",lill production {I} Hyp",i",ulin~mia is exptct<"d with ph)"iologic hyptrgl~mi. breau.. hy~rglyamia stimulal.. the produclioll .nd rdnK of illsulin. {2} Hyptrillsulin~mia is exptct<"d with palhologic .nd ph ..macologic hyptrgly_ ""mias if inmlill rd~= from IJ-ctll. i. not d~f=iv~. Also •• 'UI~ of insulill =isun"" may illitiat~ or augm~m • hyptrgl~mi" sUI~ and rollcurr~nt hy~rill!ulill~mi .. 2. Anti_insulin amibodies: Th~ prestlla of ami_insulill .ntibodies may resull ill, falsdy iner .... sn1 [IRI] in some >.<Says (-= Fig. 17.2 for th~ conapt). Anti_illsulin amibodies may ari", from spontalla",s pathologic proce.... or in",lill th~rapy. [v. Hypoin",lill~mi. A. Dorum~millg hypoin",lill~mi. could hdp in classiry;lIg or ch.racI~ri:r.ing OM states: for ex;;ompl~. collfirming typt 10M. staging ~ 2 OM. or """';lIg insulin nacus ill olh~r di.bHic .rates. I. M~.!Urillg [IRI] in hyptrglyamic allimals is 1I0t common. Th~ illfr"'lu~m m.... sur~_ m~m of [[RI] ill DM "''''S is prob..bly due to many f.cton. ",ch as cost. l.d: of stalldardiud ......ys •• nd lad: of diagnonic or prognonic erilu;, :woc;'I<"d with vari'liolls in [IRI] in diff~ .. nt types of OM. 2. A d~fici~ncy ill illsulin c;;on k defill<"d ill two way" {I} .n absolut~ d~fici~ncy in which [[RI] is below all approprial~ .. f~r~II"" im~rvaI. alld {2} • rdalin d~fici~ncy in which th~ amount of insulill i, ill!uffici~nt 10 mailluin a normal "'rbohydral~ meuboli,m. Fasting pla,ma [I RI] has kc:n.....d.' a mahod of da=illg ill!ulill resi,un"" in ",I, filtillg Ih~ ",rolld d~finition.m B. Oisord~n or conditions wh~rr [IRI] i, exp<et<"d to d<e ...... I. Pathologic hypoinsulin~m;' a. Typr 1 DM: [1I,ulin produclioll is d<er~.sn1 breaust of Ih~ dmructioll of IJ-ctlls. b. Typr 2 DM: Advan=l nages of panc...lic amyloidosi, ill""l"" IJ-all datuat<' and Ihus d<erras.-d in",lill productioll. 14 1 GLUCOSE, KETOAMINES, AND RELATED REGULATORY HORMONES 129 2. Physiologic hypoinsulin~mia: Animals with a variety of hypoglyo::mic Immuno.. xtive: insulin to glUCOK (IRI: G) r>tio A. Ba:au.. insulin pnxluction and rd~aso: from p-cd h d~nd on pla"n" [gluco",], an lRl: G ratio should indicat~ wheth" the measured [lRl] is appropriate for th~ degr.., of glUCOK stimulation. Wh~n uK IRI:G = [IRlJx 100 [gl ucos. ] (14.3.) with [IRI] in ).lV/mland glurosc: in mgldl: thus IRl:G ratio unit i. ).lV/mg glucose l. d: of analytical agrttment .mong insulin =Y' requir .. that r~fe .. no:: intervals for IRl:G ratio. ~ osublished for ~och .....y, which is a task th.c is not commonly accomplished. C. Interpreution of th. fasting lRl: G ratio {assuming valid cono::ntrations of I RI and glUroK .nd comparison to . n appropriat~ ref~reno:: int~rval for lRl: G ratio I I. Increaso:d a. If associated with hypoglycemia, then illlulin is rontributing to th~ hypoglycc:mi •. b. If associated with normoglycc:mi. or hyptivdy rdiable method for assessing in""lin sensitivity compared to "",eral oth" methods. m 2. Within the ref.rence interval a. If associated with hypoglycemia, then. pathologic state oth.. th.n hyperimu . linemia i. causing th~ hypoglyo::mia. b. If associated with hyperglycemia, then. factor oth.. th.n insulin deficiency is causing the hyperglycemia. 3. Decreased: If ow;oci. ted with hyptio may ~ difficult to interpret for reasons oth~r than the variations c=ted by diff~ .. nt assay•. I. Subsuno::. oth" than gluco .. influ.no:: in",lin rde..,. from p-cd ls. 2. Much of the rd~aK Th~ 730 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY [glum..,]. If th~ modification was valid, Ih~ .. would bt a dirttl rd.tionship bttw~.n insulin .nd g1u= con~mra{ions ov.. 30 mgldL; thot is. x IlU of inmlin for rv0l}' J mg of gluoo..,:> 30. 2. [n 1973. Tum,r, Oakley, and Naoorro ,. ponrd changes in plasma [insulin] during .thanol_inducal hypoglyc. mia in ob...,:md nonol><.., peopl •. l1'I a. They did nol m.ntion th. i, "< 30 mgldL" th",,'Y or propo,rd am. nd«lIRl: G ratio of 1971, .nd Ih.;, publi,h.d Insulin was not d"t.] of 4O--6S mgldL TIm. w.r. only thIN .. mpJ., with a [gIurosr] < 30 mgldl; insulin values in thOst sampl.. nngn! from Q.-2.0 ).IV/mL. b. They did writ., "lb. fall in plasma insulin w>s • function of Ih. fall, nth.. than of Ih. abwlut. values of Ih. pum... g1uco .... " c. Anoth., va,i.bl. not oomjd.rffl was Ih. diff~ .. n"" bttwttn insulin ron""ntn_ tion, in portal and ~riph~ral blood. Ba:au.. much of the "",..,,,"d insulin it rrmov«i &om ponal blood by h~p'IIOCyt .., asst:SSm~nt of insulin ,a;retion ,timuJ.ted by gluco .. i, bettu ,"",,-lmu«i by m~asuring portal blood ron""nt .. _ tion" which i, a technique gtneraUy limited to ex~rimental im~tig.tion •. 3. Many ve{uinary publicnion, have indud«i the use of the amended [RI: G ratio to evaluate [IRI] .nd [gluco..] in dome'tic and nondomestic .nimal•. Thu~ h.ve .ho bttn se-ve..l.ttempts to squdch the use of the .mend«i IRI: G ratio. but it .... m' to h.ve. life of it, own., ..... ,,.. The amend«i IRI: G mio .hould not bt usa!. [MMUNOREACTIVE GLUCAGON (IRG) CONCENTRATION [N PLASMA I. Phy>iologic process~. A. Pilncrrilfic g/u('(lgon (M, = 3485) is a 29_amino-acid polyptide hormones (coUecrively call«i GLP.) that influen"" or regulate """ral digestive and m..r.bolic proct ..... Oth~r than glucogon. the IIUjor GLP involv«i in glu= meuboli,m i. GLP_I.'" I. The major rol~ of glu<:aJ:On i. to maintain blood [gluco..] during fa,ting. Stimuli for p.ncreatic glu<:aJ:On secretion indud~ hypoglycemia (which may bt induced by exerci.. ), incr~as«i amino acid" hypc:rrortisolemia, and probably hypoinmlinemia. of the intestinal mucosa 2 GLP_l, which is releasa! &om L ""lis (brgt granule after fttding. ,timuJ.tes the rd~.... of insulin and r«iuct::l po'tprandial hy~rgl}'u_ mia. Prior to the id~ntifiQltion of GLP_I, the hormone was referrffi to •• gut glu<:aJ:On, which crOSl-reacred with glu<:aJ:On in some immuno .....Y'. B. Proglu<:aJ:On i, produced by pancreatic ct-all" inteninal L and th~ paratympa_ th..ric nudru, of the V>gU' nerve.''' A variety of factors rontrol the cleavage: of proglu_ <:aJ:On into glu<:aJ:On (primarily in the pann=), GLP_1 {primarily in intmine}, and oth~r GLPs. C. The major action, of glu<:aJ:On a.. illu,trnt«i in Fig. 14.1. ""n,) ""II" [I. Analytical oon""pu A. Term, and units I. The term IRG should bt .. strved for th~ glu<:aJ:On that i. measur«i by immunoassay. that arr ~ific for pancr'""-tic glu<:aJ:On. Unfonunately, ,om~ 14 1 GLUCOSE, KETOAMINES, AND RELATED REGULATORY HORMONES 131 radioimmullo~~ .ntibodi.. also d",,«t oth~r GLP., .. ptcially GLP_I or gut glU<4g01l. Some .uthor> r~f~r to [RG a. g/urllg,m_uh immuno""ctMty. 2. Ullit conv""ion: pglmL = nglL (SI ullit. lIe~rm lO nglLl' B. As.says l. For many y~ars, the gold .t>nd",d =y for eonin~ IRG wa •• radioimmuno:lS""y with Ung~r's 30K antibody, all antibody that W:lS colI,id.red .ptcific for th~ C t~rminu. of glucagon.'" Oth.. investigators h:IV~ .hown varying dog .... of antibody sp«ificity for pancr .... tic glucagon and glucagoll_lih immunor~activity."" 2. One group of in=tigato.. d..crib.d validation ,tudi.. ming .n anti_{bovin. glU<4g01l) antibody to asse .. [glucagon] in dog., em, sh, cows, .nd horses. In [he f>Sting lIat~, .bout 30 % of the glucagon_lik~ immunor....ctivity w;u pancr~atic glU<4g01l. Th~ir .ntibody did croll_react with l"g~r mol«ular form. that had glu<4goll_lik~ immuno ..activity. ". 3. Some in=tigato.. us< comm~rcial glucagon =ys.'" R~sult. nom two comm..cial glU<4g011 .lIays difi'tred colISid~rably {> lO_fold ill ""m~ pla.ma ... mpl.,} ill • ",... nillg Muation. ,.. C. Samplel I. Mon investig.to.. colISid~r IRG to b. v~ty unstabl~ in blood, .nd thu •• ptcial handling i. =mmended." [mm.diatdy aft .. collr<:tion, EDTA_blood i. immer"':! in an icc: bath and. prot..... illhibitor {aprotinill} i. added. Plasma i. q>.rated nom nythrocytel in a 4'C a:ntrifuge .nd th. n froun at - 20·C. Sampl...hould b.: prot=ed from ligh!. 2. s.:rum h., bttll u.ed in £om. studi...''' Th..~ i. ~vid.ncc: that the .tability of radiol.bded glUcagoll i. 1I0t th~ ... m~ ill all sp«iel.' '' [n som. =y system., d 1Il. Hyp<:rglucagon~mia :wociatal with glucagonomas A. B«aus< glUcagoll is • homlon~ illvolvrd ill corbohydrat~ and lipid m"".bolism, th ... are mallY ..]>Om of immunoreactive glucagon rona:ntration. in various physiologic .nd pathologic nat~'. However, [IRG] h:lS very limitrd us< in vet~rillaty di>gnostic dfom. B. Some dogs with pallcreatic glucagonomas have a charact~ri.tic m~rficial n«rolytic d~rmatitis, but not.ll dog. with th. d.. matologic di""rd~r hav~ glucagonomas. l. [nc.....d plasma [IRG] h", bttll rq>Onrd in some dog. with th~ disord~r.'~l""""" 2. [n. study inmlving 22 dogs with m~rficial n«rolytic d .. matiti., pancr~.tic nroplasm. we .. not found (19 of 22 had histologic pallcreatic a.minatiom), alld immunor~.ctive glucagon concc:ntratiollS w~ .. withill th~ rdi:r~ncc: interval in th. fi"" dogs that we .. .val""t...!.''' References I. Adamo HR. 1995. AJ..n..p, "&",,in and ""<>e<> Ado.....vc 732 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY J. Th""""" je. T,""'Iv.ilU wI, Bnuo.. G/. 1996. P"""".tI.rtko .,.J ""........., adjw.- ]'" Thw""", Ie. Tn<>n>k n, 69--90. Ame" [owa 5..", V.o....ity v,,.. ......, ''" S. McMahon CD, R.dclilf RP. lookincI...d KJ, Tuck.. HA. 2001. Nn. ..."~ati.on of ~ bo"""", a«... HA. F<>«l EJH. 19'91. GIu.ro",'Vnooi. from .,..",;.0""" p..duc.d in "'" ",Ion of "'" bo .... B. Vrt J 147,J40.--J4S. 7. l.w.d/"" GD. ]"""'" C, R.duI'Ku G. 1986. No.. ",d OU_ ....., .. an into-modi", ""'"C' d...k. ..it!.in tl., I"bo..",,'},. Am J Clin P.tW 66,6Sl-·-t;S7. 9. Bu,k VJ. j,tl9- IH. 10. o..; ..opb", M.\1. O'N,iIl S. 2000. E./foct of op=te ia ...,.;at«! ,.;th ",oglin~ and """,,«<1 """""tratio... ef I",,,,, and .... ,pioqlui"'. I Y.. Int<m Mod 16.12.J.-1J2. 2.1. M.ock A. N."J'"F';' ..T6troUod ~ Iootmont <>'""production and na"'..u, <> =,.. = = = cs. 141 GLUCOSE, KETOAMINES, AND RELATED REGULATORY HORMONES 133 ll. lutt TA. Rand IS. 1m. P.""""", .. offdi... di.brt.. m,IIi .... v" Oin Nonb Am. Small Anim p,Kt 2S.S27- H2. l2. H_ "tk...AU ... Rl. T ' pr, L. Blwn JW. 1994. [noulin b1P""~" ond duooowia in in_.ivdy milk· fed "Iv< •. I Anim s.; 72.160-17}. H . B,,,,,,,,,, CL. H<>«<>mrti.ootropb pinoitaty «knoma (doubI, ""-'ma) in • = ..ith diabrt" mdlitw ond byp« ond inoulin oa=tio~ in ' pontanOO •• [,loyp6d ",,,. I E.ndoctin.oI 12h:M9-2j I. .(j. rn.o.. RM. R.id SW. Moo.,..,. cr. 1m. Ep~ dinicd ..........toIocialond biodt...niad cb ....-iatia of ~ k.rp<>thp<>6di.n. V" RL< 145.'-11-487. 4~ . R.ucb CE. G")'" B. Botrtti FS. 2002 . s.. .... f_min, «>oem ... tio ... in do, • ..itb Ioypotb,..,idiun. V" Rto Coanm"" 26.SJ I- H6• .. S. H..v. AM. St< ... m,IIi ..... "' ... [ ....yIoidoo .. ond thrombooi. in pw"-d l><.p... Am I V.. R.. "h 1077_ 1081. .. 6. Fold...... EG. Nd.~n RW. 1996. Hypodt.,..,idi ..... In. c...;... • ..JF"'- E~.!.o ",,,J &, ~ . 2<>d odition. 68_ 117. PkiJ..d,dpl.ia.. WB 47. llattI... rY. M .. LSL. Woo I. Fddnun EC, M .. t1ytk mi"..,..,. <>ytk, .... in 6d.!.os;. ond in nrtI.yroid """u..J •. M.", Diabrtol La, u;, ).01- ).07. SS. r" ...... ME. 19S7. E.If""'" diabrt" mdln. • .-.dated ..itlt. p<";""" bovin, .0.[ diatth" (BVD) viruo inf«tioo io 1"""1: ""d,. V.. Padt...J l2m 1- 229. j7. Muroodoti A • ..., dot Knlk IH . .... dot Uodo-Sipman IS. 1999. T 1P" I ",dli... in • P''U'''''' kdf" p« ..""tly iokocted ..it!. lx>,;"" vica.I dianlooc. ,;"... V" RL< 144.268-269. ""'otan So"""". So""""•. So""""'•. 734 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY 58. Mid.i< HR. 19'96. M ... boIian of ....... and multipk orpn f.ilw,. W~MJ S" 'l: 2o,~ . 59. Hintt HF, (.,... JE. CliffoNi Aj, Vl",,", WJ. 1970. Ammoni. iota6 J--966. 60. Knam .... SS, Ant~..d" AC. M • ..,,. CA. .loa", PC. Su.n.l'in Me. Mort cs, Mi....dol. RM, Ortolaai EL ZOO" A modd for """"""ia poi •.,.,in, in attic. Y" H ..... T oxi.roI 4:\,274-277. 61 . Stidk IE.. McKnido' CA. Willi ... , K/, c. .. EA. 2006. Di ... b" and byp<. .... ",,,,,,"'ia in. h,m,..ith ~, ..... ....;" ........Jocic .i_ Y" CliD P.1hoI. JS,2S<J.--1H. 62. P,ok SF. 0;.",. 1), CJ. 19'n. H1P"' ...."""'.."'ia ...,.;at«! ...it!. «>«P~"""""1 ....1 obdomj"al paiD ..id.ou., ~ of Ji.... <10. £..,,6..,.J J""" V.......m"'"J A""th.,;". J ..ttl. ,dorion, drool ;o,..nc.';"'. I Am v ... Mod A.ooc I g.j,J7-41 . 70. 0.. ......." . A. P"""U SH . P....ky I. I ~ S. Aw .... tio~ by P""P,uooIoI"( hyp<.p,,,,,,,,,,,, ,ffect of k~ ... .kk in ""'" II di.bni ... and "" .. ~ ~ I~ , Ad ..... HR. ed. V..",'_., I'/,.""","'-O • ..J n.....,.-,.,;". 7th «Ii';"'. Zl .....JIO. Am .., 10... S .." U..;...".ity P. ... . 72. Kn.j, AC. 200}. H~. and ;"'"",oibk o,woIock«>mpliati.o... io. a • ..itb inndinoou.. I Am Y" Med A.ooc 223,~1;4I14 . 7J. Rd...,. SB. Pdooi A. F""k ID. Stdic H. A. ..... T. Kat.u.k.i S. 1966. Blood ~ and pi ..",. io . uli~ 'OOPO"'''' "' ,.,-lito] odmini .."tod intra"""""",;o dop. Biodo."" Biopk,.. R.. Commun 20(,471-47 5. 75. K.",,.. T. Kanaza... Y. K.ooab. K. 1969. Stim.lati.o",,(;orulin oeartion by,.,-lito! in do, •. &d.ocoiDDlou 84aOO-207. 76. Duo.,... EK. 2OOl. H~ &.l"""int: ~ ;op';'" of .,.titoI-«>ntaini...: ,""'. V... H ..... T osirol 46087-11 8. n Duo ...... EK. c....Jtn..y. B..ot SM. 2006. Ao." J..polk failw, and oo"P,lopodo,.-ci.tod..ru.,.,-lito! ~ io ,; ~ dop. 1 Am Y" Med A_ 229,111J-1117. 78. Sym< HM. Srott·Mooaidf IC. 1998. Ckronk kyp<>&lya<mia in • kwrtio, dor; d... '" ««>n9-7 1. 84. R"by KAW. Beed I. Bloom. Ie Blad. M. 1990. H<paOO" BR. 1995. ObKn-a';"'. on k~ .-.dated ...it!. • J..po"' .... io. at. NZ V... I 4},1B<>-189. ,.eb"" An'"""'. ""p. ""p. 14 1 GLUCOSE, KETOAMINES, AND RELATED REGULATORY HORMONES 135 B7. s...in 1M. p~ ;., RS. H..dooo NP. EJ.. IlW. E..... H. 1>kGon.", Be. 2005. 1n •• Iin·Ii"", v-tl. r-.." ..,d ............ h,pou p",ia....datod..ith.....! cdJ ~"'. j.,. 100.... I Yrt I""m Med 1~61J-.616 . BB. Bim.i1 6:/'297- }07. B9. G..id.. IE. 2001. H~ . [", !kG""" LI. l...-,nlJ.. ed •. E..J.m".ioo- 4th odi tion. 921 - 940. l'biJ.de!. phi .. WB Sowtd .... 90. S",ith IE. Cipt";"'" IE. Holl SM. 1990. In vi"" ..,d in vi", d ""'" """.. mpoioo in ....;..., ytl...,,,,,,,,,,,;,. Z,n'nlb! y,,,,,j.,aancd [B[ J7,587- 592. 91 . lachon RF. Bru .. MJ.. Gro..n.y PI. s.,....... WG. I~O . H~.k<",o"";' j.,. P"P'"'" bitch. I Am Y" Med h - 177,1 12.J.-I IZl. 92. O·B~'n OP. B.cd.op BA. F...... KK. 101.<>000 GC. Gib"", KM. Slt.h... GO. 1999. I>Wooic acidw1. in Malt_ do,,, Nonaal ntrthyImoloaic acid oooem ...oio ... ...d m.Jooyl.c..A d.",boxyJ." ",civi.,. in 6brob1.." . I lob ..it Mrtab Dit n,BBJ-39(l. 9J. Gi",n BO. Mottrom. MS. Tully R. Dttlo...k, N. Ilubm....n..AH . 1~8 . s....", ~~ ... tributabI, to .",,1 10,21_24. 104. S",ith IE. Wood PA. Moo" K. 19~2. E... lwotio" of • ccI.o.i",,";" mpIa .... k1P"'odrntoco.>ki ..... • ..J .0"";" I Am Yrt Mod h - 211 ,72.J.-7Z7. 109. Elliott OA. Nd"", RW. R..u.d. CE. F,ldman Ee. Nct.l L\.. 1999. Compariooo of __ m f~ and blood dr=.,.t.ted ~'modobin """",trati.ooo f." _"""'" ,,( &lpmic ",nt..,1 in ca .. ..tth di. ....... ",dli..... I Am y" Med h - 21.,179.j....1798. 110. H"",is: M. F«p" "o OC. 1999. 0ia&noo'" utilit, of dJ=tyJ"od ~ oooem ... tio", in th, "'. Dom .., Anim Endoain<>II6.11_ 17. III . Ka.rto 1/. Ka.........to M. H,.""" AA. F,Idm", EG. K..izumi I. 1992. &aluatioo of " rum fn.c",.amin. ",,,,,,,,,,lion .. an """" "(bloc.J &100000 ",ntroI in.-. .. ru. ..ith diabo". mdJi .... ...d .",.. k~ . I Yrt In"m Med JI),}60-.J6.j. 1lJ. Elliott DA. Nd"", RW. Fdd", ... EC. N"l L\.. 1997. GlycooyIated """""""'0 ootttnti... fo. .... urn ... , of ~ oontroI in u.. K." 73. lIt. lIS. 116. 117. 118. 119. FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY w"",""" Mj, Milb RJ. 1988. s...... fn.c~ in patim. .. ..it!. ......., ...d """od thyroid di...",," ""0 Clin Biod.<m 2S(Pt S),S87- S.!8. J"" .. n AL 1993. Vciow pro ..... aod albumin «>noaiono of th, ........ f"""....... io, «>D«nmtloo in th, dia&nooio of =in< di.bm.o mdlitwo. V.. It.. Coonm"" 17,IJ--lJ. Rnod.. CEo H ....... B. 2001. &aJ.";"n,,( fn.c~ in d.op ...d ca.. .,;tb bn- o. kyp<rp«>thilirubj., .. mia. Y" RL< 148,}7n«ot. . tiono ... ~ Grond"'= frurto""';"" ronc 120. G.aham. PA, Moo"", cr, c ... J Vrt R<. 63, [}I!-ltl. s....", &uctoouni ... roncid "'t>. R.. V M""" M. 1m. 67,171- 175. 121. Rn..n CE. T ...... K. 1999. s. ...... fn.c..........., ",..aut..';".. in "''' ..ith 0Y, A.c.9. 126. S",dd"", S1.. Nod..d.", RF, K.d.bi,1 JD. I~J. C&nin. immw.,,,,occi.. io... lin q .... ,;tati." •• ;", 1M 9S 1- 953. l}1. Kno..]", GG. Sd.& WD. 1984. n..,.......dod ;".uli.,.~ .. ti., J. it «ally brtu.1) Am Y" Mod A.... 18S,J<;17- l99. l}1. M.e... D. 1m. p""""atic ad~.) Am Y" Mod A .... 175.1017- 248 (I."",). l}J, Bro .... SA. E.d..aed. DF. 19M. Fi .. al ro",,,,, .. " on.......dod i.....tin-duoo .. utio. I Am Y" Mod Aoooc 189,008--409. l}0. Sd.." M . 1986. W....tinc;d.o..... 0.. "".......dod i.....tin,~ ..tio. J Am Vrt ModA..o< 189,2S9. l}S. Kid!<, Tj, Hal,."". JF. 1999. lb, d~ ... ~Ju, poJoVc. Mrtaboliun 2S(Suppl II)dJ27- Il29. l}8. M.c.....)P. ~ EN. Aal.rth DL 1989. Validation of ..dioi"'m .........y (RIA) fw dua_ in """"'otic .Di"'.... I Aoim Sci "',2W (obou",,). 1}9. Bond R. McN.il PE. E..... H. Stoi. in """ doc • ..ith di/f", ... ~di ....... Vrt Rrc 1}6~71. 1.. 0. Soockb .... S1.. N",h.,;"" RF. K.d.bi,[ JD. I~O. R..dioim"' .........y of"""", inrulin. ~.. and Cp p.c._ 14 1 GLUCOSE, KETOAMINES, AND RELATED REGULATORY HORMONES RJ. Sa.p NG. 1992. &id.nc< that tt.. 1"""«>« of..p.in< an t..d to OY<..-inu.tioD of l...d . ........ ...t by ..dioimm""""""'y. Clin Cltim Acta 210,211 _2 19. T onet S. IO~""'D K. McKen-.. P. Hanly R. 1997. S~p<.6cial oocrol.,..;c -2S1). Mill" WH It. And"."" WI. McCann IP. 1991. NocroI.ytic mig..".,. " yt!.,ma in • doo, ..it!. • P oapoo.t« 144. L.«y ~ I·U . 1.(6. 1.(7. 148. 137 This page intentionally left blank Chapter 15 EXOCRINE PANCREAS AND INTESTINE Exocrine Pancreatic and Intestinal Absorptive Malfunctions .... . ...... . . . ........ Trypsin-like Immunoreactivity (TLI) Concentration in Dogs, Cats, and Horses ........ Pancreatic Lipase Immunoreactivity (PLI) Concentration in Dogs and Cats .......... Trypsinogen Activation Peptide (TAP) in Dogs .. . ...... ... .... . ...... ... ........ Cobalamin (Vitamin 8,,) Concentration in Dogs, Cats, and Cattle ... .... ......... .. Folate Concentration in Dogs and Cats ........ . ............. .. ....... .... ..... Fecal ",-Protease Inhibitor (",-PI) Concentration in Dogs and Cats ........ .... ..... D-Xylose Absorption Test in Dogs, Cats, and Horses............................. Glucose Absorption Test in Horses ...... . ........... ......... ....... . ........ Lactose Tolerance Test in Horses ...... . ...... . ...... . ...... . ............. . ... Urine Sucrose Concentration in Horses ........................................ Other Methods of Evaluating Digestive or Absorptive Functions........... .. ...... 740 741 745 746 747 750 753 754 755 756 757 758 739 ,. FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY Table 15. I. Abb...,,,iatiom and symbols in {hi.. chapler [xl Con"",mration of x (x = an. Jyt<) AMS EDTA EUSA EPI GFR HCO,LPS Pll AmyJasr Ethylcn«li.minctet=ic acid Enzym~linkffl immunosorwnt ..... y RlA Radioimmuno:assay SI Sys{~mc TAP TAP:Cn Til URL a,.PI Exocrine pane"". !;, inmfficicmy Glomcrul", filtration n le BicnbonalC Lip"'" r.ne,,,,,!;,, li~ immunor....ctivity Imcrnnional d'Unit" Trypsinogen ac{iv:uion p"ptid. Trypsinogen activation ptptidc to creatinine T rypsin_lih immunoreactivity UPI'''' ""ferena limit o:"Prol<= inhibitor EXOCRIN E PANCREATIC AN D INrFSTINALABSORPTIVE MALFUNCTIONS I. Exocrine pallcr.... tic insufficiency {moldige;lion} A. F=rri~ pancmltk insufficimcy (EPI) i. a pathophysiologic sm . in which in:od and, =ndarily, i ... dequ>te abwrption of nutrients, I. Dog. and alU with EPI typically ha"" la'll weight .nd produce malformed f=., Pancreatic maldigmion Jruly .. mit from inadequate srcretion of LPS, AMS, tryp>inogen, chymotrypsinogtn, corboxypeptidases, or combiruotions of the .. zymogtns .nd enzymes, 2, Thr.., pancreatic ronditions are recognized •• couso; of EPI in dogs :md cots, EPI is not recognized in cottle or ho ..... The.. must be extensivr loss of .cinar cdls before there is clinicol evidence of m.ldi~tion COUsM by EPt a, Pann7atk acinar atrophy (or juvenile pancreatic atrophy) in dogs: Current evidencr indicates this condition is caused by. hertditary immune_mediattd lymphocytic pancreatitis (atrophic lymphocytic pancreatitis) in German shep_ herds and rough-<:o.>ttd colli..," When presenttd for w.ight loss, there is nearly • rompl",e .~nce of pancreatic .ciruor cells, U.ing • decr.....d srrum [TUj value as • marker of affecttd dogs, a study involving 134 German shepherds indicottd an .utosomal r=ive inheritance pattern,' b, Chronic J>'Increatitis in dogs' and cats:' This disorder is typically.n idiopathic, recurring pancreatitis that causo; exrensive destruction of .ci ... r cells, If there is concurrent destruction of islet cells, the dog or cot can develop diabetes mellitus, c, Pancreatic duct obstruction in dogs and cau: Impaired secretion of pancreatic enzymes into the intestine rould GlU,. nuldigtuion, However, the obstructive lesion would probably lead to a.cute inflamJrultion, and thus the .nimal would be p"""'nttd for an acute illness and Jruly not develop a maldi~tive state that cau... chronic weight loss or chronic di arrhea, 141 15 / EXOCRINE PANCREAS AND INTESTINE B. Dogs:rnd cm with EP[ m"}' drvdop ~rx!.ry intOSlinal . bnormalilies. such :as inc=std muco..aJ maltast and suc'""'" activiti"" ioc=std microvillar membran~ prol~in', or bact~rial overgrowth. Th~ lmor could Iud to muro..aJ m~ thaI "'.... malabsorplion.' 11. Pancr~alilis A. E""n though Ih~ iniliating factors for pancr~>Iic inHamm.lion . '" not Ihoroughly und~mood, it i. known Ih . 1 p.ncr .... lic acinar cdl d.magt i. a mojor roll5~u~n"" of Ih~ inHammalion. B. [n .cut~ p:mcrntiti, (from mild td~m'IOU' to ~ .. n,""rolizing or h~mollh"1:ic) , Ih~ rd~",~ of cytoplasmic ~nzymes from th~ dam>gffi :>cinar ",n r~sull in inc=d ..,rum aClivilies of AMS :md LPS (..,. Chapl~r 12), incmutd .. rum [l1l] and [PU ] (Ott Chapler (2), and increas.d urin~ and plasma [TAP] (... Trypsinogtn Acliv' lion Pq.tide (TAP) in Dogs, 5tCL IIl.A). Among domOSlic mammal" .cul~ p. ncr .... litis is mon common in dogs. Alfecttd dog, frequ~ntly h. ve an .cul~ onsol of clini",l sign, ,uch .. .umiling and .nurior .bdominal pain. e. Chronic p:mc",alili, may resull from ra::t1r",nt ~pisodes of acule p>nc",alili, 01 slowly progressive destruction of pancre.lic acinar ""II.. Wh~n """..~ . the p:mc",.. may not bo able to ,,",,ret. ,ufficient ~nzym .. 10 digest food, .nd Ihe animal may devdop EPl. Chronic p:mc",alili, occurs in alU and I.".. commonly in dog" and moy ... uh in EP[ wilh clinical ,ign. mch .. progressive weight loss :md soft or malformw f=,. Endocrin~ p. ner .... li, d,..filllction moy .lso .ri..,. ""U, Ill. [ntoninal mal.bsorplion A. Sevoral .mall intestinal d~, "'.... inad"luate absorplion of nutrient' .nd thu, inteslinal malabsorption. Th. nuL.bwrptive nale amid bo locali=:l {e.g., proximal ,m.ll intenin~ 01 il~umJ or diffust.-. il oould involve malabwrption of mony nutrient' (e.g., .ugars, proteins, .nd f. I,) or bo very specific (e.g., cobalamin). When intestinal malabwrption i. not ",,"u,ed by. specific absorption d~fect, the .nimal i. prestnted beau.. of w.ighl loss 01 malformtd f'""t>. Acule enteric di ......... lhal ",use diallhu for a few daY' prob.bly ""lISt. I~mporary mal. bwrptive nale, but .uch disorders .'" not Iypi",lly ron,idorw in discussioll5 of mal. bwrptive disord~" beau.. Ihe .. i, not • concurr~nt malnouti.hed ,Ule. B. Intestinal di ......., lhal l~ad 10 mal.b,orplion occur in mo,1 .nim.l species, but laboralory tests are u.td mo,tly to <"V:Ilual~ the disord ..s of dogs and occa,ionally of cm .nd horst" Specific diagnOlis of su,pected primary intonin. l di ..... mually r"lui ... histologic examinalion of inteninal lissue. Examples of intestinal di ......... Ihal "'''''' malabsorption include Ihe following: l. [nflammotory: hi!lop!a!JIIOli,. lymphocytic TRYPSIN_UKE [MMUNOREACTNITY (TLl) CONCENTRATION IN DOGS. CATS. AND HORSES I. Physiologic processes (Fig. 15.1) 11. Analytical con""pu A. Term, .nd units 742 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY Kidney Urine Fig. I S.!. PhJ"iologic p~ .bot in8 ... nc< pw.n. or .. rum ooDC th< I"'n= .. :ond .n"'''' the bLood, in which it ern be m.....""' .. TLI. Also, srrWl:amounts of trypsin may be form«! in d.. pm" ... (... TAP): this trypsin m>y ..,"', th.e bLood, bind to morro",...,., and rontribut< to [lU I. PJ.,,= tJypsin<>g md by th. mononud.., ph:.gocytr 'l"'''''' • TAP: [n ",.lth, sm:dl .."oun'" oftrypsinog"'" of it is d.>ffif vi. th. lcidn.y> and is ""Cl-.! in tb. wi .... Actin.ion Oftrypsin"ll"D to trypsin by..,"'rokina>< in the in... ti"" aho result. in th. form. tion of TAP, but this TAP is no. m.orb.d :md tit"" does not ..,"', th. pl .. m .. • PLl: Most ",ncr.. tic LPS it =:",,,,d in rnzym<.rich P:UK7U,ic >«rC'pes tt.. patten .. :md rnten thr blood. in which it crn be m..... ",d .. PU. Thr lci<\nry> .,. invoJv.d in thr "monl of LPS from thr pl .. ma. J. Immunologic assay. detect cationic trypsinog<'n, trypsin, and trypsin bound to prot.,... inhibitor> (probably cc, ,,,mtitrypsin); thll'l the nam. TU. In h....hhy animal •• nearly all TU i. trypsinog<'n. 2. TU is reportedly !"Inc",.., specific, and valu .. ~ .. rruorhdly decr ....al aft .. panc...., removal; howrYer. TLI was still measurable. Trypsin i. presrnr in the human .mall inrestin. (paneth crU.), in bile rpithdium. and in ovarian and hrpato_ biliary neopl.,m,. 3. Unin: jlglL {SI unin not found} 143 15 / EXOCRINE PANCREAS AND INTESTINE Table 15.2. Discas... and condition. Ihal cause increased [TLlI, [rLl I, or [TAr l in dog. and cal. Inc..ascd rd~~.., of u}'psinogtn (or uy]>'in), LPS, or TAp· from panc""~tic acina, cdls 'Acimor ""U damat<' cau...! by p~nc, ... titis or oth.. di""rd~ .. Stimulatw by food imak~' or mol..cystokinin .nd stCretin adminim.tion D=... scd GFR (>ft p,.""nal, '~nal, and pomena! :rrot~mia in Chap, 8) Cobalamin drfici.ncy in cats ((TUll • A rel. tively oommon dioe ... or condition 'In"",....:! winary =r .. ion of TAP i, ilio IOund ..ntb d:ur..g. to pancr •• tic .cinar ",It. (.... be _t), • Th. [Tlll b .. oo.n mo"",, to inen= after food intake, 1br '""'" dung< b .. "",,,ted for [PUI or not oo.n docu- [TAP] ~, B, Assays I. Canin~, fdin~, and equine [1111 a.. m~~m,ed by sproes_spa;ific immun=ys, Trypsin's enzymatic aclivity is not mnsu,cd ba::au.., of Ih. p ....,n"" of Iry]>'in inhibitors in .. rum, 2, [n cat" ""r.r~n"" imerval, for [TUI m...sured by a RIA (17-49IlglL) v.."" diffe'~m from those m~asurw by an ELISA (I2---821lg/L).' C. Sampl~ Serum is Ih. p",ferrw .. mple, but EDTA pla!ma 0' he"",iniud plasma can be UlW, Sampl.. mould be nored at 4 °C or - 20°c" ilL Ina"""'! [trypsin_like immunoreactivilYI [TLiI {Tabl~ 15,21 A, [nc..a""" ,d~ from p.nc, ... tic .cinar edl, I. Acinar ceU damat<' cau...! by p.nc, ... titi,~l. a, Trypsinogen or tlYP.in rd ...oed from damag«i acimor cdls ~m~" blood, probably via lymph and p..iloneal Auid," b, Dogs {I} [n an ""p",im~nt;;ol canin~ panc'''''lili, nudy, ..,rum [TLiI t~ndw 10 paralld .. rum AMS and LPS 'Clivilies; that i" all na'ted to inc",,= within I d, .. mained inc, ..."'! through day 5, and had murnw 10 n..., baseline valu ... al 2 wi<. However, Ih~ p"ak in [lUI ocrurrw 1- 2 d prior to Ih~ p"ak activilie, of AMS .nd LPS," U,ing dttn rq>Onw 10 be about 33-50 % (I."" tru.n tho.., fo, .. rum LpS activity), {2} D=om.tha",n. {Q,25 mgllq: P'" os daily} v..a, associatw wilh inc, ...,cd [TU I by day 7 of t..~tm.m .nd was Wilhin the ,.r.r~nce im~rval again 7 d aft~r withdrawal, Th. m.~n inc' ..... wa, Ih,,,,, lim... baseline m~~n, bUlonly 2 of 12 dogs had [1111 ;> Ih. URL, Th. authors SUggt'lIW Ihat Ih. [TLI] was inc=d ba::au.. of toxic df=, of dcxamethason~ on Ih~ ""nct.as," c, C.U {I} [n. pro,pecti", nudy involving 28 cats with clinical 'igns rompalibl. with panc""atiti" the .. v.. .. nol . 744 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY with acut~ n«rotiz.ing panereati!;, (S). " Th"", conclusions w..~ questionffl by oth~r invrnig. tors who wrote that inc",~ [TLI] is 'pacific for fdin. pancreatitis! {2} Oth., studies d.,crib«! ch.t [TIl] h .. an 80-86 % diagnostic stnsitivicy for modem. to ~'" pancreatitis in em when empirical d«ision Ih""hoJd. ~a{" d. th,n th. URL.", Ulffi."·l. Horses {I} Ho", .. that han ,tranguloting intestinal obstruction and endOioxic mock can ha"" incr.a=! plasma [trypsin] {as m .... surM by an activity ",..y}." Th. t'YP.in is probably rd~ from p"ncr 100 IlgfL {ref.renee interval = 12- 82 JlgIL}, Th. reasons for the incr..sed [TU] in these cats w.re not det"mined, Serum [TLl] d«r..... d in 9 of 19 em wh.n they w". given parente",l cobalamin therapy," 2, Of nine cats with V.riOUl type. of inflammatory bowd dise ..., fin ru.d mild to mod.rat. d«f most IV, Decr.....d [trypsin_like immunorea.ctivity] [TLl] (Tabl. 15,3) A, D«r••..d rd .... from p.ncr..tic .cinar cdls I. Chronic p"ncf<.titis th. t leads to d.. truction of most . cinar cells' 2, P.ncr..tic .cinar .trophy in dogs B, In dogs with dinical signs of mald~estion (due to EPl) or malabsorption (due to small intestinal di ..... ), the diagnollic .. nsitivity, specificity, and ,ccu"' 15 / EXOCRINE PANCREAS AND INTESTINE Table 15.3. Discas... and condition. Ihal cause decreased [Ttll and cal. 745 Of' [rUI in dogs D,""rra'M rd~~", of Irypsinog~n (or uYl"in) or p:mc ....uic LPS from pancreas Chronic p>ncr~~titi, • P. ncrratic >cinar atrophy • A ,.tui""ly oommon dio.... or condition Not<: Tn. [TLl] 1= been .h""", to dec ..... in tt.... diso,d.n in both dogs :ood cats. A dec .....d [PU r b.. not t-n d<><WJ> that d,""r~asal [TLiI may r~pr=nt subclinical immun~_mMiatM lymphocytic ""ncre_ atili, (.trophic lymphocytic p>ncr~~tili,). D. [n a group of20 e>lS wilh clinical 'ign> ,uggcstin ofEP[ (~ .g .• weight loss. 50ft or voluminou, nool. :md polyphagia). 17 had d,",,==' .. rum [1111 valu~ .. ' Additional diagno,tic Naiuations and/or r~.pon .. 10 "'zym~ rq>la""m~nt U~.lm~nts supponM Ih~ oonclusion that th~ em did ha"" EPI. PANCREATIC LIPASE IMMUNORFACTIVITI {PLI} CONCENTRAT[ON [N DOGS AND CATS I. Physiologic p""""sm (Fig. 15.1) [I. Analytical ooncrpu A. Tum, .nd units l. Breau.. th. immunoassaY' au d•• ignM to measure th~ ""ncrealic LPS prol~in (M. = 51.000 in dog.) and not its aclivity. the .nalyte i, callM pannYdtic lipmc immUlUmdctivity (PU ). 2. Units: Ilg/L B. Assays l. Spn;ics_spmfic immunoassay' for p>ncuatic LPS han bttn devdopM for dogs {RIA and ELISA)"-'" and cats {RIA)." Th.", assay. m= .. the accual ooncentra_ lion of th~ lip... prouin. not Ihe 'Clivily of the .nzyme. Th~ polyclonal .ntibodies localiud only 10 p.nc..alic acin .. cell, in immunohinoch~mical ,cudi... of canine rissu .... 2. Th~ 5Our= of Ihe standard 501ution, for th. assay. w~ .. nol spn;ifiM but .!SUmM to k LPS i50latM from canine and fdine p>ncreatic tissue ...spcctivdy. by th. investig.tors. 3. Refc:r~nce interval, vary wilh the mffhods usal and .mong laboralories. C. Sampl~ ~rum is Ihe p ..ferrM .. mple. 111. Incr.-..M [p>ncrralic lip... immunorraClivityl [PUI (Table I 5.2) A. [nc... ...:1 rd~ from damagnl ""ncreatic acinar cells l. During ",!",rimental and spontaneou, p:mc ....uiti •• LPS i. rd.-..M from damagM panc'"". tic acinar crll. and ~nters th~ blood (probably via lymphatic ""...ls). 2. PLI te,ting has the . dVlUltagt over moll routine .. rum LPS assay, of king specific for ""ncrcatic lip .... and testing i, OffcrM by some la,!:" '~terinary ..fcrrallaborato_ nes. How~~r. rcsu!ts may k too delayed to be u..ful for diagnO!ing and managing patient, with acut. pancreatiti .. ". FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY 3. Dog" Wh~1I d""j,ion th ...hold, of th~ URL or of.n ~mpjrical ...Ju~ g=t~r than the URL w~re u~, the [Pll] had a di.gnonic .. mitivity for acut. pancreoltitis of]oo % and 82 % , re'Jl"Ctive/y. " Diagnonic ..,nsitivities of LPS and TU were I.... 4. Can: Th. [PLI] (with dros.ion thro;hold of 10 IlgIL) j. ,.portal to han ~{{.r di"i:"o,[ic stnsitiviry for P>flC'''''{;lj, than d"". the [I1.I] in cats." Aft,r uperirnen_ tal initi.tion of p"ncrratitj. in mIS, the [PUl had g,rater incr= {SOx basdine '" 35x baseline} .nd more penistem increases (IO d vs 3 d) than did th. [TU].'" B. D«r••.ffl renal drannc;., in disorders chat au .. da:,rasnj GFR l. The kidneys at. involvffl in the ,.mov:.! of pancr",,{ic LPS from plasm. {= Chapt.r 12}. When th ... are disordm that muh in d,"""asM blood flow through th. kidn"Y' (i.•.• p",.."al, .. nat or posIten:'! disorders), th~n LPS h... a 101lg~r circulating h.lf_lif~ alld thu. COli accumular. ill plasma. 2. Usillg the collin~ PU ELISA. the [PU] was mildly illc",asal ill dog. with aperi_ m~mally illdu=:! chronic renal f.ilur~. but 1I0t above th. suggested diagnostic threshold for p""u... titis. "." Lock of illcr .... ed .. rum lPS activity in this srudy suggests that the modd =y not have bn,1I .ppropriate for ...... illg changes ill [PLI] cousal by rellal failure. [v. Decrnsal [p""creatic lip"'" immunoreactivity] [PU] [Table IS.3} A. Rd.... e of LPS from p,"creatic acinar cells may k dec",... ed ill disorden associated with redu=:! functional p"nc ....tic aci""r tissue. :md ther.fore .. rum p,"creatic li~ immulloreactivity oollcemmiollS =y k decreasal. The primaty disorders ill dog. and cots .'" chrollic p,"creatitis (which l... d. to destructioll of most aci""r cdls) and, ill dogs. p:mc",atic acillar .trophy (an immune-mediated disorder of ~rman shepherd. and rough-coated collies). B. [n. study usillg an ELISA. .1125 dogs with EP[ (... d.fined by havillg a decreasal [TLI]) had decreasal [PU]." However, TIl i. prefer=' over PLI for diagnosis ofEP[ kc.u .. TLI .ppe.n to differ.miat~ affected dogs from healthy dogs more d ... rly, and it may have ~.ter diagllostic .. nsitivity for EPI. TRYPSINOGEN ACTIVATION PEPTIDE (TAP) [N DOGS I. Phy>iologic processe" After ingestioll of. meal, ttypsillogell from p"ncreatic aci""r "",Us emers the intestillal lum," :md is de.ved by the .ctioll of enterokinase to produce trypsill and TAP,." ~ight"""mino-a.cid N_termirud deavage peptide. [n theory, the TAP is not absorbed and thus d~ not ~nt.. pl ... m• . A millut. [TAP] con k foulld ill plasma .lId urine of healthy dogs, p remmably from .ctivation withill the p:mer......" [I. Analytical oollap" A. Ulli" l. Plasma: nmollL 2 Urille: nmollL; also ..""ned as • TAP: Crt ratio B. AssaY': An immuno......y (either enzyme immun<W.Sayor RIA) detect. TAP; ftve highly ooruc:rv.d amillo acids .llow for cross_speci.. "nillg with a sillgl.......y. Th. analytical detection limit of the enzytU~ immunoassay is near the lower pla,= oolla:mrations found in healthy dogs and thus canllot k used to detect decreasal pl",= [TAP]. """"Y' .re lIot widdy available. 15 / EXOCRINE PANCREAS AND INTESTINE C. 147 Sampl~ I. EDTA blood is collttted and a.mrifuged at 4°C. and pt.",ru. is ..""rated within 1 h. EDTA pl .... /rul is frozen {~t - 20°C} umil just prior to analy>li •. 2. Urin~ is rulb:ted into EDTA_containing vacuum tube; .nd frozen {at - 20°C} until jll'lt prior to analysis. Ill. Inu~ pl:.S/rul [TAP] or inueo.ed urinary TAP: Crt ratio"''' (T.bl~ IS.2) A. Acinar cdl donug. caused by pancre~titis: During paner .... titis. trypsinogtn is cluved to form trypsin:md TAP within th~ panc ...... through a.ctions of cath~psin B or through autoactiV:l1ion. TAP prob.bly ~nt~rs plasm. vi. lymph. and some of it i, cle.red via kidneys. I. [n dog, with inu~~K with a histop.rhologic di. gn with renal di ..asr /ruIy han increased plasma [TAP]. In four dog, with plasnu [TAP] incr...... d by r~nal di ..as., th~ urin~ [TAP] was d=~ • .ed. [n two of th ..~ dogs. th~ urinary TAP:Crt was within th~ ..f..~ne<: im~rval. COBAlAMIN (VITAMIN B,,) CO NCENTRATION IN DOGS, CATS, AND CATfLE I. Phy.1hw~y>l involving folate {Fig. 15.2).'" B. Con..lamin i, also a r«iuired cofactor fur th~ convtrsion of methyl malonyl eo<:nzym. A to succinyl eo<:nzym~ A Without thi, connrsion, pl.. ma [methylmalonic a.cid] accumulat.. (mrthyhruolonic acid.mia and .ciduria), and a neurologic disra .. d<"Vdops b«:au.. of d~fet:tivt formation of n~uronal lipids. II. An:olytical cone<:pu A. T . .., Pancreas IF c :::> Blood Erythrocyte , D \ ''''. , Cell Fig. l S.l. Phyoiologic ~ th.t infl"...o, pw.n. at >lophi~n or haplOCOlTin; R rn...-.piftS through the um.II int~ti.,.. When it r~"""" the iwm, the ooI>aWninlintrinsic r..c- rompk:r. binds to ~fic JJWooo.aI reapto" mv.,n.in, atlwn (atbilin ond :omnionless) and meplin, and "'''" "'''roqtes. When ODbalamin . n'on ,ho poJ<XJb.bmin 1 (fnDII). a '48 15 / EXOCRINE PANCREAS AND INTESTINE 749 2. Unit con""rsion for cyanocobalamin: pglmL X 0.7378 = pmollL; .nd ngidL X 7.378 = pmol/L (SJ unit. n~are;t 10 pmoIIL)" B. AssaY' l. Th~ asiaY' ar~ d~signal to m=r~ [cyanocobalomin]. Com~itiv~ protein_binding """Y" ar~ mot~ common than bioassay. that at~ t..ronically difficult. 2. Assay, d~signal fot human "'r:I may not k rdiabl~ fot othet ,pa:i~s ~u", of cobalamin_binding prot~illS in cmin~ .nd fdin~ "'r:I. Som~ comm~tcial ....y. d.. ignal fot analysis of hu/IUII sampl.. u'" porcine gasttic inttinlic facrot :as th~ binding ~nt. [f th~ binding t~ag~nt contain, contamin.nt R prot~ins. th~y am bind to ina.cti"" cobalamin metabolites .nd gi"" fal,dy inc",=d value;. .. C. Sampl~' l. s.:rum is th~ pt.f.rted sampl~ . Th~ u'" ofhqmini=:! ot h~molyz [non=! ",rum [cob.lamin] A. [ncr~a ~ ",rum [cow!.min] is unrommon. HOW""~t. disotdw; or conditions that could inct~:as~ [cobalamin] should k ronsid~tal wh~n int~tpming ronuntratiom that ate drct...,.,.j ot within th~ tefet~n"'" interv;;oJ. B. D~ ot ronditions that cau", inct",sal ",rum [cobal.min] l. Cobalamin suppl~m~ntation: parem"al ot oral 2. Rde= from damaged h~patocytes: Beau"", hepatocyte; at~ a nong<: site for cobalamin. hepatic necrmis could inn~= ",rum [cobalomin]. The magnitud~ Ot duration of the inct..... is not known. [v. D=-eatM .. rum [col>alamin] (T.bl~ 15.4) A. S~rum [cobalamin] will not k drcr~=d until th~ body t~"'rv~ is d~pletal. Depletion typically occurs ~u'" of drct~:asal il",l .bsorption of cobalamin. but the primary defrct may k pr~.bsotpti"" {i.~ .• kfot~ cobalamin absotption by th~ ileum}. =';nwa Fig. 15.2. • Fob te: Fob« i.s prestioa ",Ie...,. it from food. the polygluuma'" foh.. i.s hydrolyzed to monoglut..ru", fob« .. tb. brusb bord., of the proxim:ol mull in ... t;"'. Aft .. ooUuhr uptah. it i, <X>nv ~ during • .."pJ. <x>llectioa con inc", ... mu,w,"" .. rum lfol". I. • Rot.tiomhip of cobabmia rnd fob ",. Cob:ol:omia is • nquired roh e"aly= tb. oo.",,,,ioa of N' _methylt .. riliydrofoht. (tb. primory molecul. in pJ.UJl1) to "'trahydrofoI.",. which i, tbea avail""J. for D NA .ynth"'i' in ",,]k If tb... is • cobol. mia ddiciemy. tb. methyl group of N' _methyl«tuhydrofoht. i.s not trrnd'erred to cobalamin ("",thyl trapped). :ond thWl • ""llul., deoei=<}' ia t .. rahydrofoht. ocruJlI. 75O FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY T able 15.4. Di""a..es and condition. thai cause decreased ....... m [cobalamin] Cob:'!t ddici~IICY in c>ld~ Preabsorptin d~fecl in doc' and cm £PI: pancre>tic atrophy, chronic panc'''''litis Intestinal oocc"i;d o,,",rgrowth: EPI, impaired emrie acid stCrelion, em,,;c di,ordw; (Stt the text) Dd'r<:lin absorption of cobalamin in ileum of dog> and caU 'Ileal di~ inflmunation, I=;on, villous atrophy (viral, hy~mnsitivity, cytotoxic drug.) Coo/:"nirai ddicicncy of r~{or in giant Khna~rs and Border colli." ~ve"" cobalamin ddicicncy in a cat (probable congtnitod malabsorption d.f= ) • A rel.tively common dioe .... 01 condition Noto: lins of .p B. Di~ l. 2. 3. 4. or rondilions that ,...iu"," oobabmin .b50rptioll in Ih. intestine Cobalt ddicirncy: A di...:ary rob.lt ddici~n 10' colony_forming uniu/mL of duodenal juic;.,),"" .. p«iaUy involving obligate anaerobes, can lo~r .. rum [cobalamin] by incrrasing th. amount of cobalamin bound to inteninal bacteria and thereby d",reasing th. amount of cobalamin that i. frtt for .bwrption. Def"'tin .b..orption of cobal:omin in th. il.... m of dog. and cats a. Di..,,,,,,, th at damag< th. ileal muco5:l.; inflammation, viUou. atrophy, cytotoxic drug., and ,,"""tion b. Congenital deficiency of th~ cobal:omin/intrinsic factor compln r=ptor has oon reported in giant .rorulUze".~'" This r=ptor 'PP"''' to involve culM", (a compln of cubitin and .mnionl.... ) and megalin. A similar disorder occurs in Bord.r colli"" Australian .heph",Is, b.agl.., and komondors." ~ FOlATE CONCENTRATION IN DOGS AND CATS I. Phy>iologic process.. A. Stt Figs. 15.1 and 15.2 for basic p =..... B. Cobalamin and folat~ m.taooli,m a"" linked in a r....ction in which a methyl group is transf.rr~d from N'_methylutrahydrofolat. to cobalamin in on.-caroon pathv..ays. [n 15 / EXOCRINE PANCREAS AND INTESTINE 751 Ih~ abs.na. of cob. lamin, Ih~ m~hyl group is lraprffl in N'_methyltelrahydrofolat~, and the ... uh i. a funclioruol folat~ deficiency, B.caUst N'_m~hyll~trahydrofolate contributes 10 th~ toul m~asurw [folat~], strum [folat~l =y not ~ decreo.w even Ihough Ih..e is. functional fol'l~ d~fici~ncy {i,e" Ihe manifestations of. folat~ ddici~ncy are pr ..~m}, [I. Aruolytical cona.pu" A, Turns ~nd units I, Spn;ifically, folale is the anionic form of folic acid, which is compost III. ~.ted [ncreaKdstrum [folat~l (Tabl~ IS,S) A, Incread .bsorption of folat~ in the small intestin~ L SmaU intestinal baet.. i:d overgrowth: Excessive folate produced by enteric bacteri a is ab50rbed in the small intostine, The overgrowth could ~ KCOndary to EPI or cu.ed Tahle 15,5. Di..... es and condition. that au ... increased 5el'1Im [folate) IneteasM absorption of fol. le in MillIll intrnine Inteslinal ba.cterial overgrowth: EPI, imp~irM gaslric acid stetetion, enteric disorder> (.... tat) Low intestinal pH: EP[, excessive gaslric acid production High dietary inuke p.",meral mpplellYnution Cobalamin deficiency in cats • A ,.t1tiv<[y oommon di..... or condition Not<: Lim of .p 752 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY T able 15.6. Di""a..es and condition. {hal cause decreased ....... m [folate] D""....cd absorption of folatc in "'tall intminc 'SmaU intestinal mucosal di..,,,,,, (proximal or diffuse) Di,wy ddicicncy • A rel. ti",[y common dioe ... OJ a",dition by impaired gastric acid ,,,,,mino, da:,nsN inuninal ptrisulsis, a defn:tin mucus barr;«, or da:rtaSft! pnxluction of immunoglobulin by im""';na! lymphoid c;.,lls." A rch livc 19A ddicicncy ha. bttn >s.soci. lcd with inte'tinal bact«;:.! overgrowth in w,rman shq.h.rd,. '" 2. low ;mmin.l pH: Mnimal folate aboo'plion occurs in .n acidic cnvironmem. D~",..ro inte'tinal pH (inc,rasN abwrption) moy occur with EPI (irnpair«i HCO,- "",mioo) or excess;"" gastric acid production. 3. High dietary imak.: Folate .bOO.plioll usually occurs by" f:..cililated tnmpon ,)'Sum, but, OIl high inteninal con~ntrations, .bsorption occurs by pas,;n diffusion." B. Par~IItis con cou'" fal .. ly inc",asaI ",rum [fol . te] beca"", ~ryd"ocyt .. contain folate. IV. D=-....ed .. rum [folat~] (Tabl~ 15.6) A. ~re~ absorption offolat~ in the ,mall intestine l. Dise....,s of the proximal.mall int.. tinal muco", can cause impaired . bsorption of folat~. 2. Dietary deficiency: Comm~rcial diets should b.n more than minimum folate ""Iuirem.nu, and thus folate-defici.nt dieu are uncommon. Feeding . n all.ch= diet to dog. created a folat~ d~fici~ncy." B. Othu con""p" l. Prolonged phenytoin {Dilantin} trratment i, known to low.r ",rum [folat~] in people. [ Folat~] was not .ignificontly diff..ent in ph~nytoin'''''ated and nontrrated dogs tb.t w~re fed • folat~-d.fici.nt diet." Th~ dr.en of phenytoin on [folat~] is not known for dog. fed • folate·. d"'luate diet. 2. Sulf..alazine also i. rq><>rted to low.. [foute] in Jl"OPl~, but similar findings have not bttn r~ported for dogs or cats." 3. In thwry, st~rilization of the int.. tin~ with . ntibiotics may low~r .. rum [folat~] beca"", a major source of folate {int.. tinal floral would bt removed. Also, fol . ", is involved in DNA synth.. is, 10 """,nsin neoplastic ""J] growth could potentially deplete fol.te levd,. 15 / EXOCRINE PANCREAS AND INTEST INE 753 FECAL CI.,_PROTEASE INHIB1TOR (CI.,_P1) CONCENTRATIO N 1N DOGS AND CATS 1. Phy,iologic process.. A. u,.PI is a protein th~t inhibits the activity of prote=. (e.g .• trypsin) in fan. It is "port.d to han n.-arly the same mola::uJ.r m.., ., albumin. CI., _PI is normally p""'nt in plasma. inte"titial fluid • • nd lymph but not in the im.. tinal lumen." B. The a a.ct identity of this protein has not bttn found. B=UK it i, considered to b. of blood (plasma) origin. the antiproteaK a.ctivity may b. due to u, -.mtitrypsin {humm M. = 54.000} or CI., _antichymotryp,in {hum.n M. = 68.000}. The "porta! mola:ul.. m.., of dom.,tic mammal u,-PI mol«:llies v",i.. .lightly with the species: canine M. = 59.000. feline M. = 57.000. and equine M. = 58.000." 11. AnalyticaJ concepts A. Units: llg/g of feces B. Sample l. Becau.. of the VlU"'tions in [CI.,_PI] fecal "'mples. thr.. q>:Irate void.d fecal sampl.. are colla:t.d. Colla:tion of feces by digital ot ma:MnicaJ .. moval of fan m. y introdu"" blood into the sample. which will =ult in falsely incre",.d [CI. ,_PI ]." 2. After collection. the fecal "'mpl.. should b. imm.diatdy frozen and shipped with dry i"" to the "feren"" loboratory. When L:.pt at 37°C for 72 h. the [CI.,_PI] da:r.-aonl by . bout 30 % : when k.pt at 4 °C fot 72 h. the concemmion da:r.-aonl about 5 %."' C. Currently. ther. is one ELISA for measuring immuno"acrivity for [CI. ,_PI ] at Texas A&M·, G"'irointestinalLabomory (Colleg. Station. TX}."' D. An =y for fdin. [CI.,_P1] in .. rum Ju. bttn d~loped. but docum.nt.d evidence of clinical application wa.! not found." 111. Incr.....:! f=l [CI.,_PI ] A. 1nc... ..d plasma protein loss into intestin.. I. Protein_losi"l: enteropathi.. cauonl by ate",i"" intestinal disease such as lymph.ngiect",",. idiop>thic inflamm. tory bowel di ....... {including lymphocytic, pwmacytic. or eosinophilic disorde,,}. intminallymphoma. int.. tinal carcinoma. hi"",,viral enteritis.'~" 2. Blood loss into the alimenury trace: Blood loss may b. :woci.ta! with protein_ losing ent.rop>thi.. (•. g.• ulceration ot cminoma). but it may .lso b. due to hemorrh"J:<' from nonenteric dist",. (i .•.• thrombocyto!",ni •• coagulopathy. or gastric ulcemion). B. Contamin. tion of feet, with blood or plasma during collection of the fecal sample. C. Inc..ased fecal [u,_P1 ] may b. mote .. nsitive to early protein_losing enteropathy than .'" .. rum albumin. globulin. and total protein con""ntrations. 1V. In. study involving seven dogs that were b.ing tr.-ata! with the nonsteroidal anti_inft.mmatory "i:ents mdoxicom or c"profen. fecal [CI.,_P1] did not incre=." 754 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY D_XYLOSEABSORPT10N TEST IN DOGS, CATS, AND H ORSES I. Phpiologic proa=> A. D-Xy{"u is •• jmpJ~ ~nto .. thai j, not commonly ingm""', and nry li{d~ is pr...,m in blood or livtr. If ingesta!. jt is p:wivdy absorb«! in th~ duod~num and proximal j~junum, from which it ~nt~'" porru blood. Aft~r byp:wiJll: th" lin•. it p"'" through the glomerular fi]tr:uion barr;" and is aa~M in urin • . B. In pn>pl., xyJos.. Ih. glucuronic :ocid- xylulo .. pathway in h.p"[ocyt ... It i, oma/ly :assumM that "''1 liul. of Ih. absorb«! xy]"'" i. dq:rad..d by hq>.tocyt~ of domestic m:omm. b. .nt." [I. Analytical oonctpu A. Unit 0011"""';011: mgldL X 0.06661 = mmolfL (SI unit, nr. rest 0.1 mmoUL)" B. A..ays I. MllhipJ. xyl= =ys are .vail.bl., but clinicalloboratori.. do not commonly off., thrm. 2. A oo]orim<'lric ..... y using phylorog/ucinol ~.n u...:! fur '"'Iuin. and fdin. Th". also "'~ assap that involve dq"o,t~ination of whol~ blood, ga, chroma_ tography, or h., ""r;o." Ill. Xylo .. absorptio,n tests A, [h,ia; o,f proc.dure L Th~ D-XY/Oll "bwrption "o:t comins of administ~ring D_xylose o,rally and th~n coUecting multipl~ tim.d blood .. mpl .. 10 ...... th~ intestinal ability to, .bsorb D_ xylo .., Man of the .bsorbtd xylose paMCS through the liver and ente", 'J"1emic blood, Th~ pffi< blood [xylose] should reflect the amount of xylose absorkd by the int<Sti"", 2 Xylo .. is cl~.red from plasma by passing through th~ glom.rular filtr:nion bmier, The .mount of xylose in urine i. reln.d to the :mlount of xylo .. . bsorbtd in the intestine, B, [n dog. and Qlts L Xylo .. absorption Ust. have oon u.s.d to, evaluate the ,mall intmi nal function of dogs .nd cots,"'" In some .nimah with small intestinal di,.=, xylo .. was poorly absorbtd .nd thu. the absorption curves ~r~ fLn, 2, [n most of the current tntbooh, smaU animal intemiS{s no l0lli:" =mmend xylose .bsorption tests, p"rhap. btc.u"" of the limit.d availability o,f xylo .. :ossay. or btc....., other methods of ch.racterizing intmiruol di ...... ",e more fruitfuL C. In ho,,,,e,, Xylose absorption test. h.ve oon u.s.d to, """llIate th~ function of the .mall int.. tin~,70-" [V, Dis...... and conditions thot ah~r xylose absorptio,n cu"",. (fable IS,?) 15 / EXOCRINE PANCREAS AND INTESTINE 755 Table 15.7. Di""ascs and condition. Ihat alter xylose absorption curres Flal xylo .. abwrplion cur"" (d,""r GLUCOSE ABSORPTION TEST IN HORSES 1. Phy.iologic process.. and imroductory concqltS A. The glucose absorplion lesl i, ustd priJrulrily in horus truot .re presented b«ause of chronic wnglll Ins,. Th. ho .... may be passing Jrullformed feces {e.g.. cow_pie}. If Ih, muco,al di ..= i, limited 10 Ih. mtall imenine. f~. m.y ha"" normal consi'lency. B. Glucose i. a 'impl. hao ... Ihat is .bsorbed in Ih. duod.num and proximal j.junum. from which il em... portal blood. A largt portion of the absorbed glucose is removed from Ih. porlal blood by h'palocyte, .• nd the remaind .. eme .. Ihe peripheral blood. From the periph..al blood. glucose Jruly ent.. melabolic pathwaY' of cells (•. g .• glycolY'i' or glycogen .ynthesi.) or be excreted by kidneys if Ihe renal resorplive JrulXimum for gluoou i, aettdai C. In ho ..... microb.-. of thelarg. intCSlin. digest cubohydrates and produce vol>!il. fmy acids Ihal a", .bsorbed by the muco .... Th.""I0 ... disord ... of Ih. lugt immine in horse, am coUR dinical ,1. 1el in which ingtned corbohyd..les ue mal""'imil>!ed. D. Wh ...... , the .. mlu of Ihe xylose absorption ICSI depend primarily on the intestinal absorplion of xylose. the resull' of the glucose absorplion lesl depend on imeslinal absorplion and gluco.. utili7.;OIion by h.patocyt .. and periph.ralliuues {primuily mu.d. and adip"'" li ..sue}. Th... u. thr.., m.jor advamages of th, glucose absorplion 1m comp.red to Ih. xylose absorplion ICSI: {I} gluco .. is more readily availabl •• {2} gluco.. i, leso ape",i"". and {3} ..... Y" ... mo .. readily .vail.bl. for m=uring serum [glurost]. E. The glumse absorplion tesl con be ",ed to orness the intestinal absorplion of mono ... c_ charid.. in monogaslric Jrulmmal •• but cannol be used 10 ass ... inteslinal absorption in ruminants. It is "lfdy u,ed in the evalualion of dogs and cau. probably for lwo r....,nJ: {I} other diagno'lic methods ar. availabl. (e.g .• Ih"", u,ing IOlale and cobalamin). and {21 dilOrd ... Ih. t cause glucose intol.rano:: u, mo .. common. When used to assess corbohydrat. lol.rano::. Ih. procedure i, called Ih. ~r"l g/UCflU ro/nrma Wt. 11. Analytical concepu {Ott Ihe Gluco .. Concentralion. leel. Il in Ch'pter 14} Ill. Oral glurost absorplion lesl A. B.,ico of procedur~ Glucose is admininered via a noJrulch lube. and Ihen multiple blood ... mples ar. coll,""led 10 assess the inteslinal abilily 10 .bsorb il. A!.suming normal glucose m.laboli,m. Ihe peak blood [gluco..] should refl«1 Ihe . mount of glurost absorbed by Ih. intCSlinc. 75. FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY T able 15.8. Di""a..es and condition. {hal alter oral glucose ab""rption curves Failu,", to m..,t cril~ria of .d'"'lua{~ absorption 'Milibsorption c;ou~ by ,null intestin:ol muco ... l dista5t (inll:murullion, n..opi.,i., villous atrophy) [ncompJ",~ ddinry of g1uco.., to i,lt B. pro<:ffiurc" l. Fttd is withhdd owrnight prior to the ploudur • . 2. A blood ,ample is coll=«l for a b.osdinc (or 0 time) [g1uC()Uj. 3. GluffiSt (l glkg lxxIy _igbt as a 20 % soJution, weight/volume) is .dminisw«i via stomach tub.. 4. Blood samples are coll.cud at 30, 60, 90, 120, .nd 180 min .flcr g1uro", j. adminiswal. 5. Blood samples .", proa....J .ppropriatdy, and g1UC01lt con~ntrations a", m=rM in the ",rum or plasma .ampl... C. Ex~«I ...lu.. in h...Jthy hor",s l. Two criuria ..e usn! a.s mden~ of .drqu.re intmiruol .b50rption of g1U(:()R: [g1uco",] at 120 min is ~at .. than either 85 % '" or 100 % n above the baseline [g1uco",] 2. The guiddines ..e b:.sed on data from II healthy ho ..... The peak [gluco",] at 120 min wa.s approximately twi~ the cona:ntmion in the basdine .. mple: th.r is, 178 ± 29 mgldL at 120 min .nd 83 ± II mgldL at 0 min (m<\)." [v. Dis U.CTOSE TOLERANCE TEST IN HORSES I. Phy. II. Analytical cona:p" {Itt the Gluco .. Concentration, =t. II in Ch apter 14} Ill. Lactose toleran~ test A. Ba.ia of procedure: LactOst i. administerffi via. stomach tube, and then multiple blood .ampl....e collected to ...... the intestinal mucosa'. ability to digest lactOst to 15 / EXOCRINE PANCREAS AND INTESTINE 757 g1uco.., .nd gaJ.cto ... Assuming normal g1uro", absorption and metabolism. the peak blood [glu=] should refl«t the amount of locta.. . ctivity in the intestine. B. Procedure" l. Fttd is withhdd for 4 h prior to the pro=lure. 2. A blood .. mple is collected for . baseline (or 0 time) [gluco..,]. 3. uct"'" monohydrate {I g/kg body w.ight as • 20 % solution. w.ightlvolume} is administered via nornxh tube. 4. Blood samples are coll«ted at 30, 60, and 90 min .fter locto", i. administered. 5. Blood samples are proce...d . ppropriatdy, and glu= concentrations . ", measured in the ",rum or plasma "'mples. C. Criteria for .dequate locta", .ctivity I. [Glucose] i. 15()....2~0 % ofb"",line concentmions at 60 or 90 min." 2. A peak [glucose] i, at least 35 mgldl abo"" the b",dine cont,entration!' IV. Dis= and conditions that alter the mult. of the oral lacto.. tolerance tm A. Maldigestion cau=! by lactase deficiency l. Viral enteriti, {e.g., rotavirus} 2. Clostridium difJiri/r enterorolitis" B. Malab5Orption of glu= because of dioe= of the .mall intestine. C. Excessi"" cellulor utilization of .bsorbal g1uco", URINE SUCROSE CONCENTRATIO N IN HORSES" I. Physiologic proce" ." A. Suer"", is. disaccharide that i, .pHt into glucose and fruno"" by sucra.. in the proximal small intestine. B. Suer"", is typicolly not . bsorbal by either gastric mucosa (impermeable) or enteric mucosa {degraded before absorption con occur}. n. Analytical concep" A. M5aYS I. For the equine study cited, high. perforJruln'" liquid chroJrultography WlI.I u.ed to ..,~te sucrose from other sug..., and pul=! amperometric det«tion was used to quantifY the "'P.rated suga ... " 2. A spectrophotometric sucrose :way is av. ilable, "' but .. pom of its use in domestic JrulmJrulh we .. not found. B. Sample: Urine umple. were coU«ted, immediatdy mixed with sodium azide .• nd then frozen (at - 80°C) until analrsi'. C. Urine osmolality was also measured 50 th. t sucrose to osmolality ratio. could be calculated. 111. Sucrose .bsorption tem A. S."ic. of procedure: The urinary bl. dder WlI.I emptied, and then hofWl were fed 1 kg of Omolene .nd then given 454 g of suero.. ( to % solution in H,O) via • nouogastric tube. Urine .. mples were collected 2 and 4 h bter. B. Ho ..... with gastric ulcers h.d incr.-ased urine [suero..,] .nd inc",ased sucro.. to osmolality ratios. Tho"" ho .... s with more "",ere gastric ulce .. exereted more sucro'" in unne. 758 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY Table 15.9. Other laboratory method. used fO cvaluate fur exocrine panacatu: insufficiency Laboratory m~thod Abnorm:d ... u1t Sue;eslffll"thologic ,Ute Arocas.in digestion test F= fail"'" to digtn [kCf'~.ffl azocastm Fa:;;o/ fat excmion [nc",,~ f=>l fat acme f,",,", prola>lytic activity Moldige;tion of ingtned lipid, 24 h sample F«:al fat, microsropic exam i Sudanophilic dropJ." in M:ddigestio,n of ingtned lipid, F=>l storm, microlcopic i M:ddigestio,n of ingtned starch f=, =m Gdalin tube, digtnion Muscl. fikrs, microscopic =m Starch grallul .. nain F= fail"'" 10 liqud)' gdatin .dequaldy Undigestal mu.d. Ii"-" PABA· pr=nt D«reastd plasma [PABA) or d"",..asN urinary PABA P[",ma turbidity un excretion Absen"" of plasma turbidity .ftc, inge,{ing lipid meal, M:ddigestio,n of di=ry mu,cl~ {protein} [kCJ~~.ed chymotryJ>lin ",l~~,ed nom pancr..,.. [kCJ~a.ed pancreatic LPS activity in intestin~ but turbidity p.uent .ft.r ingesting r=iigellallipid. IUdiographic film digestion Starch digtSlion res! F= failed to de .. gdati" from film Fl at g1UCOst .b50rption curve [kCJ~~.ed f~ prota>lytic activity M..ldigtstio,n of SUM or nul.b"''Ption of glu<X>St • PABA: N.ben~J.l-tyro .. rum .ctivity within ,be ref.,.""" interv:ol in EP[ aseI. &rum AMS rnd LPS 1CIivity c:on be d.cn...d .. ben rn wnW doos no' b""" EPt OTHER METHODS OF EVALUAT[NG D[GESTIVE OR ABSORPTIVE FUNCT[ONS I. M~ny oth.. labo.. tory assays or mffhod, ~r~ used to, ",,:dUlaced other tests th at w..~ u,ed to ",,:duate pot~ntial maldig.. ti"" disorde ... B. Mnn of th~ absorption t~m ha"" not ~n found to k clinically valU [I. F~ :ways used to, det«t imminal parasiti,m .'" common in v"'~rinary medicin~, but discUSlion of th~ procedur~' i, ~nd th~ scope of thi, textbook. Th~ read~r i, r&rred to diagnostic parasitology "",ura:s. 15 / EXOCRINE PANCREAS AND INTESTINE 759 Table 15.10. Other labor-nory melho.!. u....! 10 cvaluate intestinal disca.... Laboratory method Abnormal Bacwial culiu", (isolation ) Sp«ific r~sult SUW!!al p>thologic isolat~ (~.g., .tu~ Bact~ri.l ~nuritis s..lmonr/Ia sp. ) Bact~rial cuhur~ (qu . mitati",) Biopsy procedures {incision or acision} 10' colony.forming units/ mL of duod~nal JUI"" Abnormal edl popuJ.tions or abnormaltissu~ ;> S!ructur~ Motil~ Dirrct f.-cal H . m F.-cal flotation test !",rasit.. Parasitic ova, ooc)'l~" or SmaU intestinal bact..ial onrgrowth V ..ies with th~ hi!lologic lesion; rypica.lly inflammation or neapl"'i i_ F.-cal occult blood F.-cal .. dim~mation u n Hydrogtn br....th un Plasma turbidity un with pralig Positi"" ",action for Fluh ova lncr .... snl hyd~n production Ab...n"" of plasm. turbidity Acid.fast organisnu h~m~ Crypro,poridi mm Sturn digtstion uS! Blood in alimontary traet r ..asitism Bact~ri Gi:udi Giardia trophozoite:! His,qplaJ"", organism. lncr .... snllalkoc)'lel N."plastic ""lls Uniform b. cilli Fiat g1UOOst .bsorption Ent~ritis Neoplasia Bact~ri.l onrgrowth Milibwrption of g1u~ curv~ Unronjugatal Urin~ bil~ acids indic>n tm lnc",asal d=>njugation of bil~ .cid, by an onrgrowrh of imestinal bact~ria lncreasal dq:radation of tryptophan by ~nt~ric lncr .... snl ","rum ron"",mrations P""iti"" for indican b.ct~ria Urine nitrosonaphtholtest P""itiv~ '"' lnc",asal bact~rial degradation of tyrosine by .nt~ric bacteria nitrosonaphthol No..: This is no. 1 comple'" Ii ... Other >p ... ....d for .p References .oroph., l. Wi."., ME.. s..,; SA. W"",rmard E.. 1?99. E-rio, pao<>"'1ti.c io Grnaan .bryti.c I""'""titio. V.. P.tl.oI. J6:HO- S4l. 2. Wtbrq; ME.. lOOt . P""""tic odo", atroph, ;0 Gr"""" aIr.pI..,.J do", ...d ""'rh..... t«I oollier: E.q.,drovn<.; •• d~ ;' ...d _"""'o--A...;..... v.. Q 26:61-75. J. Mod"" EM. So,;"", 1M. auk LA M wp.y KE. F....ul.lll;. Wtllia.... DA. Stanla..ia ME.. Vo", AS. 2002. w.,,;......,,,( paocn:r.tic ad ..... trop!., in G",,,,,,,, rIrrp/rrrd <10" . Am I Y.. Ro. 6J: 1429-1t.J4. t . W"""o PI. 2003. E......in< P""""tic io •..J6ci.ncy .. an ",d '''C' ,,( p""""citio in "'" do, •. I S",alI Ani", P_ 4-l:J06--}12. ' 01 s. FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY s.,;"", JM, W,1liamo DA. 2000. s......, fdin< ~ _ lih imm~tty in ",,,...ith...,..;..., !""",,,.tic in,.ffici7. Philaddpbia. WB Saw.d"L 7. s.,;"", JM, Williamo DA. 2000. D;,ap'" ..ru. cd"";, fo. dia&oo.inc J>ODG',atili. in co ... J Am Y" Mod A .... 21 7,816-1!17. 8. Ti &ocr",.,......,.tk v., ..,.J Qi,.'"J 5..""""',. ....... ,atic ~" J Am V.. Mod Aaooc 196062J--626. 10. s.,;"", JM. ZOO}. Di~ . of paocreotirio. V.. CIiD Nortl. Am Small Ani... P I"" 27,2J()-23J. 12. Simp"'" KW, Bort RM, McL..D L. Morton DB. 1989. Ci ........;", """"",tration. of "YP.i..-1dr Own"",,<eo. " -Iikr Onm~'" in k"ltby doc •. Am J Y" R... lJS7- lJ59. 14. s..ift NC. M..h SL. M""L-Jdan NJ, No"";, CR. 2000. &aJ.";"n,,( ....... fdi... "J'p.in.lik, Onm"""",",tirt.,. fo. th, diau-i. of panc=.tici. in "'D. ) Am Yrt Mod A.ooc 21 7, 51--42. IS. F~nn ... MA. Mark. S1. 0. Co.i HE.. H"",odl E). W,,"" ER. B.k.rTW. Kau PH. S"in" )M. Willi ..... DA. 2004. Enl.. tion of ..... '" kti", I""""otic ~pa>< immwoo.,,,,ci'; J....,!t in doc •. Am) p~ U60GS4J--GS'9. ZOo G.ob.. MC R.iddl-Z" R. O'Rouckr M. P....... E),. La.cman C. 19S2. PIa ..... p-=otic "YI'tinop>. in cl...,nk ...,.[ fail.", aod luoe ... ...,.. Am ) Ploytio[ 242,G 177-GIS2. . S""'" ".tt ano" s.",,.. s.....,... """""''''ki_ af", ..... 21. R.taux CG. s..;",., )M, W,Uian.. DA. 2OOS. Euly biodtbo.l.",in tuppktn=tati.on in "'" ..ith ti&no of """,,""nirutJ dioc.. .. and ..... '" b~amin"";" ) Y" [.. """ Mod 19,I S5-160. 22. M,,,pathy in "'D' A "''' «><>""'[ .tuody. B..J Mwoob Tk.arrtJ Wodtrtud.. J 16,J40-J4 S. 23. Can<> T, William. DA. 1989. Rd,,;omltip brtw .... di,....,. "",«in "''''''' .. ..;.,n aod " rum trypti ...Jikr inua."",,_ in dop. Am ) Yrt Rr. 50,2105-2107. 24. William. DA. Bat, RM. 1988. S....ici';«in< l""""'tic i.,...ffici,,"'l' in <10" . ) Am y" Mod A.oc 2ZO') 18.}-1137. orti.i.,. 26. Sod"" )M, WiUiamt DA. 2003. D...d.op""n' and valid ...... of. "dioi"''''w."....y fo, .... """'"""""" of ~ paoa",otic Upao< inua.""""ci'; JI. J2. ~f • • adioimmwooa ..., fo. th, """"""""'" ,,( fdin< p""',.tic lipa" i",,,,wt<>"'trti.ity in " ....... Cut) Y" R.t 6I!,J09-JI". Sod"" )M. Bro...tu,otic Upao< Onm~'" (cPU) «><>«",.. cio,," in dop with ""'...... ""u. I""""atiti •. ) Y" I,,"m Med IS,Il' (abttt".). William. DA. S"';"'" 1M , R.""" CG, bvro. N. 2OOJ. [<>,otic Upao< inua."""",ci';, of .... ~ Socirty of Y'''rtnary Clioical Patbo~ J!..,IJ, AL.nt. 6-12. Sod"" 1M. ","00 DR. ~ SR. William. DA. 2001 . Saum ",,"n, pa<>« """..J 15/ EXOCRINE PANCREAS AND INTESTINE 761 33. s..;..,. 1M, Rntt GM, Willi ..... DA, 2006. s.n,,,, lipao< .ai.hl" • ..d .,......,.tic tipa .. lmro"oc _ti.i'1 OOD«n"' _ Ii"'.... in d.or; • ..ith ",,<>«in< .,......,.tk i ... ..£foci,D<J'. Am IV .. Rr. 67,84-87. 34. M.... r.dd cs, I""'" BR. 2000. Pl."". uod...inuy ~~n oaintion pl =in< pana"titi.. Rr. V" Sci 7'(,]51_ 144. 36. AlIm. HS. So,;"" I, &cu. ....d I. M....6dd C. Williamo DA, I~"'" B. lOO6. Sawn uod "";0' ronc-T .yIo, I . Rrb., HA. 19"93. " - , <>l uyp,;""V" ",ti.. tioo iD th, at <>(><"'"im,otal modd of K~" pana"titi.. P...,.... 8,189-195 . 38. R.t. Now.eod thj" TIt. SI ""it. "'" b"" . I Am M«I Jw.oc 2SS.lJ29---2.H9. 40. Faitbaob VF, K1« GG. 19"99. Biol ~n' pao<>',ati.c on rob.lamin ab""pl can'" B, I N.n 83,)---6. oW. Fyf< IC. G~, U . H.l1 CA. 1''11 PF, Klumpp 5.A.!..vi", IS, P"",...,n DF. 1991 . IDlt.ritod..kti... io".tin.1 ooI.alamin malab><>tion ohitunin 8" in ri- dD.""" •. I Am Anim Ho"" Jw.oc 2S,SJJ-Sl9. 46. o..".b.idu CA. M, ... SI.. Gi, .. U. 1996. H .. odituy cobalamin "'f"'''K) in Bood" «>IIi, d.or;o. I V.. In"", Mod ina.ffic""', )0,169 (....... "'.). .fl. VadtnSI.. Wood PA, kU'1 FD. Co. ....... n PE, Mill .. RT, Pag R. 1992 . Cob.lamin ddici.tno,-'atod with ....thyImalooic acid"";. io • at. I Am V.. Mod Jw.oc 200, 1101_1103. 48. Batt RM. M<> 10. 1982. R.,&., <>l ..."'" fola" ...d .itamio B" «><>«D'''dooo io th, dilf.. ,otiatioo <>l "".II intminal abnonn..liti<. in th, d.or;. R.t. V.. Sci 32,17--22. 49. Rnq; ... HC. B..n RM, EJ..ood CM. Umpo.. A, 1995. SmaU int.-inal b.ct .. ial "'"'~ io.lop wid. d.ronic intminal dj,cut. I Am V" M«I A""" 206,137_ 193. SO. Bat. RM, Ba...... A. Rut"". HC. Cart .. SD. 19"91. R.tJ.';"" I~ dt6ci.ncy ...d "".II im";n.l ba.ru.i.l "m p owtit. in G .. m"" d.tpI.«ld., .mall in."clnt. ID' Eninc.. SI, Fd.dmo.n EC. od.. T""" .... ofV~ ft.""",/ MuEfflu, Dis...- of th. D.r ..,.J Cott. 5th edition, 118l-1238. l'bii.oddphi" WB Sawtd .... 52. Bw.cb Sf.. £ ..1., IR. C...u", 1M. 1990. H"".",i.oO< val ..... and pi ...... ...d tiuu, faI,,, ",oem",tio ... in do,. VvtO p/Kuytoin on • 100, .""" b..i •. Am I V" R.t. 51,IM5_1868. 53. William. DA. 1m. Ddioio,.....u in"ttina] dioca ... In, P~ <>ld., 17'" ACYl:'J Fa"" ... a.~ ~. 54. M"""" KF. G"",ao AI, Ru."" CG. Sttint, 1M. Wio",tin.1 di...... V.. Clin Padt.oI32..67_72. 55. F"" K. Ru,,,,, CG. Sttio" 1M, S.cl.odobki IS, Willi.m, DA. 2004. l'urilicatioo ...d pattial ""' ..... ..nation ~f "tin, o,-P"""io ... ioh.ibi"" (fo.,_PI) and ... , """"lop ....... Dd validation ~f • ,adioimmw>oo.", /'0, th, m"",,,,,",,,,,, <>l 1a,_PI io .. rum. Biodtimi, M..67- 7S. 56. K...ni,A. SI,,,, M, R.w..o. CG. s..;..,. 1M, William. D. 2002. Compariooo of fecal a, -pt<>t';n ... iohibi"", oonc 57. Mtlp..;o T. Willi.m. DA. A"", EK. 1998. Eo'1""'linW immw.<>toD in-nn.l p.i..! """,iti •. I V.. 10""" M«I 17,791 - 798. 59. M"""" KF. Go""", AI, Rw. ... CG. Sttint, 1M. W, ...... p,. V.. Clin Padt.oI 3hlJ.6.--lJ9. 762 60. 61. 62. 63. 601. 65. 66. 67. 68. 69. 70. 71. 72. n. 7.(. 7S. 76. 77. 711. 79. so. S I. Sz. FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY a.n. Tj, Sampl, RH Glick Mil. Ellio GH. 1979. A oimpli/icd, «>i.orim,,,'" ""=m,thod fo. ..po.. in ........ o• .........hl. pklorocIociDol. Clin a..... 2s.J44I>-144J. H...d,non AD.. RinIu. AD. 1999. G .. tric, pao=atk • ...d in",nina] function. 1m Butio CA, A ... ....,.,d Ell. ..... 3M cditioa, IZlI - lJZ7. Ph.lI..kJphi .. WB Saw.d .... Ti, .. T,""'-/, ./C{;,,;.'" Bau", JD. A.ch.mana!'G. Too<> G. 197.(. Q;"".ILI ", • .,.M~, Stl. odin..... St u,.,;" CV Mooby. s.,.'M and aboonnal xyloo< .boortl. Am E.quin< [',"', ),S07- S14. R..bm. MC. 1975. CuboIoyJa.", diVnioo ...oJ aboorptioa in th, oquin< .mall int"tio,. ) S Ah V.. Jw.x -16,19-27. M.<>ttman G. St a..........,. ""'p.' i" Ubo.....,. JI.." Chapter 16 LIPIDS Lipids .................................................................... Overview of Lipoproteins ................................................... I. Lipoprotein Classification, Content, and Properties ...................... II. Lipoprotein Metabolism ............................................. III. Lipases ........................................................... Cholesterol Concentration of Serum .......................................... I. Physiologic Processes .............................................. 764 764 764 766 767 767 767 II. Analytical Concepts ................................................ 769 III. Hypercholesterolemia ............................................... IV. Hypocholesterolemia ............................................... Triglyceride fTG) fTriacylglycerol) Concentration of Serum ....................... I. Physiologic Processes .............................................. II. Analytical Concepts ................................................ III. Hypenriglyceridemia ............................................... IV. Hypotriglyceridemia ................................................ Hyperlipemia, Hypertipidemia, and Hypertipoproteinemia Disorders ............... I. Terms ............................................................ II. Classif ication of Hyperlipoproteinemia Conditions and Disordel"ll .......... III. Physiologic (Postprandial) Hyperlipidemia ............................. IV. Primary or Possible Primary Hyperlipidemi a Disordel"ll in Domestic Mammals ......................................................... V. Secondary Hypertipidemia Disordel"ll .................................. Other Assessments of Lipids ................................................ I. Lipoprotein Electrophoresis .......................................... II. Nonesterif ied Fatty Acids (NEFA, Free Fatty Acids, and Fatty Acids) ........ III. Standing Plasma Test for Chylomicrons ............................... 770 770 771 771 771 772 772 773 773 773 773 773 775 778 778 778 780 763 764 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY Table 16.1. Abbreviations and symbols in {hi.. chapler [x] LDL LPl LPS NEFA 51 TG T NF Con""mmion of x (x = . nalyte) Cotnzyme A Ethylened;"minmtr:oac<:{ic acid High.denlity lipoprotein Imn,ftr ... Low-dcnsity lipoprotein Lipoprotein lipa« lip"" Nonesurifial fmy . cid, f,re fany a.cid, fallyacid Syst'mc lnurn.lion. J d'Uniles Trigly"",id. (also called triacylglyctrolJ Tumor necrosi. f.ctor VLDL V cry [ow-dcmity lipoprotein CoA EDTA H DL IDL LCAT UPIDS I. A lipid i, a compound that is soluble in organic ..,lnnlS and rdativdy insolubl. in H,O. Of the lipid compound. in blood or oth" body Huid., serum [cholesterol] .nd [TG] "c the most fffl:J.ucntly mu.ured .nd [hu.... Ih. focus of Ihi, chapter. n. Lipids found in IIUmmal,' A. Sterols: cholmcrol, molesterol cstw;, bile acid., suroid hormon .., and viumin D B. GlfO'rol <SI~rs: pho,phoglfO'rid.. (phospholipid.), monoglya:rides, diglfO'rid .., and triglfO'ride, C. Fmy . cids: mort_, mnlium_, . nd long_chain fatty acids, .nd prostaglandins (f. tty"""cid d~riv:lliv..) D. Sphingmine" sphingomyelin (phospholipids), .nd glyco.phingolipids E. Tu~nu: vitomin.A, E, .nd K Ill. Lipids h. ve many function! in th~ body but .'" particularly important as .n ~n ...gy Klura: (triglya:rides and fatty .cids), as structu.,l compon~nts of all cdl m~mb.,nes (phospholipids and cholest~rol), and as substrates for hormones and =nd messc:IIi:~rs. OVERVIEW OF UPO PROTEI NS I. Lipoprotein Classification, Cont~nt, and Pro~rti.. A. A basic underst. nd ing of lipoprotein compolition and pro~rties is nttdnl to und.r_ nand .. rum [TG] and [chol",~rol] b.au.~ all TG . nd most cholest~rol mol«:llles .'" transportnl in .. rum within lipoproteins. Formation of lipoproteins .""bl.. lipid. to form small partides that am circula te through th~ vasculature without roal~scing into large lipid masse. that would ob.muct blood flow. B. Lipoprot~ins are dassifinleith.. by their dectrophomic mobility or by th.ir d~nsity rdative to H,O. T heir pro~rties d.~nd on composition (fabl~ 16.2). _..... --: l'!f"'-"-.. ., ~i i <, H.pm I, r, ".,..., £kb ,100 ,,;.,..,_ 0;.""", (am) Coo_ .. , ...... I", .,,' .10, A, I. C E """ > 70 (""" 500-1000) y ... _..;.o.n- " o< dde_oI .... [0 , [ .. " , ' 8, C, E ~. ,.~" " ["'0,; ... " I I ,C, E ~ ....... ..... .... C. D.E • ~ u~ ~ PIo_ (_ M. jo< .... 01""_ ,~~ M.jo< .... 01 d",>do ". f -~ ,., ,otl t' "'""' Otfo""',.·· hI\' ,;.","" .... -" , ...."',. ""'.. ~ FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY '" oomp",ition of lipoproteins in dometioll of lipoprouill f"'ctions and subf",ctiolls .mong s~~ •. a. Dogs, cats, alld horso; ho"" predominalltly HDL molest~rol "'th~r thon LOL cholmerol. LDL mol~nerol pr~dominat .. in peopl~. b. Predomill.nct ofHDl cholm~rol is associated with decr......d or .b.. nt moles_ t~rol est .. tran,f.. activity (t"'nsf~rring molesterol from HDL to LOL) alld d,""rease [I. Th~ gen~ral Lipoprouin m~abolism {Fig. 16.1} A. III the colltat of .. rum [molesterol] .nd [TG]. disorden involving lipoprotein m=bolism involve aass synthesis, defective lipolysis, or def,""tive clramll"" or allular uptake of lipoprou ills. l. Nas""nt (llewly made) lipoproteills,,~ produced in .mall illtestillal enterocyteJ (mylomicrons) and in hepatocyte. (VLDl.nd disroid HDL). 2 Lipolysis of lipoproteins {mylomicrollJ, VLDl, .nd IOl} occun on the lumillal surfact of capillary elldothdial called lipnnu. elta,ing/Mto,. 4. lipoprot~in ",mnallts ar~ cl.. red from pla,m. by hq.atocytes. LOL molecul .. are ",moved from plasma by many types of all .. LPL incr..,..." LDl uptake by promot_ ing lDL binding to ""U recq>ton.' 16/ LIPIDS 767 B. Apolipoprouir" are criticol for normallipoprot~in m~abolism. inHu~nciIll: lipopro"in .tructur~. acting as cofacton for ~nzym .. involval in lipid m~t>bolism. and .cting :as ligands for r«:q>tor_m.diat.d cdlular int n prot~in. B dass. from intestin~ .nd livnd coronary h~.n di ..... and some primary lipoprotein disorders. Bea"", ouch disord." are uncommon in domestic Jrulmmal •• lipoprotnn information will ~ limital. 2. 111. Th~ Of th. sev~ral lip"'" in th. body. th~ major on~' that p CHOLESTEROL CONCENTRATION OF SERUM I. rhy>iologic plO ""U, Exogenous Lipid Pathways Endogenous Lipid Pathways s~~il- -'. --',-',... -;.;.- '" ~ttG Intestine;:" I.;" FA ~ ----- ___ ii!"," .', _----...;.., :,;-o.TG CE -~'\.--' _'l_ '. Adipose tissue o TrigI)'C8rido mokt<::ukos ., lipoproteins • ClIoI&steroi 0&\9< moIecuIos ., lipoproteins ABC 0 E Apolipoproteins A. C. D. ~nd E in ~poproIeins a Fig. 16. 1. Buic phJ"iologic proce>oe, in ~popro,e:in m.,,:,bolism. Th. duN m, jo, p"""'.... ,b, t affect I ~popro"'in] .,.d th"" [TG] md [cbolos",rol] .r< (I) srntl=is of cbylomicrons in . ntorocytes md VLDl in hepatocyt ... (2) LPL-""ulyzed ~poJy.;' on .ndo,he~:d c'ocyt. cJ.:u:mc< of lipoprotein I.."nan". Tbe", .,., thW>Jl" for lipids. one fOlendog<now md on< fol uogenow ~pids . • &ogenoWl 01 ~ary ~pids: Ingest«! TG in the p'." " "" of bit. ""ids and lPS undergoes ~poJy.is to fonn MG md FA. Aft.,. . !>sorption by en'orocytes. MG md FA:or~ '..... mbkd into TG. Entorocytes:Wo produce CE, phospbo~pids. and .poIipoproteins A.,. lPl (on endothelial ""J[ membnnoo) ,b", ",ulyus the ~poly>is ofTG to gene... ", FA. n.. FA ente" . dipocytes to he "0",", in TG 01 ente" muscle fibe" (Ol otber ""lis) to undergo oxidation to gene .... energy. After «moval of mos' TG mokculos. the cbylomicron «mnanu .,., ct. ... d from pmm. by hepatocyte> in. p = involving B apolipopro"'ins. '68 16/ LIPIDS 769 Cholm~rol .. t~rs ~nUr cdh by ra::qJtor_mMiatM endocytosis of lipoprouin fragrn~nu. Thq ~r~ ddinrM to lysosom... wh~", acid lip....., promot~s th~ir hydroly.is. Choles_ r.rol can th~n "" rd.-astd for ""'" by the ""U. F. Wh~n cholmerol ruch.. hq>.rocyt.. via lipoprot~in ",mn:mIS, il may "" reu~ for lipopror.in .ymh..i., ",crerM unchangM in bile, or degraded 10 bil~ .cids. E. II. Aruolytical oon""pu A. T urns ~nd units l. Serum [cholmerol] r.p .... nts the total [chol.. terol], including cholest~rol and cholest~rol est~n that ar~ hydrolyzed during .nalysis. Th~ r...cting chole;{~rol molecul.. are within Jipopror.ins. typically in chol.. t~rol_rich LDL and HDL molecules. 2. Units: mgldL X 0.02586 = mmollL (SI unit, nearest 0.05 mmoUL)' B. Sampl~ l. Serum e. is the p..fertM sample. Total [chol.. t~rol] is suble at 4 °C for 5- 7 d, al - 20°C for 3 mo, and .1 _70°C for y.ars. R'p"'tM tluwing ~nd refrttring should "" avoid...!" 2. Dog. and caU mould"" f:lSI...! onmight or for about 12 h to avoid ponprandial h~rlipidemia, which may ~ffecl [cholesterol]. Methods l. Mon automata! ous;oys are .nzymatic methods that hydrolyze cholm~rol esters :md then US< cholenerol oxida", to oxidize chol.. r.rol and geneme hydrogen peroxide, which then reocts with .n indicator dye. Some assays us. .n O,_s.ming dectrode to meam .. 0, conmmption. Fig. 16. 1. «mtinwa • Endogenow ~pids produ=l by hepato: H'pat<>«Rted into sinusoidal blood. In 110. p,esen« of inru~n, .poIipoproteins C_1I on th. VLDl .ctivale LPl on .ndothe~al ""lis to initille ~poty.is :ond m,.mion of FA from TG. k VLDll ..... TG. it b.co""", <1< ..... to form lDl, .mim auy also undergo ~d.itional lipolysis to form LDl. LDl ,_. • Discoid HDL puticles consisting primarily of apolipoproteins ond phospholipid:ore produced by hepatocyt'" :ond h.ve two m ojo, functions: (I) they ...... as a SOUl"" of C 10d E apolipoproteins fo, other lipoproteins. and (2) th..y .ccepl mo!..t...,l from pl .. ma """,b,..,., or lipoproteins ond tnn.port it fo, reuti~ ...tion (in hepatocyt .. ) or d.gndation (in hepatocytes or maaoplug .. ). Chot.._ terol is captured by the LCAT-cau.lyud formation of moJ..terol este, 10d lywlecilhin from J.cithin and cbot..terol. Thi. is mppo,ted by apolipoprotein A. k HDl ""rumula"'" chot.sterol in cirrubtion, tbey b.co"", ~r 10d ""qui", th..ir spherical form . • Th. outer ",rf.=< of lipoproleins contain phospholipid., noo<'teri&d cOO!..terol.:ond apo~poproteiru. Shaded lett .... A. B. C, :ond E "'" A, B, C, ond E apolipoproteins: AcCoA, . ""tyI-roenzym< A: ATP. ah. te: cr, cholesterol Oller, Cool, molenerol: FA, f..tty acid: lPl, ~poprotein ~P""": Ll'S. p:onc .. atic lip""": MG, monoglyceri FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY Table 16.3. Di""a..es and condition. thai cause hyperchol esterolemia Inc""asal chole;t~rol produCiion By h~pat()(yu. 'N~phrotic .yndrom~ or protein.lo.ing nephropathy By ~m~rocyt .. • Po.lprandi.l hJ'P"rlipid~mia D..:""asal lipolysis or imravascular processing of lipoprol~ifll • Hypothyroidism 'N~phrolic ,yndrome or prol~in.losing nq.hropathy lipoprot~in lip,... ddici~ncy (""'1 rar~ in dog,) Oth~r, unknown, or mulliple mMani,ms Acute p:mcreatiti, 'Chole;tasi, (olmruCli",,) ·Diabet.. mdli{U, Hyp..:od""nororlici,m or exa:s.s glucocorlicoid, Hypercholcst~rolemia in briard, Idiopathic hJ'P"rlipidemia of miniature .ch""u",,, or oth~r brttds • A lel1tively common di...... 01 condition 2, High [bilirubin] .nd [aocorbic acid] /lUy callS< neg.ti"" im..f..en~ with the enzymatic =ys,"'OAscorbic oxida.. may ~ indudal in .n assay', "">genu to ""mo"" th~ df":l of ascorbic .cid, III. HJ'P"rchol~nerolemia A, [ncr~ • ..d .. rum [cholesterol] indicat.. there arc ineru...:! ron"",mrations of lipoproleins thaI contain chole;leroL B, Disord~" or conditions (Table 16,3) C. Th~ pathologic P""'"'SS" Ihat cau.. hy~rcholest~rol~mi ••"" describal in • lat....ction on lipoprot~in di50rd~", (Hyperli~mia, HJ'P"rlipidemi., :md Hy~rlipoprol~inemia Disord~,,) , [V, Hyp<><:holmerol.mia A, Hypochole;lCrolemia i, an uncommon finding in dinical """', B, Disord~" or conditions thaI cau.. hypocholesterolemia (T.ble 16.4) 1. PorlO.ynemic mums: It is rcponal th aI .bout 60- 70 % of dogs:md caU with porlosyst~mic mums h."" hypocholmerolemia," Two pathog.n=s arc propo...:! for Ihe hypocholest~rol~mia: {I} d..:r~• ..d cholcsl~rol production ""cau .. of d~er~...d functional hq.atic mass, :md {2} inhibition of chol~'I~rol 'fllth..i, by bile acid., 2, Prot~in.losing em.ropathy (especially bccau.. of ime;linallymphangi..:tasia}:'l-" Th~ pathog.ne;is of Ihe hypochol .. t.rolemia may indude d..:""...d lipoprolein production bccau.. of th~ catabolic stalc or, in the ca.. of Iymph.ngi..:ta,i., loss of lipid. that arc nor/IUJly produced by em~rocyt .., 3, HYP""d""nocorlici.m a, In one study involvillJ: 17 dog, with hypoadrenoronici,m, 13 had hypochole;ter. ol~mia," [n .nolh.. !Iudy involving 20 I dog" 14 ""''''' hypochol .. t.rol~mic, but 29 had hJ'P"rcholcslCrolemia," 16 / LIPIDS 171 Table 16.4. Discascs and condition. Ihat cause hypo<:holcslcroleoua O,""rra,aI cholCSl~rol production Pon""ysumic shum, in dogs and cm PrOl~in_lo,ing ~m~rol"thy Olh~r. unknown. or multipl~ m,""hani,ms Hypoad.. noconici,m =tion on hy~radrenoconici,m in dog. {Hyp b. As aplainffi in th~ Hy~rlipid~mia. TRIGLYCERIDE (TG) (TRIACYLGLYCEROL) CONCENTRATION OF SERUM I. Physiologic process~. A. [n gen~ral. lipoprot~im lrampon TG from immincs and Ii""r to mu.d~ for ~n ...gy or to .dipo.. ti",u~ for stotag<'. B. N~..ly all TG in • fasting strum sampl~ i, wilhin lipoprouim that w~ .. produced in hq>>loc)'ieo. Much of th~ TG in a ponprandial sampl~ i, in chylomicrons thai w~ .. asstmblffi in em~rocytcs from dietary lipids. C. TG .ynthai, begins wilh f>try .cids (short. medium. or long chain) and gl)'C"rol from carbohydrat~ m~laboli,m. TG synth"'i' for lipoproteim occu'" in hq>atoc)'i'" and ~m~rocyt ... O. Wh~n lipoprot~im conl:lining .polipoprol~in C-11 bind 10 LPL on endolhdial "",lh. fmy acids .. ~ cl~'vN from TG mol,""ul", and ~nUr cdl, {e.g.• adipocyus .nd myo_ cyt"'}. With .. ~ated proces'ing. th~ lipoproteins krom~ TG d~pl<:{ed and Ihu, strum [TG] d,""r~...... [I. Analytical conctpu A. T~rm' .nd units l. Trigl}ct,;d( i. the mor~ common t~rm for. group oflipids that comain thr"" fatty ""id, m""hed to a giya:rol b.ckbon •. The mo", corr~ t.. m for Ih~ mol,""ul", is rriluJlg/y«rol. but il i, not used as fr'" m FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY 3. Inn,,=<:! OOflctntmioIU of chylomicrons, VLDl, and J>OS'ibly IDL moy caus. la.cte'''''lIct or turbid ",rum and may im.rfore with spa:trophotomet,ic """p. Thu., laboratory ~"onnd =y d •• , the .. rum {by ctntrifug.tion or .ddition of clearing "1:,m.} prior to chemical analy.is. When the ",rum is d •• rffi of the TG_rich lipoprotein>, the [TG ] in the sample will bt d"",rasffi. 4. H. "",,{ing .. rum btJow a .up<,ficiaJ "c'''''m" layer after routine ctmrifug. tion may also f:dsdr dao,.,... ,erum [TG]. 5. Dog. and em mould bt f..,IM overnight or for .bout 12 h to avoid ph)"iologic postprandial hyp<,jipid.mia. Prolongnl postprandial h~rlipid.mj. indicat.. the pr=na of. pathologic nat •. C. Method. lipa.. tru.t liborale! glycerol from TG. Th.libuaud g1yctrol is then m.am."! in coupl.d r~ctions ch.c ..",It in l. [n most automated :ossay', the initial ",.ction involve! a products th at em k d~t«t.d by .p«trophotom~try. 2. High [f= g1ycuol] in .. rum v..ould produ~ faJ ..ly d<"V:It.d [TG]. but mch conditions ar~ rardy r..rogniud. 3. [TG] in a grouly lip<mic sampl~ mayexcttd an assay's analytical r:mC". and thus th~ sample may n=:l to k dilmal prior to :malysis. Ill. H~migly~ridemi .. rum [TG] indicat.. th~r~ ar~ incr~• .al ron""ntrations of lipoproteins that contain TG. B. Disord~n or conditions (Tabl~ 16.S) C. Th~ pathologic process<. that ca...., hyp A. IV. Incr~a.al Hypotriglyuridemia: no significant pathologic sta t.. T ab le 16.5 . Di""aseJ and condition. that cause hypertrig1yceridcntia lnc..ascd triglyuride (ui 16/ LIPIDS 113 HYPERLIPEMIA. HYPERLIPIDEMIA, AND HYPERLIPOPROTEINEMIA DISORDERS l. Turns A. Hyptrliptmia. hyptrlipid~mia. :md hyptrlipoprouin~mia ar~ n~arly synonyms. I. Hyp"{ipnni,, .nd hypn-/ipid(mill r~f~r to an incr~asffl Oipid] in blood. wheth.. it is from hyptrlipoprot~inemi •• hyptrcholesterol~mia. hyptmigl~rid~mia. or even incr""sed [fmy.cids]. 2. Hyp"{i~protrimmill i. an increased con~ntration of one or more lipoproteim in blood. It i, the pr~f~rrM t~rm when a meamrM con~nt .. tion of. lipoprotein (~.g .• LDL or HDL) i. increased. 3. The t.. ms ..~ ",metimes used to id~ntilY clinical disorders {e.g.• equine hyptr_ lipid~mia or equine hyptrlipoproteinem;"}. B. Lipnni" {.lso lipidemi.}. by most dictionary definitions. i. a synonym for both hyptr_ lipid~mia and hyptrlipoprot~inemia. [n routine clinical u... liptmia is usU ll. Classifiation of hyptrlipoprot~inem;" conditions .nd di",rders A. Phpw/qjc (postprandial) hyp"{ipiJm,i,, is the hyptrlipidemia that occurs aft~r ingtnion of a meal conuining TG. B. PrimflTJ (or f.mil;..l) hyp"upuprouinnni 1Il. Phy.iologic (postprandial) hyptrlipidemia A. The hyptrlipidem;" occurs in monogastric mammals and is caused by a hyptrchylomi_ cronem;" that occurs .ft.. intesciruol ab"'rption of monogly"'rides .nd fany .cids thac have formed from digenion of ingestM trigl~rides (Fig. 16.1). Hyptmigl~rid.mia wiU k present: hyptrcholesterolemi. may not k pr=nt. B. In dogs and cats. the hyptrchylomicronemi. ptoo by 2-6 haft... me..! •• nd th. chylomicrons are mu..!ly cleared by 8- 16 h.' Dd"}-ed cle.ring of chylomicrons india". def,""tive lipoprotein m"'abolism. eith... primary or secondary hyperlipidemi •. C. The prosen", of postprandial hyptrlipidemi. with and without .ddition of pancreatic extracts to food has w"n used to ....... for exocrine pancreatic insufficiency {plasma turbidity ten: !l« Table I 5.9}. Howevc:r. variation. in gastrointestiruol function a"",,, var;"tiom in the development and d earing of the ponp.. ndial hyptrlipidemia. thus complicating int..pretation of the test. which has been largely replaced by measurement of .. rum trypsin_like immunore.ctivity {see Ch apter IS}. [v. Prim.ry and possible primary hYl"'rlipidemia disorders in domestic mamm. ls A. Idiopathic hYl"'rlipidem;" of miniature schruouzers~' 774 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY l. Th~ p"thogtnesi, of {h~ hr~rlipopro{~in~mja js nol mabJi,hal. It may ~ cau..,d B. by. ddici. ncy in LPl activity or.n apoJipoprot.in C·1I ddici.ncy. 2 Th.", it hyptrtrigl~ridemia with or without hyptrmole£t.rol.mia. Th... =1 bt concurr.m glum .. intol.r>n~ du. to insulin =;'UDct. Hyps SlI,~aI to h • .,.., "hyptrliptmi. du. to • proem;sivr j""crivat;on of lipoprouin lip... with aging." Hypr'triglyct,id.mi. WOI.'l found at 5, 7, :md 9 yr of .gr, md hyptrcholm.rol.mia was not pn=nt. ~rum [TG] did not daornR h. po.rin administration. 2. Th. hyps not found and ch. dog', KID (3 Y"'I':'l youngtr) also h.d" f>Sting hyptrtriglyuri. d.mia. HowrY,t, ""pon.., to h' p",in in Ih. son w;;u limi!." to" control dog', =pons<. Idiop.thic hJ'P<'rlipidemia occurring in dogs of oth.. brttd. (e.g .• Sh .ft., C. D. E. F. 175 16 / LIPIDS v. &condary hyptrlipid~mia dilOrd~rs A. Acut~ pancreatiti.... ,. I. Dog. with xut~ pancrutiti. fr"'lu~ndy ho"" hyp.. triglyc~rid~mi hyptrchol~n~rol~mia. Th~ hyptrtriglJ'C"rid~mi IIUy ~ ~ith .. an initiating f:octor or a co~u~nct of th~ pancr~atiti •. 2. Th~ pathogtn~sis of th~ hy~rlipoprot~in~mia i. not firmly ~subli.hffi but appears to ~ • d~fa::t in th~ intravaocular proct.. ing of chylomicron. and VLDL. a. On~ thalry is thot da:rrastd [inmlin] au.." dd"a::ti"" LPl :octivity ~caus~ mo""m~nt of lPL to th~ lumin:d surfact of ~ndothdial cdls is dq>tffi with rd..,... of cytokines during inflamllUtion. c. B«>IllSt pancreatitis may em", obstructi"" cholestasis. p:m of th~ hy~rlipid~milffi that a rommon positiv~ df~ctor induas synthesis of cholmerol and bil~ .cids. c. In ex~rim~ntal cholestasi, in dog•. incrrasffi [Wl] .ugge;ts. def~cti"" uptake ofLDl by h~patocyt ..." 3. Cholestasis has bttn as.IOCited by :dbumin within th~ ror~ and apolipoprot~in C on th~ surfact. b. Lipoprot~in X may form from a physicochemic:d. nonm..r. bolic interaction of pla,1IU lipid. with bil~ :ocid•. Anoth~r thtoty is th at reduced LCAT .ctivity from hepatic disrase may play. rol .... this is co",i"~nt with the pres.n"" oflipoprot~in X in hUllUn f.mili !ition characteristic of human lipoprouin X."''' C. Di 716 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY 2 Th~ hyp may ~ COUsM by mul{ipl~ ffiMan;,ms: a. Def.ain imrav:lScular processing of chylomicron, and VLDL ImIy occur b.caus. of low [inmlin] .nd thus def,""!;",, LPL .ctivity. b. Inc"".."! YLDl .ynth.. is by hq>. tocytel nuy occur due to mobilization of body lipids that is causal by inc=sN hormonNensit;"" li~ activity in .dipocyt... Th. influx of fmy .cids into hq>.tocyt<::'l and th.ir oxidation caus.. = formation of .e ap 16 / LIPIDS 177 b. Th~ po ... ibl~ incr~~.ffl production of cholm~rol h"" oon qu~stionffi in ~pl~ b.-caUst cholest~rol 51nth..is did not appt~r to ~ incr~aKd in n~phrotic p"ti~nts." data in ms indicot~ that th~,", is not. down.r.-gulation of th~ LOL r~tor-rdat~d protein." 2. Dog. and caU with prouin.losing nephropathy =y ha"" hypoalbuminemi a .nd h~rchol~nerolemia but not enough wdium .nd H,0 ret.ntion to COUst the ""cites and we= of the n.phrotic syndrom • . G. Equin. h~rJiptmia ofhorsn. ponies. and donk."., I. Th.st .strondary equine hyptrlipidemic dilOrd." or syndromes have oon gi""n [he name hyptrlip'mid. which occurs .strondarily in ..""ral metabolic dilOrd.rs thaI ah .. lipoprot.in meubolism. 2. Some authors divide the condition into two forms:" .... a. Hyptrlipid.mia: Plasma lipids are increased but with litd. clinical significon~. ~rum [TG] is usually < 500 mgldL. b. Hyptrlipid.mic >yndrome: [ncr....w pw= lipids ~re associ.tffi with hq>~tic lipidosis and marked clinical chang... s.:rum [TG] is usually;> 500 mgldL. 3. Th~ disorders th at produ~ changes in lipoprot~in m.uholism are v.ried. bu[ [h. ch.nges ~re .imilar. a. Physiologic or pathologic proces=; resuh in mobiJiza[ion of fatty acids from th~ TG mol<"Cul., of adipost ti«u~. b. Hepalocytes iner.""" the >ynthesi. ofTG.rich VLDL molerul~s that ent.. blood to COUst hyptrtrigl~rid.mia. Th= VLDL molerul~s have altered apolipopro. t.in cont.nt. c. If the condi[ion is """,re enough. th.n TG .ccumulation in hep.tocytes causes hepatic lipidosis .nd subsequent damag<: to hq>atocytes. 4. Condition! thai are ousocia[ed with equine hyptrlipid.mia includ. anoraia. o~,ity. pregnancy. I.ctation. renal failu,",. ~ndotoxemia •• nd oth.. st.tes that cr.. " a nq:.tiv. en.rgy balan~. Hormones or oth.. subsun~> can conlribu[~ [0 the d. fecti"" metabolic pathways." a. C.t.rnolamin.. and glucagon stimula,. hormon ...."'itiv.lipa.. in adipocytes to inc ...... rd.""" of fatty .cids. Insulin [ypicolly inhibiu thi. enzyme. and thus d<"Creased inmlin activity may remo"" a netati"" effector and promote fally.xid rdeast . b. Glucocorticoid hormones allO stimul ate hormon. ... nsitin lip"". c. Insulin stimul ates LPL for hydroly.is ofTG in most lipoprotein>... p«ially the TG.rich chylomicrom and YLOL. Decreased insulin activity rwu~s the .ctivity ofLPL. and thus the intravascular processing oflipoprot.ins is rwucW. d. Prog~steron. (when incr....w in pregnancy or lactation ) nimulat.. growth hormon•• which c,"". tes imulin ... ist:m~ by cousing insulin r«q>tor and postr"""ptor d.fects. .. e. Hypogl~mia. when pr . .. nt. stimulates glu~ rei ...... Glucagon nimula[es hormon~ ... nsitin lip"'" in adipocyt .. and thus promot .. theli~mion of fatty .cids. f. Either directly or through cytokine rd ..... endotoxins stimula[~ TG .nd VLDL synthesis by hep"tocyt... and they .lso can inhibit lipoprot.in cotaholism."·" g. Rau in renal failu,", (cr•• ,.d by panial nephrectomy) had d.fecti"" lipolysis of VLOL molecul.. and thus h~miglyc<:ridemi .. Th~ .gent or "!:.nts thaI c. Exptrim~ nul 77B FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY produ~ Ih~ d~f~tjvt lipolysis w~r~ !lot id~ntififfi." It jt nol known wheth" • sima" d.fa:! occurs in hon~ wilh ""ruoJ f~ilu",. 5. Con~nit;;oJ hyp in ~ Shetland pony fwl" a. A Shetl and pony o""n. te had vimally liptmic .ua, incre.w tou] [lipid]. and hypIM with prq;n.ncy), so th. foal'. liptffii<> Im}' hovt bttn Clu.sN by the pb"""uJ {ran,r.r of fmy acid. from Ih. rna ... Th. inflUJI of fmy acid, may han st;rnuJ.tffl YLDL synthesis sufficiently to COlO"" hyptrliptmia in the fo:ol. H. High di...:.ry fat inuk. in docs I. Ex"" ..;"" di...:.ry lipid ".."uld ... ,,It in norage oflipids and potenti<>lly obesity. Such dogs may ha"" prolongnl postprandial hy""rlipid.mia ba::ou", of insulin resist.""" or oth~r f""tors. 2. Ext"'mdy devat~d fat inuk~ .nd concurrent in:od'"'Juate protein inta~ con le:od to defective lipoprot~in met.boli,m .nd hep"tic lipidooi •. OTHER ASSESSMENTS OF LIPIDS I. Lipoprotein d«tropho=i. A. Lipoprotein d«trophor~. is h .. !>ttn u...! '" an .djunct qualitativ~ ass~.. ment of lipoprotein abnormalities in dinical veterinary .amples. However. methods V'ry. and ten availobility .nd interpretive npeni"" .r~ limited. The following i. a brief JUmmaty of th~ procMU": l. EDTA pl...na {or .. rum} is applied to a gd {umally :.garost}. 2 An d«tric current cou ... lipoprot~ins to migrate through the gel b:ued on th~ir d u rge •• iu. shape. and inte..ction with the gel. 3. After "'P.mion. lipid stains.", .pplied to vi,ualize the lipoprotein bands. which are named according to .. rum a- .nd ~.protein band. of cor... ponding mobility. a. Chylomicrons. if p.... m. remain at the applicotion origin. b. HOL molecules migr.te fimhen toward the .node la-bands). c. LOl .nd VLOl molecules migr.te intermediately {~. or pre.p.b.ndsj. 4. The gel .hould bt namined qualiutivdy for dyslipoproteinemic pmerns. In dogs. incr ......! oc,.mig.. ting lipoproteins .'" associated with hypercholesterolemia. and iner ......! p",.p.band •• p.bands. or applicotion .it. band. are '!JOCiated with h ypertr igl J=ridemia." B. Although gtl banding patterns can provide useful information about lipoprotein .ubfractions. bands ... not coII!istently :wociated with certain daISeS oflipoproteill! '" defined by their densities. Therefore. lipoprotein electrophoresis is most u..ful when combined with dell!ity fractionation studies or to monitor ... pon"" to therapy. II. Nonesterified fatty .cids {NEFA, &tt fatty acids •• nd fatty ""ids} A. Physiologic processes l. N EFA. and glycerol form from hydrolysis of triacylglycerol in adipose tissue. li""r. and mammary gland. 2 Hydrolysis in :odipocyte< is mediated by hormone ... nsitive lip .... which it stimuloted byepiMphrine .nd glucagon. Insulin i. inhibitory. 179 16 / LIPIDS hydrol)"is, NEFAs >r~ rel ....sN to blood, whr~ transportal to othjor t .. ~ organs ~r~ mu.d~ and livmm~nda! term is fotfJ adds. b. Units: mEqIL X I = mmol/L 2. £ample a. ~rum or plasma from EDTA_anticoapdata! blood (not hq>:uin or coll,"",ion from hq>arinizal p.tienu) mould be usN." b. ~rum or plasma should be "'p"rata! from ctlh as won as pos.t cool, or if "sting of sampl.. kept at 4 °C is ddaya! bqond 24 h." c. Significlflt fal.. inc",ast's also occur with moderat~ to !JeVtre hemolysi .... d. For monitoring prq>anum cotde, sample. should k coll,"",a! shordy befo.. f.aling time .nd 2- 14 d kfor~ calving. Th"}' con k processa! and stora! frozen until calving occurs, at which time it will k known wh~hre usually ousessed in ruminanu, particularly to monitor transition rows in the last 1- 2 wk prq>artum, when the .. are high energy demands for the f~us and milk production {Ott H< ar~ in a negati", energy halan""" and man"l:ellYnt changt. are n.ala!. 3. Aft~r 700 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY {2} InC,,",asffl pr~panum [NEFA] is associatffl with rno .. po'{p,muri~nt ketosis, fmy Ii"", and di,pJ.cffi abomamms. c. [NEFA] Im}' also bt incr~~=' with diaktes mdlitu., h~p"tic lipidosi., onrf=!_ ing and ol>.,,;ty. food d~privation. and :oft~r ~"'rcist. Ill. Standing plasma uS! for chylomicrons (aho call'"'" ...fril:"ration t~n) A. 8«;oUst of th~ir high TG runten! .nd thul low density, chylomicron, will float to the 1U'&", of a strum or plasm. umple if th. sample is undisturbal. B. A 16 h- standing test has bttn Usffl to det<'CI chylomicron •. [n th. p~ur" 2 mL of plasma is placal in a small glass tub. .nd then n.minal aft" 16 h of r.fr~ra{ion at 4°C. l. Th. P'"'''''''' of. cr.... my layer on the pWIlliI sample indicates the pr=nu of chyJomicrom. Chylomicron> indian. ponpnndi:d hYJl<'rlipid.mia (which could bt pathologic jf jt r'pr=m, dday.d draring). 2. Turbidity ~low or withour • surf= c"'~my lay~r suggests th.t olh~r <:au.., of hYl"'rlipidemi<> m~y n=i to ~ ronsid~ral. C. How"""r. th~ 16 h- ,tanding t~st hal a rd.rivdy poor C>l"'bility of detecting chylomi_ cron, wh~n roml"'ral to lipoprOl~in d=rophoresis and uh ..""ntrifugalion. In on~ nudy. only .bout 20 % of th~ chylomicron_po,itive sampl.. (a, det=a1 by d«tropho_ resi,) had a po.iti", ]6 h- nanding test." Similar result. w~'" found wh~n th~ ,tanding un was romparal to ult ..""ntrifugation." Th~ pr~"'n"" of. c",.my lay~r in the nanding Irst i•• p«ific for chylomicron" but many .. mpl~, th>t ronlain chylomicron, do not have a detectable lipid lay~r. References IJ. 1999. Lipid •• ~P"P""'tt<"~pid'mia •. In, Ettin~ 5J. Fddman E.C. cd •. T,xti-/t .(y,,,......, I""'""'" M.Ji';_.. [)U".", .jlln D., • ..d c., Sob odi';"". 28.J-292. l'biJ.Jdph.i .. WB Sawo-. hl odition. 8.J-II S. s.., Acad.mic. G..,.. Kt.. WcddoiDd KJ. CowdJ CS. Sdoonob ... WD. Jnvdl DE.. Zki .. SC. Dd>c...kd", J. F...,. RA. 2000. N."","". [0' H...d MS. notd." CD. Rrmilt..d RL. ~ P. cd •. S-Ilkm../Cs..;,,J N"",·';"' . 4d. cdition. 21_ 107. T opeb.. KS, Mad. Mo .... Inrtitut<. W.... n nx;. s...;, J. I?9J. Lipopo<>t<"~pid .. mia in tIoo do, ...d ca" A...;..... J Small Anim Pract j.(,.j79--487. !laOn.ky Mt.. 1981. M,. .",m,o, of ~p<>I,,<>tttdb...r; GD. I"" .... C. RadvJ.KU G. 1986. N_....J thl" n.., SI ""ito..., b.. ,. J Am Mod Aoooc t. Rio..; N . !l.cl.o.ik!'S. Al ..... 2. l. 4. S. 6. 7. 8. Di"", 2SS,2J29--UJ9. 9. Tiott NW• ..itb Dru.d", EL "kPh. .. "", RA. F.lum ... SA. <><10. 1m. CO",·,,J G..Nk .. ~.....-,. T_. J..! odition. Pbiladdphia, WB Sa."""' •. 10. YoW'!: DS. p..,,,,,, !.C. Gibb..m.., Y. 1975. Effirt. of druco on dinicollabo.....,. t..,~ Clio Cb"" 2h ID-(JW (opk .--...I.............,. in tIoo do.: ...d at. 5. .. 0, Yo< Mod (S.."ll Anim) S,8l-9l. 12. Fouum. lW. 1989. P-.in-looi"l!: ,n"""pathy. s.m.in Yo< Mcd S"'I (S.....u Anino) 4,219--22S. s..., 16/ LIPIDS 781 13. Unman MP. D.mt-h DM. V>potl.y io ",ft "",ted wt...., ... "..;.. " 122 "'"'" (1~.J.--I997) . I V.. lamo Med 14,68-.W. I... W,lIud MD. Holm .. G. FndkinJM. B.d.., T . Bro..o RM. J.n.i. Be. W",h BR. 2000. lalati...t "!'P' l.. ioou • ...dated with p«><""_Iooi"ll: ,o'''''I'''thr in tI., doc. I Vrtlnttm Med 1.. 0293--.107. IS. UI'ton SI. Kin, LG. z...b. CA. W>6. Gluoocor--I99S) . I Am V" MedA_ 21)9,2076--2081 . 16. P........ ME.. Kiottn PP. K.. PH . W>6. 1'."""._ dioiad...d Iabo"",,,. findio r in d.op..ru. k~_ ti.ci ..., 125 ""'" (1979--1993). I Am V" Med A_ 208,8\--91. 17. Hotbon B, do La Fatz< F. B...... IP. M."",lJP. 1987. H,p<"~pntti. in th, 1"0"11: popl')'. Pro< Soc E..t> Biol Mod 13h 18.J.--IBS. 19. P"";n LN. BrunuIJ ID. H • .....,.--Clark, C. Ptitdtatd PH. }on<. BR. H.,-dm. MR. 1990. Chu",,,.ization "f. tipop..-in lip... 01.... III typ< ""foo' io byploR)' of d.omdooI«to=laomi. io botiaod. io th, U.,;t«! Kio~ ... R.. Vrt Sci SOU(l_I! S. lJ. W"hito'Y MS. 1992. &aluation of b,palipid..niat in doc. and cat ~ S.",in v .. Mod Sour; (smaU Anino) 7,191r-300. 24. W"hito'Y MS. &00 GD. Rrb.u AH. J\o.d RB. 1987. Effect. of "'''''' pan<>",atiti. on drculatinc; lipid. in d.op. Am I V" R.t " ,10I91r-1497. 25. s",.h K. Catb.1lo I. Appnt HE. Ho....d 1.\1. 1973. looulin kvd. io 067-471 . 000«1, ina",•• pt.tm. l...d. of 26. Krauu RM. Grunfdod C. Donrt., WI. F<>Oi. _ "''' Jo.. cp«im ... tal d.oi< ... ~. in tit. doc. Clio a.im Ao .. 80,157_ 170. JO. Rid...d S. 11<,,'0 A. 1975. 1'1 ...... «><>ctp«im ... tal diabrt.. in doc •. m.b<'.", .«),16}O-16}9. 32. Fdodm ... EC. Ndooo RW. 1m. Hypothp<>idimL Gt,.;_,.,.,J F,' ", £"""""""'0 ,.,.,J &fr"'/"";-. 2nd oditioo. 68_ 117. P~iladdpl.ia , WB s"o"""' •. 33. Soott.MonaidfICR. GuptiH_Yo.." I., 2000. Hypotbyroiditm. 10, Ettio"" SI. Fddman EC. ed •. T,~ of V.............,.I""""'IM.JK;,." DY_ oft/" Dot ,.,.,J c.... StI. edition. 1419-1 "29. Pkil.ddpbi", WB Sawtd .... 34. .\1aonin& PI. 1979. nyroid rJ •..! ...d ut«i.J l"iono of b",~" . ".;tl. familial J.ypodt.yroidi .... and J.ypt HK. Mi" I. 1977. F"",ili.1 b,palipop«>tdn..nia in h<.p, •. Lifo s.; 2(),999-IOOS. 36. VaId"".noon S. H....-... P. Hco·Eh[, P. 1983. Rdatiooo 1.."""", tl.p<>id function. ~'patic ...d lipop"""" lip.t< occi,;ci"" ...d pl ..",. lipoprottt 782 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY o..llaart RPF, Ganon..ort RT. 5J ..... wI, ! oynt!.<.i., io "'" d .... ted in patit1ipi <. y" Q,S}21_SJ26. 46. /dJcon LB. Field JR. 1985. C"",nt ronc 49. SO. 51. 52. 53. 54. 55. 56. cat~ J V.. ]nt<m Mod .. , 192_ 201. KR. Sup"", [, M"""o RA. M~ AH. Sbip.",. /K. Do.."'" W. Dinudlo CA. GrunkId C. 1992. E.x!ot..';o "ridl, in<j"",. d.",V' in lipid mrtaboIiun .In' prod"", hyp dooa i .... ibi, d.acan«. J LJpid Reo H,[76s.-1776. G"1:E Re. Diamond A. Mondon CEo Rr.Y<><mal dop and iD dop with oD bo';D, no""otnilicd fatty odd and brt •. k~", oonctrati.oo. in blood. / Dai.r s.ci 88,llJ9-JI04. Stokol T. Nydam D . .1:005. Effect of """"lpi • .,.. btnin< t>OO...,,,,,,i6ed f.tty odd uod bota-bydro~,y... ", oonc<>pIoo",.;. oompued fo. ctioD of d..~,..itt., J.i&b .•.-J 1.1.0"'0., «ntrlfu", oompued ..ith. 16-h·.uodinc _ Iipop«>t = Chapter 17 THYROID FUNCTION Physiologic Processes ..............••..•......••......••.••......••........ Analytical Concepts .................•..•.......•..........•................ Thyroxine IT.) Concentrati on ......... •• . •• ...... •• ...... • .. •• ...... • •. .. .... I. ITotal Thyroxinel (tT,) ....... . •• .••..... . • •... .. . •. . •• .... . . •• ... .... A. Hyperthyroxemia ................................................ B. Hypothyroxemia.......................................••........ II. (Free Thyroxine].d ((fT.l ed ) • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • •••••••• 784 785 789 789 789 790 793 A. Increased IfT4111d (Free Thyroxine Concentration by Equilibrium Dialysis) ............................... ... . . • • •. ..... B. Decreased [IT.J.cI (Free Thyroxine Concentration by Equilibrium Dialysis) ............................................. Total Triiodothyronine (tTl!) and Free Triiodothyronine (fT 3 ) Concentrations ......... Thyroid-Stimulating Hormone (TSH) Concentration ............................. Autoantibodies ............................................................ I. Thyroglobulin Aut oantibodies (TgAA) ................... ... . . .. ... .... II. Thyroxine Autoantibod ies IT,AA) and Triiod othy ronine Autoantibod ies (T,AA) .............................................. III. Thyroid Peroxidase Autoantibod ies (rpAA) ............................. Response and Suppression Tests ............................................. I. Thyroid-Stimulating Hormone (TSH) Response Test ....... ... . . • • •. ..... II. Thyrotropin-Releasing Hormone (TRH) Response Test ..........•• . . . .... III. Triiodothyronine (T3) Suppression Test ................................ Total Thyroxine to Thyroid-Stimulating Hormone (tT, :TSH) and Free Thyroxine to Thyroid-Stimulating Hormone (fT,:TSH) Ratios in Dogs ............ Interpretation of Thyroid Hormone Concentrations and Profiles ................... 793 793 793 794 794 794 795 795 795 795 796 796 798 799 783 784 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY Table 17. I. Abb...,,,iatiom and symbols in (hi.. chapler [IT.J.. [xl Fret thrroxin~ ronctntralion by aJuilibrium dialf1i. Conctmration ofx {x = analytd IT, Fret IT, RIA SI T, T y\A T. T.AA TgAA TpAA TRH TSH IT, IT, WRI Fret thyroxine Radioimmuno:way Systtmc lnurnational d'Unites Triiodothyronine T riiodOlhyroninc autoamibodies Thyroxine Thyroxine autoantibodies Thyroglobulin . ut""ntibodi .. Thyroid ptroxj~ aut""ntibodi .. Thyrotropin_rd=ing hormone Thyroid_.tirnul.ting hormone {thyrotropin} Total triiodothyronine Total thyroxine Within .efuen"" inurval {rijodothrronin~ PHYSIOLOGIC PROCESSES I. Hormonal control of thyroid glands (Fig. l 7.1) A. TRH from the hypothalamus ,timulates the production and rd .... of pituitary TSH. Factors that inAu.net the =mion ofTRH .'" poorly understood. B. TSH binds to thyroid TSH rccq>tors and stimulm:. the productioll:md rd.~.., ofT. and T, from the thyroid gUilds. All circuutillg T. is produ=:! by the thyroid gum/s. but a minority ( 10-40 %) of circulatillg T, i. produoN by the thyroid gUilds.'''' Th. rest ofT, is gell.mal from T. ill cilyvariatiollS in [tT.] have bttn r<pon..d. a. III homes, the highest ooncan that there is not. pralicuble diurnal vari.tion, .lId there COlI be collfusing fluctu ations in random ha ..line [tT;.].' However, ba..d on • study of a """ll group of dogs. [tT.] was significamly lower .t 8 a.m. (m'~II, 1.75 J.lgldL) thall at II a.m., 2 p.m., 5 p.m., and 10 p.m. The highm me.1I [tT.] was.t 2 p.m. (3.54 J.lgldL).' A similar patt.rn (but with I. ... ch,"ge) was foulld in 8 a.m., 2 p.m., . lId 8 p.m... mpl ...' Thus, the tim. of sampling may illfluellC< the imerpretatioll of [tT;.]. Similar patt.rn. were =11 ill [IT.] .... 17 1THYROID FUN CTION 785 -- ' .•- -- ----- ~SH Blood -" .• -~ TJproI' a,n '. • fT, T,/proIoin_\ 6'- •. ~ 0'> ~~.~ fT, '. " , "\ ,, , , , , , , '. , "I-- ,'. Periph9ra1 ~" Thyroid glands Fig. 17. 1. Hormonal rontrol of thyroid glmd.. TRH ",le... d from the hypotbabmw "imuI:.,.", th. ",leas. of TSH from th. pituiwy. TSH .timub... the production and ",le... of T. rnd T J in pathW>J'"' involving thyroglobulin in the tbyroid glands. In blood. moot T,:and T, mo1 C. In blO<X!. most tT, ~nd tT, mol.cule! are protein bound. I. Nearly all T, rdeasffl from the thyroid glands becomes bound to plasma protein,."! F.ctors that influ.nce protein binding are not dearly esublish..d. [n dog, • • bout 99 % ofT, is bound to thyroid hormon .... binding globulin, mnstbyr.tin. albumin. and .polipoprotein,. The binding protein, in cats are not enablish..d. [n horses, T, i, bound to thyroid hormon.... binding globulin (61 %), T,_binding prralbumin (22 %), and albumin {17 %}. 2. T, not bound to plasma protein, i, call..d fT, and is the biologicolly active form of T,. fT, ent... most cell, of th. body and i, converted to eith.. T, or rev....... T, by th. action, of deiodinase eITzym ... ' Reve"'" T, is biologicaUy insignificant. 3. Like T ,. most T, (99 %) is protein bound {specific globulin. and albumin}. fT, is about 3-5 times as potent as fT,.' D. T, and T , dissociate fJom plasllU proteinl and enter ",lI" where they bind to rrceptors, induding nudrar r=ptor proteinl.' The hormone_rrceptor compln.. init;"" changes to cou.., or promote inereasffl m.tabolie rate, iner......d 0, conmmption, increased he. n rate, enhaned respon.., to cotMol. mines, bone formation .nd resorption, incre....d coubolism of muscle and ..dipo.. ti ......e, protein .ynthesis, .rythropoiesis, and alteration. in lipoprotein m.taboli,m. E. T, and T , .re ner"'..d in bile and urin •. ANALYTICAL CO NCErTS I. Terms .nd units A. Thyroxin. and triiodothyronine are commonly referred to as T, and T" resp«tivdy. It i, imporunt to different;"te tT, from fT,. B. Unit conversion' I. tT,: J.Ig1dL X 10 = nglmL; J.lgldL X 12.87 = nmollL; .nd nglmL X 1.287 = nmoUL {SI unit, ne~rest I nmoUL} 2. fT,: ngldL X 1287 = pmollL (Sf unit, nrarest I pmoUL) FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY '0; 3. T,: ngldL x 10 = pg/ruL; ngidL x 0.01536 = nmolfL; and pgldL X 15.4 = nmoJIL {SI unit, M~r~'{ 0.1 nmoUL} 4. TSH: J.Ig1dL X ]0 = ng/ru!.; 0.1.0 rq>On«i as mUlL (or J.IU1mL) 5. TI}'\A: positi"" or n. or P"=nt"1:~ of. known positi ... sample Sample A. Canine [IT..} is subl. in EDTA_plasma and , ..urn when B. C. D. E. III. n '{OrM in plastic rubes for 5 d at 25°C, 8 d at 22°C, and months at - 20°C; [IT.] inc,..,...d in .. rum sampl.. norM at 37°C for 5 d but ""main..,j stable in EDTA_pw/IU ,ampl~.'· Stability of conin. [IT.J (..rum or plasma) is ,imil •• in gl .... rub.. at 22°C and _20°C for 5 d: [IT.J inc",,~ when norM in gbs. tubes at 37°C for reawns th.t ."" not mabJishr stability is ap«tN in ,",u from oth.r sp<'Cies. Sc. bility of [IT.] is similar to that deocribffl fur [IT.]." AI for [IT.], [IT.] inc",,-.:l in both EDTA_plasm. and .. rum .. mpl~. storal in glass ar 37°C for 5 d. TSH and TgAA >r~ nabl~.r 4 °C for a fow ""ys. or long~r iffro",n. To reom rdiabl~ conclusion. about rho ondog~nous rhyroid stotm of an .nimal r~ving thyroid malicotion. a 4 wk malicotion wirhdrav..al ""riod is ra::ommondal prior to resting. Inn~~=' pl .. JruI [fanyacid] am mult in fal..,ly inc",=<:! [IT..] in i"'Opl~; th~ fatty acids di'pla"" T. 011 billding prot~ill'. Th~ illcr......:! [farry acid] and fal.dy illn~aKd [IT.] ha"" bttn .hown to occur wh~1I ""opl~ w..~ ginn o ith~r intrannous or sub_ cuC>neous h~p",in."·" Au.y principles and procalure! A. [tT..] l. [rT..] i. rho conc~nt,"rioll offrtt and boulld T •. 2. [rT..] i. usu ally m.... ural by RIA. but ~nzym~ immuno=ys {mzyme_linkal immunowrknt :assay alld m~milumin=nt ~nzym~ immulI~Y).'" also uKl hav~ all .nalytico.l ranI:" appropriat~ for hUJruln [tT..] .hould not bt usa! to evaluare amillo [tT..]. 4. In th~ common solid_ph= RIA sysr~m'. T# in ",rum will cou'" fal..,ly dev:nod me. sural [rT.] {Fig. [7.2). In less common :assay••• fal.dy low [tTJ will k musural if the =y d",rct •• ll antibody_bound radiol.klal T. (bound to ~ilh~r ~nt antibody or autoantibody). T gAA do not int~rf~", with the;., :ways. but ",ra rhat have TgAA may h."" T ,AA-17. 11 B. [IT.] l. Equilibrium dialysis a. [IT.] i. best mrasural by all :assay th.r involve! «Juilibrium dialysi •. In «Juilibrium di.lysi. proa 17 1THYROID FUNCTION 787 "'. ".,,:S 1noT~ l2 ' . @ "·"1 Dog 1 Y,. t, • ] t' lO -----------,-- •" withT~ Dog 2 '~ 't, Fig. 17.2. EifK:ts of T.AA on me .... rod ltTJ in a soJid·pb ... RIA. RIA.. >t< compS1>J'" in .. bich the p. tiont', T. rompmount of T1(liooc~ T, {,T,) fOr a ~mit«l numOO of ontibody.binding .ites (~.g .• in a tub< co. ted .. itb anti·T 4 antibodies). Tbus. a gr~~t .. con«ntr:ltion of p.ti~nt T, resul", in kss bound *T•. • In th. on.Jy.is of dog J', .. rum. i", Satod tub< (''''p a). During inrub.tion (''''P b). som< of the p. tiont T. and 'T, bind comp"ti~ly to :mti.T, antibodi .. on tb. tuOO: other T. mot.cuks r..",.in unbound. Af"'r inrub.tion. tl>. unbound T. is rinsed from the tube. and the bound T, ":OY> in tb. tub< (''''p c). no. tw,.·, ndioactivity;, tben mea.swt< :ad d). During inrub.tion. *T, binds to the anti.T. antibodies ro. ting the tube :md to tl>. ToM and thus J.s. *T. is bound to the tub< (''''P e). Aft.,. 'insing out T, not bound to the tub< (''''P I). the tube', r:odioactivity is m.,."u b. Anti. T. antilxxli~, do not int~rf~ .. with th~ <"<Juilibrium di:.!)"'i. assay b«:.use th.. antibodie, do not diffi..~ through the .. miperrn..able membrane. c. The major diwv:mug. of d .. ~rmining [IT.] by <"<Juilibrium dial)"'is is th. a pe""'. 2. Nondi:.!)"'i, IT. RlAs a. Nondi:.!)"'i, RIAl d ..ignrd for qU '88 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY b. In a romparison study of (h~ Nichols «\uilibrium di:dysj, =r and nin~ nondi:.!}"i. ~ using ",ra from euthyroid Jl"OpJ., the oonoordan"", ~r""'nul:"" vu;ffi from S9 % to 96 % when .. rum T._binding capacity w>s within th. ,d'",e""" interval, from 66 % to 90 % when .. rum T . _binding cap:ocity incr da:,easni "'''Y: Result. from a ch.milumine=nct "'''y for [IT.J w... low.. th an =ulu from the «\uilibrium di:dysis =y. Th~ w". about 50 % lowe, for e;>t ..n." Th. degr« of diff.",na was not ,eport"! for dog .. r.. '" C. [tT,) and [IT,] l. (rl,n.ralJy. the sam. basic principl.. for T , .....y. (tT, and IT,) apply to T, ItT, and IT,} assays. Unlik~ tT, assay•• assay. designed for mnsuring [tT,] in hUllUn set:> han .d"luat~ .nalyrical ran~ to m~asur~ [tT,] in dogs :md cats. 2. RIAs ha"" the analytical sp«ificity to diff~"'ntiat~ T , .nd T , mol«ules. Ba;;>...., of low.. ron""mt:>tiono. assay. that m.."".. [t T,] .. nd to be, less rq>roducible than ..... Y" that m~.JU'" [tT,]. D. TSH l. Canin~ TSH a. Th~ .. a", romm ..cial .... Y" {immunoradiom",ric .nd chemilumin=nt immunometricl for canine [TSH] . but it is not known wh"h .. they det«t aU glycosylated form. ofTSH." The canine TSH :ways lack ""fficiem .nalytical range to enable documentation of d«r...... d [TSH] .... The", WlI.I good agrttment betw«n two immuno.... Y" when the measured [TSH] w:as "" 0.5 ng/ml :md fair :agr..m~nt when the [TSH] w:as betw«n 0.1 .nd 0.5 nglmL ,.. b. The ",fe",nce interval, for canine [TSH] and the m",hod. uK E. TgM l. A rommercialenzym~ immuno .... y {EIAI for TgAA v..as developed during the 1990s {produced by Oxford Biomedical R..~.rch}. Prior to its availability. the .. w:as 17 1THYROID FUN CTION 789 a lack of =y standarda.tion. Thus. results from diff,,~m l. boratori.. wer~ difficult to im~rpret. Another EIA for TgAA w:" devdopffl. :md its results wer~ in good agretm~m with th~ oomm~rcw :way." 2. For th~ oomm~rcial :assay. TgAA results ..~ rqrortrd as • Jl<'=m~ of th~ r~.ult obtainrd for. known positi"" sampl~. with Jl<'r""m"!:,,s .bo"" • ""n.in drosion th ... hold oollSid"rd positi"" for TgAA. Prior to 2005 • • positiv~ ",.uh w.. . n optical d~lISity v:.tu~ ~at" than 2 st.ndard deviatioru .bo"" th~ optical d~lISity v:llue in. n.-gati"" oomrol .. rum. 3. As with any diagnonic ....y... rablishi"l: th~ d«i.ion threshold that s~parat .. positi"" .nd n.-gati"" r~sults .ff«ts th~ diagnostic s~ruitivity and sp«ificity of the ..... y. A low~r d« ision th ... hold for a TgAA .. mit ltd to f=iu~m f:.t .. positive.. A high~r d.-cision th ....hold would cau .. mor~ faJ....nq:.tiv~ r.. ults. 4. Bw l.U.. nomp«ific binding of immunoglobuliru to ..... y pl.t~. can f:.t ..ly incrra.. T gAA values. {esting with and without """"ing :md subtracting the norup«ific binding yields diff~",m ... ults tmot mun b. im~rpr<'ttd with diff~rem d«ision thr.. holds. Som~ laboratori.. h."" l.b.ltd th~:assay , TgAA {.p«ific TgAA} to differ.mi .t~ from NSB TgAA (nomp«ific binding TgAA) whennorup«ifi" binding is assesstd and excludtd from th~ r.. ult. F. T oAA and T,M: AssaY' for T oAA .nd T,AA h."" bttn u5«l in sp«w laboratories for /ltany yrars. Th~ diagnonic ..",itivity of th. T gAA values for Cllllin~ lymphocytic thyroiditi. i. gr~.ur th:m diagnostic s.ruitivity valu~s ofT.AA .nd T ,AA.'u, but the.. =)" help id~milY f.l .. T, .nd T, .. mit. causal by im.&ren"" from ToM and T # G. TpM: Canin~ TpAA mov. bttn d<'t<"Cttd in .. ra by mi"l:.n immunoblot :assay." THYROXINE (T,) CONCENTRATION I. [Total thyroxin~] {[tTol l A. Hypenhyroumia (Tabl~ 17.2) l. Inc",ased production ofT, by thyroid neoplasia a. Thyroid adeno/lta {or "ad~nomatO\ls thyroid hyJl<'rplasia"} in cats {I} Wh~n pr..emtd with th~ clinical signs of hyJl<'rthyroidi,m • • bout 90 % of hyperthyroid caU wiU h."" increased [tT,]." Th. moJi common pr... ming problems includ~ w.ight loss .nd polyphagia. Others include alopecia. polyuria. polydipsia. :md nomp«ifi" g.moimestinal or n~rvou.s syst~m findings. uboratory data may includ~ th~ following: erythrocyto.is: Tahle 17.2. 0; ....... and condition. that a u... hypenhyroxemia {increased [IT,]} lm:r... td production by thyroid neoplasia "Thyroid ad.noma (common in cats. uncommon in docs. rare in ho .... ) Thyroid arcinoma (dog•• cats. horses) Muhipl~ endocrine neoplasia {type II} Administration ofT•• TSH. or TRH Administration of compounds comaining iodid~ • R.htivoly common disc..., or condition Note: Owing di•.nrus ""d p"'gnancy. <S lun g<'~"" [tT.I 790 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY incr.,.snl alanin~ tr>n {2} [tT,.] m.y Huctuate sufficiently in hYJl"nhyroid cm so th at th~ [tT,] may be WRI on. random .. mple," Also, nonthyroidaJ jJJn~ss may low~r the [tT,] in hYJl"rthyroid aUS 50 that the [tT,J it WRL" A T , suppression te'lt may ronfirm the presence of hy""rfunctional thyroid ti .. u~, .nd a SIl~uem [t T,.] may be increasN, {3} Mutations in the TSH rraptor causing ronnituti"" activation and thyroid hormon~ production m.yalll" hypenhyroidism in cat .. " {4} Thyroid carcinomas ha"" l>ttn reponed to occur in .bout 2 % of hYJl"nhyroid m(S," b, Thyroid ad~noma or .denocarcinoma in dogs; About 25 % of canine thyroid neoplasm. produce sufficient T , to muse hyperthyroxemia and diniml .igns of hypenhyroidism, I c, Thyroid ad~noma or adenocarcinoma in hors..; Hypenhyroxemia is rardy reponed in horm with thyroid neoplasia, In one ho"",,, the [tT,J ~ about doubl~ the upper reference limil," d Multiple endocrine neoplasia {type II} in dogs; Thi. rare dilOrder involns neoplasia of APUD (amine p=:ursor uptak~ and decarboxylation) ceUs and is characterized by thyroid medullary ao.rcinoma, ph~romocytoma, .nd p:mnhy_ roid hyperplasia or neoplasia," 2, Administration ofT" TSH, or TRH 3, Administration of rompound. comaining iodide; Hor..s may have temporarily incr.,.snl [t T,.] beau"" of inn~ased production ofT, .fter ""posur~ to iodide in ""!"'Ctorant!, coumerirritant!, comrast medi., leg paints, or .hampoos,' 4, T"AA may cau.~ po.iti"" imerference in some tT, :mays {Fig, 17,2}, B, Hypothyroxemia (Table 17.3) L D«re...,d production ofT, a, Primary hypothyroidism (c;ousnl by thyroid gland dis.,. .. ) {I} Primary hypothyroidism is a common di""rder in dogs, {2} The most common pre""ming problems indude weight gain, lethargy, .nd symmetric :dopec;" The more common .bnormallaboratory data indude mild anemia {nonrq:<'neratin}, rodocytosis, hYJl"n:hol~st~rolemia , and hy""rtr igl yceridemia, {3} A b=.J [tT,J i. the nandard ",r..,ning ust fur hypothyroidism in dogs, D«re...d value. support hypothyroidism, but ther~ .re "",~ral ruwns for hypothyroxemia other than hypothyroidism, Converuly, it i. very unlikely that a dog with a [tT ,] th at is WRI has primary hypothyroidism (if the [tT,.] is valid; I t t Fig, 17,2), {4} Lymphocytic thyroiditis, considered an inheritabl~ .utoimmune disorder in many dog breeds, is a rommon cam" of thyroid gland denruaion .nd primary hypothyroidism in dogs {at least 50 % of =)," Th~ damagrd thyroid gland exposes .mig~ns from immunologically privileged siw 50 that the immune 'Y't~m produce, auto.ntilxxlie'l such as TgAA, T M T,AA, and TpAA. Idiopathic thyroid gl and atrophy i, another rommon finding in dogs with hypothyroidi,m, It may be the termin:d stago' of lymphocytic thyroiditi" 17 1THYROID FUN CTION 791 Table 17.3. Di.eaoelO and conditions thar cau"" hrporhr roICmia (decreased [tT,1l ~er~a lffl production of T, 'Primary hypothyroidism: lymphocytic thyroidit"" idiop.thic thyroid atrophy, congenital thyroid gland dR.nesis, d.muction of thyroid gund lnwpwia, "'-'rgtry, ndi"'ctin iodid~ tr.. tm~ms, or other m..",) &rondary hypothyroidism: TSH ddici~ncy c;ouKuphala (eml~) • A ..hti""ly <X>JIUDOn d...... or oondition Not<: Hr.lthy hrge.broed rnd modium_brerd dog, t (5) Thyroid nwpu,i . may destroy .nough functional follicular edls to ern.. a hypothyroid stat., Conversely, old, untrnted, hypothyroid beagles had a rd . tivdy high incidence of drvc:loping thyroid nwpl .. ia, pos.!ibly bcco"", of chronic TSH stimulation,'" (6} Thyroid gl.nd, c;on also b.: damaged during "'re'ry, during radi~tive: iodide t",. tm.ms, and by other phY"ical mean., (7) Con~nital thyroid gund dy.genesi, with hypothyroidism has bttn reported in dogs,'" (8) Nongoitrou. hypothyroidi.m was found in two Scottish dttrhound puppies, Th~ con~nital disorder was c;ouKtions, . hypothyroid kitt.n may have [tT,] within the .dult ",frrene<: imrrvalY b. &rondary hypothyroidism (em=' by TSH deficiency) is on~ form of c.ntral hypothyroidism. (I) Ra .. cases cau..d by pituitary neopwia or malformation are reponed in dogs and c;ots. I t may also occur bcco"", of pituitary gund damage: caused by surl:"ry, radiation, or trauma.'" (2) Thyroid nwpu,ms may producr abnormal hormon .. that do not stimulate physiologic r"pon .., (th.refor~ hypc:nhyroidi,m does not develop) but do suppress TSH rd .... (th.refore hypothyroidism devdoJ>l)." 792 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY {3} Lock of TSH ',",,"'Iioll caus.. atrophy of thyroidal follicular cdb and thus drc,,,,,sal T. and T, production. c. T~r{",ry hypothyroidi.m {caUoN by TRH ddiciency} (on. form of umral hypothyroidism): Th. alImO," are not awn, of documemM cas •• in domestic mammals. d. D.f«tin T. production {I} In dog., iodin. org.nification d.fecr'" or roDl:"niral thyroid p,",oxidase drficiency in toy fox urri",;"" {2} [n foal" iodin. ddici.ncy from drctrasN dietary incako {fool :md dam}" {3} D.frctin thyroglobulin synth .. is in M ..ino ,h, Dutch goats, .nd mj"", prod"""d hypothyroidism. How"""., d.frctive thyroglobulin synthesis in Afribn.r (Afrikand,,) cattl. and Bongo .ntdo"" produaJ euthyroid con~nital goit"';'" 2. Multifactori.l {may include d.crUsffl T. production} or unknown ma:runisms a. Nonthyroidal disrases {I} '\hny infLommatory, neoplastic, metabolic (e.g. , rrnal f. ilurr), and endocrine di",rde", {.. p«ially hy~radrrnoconicisml a", known to d",,,,= [tT..]. Sick euthyroidism {ru from Grttk, meaning · w.U"} is th. condition in which a di,.a", outside of the thyroid hormonal .ynem cr•• t.. hypothyrox.mia. {2} Nonthyroidal di,....ses =y lo~r [tT..] by on. or mo .. of the following processe" (a) O«rra..ffl protein_bound T. beau.. of d"'r~ concentration or affinity of binding proteins {b} Inhibition ofTSH ''''rHion (c) Inhibition ofT. production {2} Ass=ment ofTSH and [IT.] .. =y hdp diff... nt iat. nonthyroidal illness from hypothyroidism when [tT..] is d",,,,....:! in dog. with clinicol signs of hypothyroidism. b. Many drug> (esp«ially glucocorticoi.u) a", known to low.. [tT.] . {I} Dog. with hy~radrenoronicism f=iu.ntly h. vt decreased [tT..]," but changes in [tT.] in dogs receiving glucoconicoid th.rapy a.. variable. Th. [tT..] did not changt in one study,'" wh .....s the [tT.] dec",....:! in .noth.. study.' Th... is evidence that the changes in [tT.] are COU..ffl by multiple f. cton, induding reduced TSH KCrHion," and th at the thyroid gl and. are Ie .. responsivt to TSH." Th..e i, also evid.nce th at glucocorticoid. reduce the oonvtrsion ofT. to T , in ~riph.ral tissues." [ncr~ [TSH] is not ap«tni {2} [n dog., prednisolone,' trim.thoprim_sulfadiazin. ," trimethoprim_ sulfamethoxazol.," and phenobarbiuP'" treatm.nts havt bttn shown to couse hypothyrox.mia. Sulfonamides int.rf... with the iodination of thyroid hormones by inhibiting thyroid ~roxidast- activity,'''' so [TSH] =y bt incr ... sed. {3} [n horses, phenylbutazone and glucoconiooid trratments ru.vt b..n shown to cou.. hypothyroxemia." {4} Clomipramine (ClomiCalm) administered to dog> for n... rly 4 mo d",rr....:! [tT..] and [IT..] ... Th. dec ...... in [tT.] was detectable .ft.. I mo of treat_ m.m, penisted for the duration of the aperiment, but did not drop bdow 17 1THYROID FUNCTION 793 the low.. r,f... nce limit." Clomipnmin. is usa! for. v. ri"y of bdt. vioral disord .... c. Th. following h.ve bttn mown to produce low.. [tTJ in horses: (I) Diets high in . nergy, protein. copp'r, or rinc {2} Ingestion of endophyt._inf«1ed f.",ue grass" {3} Food d.priv.rion for 4 d (m • • n [t T J decr",,""" from 19.9 nmollL to 7.6 nmollL)" d. C.nle fed foliage from uurJlma !tu~«pha'" (rommon ruome", mind. trtt, ipil_ipiJ. uazin. pje, and kub.bul.) developed hypothyroxem", .nd hypothyroidism." 3. c.n:ain dog brttds t.nd to h.ve lower [tTJ th.n oth.. breeds. In one study, the m•• n [tTJ in !.rge_breed and medium_breed dogs v..as about 0.5 j.lg!dL low.. than the [tTJ in the .mall_breed dogs." Grryhounds, . nd pouibly dogs in oth.. sight_ hound brttds. ha"" low....f..ence values for [tTJ . nd [IT.] than most oth.. dogs." Th. di"i:nosis of hypothyroidism in sighthound dogs may ..qui .. d. u oth.. than [tT.] {e.g., increased [TSH]}. Unpublished dau for dogs in [h •• ighthound brttds indico" that diff... nt ..f... nce interv.ls are need.d for [tTJ and [ITJ. but not for [tT,] or [TSH]. [I. [F= thyroxin.].. ([ITJ .. ) A. [nc... ...:! [ITJ .. (frtt thyroxin. roncentmion by aJuilibrium dialy.is) I. Th...me disorders that couse • truly increased [tT.] (Table 17.2) a.. apectal to ao.use a concurrently incr=d [IT.]... About 98 % of hyp'nhyroid cots haY< incr",,""" [IT.] .. when tested." 2. [IT.].. does not al"",)" mirror [tT.] . a. A hor.. with diniao.l hyperthyroidism h. d a normal [tTJ but an inc... ...:! [IT.]...... b. Concurrently finding decr",,""" [tT.] and iner..""" [ITJ .. in cm suW'u the p.... nce of. sick .... thyroid state." For unknown ",asons, sick ao.ts occasioruolly ha"" an increased [IT.].. but • [t T J that i. WRI. 3. T,.AA may com. p",itiv. int..f... nce in some IT. as",ys. but ""lues for [IT.].. a.. not .ffeclal. B. Dec..ased [ITJ .... (f= thyroxin. concentration by aJuilibrium dialysis) I. Thyroidal disord.rs that couse decrnsed [tT.] by dec",.,ing production ofT. are apeclal to have concurrently dec",.,ed [IT.] ... 2. Oth.. disord ... or ronditions that =y couse dec......d [ITJ .. include hYP'",d .. noronici,m, infLommation (interleukin I, interl.... kin 2, int..f.ron." and tumor necrosis f. ctor m.y cou.. decr=ed [ITJ..,), and """tmenU with prednisolone. sulfon. mid... or phenobarbiul. 3. Dec..ased [fT.] .. was found in horus with arerim.nully induced hypothyroidism and in ho .... with • v. ri.ty of illne,s.. {especially with s",ere disorders}.'" TOTAL TRI[ODOTHYRON[NE (tB) AND FREE TRIIODOTHYRON[NE (fT3) CONCENTRATIONS I. B.,al [IT,] .nd [IT,] ...... ronv...ion ofT. to T, but have less diagnonic .. n.itivity for hypothyroidism .nd provid.linl. u..ful additioruol inform.tion 0"" [tT.] . nd [IT.]. In dog., diurnal vari.rions in [tT,] we .. less th.n the variation. found in [tT.].' 794 II. FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY ItT J is m=urrd to mOllitor T, supp.... ioll test. th~npeutic OOllctlllr>tiollJ alld to asses.! oWII~r compli.llct ill th~ III. If a ",Iid.ph ... RIA is u..d to mum", ItT,]. th~1I T,AA call cau.. wOllmusly illcrea..d values just lik~ T.AA in tT. assays (Fig. 17.2). [II some =)'1. T,AA f:.!.dy da: ...... [tT,] alld f:'!sdy illcr ...... [IT,]. THYROID.STIMUU.TlNG HORMONE (fS H) CONCENTRATION I. Dogs A. [lIn~~..d emilie [TSH] I. Primary hypothyroidism: Lack of lI~tiw fttdb.od: b.-caus<: of d==rd [IT.] .nd da:r ......d [IT,] should illcr~~s<: pituitary .ynth"is:md rd~= ofTSH alld thus cous< all illcr~.sffl [TSH]. Studi" illdiem that .bout 60-75 % of hypothyroid dogs haw illc.......d [TSH ]."·" 2. Compellsatrd hypothyroidism: It appears that som~ dogs with thyroiditis call m.illlaill [tT.] or [tT ,] WRI but ollly with gtt.t~r nimul>lioll of ",maillillg thyroid ctUS by TSH. 3. [TSH] is apectM to incr~ase durillg th~ TRH respollS<: te" (Ott Respoll" and Suppressioll Tests, Ket. II). B. Ba:.u.. [TSH] in ruthyroid dogs COli b.: at th~ d~t«tioll limit of th~ collill~ TSH """y . • da:r.asM [TSH] COlI!lOt b.: rdiably diff~"lIIi.trd from. [TSH] that i. WRI. Th...fo",. [TSH] will b.: WRI ill dog. with .a:ondary hypothyroidi,m. The hypothyroid dogs without illcrUsffl [TSH] may haw S [I. C~ts A. By usillg the CIDin~ TSH :assay to .ttempt to m.... su'" fdin~ TSH. th~ fc:lin~ [TSH] ill 50 h.... hhy cots was 0.00-0.32 lIg1ml {1I0t~ collille T SH u..d as . tandards).'" Inc",asrd conctlllr>tiollS of fdille [TSH] we", foulld ill cots ~fter thyroida:tomies or ~ft.. chrollic methimazole therapy. B=u.le the low~r ..ferellct limit is 0.00 lIg1mL, this =y COlIlIOt documelll da:",asrd fdille [TSH]. B. [f there is 1I0t 100 % cross.r ....ctivity b.:t"....,11 collille :md fdille TSH. then th~ u.. of purifird fdille TSH in th~ assay would product differelll results. How~~r. the .""lyti. c:.! illaccuracy would b.: rdlectrd ill th~ .. f.r~lIce illl~rva.l. so interpretatiolls may 1I0t b.: affectrd. III. Horses: Equille [TSH] was illcr.asM ill hOne< afi~r ""periment:.! productioll of hypo thy. roidism and as a =po"" to TRH wmillistratioll!' Additiollalnudi., .'" lleeclrd to ....." th~ di.gnonic valu~ of [TSH] ill hor=. AUTOANTIBODIES I. Thyroglobulill .utoantibodies (fgAA) A. Th. reponrd prevalellct ofTgAA ill dog. with hypothyroidism varies. but the perctlll. "l:es ..~ rdativdy large; that is. 36 % of 42 dog?" and 55 % of 31 dogs." With markrd 17 1THYROID FUN CTION 795 thyroid atrophy or .nd_s~ lymphocytic thyroiditi" th. amount of circulating TgAA Jruly diminish." B. In one study, ..Jleight dogs with a positive TgAA remit had lymphocytic thyroiditi •." TgM probably forrn when thyroglobulin is rdeasffl from a d:nn~ thyroid glmd:md th. body rracts to the "foreign" protein. A positivt TgM ","ult is evidence of thyroid di ...... and not eviden"e of thyroid dysfunction. C. Predictive v..Ju .. ofTgAA results for thyroid:d di ...... ru.vt varied among invmig. tors, at !.a,t paniaUy beau.. of different rne"thod. of .. ublishing ~ decision threshold betWttn positive and nrg. tive rractions.""" Positi"" result.~ .. .I"" found in dogs with hypuc:dcemia, hypoc:dcemia, hypoadrenoconicism, hyperadrenoconicism, or diabet.. mdlitus. It was not d.terrnined whether the positivt ..actions ~re true or f~l .. _po. itive results in th . .. """, •. n [I. Thyroxine autoontibodi.. (T.AA) and triiodothyronine autoantibodies (T,AA) A. M.ny dogs with lymphocytic thyroiditis:dso ru."" aut""ntibodi.. ag.inst T. (T.AA ) and T, {T,AA}. In a nudy of nearly 300,000 dogs with clinicol signs of hypothyroid_ i,m, thyroid hormone autoantibodies we .. found in about 6 % of the dogs' .. ra." B. When autoantibodies .re detected, TgM ... more cornmon th . n either T.AA or T,AA. However, £orne dogs ha"" a negative TgM result .nd a positive T oAA or T,AA result." III. Thyroid peroxidase .utoantibodies {TpM} A. Thyroid peroxida.. an:dyus the iodination of tyrosine rnolecules that are atuched to thyroglobulin in thyroidal roJJoid. The iodination leads to the forrnation ofT, and T, bound to thyroglobulin. Th. hormones are deavffi from the thyroglobulin in thyroid epithdi. l cdls and ar. r.leased to blood. B. The diagfi05tic v:due of the conine TpAA ..... y h., not betn esublished. In canine .. ra that rontained TgM, T,AA, or T,AA, 17 % conuined TpAA. " TpAA may be involvffi in the p.thogenesis of lymphocytic thyroiditis, but there ~r• ..Jso other propmed theories for the imrnun~mediated destruction of thyrocyt ... 14 RESPONSE AND SUPPRESSION TESTS I. Thyroid_nimulating hormone (TSH ) =ponse test A. Poor or in:adequat. ""pon .. to TSH stirnulation WlI! the gold standard for establishing thyroid hypoplasia (hypothyroidism) for rnany }..,~rs. Beau", an .pproved pharJrulco_ logic form of bovine TSH is no longer ~vail .ble, the TSH ... porue test is rardy used for dinic:d invtnig.tions. Rrcombinant hurnan TSH is . vailabl. but i, apensivt ." Ba!ic aspects of the TSH respome t.. t are provided to :win in the understanding of publi,hed .. pons on ""nine hypothyroidism. B. A TSH respon .. test ....sses the ability of the thyroid gland to produce T, .fter being stimulated by TSH. Sev.ral protocols h. vt betn recomrnended. The .mount of administered TSH and the sample collection times v. ry among fdine, canine, .nd equine protocols. C. Exarnple of a conine TSH ... porue test l. Procedure: A blood sample for a bas.! [tT,] is collected, bovine TSH is :administered int..""nomly {O.l unitslkg body ~ight with a maximum of 5 units}, . nd a blood sample is collected 6 h after TSH adrninistration. ". FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY 2 [nt~rpr~ta{ioll guiddines (.,.~aI v:dues moy "'lfy among a=p)" a. Euthyroid dogs {I} Basal [IT.] in huhhy doC' is npa:ud to b. 1.0--4.0 J.lgldL In nonthyroidol illn""", it may be, < 1.0 j.lgldL. {2} POSI_TSH [IT.] :> 3.0 J.lgldl b. Bord~rlin~ results: pos{_TSH [{T.] = 1.5-3.0 J.l{:ldL c. Hypothyroid dogl: b",,", .nd post_TSH [{T.] < 1.5 IlgidL n. Thyrotropin_rdn,ing hormon~ {TRH) =1"'0" t~n A. Th~ TRH r~'ponst em indirectly = thyroid function by c;;ousiIll: Ih~ ,d"".. of TSH ,hat ,rimula, .. rd~= ofT,. B. In dogs, th~ TRH "',,')fist test is not as good as th~ TSH respon .. em in d'Heling hypothyroidism. " ;" Some euthyroid dog> (as cl."ifiaj by the TSH respon .. em and din;",] signs) . ppta. to b. hypothyroid by Ih. TRH "'pons. un {positi"" p='iccive ",,]ue of 50 % }." Th" TRH uspon .. test d"." not reliably diff... ntittn us..!!, l. [n h~nhyroid em. TRH is a pact"! to han littl~ or no df'""t on TSH and [tT.] ba:."", of f=:lb.ck inhibition on the pituitary gland. Serum [tT.] in a h~..!thy euthyroid cot is apffi..! to double in the JlOst-TRH sampJ.. 2. AdV:llltag.. of the TRH respon'" test o ... r the T , supp.... ion test are that it is shoner (4 h ..."'us 3 d) .nd does not " III. Triiodothyronine (T,) supp=sion test A. Cats l. Mo" cats with dinical hyperthyroidism h. ... hypenhyronmia. :md thm additio""l diagnostic testing is not n=:l..!. However. some cots with hypenhyroidism han. b",..! [tT.] that is WRI or only slightly incr~",..!. perhaps ~cau .. of variable secretion of T. from hyperfunction"! thyroid glands. a nonthyroidaJ dis ..... that is lowering [tT.] . or other r.... sons. For the...nim.l.. a T, lupp",uion test mould diff... nt;"t. the hyperthyroid cou {those with d.f«:tive hypotlt.lamu ... pituiury_ thyroid regulation} from .... thyroid cats. · U1 2. Basics of the proc..dute a. ColJ'""t .. rum for bas.ol [tT.] .nd basal [tT,] :md fr= it for submission with later .. rum ",mpl.,. b. Own.", :Wmini".. T, Oiothyronine) per ot narting the next morning at 251lg q8h for 2 d. c. W ithin 2-4 h of the fin"! and seventh dosage ofT,. th. cat i, return"! to the dinic •• nd ",rum is oolJ,""tal for post_T, [tTJ and [tT,]. d. Bas..! and post _T, .. ra a.. submitt~d to a laboratory for determination of [tT.] .nd [tTJ. 17 1THYROID FUNCTION 797 Basal [tT4[ "~ '"00 00 "ro ," ~ " "" " £ ~ e Hyper_ Ihyroidiom Olho, di5Ol'dono Hypo<_ thyroidiom 0Ih di.orders Fig. 17.~. T, supp.<ssion t .. t ."ul", in cots; ~othyroni ... , 25)1g p« "" q8h for """,n d""", In co'" .ntb hyp'r dilO."" .. , tbe [.T, I in p""-T, umpLes w>s <: 20 nmoUL Hypertbyroidi,m ~ ru.gn<»«l in n c, ''', ba..d on c~nical signs, palpabl. thyroid noduLes, high_no.JIUJ to incr~ [tT,I, and ""pons< to t",. """nt for hyperthyroid_ ism, Cats (n = 22) .. itb ot!>'r di",rde" had c~nical 'igns ruggesti"" of hyp.havionl di",rde .. , n,. K"JshtuJd a"" "'p.... nts th. [tTJ found in H dinic:oUy h.althy c. "', T!>. g.aph _ oonstruc"' 3, Exp«tM ... u1u Th~ pon_T, [tT,] mould k gluter th~n the basal [tT,], If it il not, th.n f~jJur~ of suppression m"}' k du~ to failur~ to oodminist~r T, to the cot, b, Imerpreution guiddin~' of post_T, [tT.] {Fig, J7,3}: Valu .. may v~ry with th~ ..uy and labo..tory, Uling an absolute decision threshold is ron'iderM a kmr method of int~rpreting results than using a p<=nt decreast,"" but at ltlSt a 50 % decreast in [tT.] typically excludes hypmhyroidi,m, {I} Post_T, [tT.] > 20 nmollL {> 1,6 JlgldL} indicote. a lack of suppre"ion of T, secretion and thu, supports th~ pr=n~ of a thyroid adenoma, {2} Post _T, [tT,] < 20 nmollL {<: 1,6 JlgldL} d~monst ... es a suppression ofT, secretion and thu, indicote. that the cot does not ha"" a thyroid a, ood~noma, B, Horst. L Hypc:nhyroidism i, unrommon in horses, and thus th~ neM for a T, suppression test is limitM, Interpreti"" guidelin.. and optimal ... mpling times ar~ not firmly enabli,hed, 2, Ihsics of th~ proeMUr.... a, EDTA_pla.ma i. collected for booul [tT.] and basal ItT,] for 2 consecuti"" day. prior to administration ofT" Sampl.. are frozen for submission with lat~r ,ampl.. , ' 98 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY b. T, (2.S mg dilut«i in 5 mL s;llin~) is admjni,t~rffl at 8:30 a.m. and 6 p.m. for 3 d and at 8:30 a.m. on day 4. c. EDTA_pl",rua i. colla:tffl.t 6 p.m. on day 4 and at 8:30 .. m. on days 6, 7• • nd 9. d. B",a1 and posI_T, pJ:asm':lre submittal to. J.bomory for deurmin>lion of [{T.] and [tT,]. 3. Exp«tM ... uh ...• a. In thr~ dinically he..!thy ho", .., plasma [IT..} values were ouppressffl {< 4 ng/mLl for at [un 5 d .fter th. last do,", ofT,. b. In a hor.. with a thyroid adenom., plasm. [{T.] ",mainffi inc,,,,,snl (> 24 ng/mL) during and .fter .dmininmion ofT.. c. [eT,] should bt incr<'ll5«l in ... mpl .. oollrc,w on days 4 and 6. TOTAL THYROXINE TO THYROID-ST1MUUr.TlNG HORMONE (tT.:TSH) AND FREE THYROXINE TO THYROID_STIMUlA.TlNG HOR.MONE (fT,:TSH) RATIOS IN DOGS I. B=u"" of the physiologic rdatiomhips among IT" fT•. and TSH KCmion, tT.:TSH alld IT.:TSH ratios hav~ th~ potential to diff~ .. nt",t~ hypothyroid &om euthyroid lIata Lower ratios mggest that the thyroid glmds a.. not responding to ~lIdogenous TSH (prima,y hypothyroidism). How","er. th~ tT.:TSH mio may al"" deerra .. in KCOlldary hypothyroidism ba::.u.. the [tT.] d~cr~a..,s mo .. than th~ d~ecubl~ da:rea .. in [TSH]. [I. Reported results A. R~port~d ratios have bttn calculated directly from the numerical value of reported concentmions; for aampl~. [tT..] of 10 nmoUL, [IT..] of 5 pmol/L, and a [TSH] of 0.5 nglmL would muh in a ratio of 20 nmol tT.:).4:; TSH (20 mmol tT. : g TSH) .nd 10 pmol IT.:).4:; TSH (10 ).lmol IT.:g TSH). It would be better if .nalyre collcentra· tions we .. converted to ,imilar units before a ratio is calculated 50 that the ntios were unitl .... B. tT.:TSH ratiO! (as IImol:).lg) in on~ nudy mowed that hypothyroid dogs usually had low~r rollios {... lu .. of 1~6; 10 of II mio. < 30} than h~althy euthyroid dog. (values of 36-173) .nd ,id.: .... thyroid dog, (... lu.. of 4 5-2114; a ratio;> 4000 v.as .n obvious outlier)." C. tT.:TSH and IT.:TSH mios in. 1999 nud,.." l. t T.: TSH mio: Usill{: a decision threshold of 17.3 IImol tT.: J.1g TSH. the tT 4: TSH ratio had a diagnostic "lI!itiviry for hypothyroidism ofS6.7 % .nd a diagnonic specificity of922 %. 2. IT4: TSH ratio: Using a decision threshold of 7.5 pmol IT. :).lg TSH, th~ IT4:TSH ratio had a diagnostic "lI!itivity for hypothyroidism ofso.o % .nd a diagnonic specificity of 97.4 %. 3. Ratio> alld diagnostic pc:rformallce will vary with 'p«ific assays, labomori.. , and decisioll threshold values. D. The ratio> have not i>ttll widdy used, .nd th~ir di"l:nostic value compared to oth~r .....'menU i, not firmlyesrabli,hed. lnaCCllrate [TSH] at ve.ry low concentmions (the =y's detection limit is llrar collcentrations found in .... thyroid dog,) could resuh ill inaccurat~ :md pc:rha", mislrading ratio •. 17 1THYROID FUNCTION 799 Table 17.4.1nrcrprctation of thyroid profile result. in d<>g£ [rT.(] [IT.(].. [TSH ] TgAA lnt~rpreurion Rul~s WRl " " WRI-i< WRl-; ; Positi"" ; Negati"" WRl' Negati"" WRl-; N~gati"" ; Positi"" WRl WRl WRl Positi"" WRl WRl ; Positi"" WRl WRl ; Nc-gati"" TI><~",. -" tbe 10_' ~mi, of. TSH out hypothyroidi,m unle .. [tT.(] i, f.hely incr""oN by T .AA Prim>ry hypothyroidi,m cau..d by lymphocytic thyroiditi. Prim>ry hypothyroidi,m cau..d by thyroid atrophy or possibly ~nd-lI"l:e immun~ thyroiditis Saxlfldory hypothyroidi"" caused by pituitary gland dysfunction Sick .... thyroidi,m. nonthyroidal illness" Hypothyronmia caUoN by dfecu of drugsNonthyroidol illness (sick euthyroidism) Prim>ry hypothyroidi.m (lymphocytic thyroiditis) with T.AA incr~asing th~ [tT.(] (..e Fig. 17.2) Thyroiditis without thyroid dysfunction or f:.ls< positi"" (e.g.• nonspecific binding) Thyroiditis with comp"nsatory incr~a", in TSH production Pounti:.lly '""hd,~ .J "f =1""'''0",,, • ,.1'...,,,,,,, in",rv:o\ anno' I>< ..Ji.bly cstablisW. • Sul£omethaumlc_ttinmboprim. pr«!nisoJone. 0' pl><nob.rbiuJ < lfT.J ... ..-iU vary. ~",J" depend on wh..e th. illness.. or drugs intorfe .. ..-ith thyroid honnone prodoction (_ th. _,). No ..: ltT.1 """ Iff. 1.. near , .. pectin rd'"rene< Jimits (borderline r.... I") should I>< in"''P'''ted <... oo ... ly. [NTERPRETATION OF THYROID HORMONE CONCENTRATIONS AND PROFILES L Dog> A. Majo, patterns of thyroid profile results in dogs (T abl~ 17 .4}"~""""""" B. [tTJ can b. u.=I to help monitor thyroxin~ th~rapy. Fo, dog. r~iving .ppropriat~ thyroxin~ SIlppl~m~ntation. [tT.] i. expectffi to b. high_normal to inc,~as.d 4-6 h .fter thyroxin~ :administration and [TSH ] should b. WRI."' C. To "'as=< a di"l:n05is of hypothyroidism via hormone assays in a dog r~iving thyroxin~ SIlppl~m~ntation. thyroxin~ should b. diocontinu.d for.t l=t 4 wk prior to t~sting. in ord~r to ",mo"" th~ inHu~n~ of treatm~nU on thyroid profile results.' [I. Major pan~rns of [tT.] and [IT.] in caU (fable 17.5) BOO FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY Table 17.5. 1nrcrpretarion of [IT.] and [fT,l .. in caU Clinical hinory,',( signs mggest [tT.] Hypmhyroidism i Euthyroidism [IT.] .. .l..WRI WRl_i WRl WRI ; ""_WRl ~J "" WRl_i Hypothyroidism " " [m~rpre{ation Strong rv:id~n"" of hypmhyroidi,m (a ooncurmll t [IT.] provid.. additional ev:id~n"" but i. typically not nttd Nonthyroidal illness Clusing sick ruthyroidi,m Probable prim.ry hypothyroidism; rould bt =ndary hypothyroidism or rn..n~ due 10 nomhyroidaJ illness or drug thuapy No ..: ItT.I :and [IT.I....eor r.. p«1ivo ",krone< Jimits (boninli ... r.... lts) .houJd b. in.. rprrted c:wtioudy. Ref erences I. Q",tain CB. G.nj.m YK. 1986. C{;";,,J E,.,J'"'""""D do. q,w.. I""","' M.JK,·",. 916-92S. Ph.iloddphia: WB S. Ho~ WI'. Ob ill. 2006. Ci."", SD. 1991. Elf.., of o"J ..tn.inirt.. rioo of ... odniooloo, "" do,..,;.J fwKtion io doc •. Am J Yrt R... S2:416-_ Sa."""'.. Sa."""'.. 171 TH YROID FUNCTION 0" II. Stn"".,m HP. Ar. / Am Yrt Mod A.ooc 22t.ltJ_249. IS. P...",... ME.. D,M ..", CL. Sbddon KM. 2OOJ. Total tl.yro';", ,utin" Compuioon"( ." in·bo.", "' .. kit with "dioi"'m ....... ...d AK. /iORJ: Y. Whit, T. S ........ D. 1m. Y.lid.tk.> of""",.,di-tt",,~ itrunwooa ...y mrtbod. Jo. th, ....Jpl. of tl.yrosiD, .nd oortiool in blood ....,pk. obtoi"od from d.op. "''' • ...d hoa ... / Yrt Dia&n In",,, 9,261 _26i! . 17. l1oa.ck .. EL. Rdioal KR. lIull RW. 1992. P..........C< of ..,«>aotit-l...... thyropobulin. tbyrosin,. <>< triioJo. thy....;..., ...d ..Ioti"m h.ip ,,( .otoantibodi< • ...d ....... "'''''''"....,... ,,( iodothyronin .. i" doc ~ Am / Y" R... 5},4ol9-0H. 18. Skop.I M. Nodudn .. RF. 2006. J).,_tk.> of • • """'cit-l .... ~, thp<>id ~." i" ............ pl",,( hypotloyroid id ...d ",thp<>id abo / Am Aoim Hoop A..oc }20489-094. 22. P...di. M. P,V N.~, N . l\oa"in, M. 1?96. s.n.m.·f"" th,,,,xi,,, ron«ntratk.> •• mea ... ...! by ,b"";lwnin< .. «n« - 1 bd'o.. ...d afu" thyro...",o. admini.u""'n in h",hby do, •• hypotbp<>id do, •• ...d nrtbp<>id doc • ...ith dum.,_""'" < • h.ind«> In",., IJ,I06-IIO. 25. G..b.m PA. Rd'taI KR. N.d. ....... RF. r...v...d., .. BoUip AI.. 2000. Th, mcaN"""'" ofk~n' th~ (TSH) .,in, . ~ ani... immwoo"Jiom"''''.-y. / Y" In"", Mod 14,j42 (. b..ract) . 26. T .... " H. K..do K. Hooikoobi H. Mi....loi",. H. Okau..li T. 0 " " E.. 1991. I""""ted prim..., .hypotbyroidiuo ...ith thp<.tropkin ",iotanc< in / ' p'D'" ,,' ~ / E.ndoaiool 129,24S- 2S I. Il. B..uha., SA 2002. lbrmid." in".latin,; t.o.m.,'" in adul, nrtI.p<>id ...d hypothpoid 1.0..... / Y" I""", Med 16.109-11S. 28. P.nI M. Mood E.. 2OOJ. J).,,,nnination of .utoantibodi" to th,...p.bulin. thyroxi",...d uiiodothyrooin, in <=in< mom. / Y" Med [AI 50,72-78. 29. P",..... ME.. 2006. Di."",rtk "' ... fo. bJ'P'rtl.yroidi .... in at>. Clin T cd. SmaU Anim P. fl",,,,,,,, in at>"";tI. kJ'P'rtl.yroidiao. / Yrt Int<m Med h 1.(2-I~. JI. p"",,,,,, ME.. G.mbk DA. 1990. Effect,,( nonthyroidal ill",.. on ..... m th,,,,xin, «>ne. in atz 494 a " . (1988). / Am y" ModA..oc 197, 120}-12o.! . J2. P""". ME. T~p .... , EP. Mol/A. 2002. F,1in< thp<>id..knoma. '" in 1"" ..ooci"od"";tI. ro."tk.>. in tI., G.. V"'...d "",...ith pol,.....'l'hi.m. found iD th, thyrotropiD .« 57S. J}. T ..... d /M. Fddm." EC. N,b"" RW. Coin GR. 19I18.lbymid ~""m. "",,i,,, k1P"'th,midi.n in a b , 14 " ". (198 1- 1986) . / Am Yrt Mod A.ooc 19},J59-J6.i. .4. AJb,." MK. MpI ..;. in. do" / Am Y" Mod A..oc 181),1476-1478. J6. G..b .... PA. N~ RF. Rd'oal KIt ~BoII ~ , AI.. 2001 . L,....pbocytk tl.J""'iditio. Y" CJin No"," Am SmallAoim p,." }h91~J}. BIl2 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY So""""', J7. Bid ""'pLuia in b..~ •. Vn PatI.oI 33,486-49-(. JB. G..." os, P.. """, ME. CI.o DY, M"b.u. JE. I~S . J~""""-' J.ypodoyroidi .... in. do,. J Am V .. McdAaooc 1870948-950. J9. R..bimon WF. Sh. ... SE. St...J.y B, Wybum. RS. I%S. Co..&idi.m in Srotti.b o..,."""nd P"ppi'L A." V.. J 6S,}8C>-J89. on =vnitalloypotkyroidi .... in " 40. Jon .. BR. GrufIYn&<..it>! ...d oduit-,,,.... kypotloyNidi.m in ag, Clin T rdo. Son.1l Anim P"" .nod"" 2h4 4J. M"lanby RJ. Jdfery ND. G_IMS, H",~ ME. 2005. Sorond''J' kypotbyroidiom fou...inc;kcod ,........ in. cat. J F,ti .. , M,d So,!: 7, [J5- lJ9 . .... Bcanam. JE.. I..ip>.to.. RL. HoonofWJ. 19l!2. im.&inc fo. d" ovaho";"n eltl.Y""id ""'pl.... ...d Ioypotkyroidi.., in • doc. J Am Y" Med A_ 180,)("77_ lon. 4S. a. .. t>inCB, McN«ISV. G ......... CL. P,..ani", SC 19l!J. ~nital kypothyroidi.m in " dor; du, to on i<.lOd, orpni&ation dd'ect. Am J V" Reo "' ,1257- 1265. 46. F,.., IC. Kampoclunidt K. Dans; v. p""", SA. H, Q, u.,.,..;. C . G..b.", PA. F...., VM. 2OO}. Co..vnital Ioypotkyroidi.., ...it!. """" in .." fox '''' ....... ) Yrt ]'''''m Med 17,50--57. .fl. 0""" S. [chi;" S. 1994. CliDi=poth.ol.ocicol obo.rvati.on. on ....,~ed foaI • ...it!. ...1upl oby.oid pand. ) Y" Mod Sci ')6,771- 772. 48. Mookiroo-Nrto G. T ",&"vnik HM. Y..... G. 19"9J. Dd"",cl"" ~o .,.,"-lo ...d """tioon auoio, "';"" ...d J.ypotbyroidiuo. E..d.oa R... 1",165-ISJ. 49. F<>t!.yroOdiam .....;... d ...it!. trimttloop1. 199}. Elf"" of trimrtl.opoi"',.... famrtboDMIt on thyNid function in <>id ...... 0 of .ulf.uottlonin,. a...... Rr. T oxi.ooI 7, 164-169. 56. Gop .. A. Euo MC. Utt=h, )P. CdbbAE.. Dan,,,, ... D. Riotk. M) . st._ NH. Canooo M. Spi,~ SP. 1992. Dn., .;oduccd k.rpothyroOdi.ru n.. t!.yroid .. a nT' .. , on in kJ"P'"'''''';thlty <eoctio ... to anti.ron........... ..,.[ rull'ooomidt •. Clio Phaana •. SS. G..Jik, .. KP. P...dt.. DL. 2OOJ. Eval.ati.on of tl., _ of domip..min< on thyroid fuoctioon "' ... ) Y" In ..... Mod 17........ 9. S9. M, .... NT. John- PI. Rd'..J KR. Naoh ....... RF. Gonj.", VK. Kn. .... GF. 1995. Elfoct of 100<1 d,prt.-aboo on b.,,,hn< iodotIoyronint ..,.[ 00n«n"'tiono ;0 h,altby. adul, boo ..... Am) Y" R.o 'j6, 116-121. 60. )00" R). Blwrt CG. Numbt'l: Bl. 1971I. To';';.,. of Ln.c", ... '--,~ 11« ,1Jert of iodin' ...d ...;"".[ ruppltmt .." on pdo. Am) Y" R.o 62 ,IIJ(l-I]J}. 6J. Ilamj.... s, MoOu.. 1/. Moo", RM. Wolhlodm" KI. Ga~ot SD. Min.. MH. TayIo, W . 1995. H~", ....dated ...it!. • thyNid ad,ooarcino.... in a 21·,..,.. .... 1d ~Idi ..," I Y" [0' .... Mt Radioi_""" <......., 17 1THYROID FUNCTION B03 64. Pm",,,,, ME. 2000. Hypn«ntrati.on in .....". b, "'lv.ilib';"", dial,.. ... J y" 10..... M,d .IXJ.J71 - l76. 67. P........ ME. Mdi... C. Nkltol. R. 19"\17. M, .. w,m,'" ~f"""".-J ... ,....;.... ttiiodo"',...,.,itt,. fto< ... ,....;.... &nd doyrotropi .. roncou.. TSH itt ... , diap><>ti. of kypodoyroidi.m i" dop. Sd....i. A.do. Ti"boi!kd 146,18J._188. 69. Srntt·Mo"";df JCR. Nd .... RW. !In.,,,,, JM. W,u;."" DA. 19"\18. Com"";"", of """", «>oc J. W.-.. GL 1998. [', ...... """ ~f ••-...ibod.i" to "'~ . lin in d.op ...... DO""'Y""id.1 ill.." .. Am J Y" Rn 590911- 955. n. H,;n" DM. Looditt, PM. P",kak WJ. 1980. S."", of tIoyrocIob.1in •• toantibodi .. in do, •. Am J Y" R... ":5,149.J.-1497. 7... Staui G. 0. M .... R. 2002. A..toimm.", "'Y""id dioca", Nn. mod.,). of cd.I d,,'" in ... toO .. m......,.. Not R... lmmomol. :z. 195-204. 75 . .'it<>ti. of .. ild kypo.cic "''' fo. <..0... kypodtyroidian. Y" R.< I j.8,59"-595. !J. [)j..,.. R.\l . MOOD,!, CT. 19"99. E..lu.don of ..... m fr.., "'yro';", &nd "'yrotropin o." of ani... kypothyroidi.... J Son')1 Anim P." ... 0.72-78. M. P....,;".,. DL 1999. b i, pouibl, to diap>oo< a,"", kypodtyroidi ....1 J S...n Ani .. [',"'. 40,152-1 57. S5. Kaotroow .. LB. P''''toO M E. T "P"'"" ]A ""tim C. Nicl.ol. R. 1999. s...... tot,) ... ,....;.... total ttiiodo"'yro· ..itt,. fto< ...yro.;"' . ........ pctropin oon«ntn.tion. in do". d.o". do" This page intentionally left blank Chapter 18 ADRENOCORTICAL FUNCTION Physiologic Processes ...................................................... 806 Analytical Concepts ........................................................ Cortisol Concentration ...................................................... I. Diseases and Conditions That Cause Hypercortisolomia .................. II. Diseases and Conditions That Cause Hypocortisolemia .................. Urino Cortisol to Creatinine (Con : Crt). Ratio ................................... Adrenocorticotropic Hormone (ACTH) Concentration ............................ Plasma Coni sol 10 ACTH Ratio ............................................... Suppression and Stimulation Tests ........................................... I. Dogs ............................................................. A. Low-dose Dexamethasone Suppression Test (LDDSTI ................. B. High-dose Dexamethasone Suppression Test (HDDSTI ................ C. Interpretation of Dexamethasone Suppression Tests .................. D. Adrenocorticotropic Hormone (ACTH) Stimulation (Response) Test ...... II. Cats .............................................................. A. Low-dose Dexamethasone Suppression Test (LDDSTI ................. B. High-dose Dexamethasone Suppression Test (HDDSTI ................ C. Adrenocorticotropic Hormone (ACTH) Stimulation (Response) Test ...... III. Horses ........................................................... A. Dexamethasone Suppression Test ................................. B. Adrenocorticotropic Hormone (ACTH) Stimulation (Response) Test ...... Combined Dexamethasone Suppression-Adrenocorticotropic Hormone (ACTH) Stimul ation (Response) Test ............................................... Aldosterone Concentration .................................................. Other Assessments of Adrenocortical Function ................................. I. Thorn Test and Modif ied Thorn Test .................................. II. Concentrations of Other Steroid Hormones ............................ 807 808 808 810 812 813 814 815 815 815 817 818 818 821 821 821 821 822 822 822 823 824 826 826 827 ." FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY '0; T able 18.1. Abbreviations and symbols in {hi.. chapler {Co'I:Cn}. [xl ACIl-I CRH EDTA EUSA FAN HDDST LDDST qp'DDD PDH Urin~ conisol to CJtltininc Con""mmion of x (x = .nalyu) Adrenocorticotropic hormone (oonirotropin) Cortiootropin_rd=ing hormone EthyJen«i;"mincl PPID Pituitary pm intcrmffii. dysfunction RIA IUdio;mmunoa<s;>y SI WRI SJ"l' mclntcrnational d'Unit6 Within ,.I'",.""" illt.rval PHYSIOLOGIC PROCESSES I. Regulation of cortisol and :ddo,teronc 5tCmion {Fig. 18. I} A. CRH from the hypolhal.mu. nimul. te, the production and ,.I...... of pituic.ry ACTH and oth~r hormone,. Low [corti,ol] promotes on:retion ofCRH and ACTH . High [conisol] inhibits 5etl"etion of CRH and ACTH . B. ACTH nimulatr. th~ production and rd~ of cortisol. :ddost~ron~, and oth~r st~roid compounds from th~ .dr~nal gland •. Th~ .dr~nal gland cortice, produce most circulat_ ing cortisol. C. Aldost~ron~ «tor protrins in edh. Th~ conisol_receptor complex initiatr • •ymhesis ofhormon~ and cytokin~ recq>tors and oth~r proteins inmlval in glucon",,_ gen~';., protrin cataboli.m, lipolpis, immun~ responses, and H,O Wlance. G. .\ion conirol i, remm.ffl from plasma by hepatocyte<, but th~re i, .lso utinary excr~tion of conisol and cortisol metabolite>. IS/ ADRENOCORTICAL RJNCTION ." Fig. 18.1. R.w>I>tion of coni",]:and ald05tero ... ..anion. • CRH ",1.1>«1 from tbe hypothal>m"".timuJ. ... the p,odoction and "'I.... of ACfH from the pituiury glmd. ACTH stimula... the production :and ",lea>< of cortisol :and aldostOv..M.dback 'J"t<m (d..hed ~ ....), ioc, .... ing the lco,tiso[1 inhibits tbe ..aetion of CRH . od ACTH, • [oc,....d Irngiotensin UI, hyp<,kaJemia, hypon. nemi .. mel incr..oe ANALYT[CAL CO NCEPTS L Unit connrsion' A, Conisol: JlgldL X JO = ng/mL ~nd Jlg/dL X 27,6 = nmollL (S[ unit, ne~rest JO nmollL) B, Aldosterone: ngldl X JO = pglmL . nd ng/dL X 27,74 = pmolfL (S[ unit, nrar ... )0 pmolfL) C. ACTH: ng/L = pglmL and pglmL X 0,2202 = pmoliL (51 unit, nearest [ pmoUL) [I. Sample A, Conisol L Serum or EDTA.pl.sm. may k ustd, 2, Stability of [cortisol] in EDTA' pl",ma i. kuer than in s,,,. and ktt.. in cold sampl.. (- 20 °C k .. and 4 °C .cceptable) than in w.rm onos (25 °C or 37 °C), Th.",fo.. , sampl.. (especially .. raJ should k .hipped with ice !"'ch.' 3, It is occasionally written that serum or pu,m. should k collectffl &om blood quickly becau.. of the uptake of rortisol by erythrocytes, Erythrocytes do bind conisol trult is .ddffl to blood! but rortisol mould alr....dy k distributed to plasma and erythrocytes in patient blood samples, The", was no diff.",nce betw,""n m..!Ured [coni",l] in pl"'ma "'mpl.. removal from cd], either JO min or 40 h after blood collection,' B, Aldosterone L Serum or heparini=! pla,ma may k used, 2, [Aldosterone] is nable for a Wttk at 2-8 °C .nd for at I.. " 2 mo at - 20 °C! C. ACTH L EDTA.plasma i. preferred (.ddition of a protra.. inhibitor ouch as aprotinin h", bttn recommended to reduce dq;radu ion), Plasm. should k removnl &om the erythrocytes immffliatdy, pl.ced in • pl:astic tuk, and chillffl, The sample .hould not h,ve prolongffi contact with glass becau.. ACTH adhem; to gu u , 808 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY 2 ACTH js vel}' labil~, 50 the plasma ,«\u;re; 'p«i.l handling.' If not .naly=:! the day of collection, then th. sample mould bt f.oun and shippal (0 the ..f.ren"" laboratory in .. dry_;"" shipllYlIt. Equine [ACTH] is subl. in EDTA_plasma (without aprotinin) for 3 h at 19°C.' Ill. Principles and a=y procalure. A. Cortisol l. MY.",l commercial cortisol assays are av:lilobJ.. R1As ."" the most common and no consid.rffi the guld "'mdlwl. but enzyme immuno ... s;oy. {•. g., enzyme_linkffi immun050rknt :assay and ch.milumin=nt enzyme irnmuno:way} are aho uK CORTISOL CONCENTRATION L Disea..,,:md conditions that couse hyperconisolemia (Table 18,2) A, Spont:meous condition.: Basal plasma [cortisol] by itsdfis oflimited value because many patienlS with hy~radrenoconicism do not have increa"':! N ...l [cortisol], How"",r, th.", i. mfficient coniKlI pnxluction to couse clinic:d disorder.; either because of "J>isodic aagg.rated srcretions or because: of. mild but continuously increased production of cortisoL A [coni",l] is btn interpreted in either d"""meth.50ne supp ... _ lion un. or ACTH stimulnion test. {s.. later sections}, Historicol, physic:d, and preliminary laboratory data may clearly indicate that an anim:d h •• hyperadrenoconi_ cism but may not clearly differentiate the cou.." of the pathologic !late, Adren:d mppression and stimulation tests (covered later in thi, chapter) con be hdpful for this, and diagnostic imaging methods (i,e" radiography, ultrasonography, computw axial tomography, ot magn.",ic """,,,,,no:: imaging) con locate the pituitary or .drenal neoplasms but c:mnot determine wh",h.. the abnormal tiMue is producing ace: .. ive amounts of hormones, 18/ ADRENOCORTICAL RJNCTION B09 Table 18.2. Discascs and condition. that cause hyperoortisolemia Inc ....aI production of oortisol Spom:meou. conditions •PilUiury-dCJ'<'nd~m hyperad .. norortici!JI\ 'FunCiional ad .. norortical neoplasia: ad~noma, ad~noc"cinoma 'Stress-induc l. Pituiury.d~pend~nt hype .. dr~nocortici.m (PD H): {Hyperadrenororticism i. commonly .. f~rrMto as Cushing'. di",=. th~ nam~ originally giv~n to th~ human pituitary disorder descrikd by Dr. Harv~y Cushing.} a. In PDH, th~ neoplastic pituitary cdls rd~ast acess ACIl-I. which cau..,. bil"... l adr~nocortical hyperplasia. Th~ ACTH rd~ .. ~ mayor may not ~ inhibital by exogtnou.s glucocorticoid.. Also. individual neopl .. ms em ~ inhibital by "'m~ st~roids but nOi oth",;. b. In dog•. about 80-85 % of clinical hype .. dr~nocorticism caseo arc causal by increas<"d rd~ast of ACTH from pituitary .d~nom ... A small ~r""'n~ a.. causal by pituiury ad~nOGlrcinom .. or pituitary hy~rplasia." c. In cat •• about 75-.85 % of dinical hyper:ad",norortici.m aucs .'" cauK<:ytc--S{imul .ting hormone {p.MSH }. corticotropin.Hk. int~rmaliau lo~ peplid~. p •• ndorphin- rdatal peptid~ ( ~END peptide). :md ACTH. Th~ combination ofincreasffi p.MSH. PEND ~ptid• •• nd ACTH cau ..s act... neroid~ncsis .nd dinical hype .. dr~nocortici,m. ,. 2. Functional :adr~norortical neopl ..ia (FAN) a. In FAN. cdl. of th~ :adr~nororlical ad~noma or :ad",nocortical carcinoma product act .. cortisol. The neoplastic ctlJs mayor may not ~ ... ponsi"" to aogtnou. ACTH. A high [cortisol] is apect«i to inhibit ACTH rd....... and th~ nonneoplastic adrenal gland will atrophy. 810 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY b. In dog., about 10--20 % of din;",] h~ ... dr~nocor{icism ca= ar~ due to ad .. n:d .d.nomas or adenocarcinomas.'o c. In cns, .bout 15- 15 % of clinic..! hyptr:od .. nocortici,m = ~r. due to .dren:d .d.nomas or adenocarcinomas." Clinical hyptnd",nocortici,m may also k 5tt1l when adrenocortical neoplasms produ~ steroid hormone, oth.r than cortisol (such :as progmerone}." 3. Sm.. _inducffl hypuconisoJ.mia a. Slre:u ausa! by an illness or environmenul chang." m"y ,rimulate th. ,dust of CRH, chen ACTH. and thus stimulate th•• dr.nal glands to produ,,", rno", cortisol. b. If 1>",,;<1,[1{. the .cr... ,.spon", moy l~d to bil.{,,:d .d",nocortical hy!>"'pJas;", but it mould nol cau.. th. clinical monifesmions of p"thologic hypt cou.. clinical hy~r;odr~noconici,m. B. Iatrog~nic condition. I. Exog~nou. ACTH administration i, ex~al to em.. hy~rconisol~mia 15tt Suppression and Stimulation T"", •• Ket. I.D). 2. B.cou,. th~ anticortisol antibody in immunoassay. may cross-r~a.ct with oth~r .uroid•• r~nt administration of st~roid, could ... ult in a faI..,ly increa.al m~asur~d con""'nt.. tion. For aampl~. assaY' that list ,ignificont cross-r~activity with pr~dni!iO lon~ m. y h.v~ fal .. valu~, if th~ animal was r"""'ntly trtltal with pralni,olon~ {~.g .• p .. dni ... Tab}. pralni,on~ {~.g .• Mfficomn}. or pr«ini!Olon~ wdium succinat~ (~.g .• Solu.Ddta.Cort~f). C. Othu common rourin~ labor;otory finding' with hy~rconisol~mia l. l.eukogram: st~roid leukogr;om {5tt Chapt~r 2} 2. Ch~mistry profile: inc",as«! ..,rum activity of .Ik.:din~ phosphat...., (ALP) in dogs. hy~rglycc:mia. hy~rchol"'~rol~mia. :md lip<mia 3. Urinalysis: rdativdy uncona::nt .. tal. isosth~nuric. or hyposth~nuric urin~. :md gluoo,uri. in ho",", II. Di, .....,.:md conditions that cau", hypoconi!Ol~mia (fabl~ IS.3) A. Spont:mam, condition! l. Primary hypoadr~noconici,m (Hypoadr~nocortici,m i, commonly ref~rr«i to as Addison·, di ..as~. th~ name originally ginn to th~ hum:m ad .. nal disord .. deKribai by Dr. Thorn", Addison.) a. Causes of 'pont:mam, bilat..al >trophy or destruction of adr~nal gl:md, a", usuaUy not known. but thq may includ~ immun<,-maliat«i di, ...... adr~nalitis. or adr~nal h~morrh>g~. b. The disord .. i. r«:ognized primarily in doC' but also occur:s in cats" and occa,ionally in f""I,. " 2. Secondary hypoadrenoconici,m {ACTH d~fici~ncy}: ACI"H d~fici~fi(y could result from dmruction of the pituitary gland by di, ..... proe<::<se:l or surgery. but thi, i, much 1= common than prim"'}' hypoadrenoconicism. 18/ ADRENOCORTICAL RJNCTION ~1 Table 18.3. Discases and condition. Ihat cause hypo<:nru""lemia D,""rra,a1 producrion of rorli""l Spont>nwus rondilions 'Primary hYP""dr~noconici,m S=:.ndary hYP""dr~nocorlicism {ACTH d.fici~ncy} Atypical primary hypood .. nororlici,m (sd~in rorti""l d.fici~ncy) lalrog~nic rondilions 'lalrog~nic hyptrad .. noconicism· latrog~nic hypoad .. noronicism' ~toro""zol~ or trilost>n~ 1r~>Im~m ' 11>< rnim:d h.. clinical manifest. tions (~.g .. polyuri. or .Jop 3. Atypical primary hypoad",nororlici,m (od,""tin rorti,ol d.fici~ncy): Ra .. 'poman... ou, case, ofhypoconisol~mia wilhout concur",m hypoaldost~ron~mia, hypon.tr ... m;", or hyptrk.:.l~mia han ~~n r~porta!." In ,om~ of Ih"", Cl5e:'l, [.ldosuron~] was nor .. pomd bUI w .. only presumalro ~ WRl ba:aUst of Ih~ abRn,,", of d,""rrolyt~ ch anges." In morh .. =, Ih~ concurr~m coni""l and Ihyroxin~ ddici~ncies w~ .. co",id~ral a polyglandular d.fici~ncy .yndrom~.'" B. l.rrogenic condilio", J. larrog~nic h}'J>"rad .. nororricism a. Chronic administration of glucocorricoid drug. (~.g. , pralnison~ or prednisolon~) may product clinical 'ign' co",in~m wilh h}'J>"radr~noconicism. b. Th~ aogenou, glucoronicoids will caUst • n~g.rin f,""db""k on th~ rd~.st of CRH .nd ACTH, and thu, .dr~nal glands will atrophy and produ,,", l~ss conisol. B.. din~ [corlisol]lypically will ~ d,""r ....-d {< 1.0 ).lgldl}, .nd th~re will ~ a diminishal responst in Ih~ ACTH srimulalion tesl. Slow wilhdrawal of th~ 'r~roid rr~.lm~nts ~nables m~ adr~nal glands 10 r«:over. 2. latrog~nic hypoodr~nororricism a. Tr"lm~m of dog, with op'DDD (mirOlan~ [ly><>"radwlOOOrticism ba:aUst of PDH or FAN co ..... progr=iv~ n~crmi. of Ih~ .d",nal cona {zona f..ciculata and ron. r<'liculari,}. Exctssin n,""lOSi, can d~cr~a", ad",,,,,l funcrion .nd clinical hypood"'nocorrici,m wilh coniwl ddici~ncy .nd wmerimes min~r_ .loconicoid d.fici~ncy. b. Sudd~n withdrawal of glucocorricoid rr..tm~nts (including mrgesrrol actlat~ in cats) may caUst clinical hypoad .. noconici,m ba:aUst of adr~nal gland alrophy r.... t drvdoptd whil~ rh~ animal was r«:riving glucocorricoid •. 3. Treatment of dogs or caU wim k<'loconaz.ol~ can cou", d,""r~asal [corlisol] ba:aUst hloronazol~ inhibits st~roid bimynmesi •."'· T",.tm~m wilh rrilost>n~ ralucel [coniwl] by inhibiling 3~-hydroxrsuroid d~hydrog~n ... , an ~nzyme m>l caulyz.es an imermaliar~ r"clion in sreroid synthe,is." 812 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY C. O{h~r common routin~ labomory finding' with hypoconi",J.mia {exc:qJI with iatrogenic hwrad .. noconicism C;>U.ffl by glucocorticoid .dministration} I. leu\rogram: abstnct of suroid Jrukogram in messa!, ill p"tienc, (""niculn]y a Jad, of lrmpho~nja; and possible lymphocytosi' or eosinophili.) 2. Chemistry profile: hypon. mmia, hypochloremia, hy~rkaJ.mia, hy~rcal""mi •• hypogl~mja, and ""otemi. 3. Urinalysis: inappropriatdy low uti". sJl«ific gravity with O7.Oumia URINE CORTISOL TO CREATININE (Con:Cn). RATIO I. AI d=rikd in Chopt.. 8, ooIDp"rison of. urine .nalyt.'s oonctntration to utine [etrat;. nine] will provide a rdat;"" m. of urinary exc.. tion of the analyte (mrn :u conisol) oompara! to c,..atin;ne. [n thall}', the {Cort:Crt}. mio should d~= a hyptradrenal sUt. ktter dun a basal [coni",]] ~U'" the cortisol that accumulat.. in uriM WlU producnl ov" a long~r !",riod and thus is not as su=ptibl~ to vui.tions amsN by ~pisodic conisol « con cou"" fluctuation> in • r~ndom .. rum [cortisol]. How~nr , th~ incrrasN urinary conisol acrHion could ~ ~ith" a ph)"iologic or patho_ logic nat~. [I. The (Con:Cn). ratio should ~ calculated by ming molar conctntmions of urin~ coni",l and cr~atinine {Eq. 18.1}. A markedly diff~«nt mio would result if [conisol] and [cr~ati_ nine] we« apr......:! as J.1g1dL .nd mgldL, r.. prctivdy. Inst~ad of stating th~ true ratio val"" {~.g., 20 X Ia-'}, ",metim.. it is short~ned to "20" in clinical j.rgon. C) . urin~cortisolconctfltration ( "~ \.Alrt: n • ",uo =-'C'=========':: urine cr~~tinin~ conctnt"'tion {18.1.} with cortisol in nmollL &:: c",atinin~ in mmoliL Enmpl~: urine conisol conctntration = 200 nmollL urine cr..,.tinin~ conctnt"'tion = 10 mmoUL (Cort:Cn) mio = 2°OnmolfL • 10mmolfL Ill. 200 nmoljL lOx 10" nmoljL 20 X I 0.... Conclusions from published rq>o>rts {Fig. 18.2} A. Dog,"~7 l. A {Cort:Crt}. "'tio chat is WRl i. mong ~id~nct chat • dog d""s not h."" hy!",,,,drenoconicism. but incrrasN (Cort:Crt). mios .'" freq""ntly found in dOl}! without hy!",,,,drenooonicism. [n oth~r words, the (Cort:Cn ). "'tio has a high di"l:nosti, .. nsitiviry for conine hYl"rad«nooonicism (r~lativc:ly f~ f:d .. negativ.. ) but has a poor positive predictin v:due and poor di"l:nostic sp«ificity (r~lativc:ly frequent faJ .. po,iti=) (Fig. 18.2). 2. Basal (Con:Cnj. ratios connot rdi. bly diff"entiatr PDH from FAN, but th~ gr~atm ratio. are usually associated with PDH, as is suppression of {Cort:Cn}. ratio in conjunction with . HDDST." B. Cats:" Th~ (Cort:Crt). "'tios in 15 of 32 cots with h)'l"rthyroidism w,,~ greatrr than the up!"'r r.f"enct limit of th~ fdin. ref.",nct interval (8.0 X Ia-' to 42.0 X 10""'; n = 45 ). For th~ h)'l"nhyroid COtS, the median mio v:due was 37.5 X la-', with two values .bon 100 X 10..... The gr~.t" (Con:Cn). ratios may ~ ",fl~iv~ of the stress 18/ ADRENOCORTICAL RJNCTION '" "" ""I ~450 ~ -'00 -" '" i study A ' 'I -=':1 Sludy B ~1000 :'i ~ '00 ..,........... None ... .,.. oorII<MI ",. «I) ""...,_ I"' 2lJ Fig. 18.2. (Co.t: Cn) , " ,I i " , in to.<> studies. , In.rudy A, tb. (Con:Cn) , " , I i " , from theN grouP' of dog ...... oompar«J to. group of h..lthy dogs (b.ckground g"'y f<1Jion: me:on = 13 X lIT". n = 31). Of 2S dog...i.b hyp<: ..d",nororticism (21 PDH and 4 FAN). 23 Iud incre...d (Cort: Crt). " ,I i " , . Of 21 dog, .. itb non.ru.n..I diso,de .. (m..! insuffi· ci.ncy. liv .. dis. ..... Wlo ... phritis. bypotbyroidj,m. bronmili, . • nd di:ob.t .. i"'ipidw) but in ..rucb hyper:odrmooonicism W>S susp«1ed. only 0 ... h. d :on incr • ...d (Cort: Crt), ",tio. Ho....." in 28 dog. with mode",« to """'''' no ... dr....t diw,den (g .. troin... tin..I ... n..I. to..", urinuy tnct. liv ...... urologic. immune-medi. ted. cardiac. tr1WJl1lic. :ond inf.criow dio....",). 12 b. d incr.Um." .... and did,........ l~tu,) ""'" comp. ",d to .be (Con: Cn) , rati", found in b...Ithy dog, (background g«y ~on: mem = 6 X lIT". n = 20). All 40 dog,..ith hyp<:ndrmooonici,m b. d rn incr......d (Cotr: Crt). ratio but .0 did 18 of the 23 poIyuri:oipolydip!i. (PUlPD) dogs tIu. did not Iuv. byper:odrmooonicism. " a.<socialed with the hypmhyroid sm• •nd ,hOllld not b. comid.red "Aective of pathologic hyp I. Dogs and cats: M ... mring [ACTH] am b. v.ry hdpful in differ.ntiating PDH from FAN, and prim:uy hypoad .. noconici,m from =ndary hypoad .. nocortici,m (Fig. IS.3). A. An [ACTH] i, exp«ted to b. WRl or increased in .nimals with PDH. primary hypoadrenoconici,m. pted inc"... in [ACTH] aft...n injrction of conicotropin_rd...ing hormon • ."" B. An [ACTH] i, exp«ted to b. drc,,= 814 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY " " t" < • '" " •" " " " ~ • '" " •" " - - Fig. 18.~. ACTH coDamm.tions in od'ffiOOO.r ... dogs with FAN :md """,,,c< in..rnk Th. background gre<\ ACfH roncen'<1tions . .me",.. cat> with FAN b. Y< dec ......d ACTH ooncm""boru. n,. background w II.. r.~ ... rd'e""e< in"'rnl. D .... for tb. "",ph ""'.. en ..cted from publish«! co"""nm bom." Noto: n •• orro... .00,." th. w~d bus indic . .. tlut co"""nttotioru =y b. moch gr .. .., du n the v:tl .... sho..., in tt.. y-aus. [I. Hor~ A. Pt.",,,,, [ACTH] ,. SO pg/mL strongly ,uppon the pres..,,,, of pimitary g1:md ad~noma of hyp<rpWia. For ponie;, an [ACTH] ,. 27 pglmL is supportive.' Values and diagnostic d,""ision ,h""hold, may vary :nnong assays and l. boratori... In another study using chemilumine,,,,,nc ~nzym~ immun=y. ACTH con""ncrations in fin h~ahhy hor,." _~ 13.4 ± 2.2 pglmL, whtion w>s .lso fOund in th~ dex.:lmHhasone suppression test "mlu {= Supp".. ion and Stimulation T e;t, ,ret. [][Al. Th~ rrason fOr the ,.,...onal diffe"nce. i. unknown. Also. it it unknown wheth Clinical use of [ACTH ] i. hamperM by instability of ACTH and ex~nsiv~ dry_ice mipm~nc,. PLASMA CORTISOL TO ACTH RATIO I. Becau.., of th~ negatin fttdb.ck in the hypothalamic_pituitary"""d~nal hormonal .yst~m. d«~=d production of cortisol by ad"nal gun'" is expectM to re;uh in a high pl >snu ~5 181 ADRENOCORTICAL RJNCTION [ACTH] (thus low conilOl to ACIl-I ratio). Convendy. inc",-d production of coni",l by an adr~nal nalpl""m is exp«tal to result in a low plasma [ACTH] (thu, a high ronirol to ACTH r>tio). Th~.., rdationships may k ustd to int~rpm indiyidual hormon~ con"",ntra_ tion! (i.~ .• exp«t a high [cortisol] with a high [ACTH] in PDH). but publi,hal anempt' to u.. th~ ratios in diagnonic d~i.ion' h.ve oon limital. 11. On~ publishM study of22 dog. with pri/IUry hYP""dr~noconicism rq>OnM thot cortisol to ACTH ratios in thOst dogs (range. 0.003...{).17 nmoUpmol) {or 3- 170 as unid~.. utios} wtr~ consin~ndy low~r thon th~ ratios in 60 h~.lthy dog, (0.79- 175 nmollpmol) (or 790- 175.000 "" unid~ .. ratios}." Th~ authon rq><>rtM th~ ratios in truncotal form (~.g .• 0.00}-0.(7). wmples of th~ cokuution! are pr=ntal in Eq. IS.2. U,ing mol .. ron""'ntutions: Conisol:ACTH Ratio = 48 nmol cortisol(L 7.4 nmol ronisoi 7.400 pmol cortisol 6.5pmol ACTH/L pmol ACTH pmolACTH Ratio r~portM in the articl. for the .. data would hoy. bttn 7.4. U,ing wtightlvolum~ con""'ntratiom: Cortisol:ACTH Ratio = 17 J.1g/ L conilOljL 0.57 J.1g conisol 30pg ACTHjL pg ACTH Ratio ~ not reponal in th~ anicl~ for th.", units. III. {IS.2a.} 7.4xI0' {IS.2b.} 570.<XXlpg conirol pgACTH 57Oxlo' B1Ist of th~ lad, of .grttm~nt .mong 5Om~ hormon~ ....Y'. interpreutivc: guiddin.. may nNd to k devdopffl for ~h set of cortisol .nd ACTH .... p. Th~ cortisol to ACIl-I ratios may be hdpful in diff~rentiating primary from KCOndary hYl""'dr~noconici,m and in diff~rentiating diff..~nt forms of hype .. dr~nocortici.m. Th~ .p«ial umpl~ handling r«lui=' for ACTH samples and th~ :associatM expenst:'l will limit th~ u.. of cortirol to ACTH utios. SUPPRESSION AND STIMUUr.T10N TESTS I. Dogs" A. Low-dose dex:am<'thason~ ,uppr~ .. ion test (LDDSn:. screening test for PDH and FAN l. Procdure a. A p='ex:am<'thason. blood umpl~ i, coll~tM and proce=d. b. Dex:am<'thason~ i, giv~n at 0.01 mglkg N (som. list 1M) c. Pondenmechasone umples ..~ pro.:;.,ssffl aft~r coll~tion' at 4 hand S h ("'m~ use 3h andSh. 4~h and sh. or juu Sh). d. PrMex:am<'thason. and postdexam<'th"",,n~ pW/IU or ..rum .sampl., a", submit_ tM for m~a!Urem~nt of [conilOl]. 2. Exp«tM re",lu (Fig. ISA) a. In h~.lthy animal,. dex:ametho"'n~ is np«tal to ='u"", th. s~retion of CRH and ACTH for oevc:ral hou," .nd thus d~",ase rd..... of cortisol from the .d",nal glands. Bernuse the half_life of coni",l is < 2 h. plasma [cortisol] i, H8SHhy O Nol _ _ _ &44>.s..,d Pituilary'depe~::~,::--:~;~:::'"~""~~.;"~" .... hype~5m (n _ 181) disordonl (n _ 59) Fig. 1804. ~ ..... fOf anm. ~1us<Jn. ... ppr ...ion t .. ts. o D ... for A and F.,. r=>1.. ap«:ted for heoltby dogs. based on oommon d.cision limits. Suppr<SSion_ defin«l .. ,postdexam.dusone [com.oll <: 1.4 ).lgldl and <: 50 '" of predenrnootlu.sone ",,,,,,n".tion. o D ... for B-D. G. and H "Ol' atrxtM from I"'blished r.... lts."' In B and D •• upp=ion was defined .. • postdenmeth...,,.. loon;"'11 <: 1.4 IlgidL In C. suppression .... defined .. . p<>It~h .. o"" [cortisol] <: 50 % of th. prede""ms defin«l ... postdenmeth...,,.. [oonilOl] <: 1.4 Ilgldl or <: 50 % of th. p",deI:Omtiv< dat. for HDDST (I).....,,,, not found. In th. LDDST • .mppr... ion ...... defined .. 'p<>It~h:uo"" [cortisol] <: 30 nmoUl (1.1 Ilgldl) .• decision limit .. tUnatM from gl1phic:d d .... Nonadrenal disorders included b.p>tic. p:mc",.tic. urinory. ~ttointestirW. ""pir:otol1thyroidism. and imulinoma). Dog> ..lr<wf for this group ""'''' not SIlSpra'" supp"",ion. V.ing . <: 50 % cri",rion for suppr ... ion (0, mo", dog. with pDH h.d :odr' ''' oorsi",1 suppression tlun when judged by tb. <: 1.4 ).lgldl ai"'rion (B). V,;ng the <: IA "'Wdl cri,.,ion. 48 of 51 (94 %) PDH dog. that had suppression at 4 b had .. coprd suppr .. sion by 8 h. V,ing the <: 50 '" ",itrrion. 58 of 102 (57 %) PDH dog. tbat had 'upp"",ion at 4 b bad .. caped lUpprruion by 8 h. o V,;ng HDDST .....,ly.U dog. witb FAN (H). but . minority of dog. with PDH (G). faiW to have :odr ' ''' suppr ... ion at the 4 h and 8 h .."'pIing>. o If r.. ula from LDDST :md HDDST .,. e""mined toll"tbrr ." the", .... in:odeq"'''' rupp",uion of corsi",1 ooncrntr:otiom in both ..... in ",,:uly:ill dog. with FAN (D ..,d H: 94 %) but in . minority of dogs with PDH (B. C, ..,d G: 24 %). A f... dogr witb pDH (14 %) ..,d two dogs with FAN (6 '!O) had i",dequ,,,, Sllpprersion witb tb. LDDST but had :odr '''' suppression with the HDDST. Two dogs with PDH ppr .. sion witb tb. LDDST but inadequate ruppr ... ion with tbe HDDST. (I %) had HD. high d ....: . nd lD. 10.. 00... .dr ." .... 816 18/ ADRENOCORTICAL RJNCTION npa;t«i to bt da;t""...,j dramaticolJy >t 4 h and 8 h (Fig. 18.4A). v.lu .. . ", usually i""d'"'juatdy suppress.d at 8 h with both PDH and FAN. (I) Mon _"inarians int~tpr~t a pondn;;omecha>one [ruttisol] < 104 J.Ig/dL (< 40 nmollL) to indicate th~re was .d'"'jlIate suppression of coni""l secretion. This da::ision threshold . ppear> to h ave been calculat«i from data rulJa;t«i from 22 healthy dogs; it repr~..,nt«i th~ m""n concentration (0.5 J.Ig/dL) plw 3 sundord deviations (3 X 0.3 = 0.9 J.4::ldl).t 8 h. ,~'" {2} Some authors propo.., th at suppw;:sion i. present if the postdnamethaoone [coni",l] in the 4 hand 8 h samples i. < 50 % of th~ pralexamethasone concentration. Such .n interpr~uti"" guiddin~ i. comistem with the half_ life of plasm. coni..,l. b. If hYP"rad .. nocorticism is cou.«i by PDH or ~ctopic production of ACTH. th.n a low do.., of dnamethasone m.y or m.y not l.ad to supp ..... d rdease of ACI"H by nalplanic pituitary cdl,. and thu. cortisol production from hYP"rplanic adrenal glands mayor nuy not da;",ase (Fig. 18AB .nd C). {I} In som~ ca5e'l. the 4 h postdu.. methason~ [cortisol] is btlow th~ da:ision thr.. hold. but the 8 h postdnamethasone [coni""l] is .bove the da;ision threshold. In such the escapnl suppression i. highly indicotive of PDH if the clinicol 'igns suppon • hyperadr~noconicism diagnosi •. Nonad",nal disorders may aloo bt associat«i with this pattern (Fig. 18AE). {2} Suppr=ion i. demonstm«i in mor~ PDH """,. if the "< 50 %" criterion (Fig. 18AC) is us.d rumparal to the "< 104 J.lg/dl" (< 40 nmoUl) criterion {Fig. 18.4B}. c. If hYP"radrenocorticism is cou.«i by FAN. then low_do.., dn;;om~tharoM is not npa;t«i to da;r"".., the secmion of coni501 (Fig. 18AD). Even though suppres_ sion criteria a.. nor fulfiJl«i. rurtisol concentratiom may Huctu ate during th~ testing. btcou.., of variations in cortisol secretion by nwplastic cells. binding to prot~ins. or plasma clearance. The WDST has high diagnonic stnsitivity for FA N. d. Supp..ssion may not bt .d'"'jlIate in dog. with nonad",nal illness (Fig. 18AE). e. In two dogs ..",iving phenobarbital therapy. coni",l concentrations were not suppr~ss«i ad'"'juatdy. possibly because phenobarbital·, dfa;u on cl""rance of synthetic steroids enh.nce duamethaoon" d""rance rate. HoW<"V =. 818 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY c. If hypuad .. nororticism is CJUIM by FAN, then high-do .. daamethasone is not npa:lffl to decr..... Ih. =:retion of conisol {Fig. 18AH}. Even though supp ..... • ion nit.ria ... not fulfiJI«i, corti,ol oonctntr;otiom moy Hucr""re during the testing, kau .. of v:ui.l;ons in oortisol5tcretion by nwpJ:lStic ""lis, binding to protein., or plasma Joann",. d. If . HDDST js done in a dog with non.dren:d illn<:S!, then cortisol production is npa:lffl to b. drc,,,,,sal sufficiently to indicate a supp.... ion of ACTH = ..Iioll . • . Too ftw cases of a-topic ACTH production h. "" bttn .. pomd to establish a paturn, but th .... houJd b. a lack of mpp ..... ion of rorlisoJ in th ... animal •. C. Interpreution of d.xam""h:asone suppres.!ion un. l. Exp«IM !"'Iurns ofLDDST:md HDDST result. for individual dogs a. In healthy docs, [corlisol] will be, suppre=d to <: 1.4 J.Ig1dL « 40 nmollL) with both [edrenal illnesst., [he", mayor moy not k corti,ol "'pp.... ion with [he LDDST. The [Cortisol] i. exp«tal to k <: IA j.lgldL (<: 40 nmollL) with the H D DST. 2 T . ble 18A lim examples of rorti,ol concrntmion, from daam.th.,on. suppression t .. u. 3. Int.. preutiotlS for T.ble 18A w..e made by using Fig. 1804 •• a guide. lkst int.. pr",..!. Results .nd int.. pr c. A ]>On-ACTH umple i. coll""tal I h l. t .. if cosyntropin it given (2 h if porcine or bovine ACTH is used) to a..... for a cortisol ... ]>On ... 3. Exp«tal ..",h. (Fig. 18.S) (Sp«ific p. tient results .hould k interpretal by using ",f..ence int.rvals .nd int.. preution guidelines provid..! with result •. ) Table 18.4. En mple rcsulu of d enmctbasone suppression lests in dogs Hcalthydog> Dog I' Dog ,. LDDST HDDST Corlisol (J.lgldl)' Corliso] (j.Igldl] PrM.L ' 4 h ponda.' s h postd.x. PrM= 4h ~nda. sh postda. O.5--{;.O <: 1.4 <: 1.4 O.5--{;.O <: 1.4 <: 1.4 3.0 '.0 0.9 3.' 3.5 3.6 0.6 3.3 0.' 0.5 0.' '.9 0.6 0.3 4.5 7.0 '.0 6.5 0.' 3.5 6.' 5.5 4.5 Dog 3' Dog 4' OA o. , o. , Dol,; 5' ' Co.,....,ruon to 51 unit>: J.lgldl x 27.6 nmolll (round to neuest 10) • P,«!a., p,.daamat. supp",.. i",,;' 6f'K'.d in no,..dr.n:d w.o,dc .. , • Dog 2 had . dren:d byperpwi. co=t> PDH or nonad",nal disorders, In tbc HDDST, suppression con be found .. itb PDH and i, npK«d..ith nonadomal disorder> but ;. not consi,«nt .. itb FAN, 'Dog 3 _ bealtby OJ had PDH or a nonad«nal disorder, In the LDDST, mpp,cssion can be found .. itb h",lth, PDH, and nonadJ.n:d w.orden, In the HDDST, mppr ... ion ern be found with he. ltb, PDH, and nonad,en:d disordc ... Suppres1ion ;. not ~tesl ..itlt FAN, 'Dog 4 bad adJ.,..1 hyperpl.,i. co.....! by PDH or . no,..drcn:d disorder, In tbc lDDST, b ilu« to SUPP"'" tbe [co,tisol] to <: IA )lgldL (<: 40 nmoUl) could be PDH, FAN, or nonad,..w disorde" In the HDDST, supp"",ion ern be found..ith PDH and;' ap«t.d with nonad,en:d disorders. Supp"",ion is not ronsinent..ith FAN, 'Dog 5 had a bypoad"'nal s ..«, In tb. LDDST, tbe p,«!a...,.,th1SO ... hypoconisot.mi. indicates a hypoadron:d " ... th .. could be primary, > ,., 88 ,~,,~!!-, Hypoadmoo. corticism (n _ 225) c::::J c::::J Exaggorolodresponse - Respor-. '" hoaIhy dog. ~\e latrogonK; hypontdraflO. corliD.m (n - 28) t.jpo ., c. "'a ~ Funcb",,"] adrooocortical ""opIasia (n_ 41 ) Non"",.. ",,] dioorden (n . 59) .oopoo.. Fig. 18.5. Response! from canine AcrH stimuhtion t .. t>, , Criteria used to det .. mi". "Ppropri. «, inaa«, o. =gg< ....d ...port ... to ACfH uimubtion varied among pub~c .. ions, Fo •• ach >eO: of .. tract.d d ..., tbc antho,,' ai",ri . ..... wed, , In all bypoadJenooo.sicism c-., the ....... '" inadequ ... ""'po ...., to AcrH stimul. tion, Tbe 225 C>SeI included 220 = of p.im1ly idiop .. bic bypoad",noa>rticism and live = of oeronduy bypoad«nororticiun," In.JI.....,. of u trog«>rtic;""" od«nooo.-tio.J .t«>pby « ... b.d;n ;.,.. « «'1'.>0.,,, to AcrH stimuhtion," , In 84 % ofPDH easel and in 51 % of FAN aoes, the •• we .. .,.:ogg ... tesl ""'po ..... to AcrH stimuhtion, ,,,,,,01,7 'c;. , In 14 % of dog,..ith nonad«nal iUnes>es, tbe« ..... engger:ot BI' '2O FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY a. Hffithy docs {l} ACTH should S{iml1J.t~ th. production and rde~ of oonisoJ from th. "dren'" rottie... {2} fu~al p,<,-ACTH and post_ACTH ronumratiom of ooni50J "e diff.""m in published .. and ..f"e""", J.bonto,i ... UsuaUy, the post_ACTH [ooni",l] is < 17J.lgldL (470nmoUL). b. Hypoo.d.. norortici.m {I} Dog. with primary, =I .ll. ""m r .._ACTH and po,I_ACI1-I cortisol oon"",mrations < 1.0 J.Ig1dL (< 30 nmollL) indicOI< ad .. noconicoJ atrophy, deslruction, or hypoplasia. c. HYJ'<'rad .. nocorticism {I} From 60 % to 80 % of dogs with PDH and about SO % of dog. with FAN han aawrat.d respo"'" to ACTH thot ..f1«t either bil ateral ad .. nocorti. cal hyp"'plas", or ... ponsin !lNplanic cdl .. It is not drat why nagg~ratw {2} =pons~. do not occur in all dogs with hy~rplanic gl and •. Som~ of th~ adrenoconicol n""pla,m, may not respond ba::;,,,,,, th~ nwplastic ""II, hav~ d~frctin """ptOts or nth ... ~naling p.thw.ys. Dogs with ectopic production of ACI"H v..ould b. apa;tw to han an aageerated cortisol incr~• .., ba::;,,,,,, of th~ pr=na. of bilat~ral adrenoconical h~rplasia. Dogs with iatrogenic hy~radrenoconici,m ftom chronic glucoconicoid administration han littl~ or no re.po~. {2} Th~ da:ision th=hold that rq>re..,nts.n exagg...ted respo~ v.ries .m0Ill: ,tudies .nd umally is near 18- 221lgldL (500-610 nmolfL). d. Nonadrenal illne!SeS {I} Some dog, with di!Ord~", other than PDH or FAN may have eugg~ratw ",spon,.. to ACTH. {2} Typically, th .." animal, are thought to han mess-inductd hy~rcortisolemi .. e. Aft"r op'DDD {milolane [L}'!'O and the zona n:"tirulari,. {2} ACTH stimulation tests con b. usw to monitor destruction of .dr"nocorti. col tissu~. Typically, the goal of op'DDD m.tm"nt is to ~ the post.ACTH [coniKlI] to b. < 51lgldL {< 140 nmolfL} but not """'" .ufficient dmruction to creat" a hypoad",nocortical !late. f. Aft"r trilost:m" (Vetory! or Modrenal) tr .... tm.nt {I} Trilostan", which i. an .It.. natin to op'DDD for tre.tment of PDH, rwucts neroid synth ....is by inhibiting 3p·hydroxysteroid d..hydrogen"", .nd thm reduces the synth.. is of cortisol, aldoneron., and oth.. steroid hor. mon~'. Trilo!lan. rwuct. [cortisol] in dog. with PDH and .Iso reduces [aldo!lerone] , but to a I.... r degree." {2} ACTH stimulation test, can b. used to monitor th~ effectiveness of trilo. ,tane tr .... tm.nt of PDH. [f the dog has continuing clinical .ign' and the po,t.ACI"H [cotti",l] i, :> 91lgldL (:> 250 nmollLl, then th~ d=>ge i. 021 18/ ADRENOCORTICAL RJNCTION incr .... sn!. If th~ dog h .. continuing clinical signs suggrni"" of hypoad .. nooonicism and th~ post_ACTH [conisol] is < 0.811g/dL {< 20 nmollL}, th~n tr~>tm~nt is discontinu«l."" 11. Cats A. Low-do"" de:um",h:ason~ ,uppr~ .. ion Ie;{ (LDDSn: not recommend..,j I. Procnlu~ B..,ically Ihe same procedure as for Ih~ emine LDDSf aa:pl daamelh_ ason~ is given at either 0.01 or 0.015 mg/kg IV. Some people ..ftr 10 use of a 0.1 mg/kg IV do"" a. a LDDST in em, bUI il is oofl!ide=l a HDDST in thi, lal. 2. Expect..,j ... uln" {Sp«ifi, I"'tient =uh. should be int.. pret«l by using ",f..ena: interv:d. and interpreYlion guiddine> provid«l with ... uln.} a. In h..lthy cats, the 4 hand 8 h cortisol oona:ntralions a.. um:dly < 1.0 ~dL {< 30 nmoUL}, but Ihey ..e nol suppress.-d thi. low in about 15-20 % ofh .... lthy cats. Coniwl suppression may :dso fail to occur in cots with non. drenal illness. Therefo.. , the lDDST has poorer diagnostic specificity than the HDDST {for which conical suppression fails to occur in very few cats} :md thUl i, not recommend«l. A postdeum",hason~ [cortisol] of 1.001.411g/dL (30-40 nmoUL) is con'ider.-d a bord..line r~sult. b. In cots with hy""rad .. noronicism, the 4 hand 8 h oortisol cona:ntr.ltiofl! a", apect«l to be :> 1.5 J.lgldL (:> 40 nmol/L). B. High-d""" deum.thasone supp.... ion t.. t (HDDSD I. Procnlu", a. The initial protocol is the same as with the conine HDDST, but I mg/kg daamethasone is aho used. Som. Jl"Ople ..f.. to a 0.1 mg/kg d""" as a low dose in cats .nd oonsid.. I mglkg a high dose. Others cofl!id.. th. I mg/kg an ulm_ high dose. b. em m.y the supprnsive: dfecn of de:um",hasone fm .. th.n dogs, <0 suppr~ .. ion may be "",n only if "'mpl.. a", collect..,j at 2 h, 4 h, and 6 haft.. daamethasone ." 2. Expectal n:su.lu for 0.1 mg/kg d~ {Specific I"'tient ... ults should be interpret«l by using ",f..ena: interv:ds and interpretation guiddin.. provid.-d with .. mlts.} a. In almost :dl healthy cots. the 4 h .nd 8 h coniwl cona:ntrations.re < 1.0 llg/dL « 30 nmoIlL). A postde:t:lmethasone [cortisol] of 1.001.411g/dL {30-40 nmollL} is co",id...-d a borderline r.. ult. b. In about 90 % of cots with hy""r.ldr~noronicism. the 4 h and 8 h cortisol cona:ntrations w... :> 1.5 ~dL (> 40 nmol/L). C. Ad",nocortiootropic hormone (ACTH) stimulation {r""",n",,} t.. t I. Procnlu", (may begin al any time of day) a. A sample for pre_ACTH [cortisol] is oollect..,j. b. Synthetic ACTH (Conro.yn) is given IV >t 5 J.lg/kg or 0.125 mg/au. Por<:ine ACI"H g<:l {Cortig<:I_40} may be usn! al 2.2 U/kg 1M. c. Pon_ACTH ""'pl....e collect~d >I 30 min and 60 min or jun at I h. 2. Expect..,j ..sui,," a. S""cific I"'tient results should be int.rpret.-d by using ..f... na: intervals and int.. pretation guiddin.. provid«l with results. b. A healthy cat should have:. p",_ACTH [cortisol] of 1.0-6.0 llg/dL {30-170 nmollL} .nd a pon_ACTH [oortisol] < 13.0 J.4:/dL {< 360 nmoUL}. ="" .22 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY c. Cau with hYp'rad .. noconicism =y han a p, .... ACTH [oorti""l] WRl or incrrasnl. and a 30 min or 60 min post_ACTH [cortisol] :> 16 J.lgldL {> 440 nmoUl}. Ho~""r. only .bout 40--50 % of em with hyp Ill. Ho ..~ A. [)a,me{hason~ suppression test l. Ov~rnigh{ mffhod~" a. A ..mpJ. for pralu'ffietharone [oortisol] is oolb:tM bttWO'tIl 4 p.m. and 6 p.m. b. Dexam<:{hasone i. given at 40 ).lgliq: (about 2 mgllOO Jb) 1M. c. Pondexamech.sone ! {I} Specific patient " , ulu should bt interpmed by ming "&,en",,, interval. and interpre:ation guidelines providal with ,e,ult •. {2} H~.hhy hoJ'KS: . drqu . te suppression ... ulring in a po,tdaameth.""ne [cortisol] < 1.0 J.lgldl « 30 nmoUL) {3} Hyperad .. nororticiun caustd by PPID: iruod'"'lu .c~ suppression " sldting in a postdaameth:asone [conisol] ;> 1.0 J.l gldL (;> 30 nmoUL) 2. Cortisol ron"",mration. during a 24 h p 1.0 J.lgldL at 20- 24 h after daamrtha",ne. 3. Snsonal v.riation: Conrurnnt with the 5U>Onal variation in [ACT H] . results of the '"'Iuin~ daamrthasone suppression test " r.,,~ diffe",nt in January rompar<::d with tho .. in Sepumbe:r.Jlln a study of 10 healthy horses and 29 ponies. the .. w:as no ",..onal differen"", in the pr.daamrth.,one [coni50l] {all < 1.0 J.l gldL}. but th~ postdaam.. hasone roni501 ron"",mrations w..e;> 1.0 J.lgldL in 26 % of the Sqlumbe:r sampl.. (50m~ ;> 2.0 ).l.gidL). The increa"":! AcrH ron""'ntrations and the results of the daamrthasone suppression tests in s.: pumbe:r .. mples JUgg<:St that a ..,asonal hyp a. In h~.lthy hor=;. the post.ACTH [ronisol] i, ap«:ted to be: 2- 3 times the pr=mple·. rom:emration.' Howev... four h..althy hor.." had post.AcrH roni501 ron"",mrations th>t "'''l:ed from 3.7 to 4. 7 times the p...ACT H ron""'ntrations.' b. In most. but not :all. PDH horses. the post.ACT H [rorti50l] i, mo .. than thr.., times th~ p ... ACTH .. mple conctnt"'tion.'M 18/ ADRENOCORTICAL RJNCTION B23 B~l1St of onrlapping finding' for h~althy ho", .. and ho",~. with PDH. th~ AcrH gd ,timuJ.tion test is not r«:omm~nd«i :as a diagno,tic t~n for equin~ PDH. ' 3. Results a~ct«i for h...Jthy and PDH horses if ACTH (oosyntropin) is ginn (I mg [V) a. In h~~lthy ho=s. th~ post_ACTH [coni""lj i. a~tM to k < 1.8 tim~. th~ p..s;omple·, [rorti""lj.'" b. In PDH horses. the pon_ACTH [cortisolj i. a~t«i to k;> 1.8 tim .. th~ p..s;omple·s [rorti""lj. c. COMBINED DEXAMETIlASONE SUPPRESSION- ADRENOCORTICOTROP[C HORMONE (ACTH) STIMUlATION {RESPONSE} TFST The major advancag. of th~ combin«i tost i, that it combin~. adrenal :as.=sm~nt into on~ diagnostic procfflu",. and thu, only one ~ of s;omple; is submitt«i to a rd'trence laboratory. The major disadvantage in doC' i, that it doo; not han the ",me diagnostic power as the combination ofWDST and HDDST don~ individ .... lly. I. Canine m~thod" A. A ,ample for a ba",l [conilOlj is collect«i. and th~n daameth. lOne is :odmini'l [I. Feline m~thod" A. A ,ample for a ba",l [coniKllj i, collect«i, and th~n dexamethawne is :odmini'l Ill. Equine method" A. A .ample for a basal [conilOlj is collect«i, and then dexamethalOne is :odministtr«i {IO mg 1M at 9 •.m.}. A 3 h po,tdexametha!One s;omple i, collect«i, and then co.yntro_ pin is admini,tereti N (I mg = 100 lUI. Then, a 2 h po,t_AcrH "'mple i, collect«i. Alls;ompl.. are submitt«i for m.. sur~ment of [roni!OI]. B. The", h:as be.n limit«iu.. of the procfflure in horses. [n one ,tudy, the combin«i un did not reliably diffe",mi. te .ix healthy horses from six ho", .. with PDH.' 824 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY ALDOSTERONE CONCENTRATION I. An [:oldos{~roll~l in dinical hYl""'dr~nocor{ici,m should bt d=~aMd actp! in r>r~ atypical cases. In primary and =ond.,y hYP" ... Jd",(eronj,m, aldosuron~ COllctmrations."" jncr~:asffl. B=u"" ACTH nimul.,es Ih~ ,d....,., of conisol and aldon~ron •• aldostuonr oonctmr:uiofll btfo.. and ACIl-I .dmi"i!!,,!ion may rrH= :> .ft., n. Primary hypu.Jdonrronism A. Dog. I. Th,.., = of primary hyperaldostrroni!JII caustd by .dr'II<01 neoplasm. W I'" dr""ikd." AU thrtt dogs WI'" p,... mal ~u .. of .pi,odic w•• kness, :md rach had hypokaJrmi. and an ["'do,t.ron,] :> 3000 pmollL {rJ"."ct interval nol providal with ""'" information; = p .. _ACTH aldon.ron. coII".mution, in healthy dogs in Tabl. IS.S}. 2. Anoth".:= woo. drscrikd in which Ih. p ....ming problem wu polyuria. Oth~r finding. w~ .. mild hypokaJ~mia. marhd hypophmph.remi.> •• lkalemia. drcr ...,aI plasma r~nin activity. :md incr ...,rd plasma [aldoswone] (740 .nd 840 pmollL in two ,ample'll oomp:..aI to 30-210 pmoUL in 12 h~..!thy dogs)." Plasma conisol oon"'nt.. tion, during. LDDST we .. within r~f~ren'" int~rv..!,.. m~asur~m~nt of oth~r prrcursor n~roid ron""ntration, w;o! not m~ntionrd. B. Cats l. C,..., of primary hyperaldosteroni,m Gmsrd by .dr~nal nalpwm, ltavt bttn de""ik .nd incre.s.d "!dost~ron~ to pla,ma r~nin activity ratio,." Th~ cats we .. not hyp'rnatr~mic •• nd "'m~ d~lopal ren..! l.. ion, ron,istem with p'"i!l~m hypen~n,ion. 3. Anoth .. cot hod an .d.. nocortical carcinoma that produa.d exct.. ivt .mount, of "!doneron~ .nd prog~steron •. The resulting clinical Jign' w..e due to the effect, of progtsterone (i .•.• diabete'l mellitu,) mo .. than to "!doneron~ effects." Ill. Aldosterone con""mrations in oth.. conin. disorde" A. Th... is not a cleor "'p'.. tion of pr ... ACTH aldosterone oon"'ntrations betw..,n h."!thy dogs .nd dog, with hypoadrenoconici,m. How"",r • ..!do't.roM roncrntratiom in the pon_ACTH "'mples w..e lower in dogs with hypoadrenoconici,m than in clinicaUy he..!thydog, (Table 18.5). B. Dog. with PDH hod ..!dosterone roncrntration •• imil", to th"",,, ofheahhy dog. in both pr ... ACTH and post_ACTH sample'l (T.bl~ 18.5). C. Som. dogs with non.dr~n..! dilOrde", had. vtry high [..!don.rone]. The high con",n_ trations ..~ probably rd.trd to hypovolemi.> or dec ..... ed dfc:ctivt blood volume (T.ble 18.5). D. Result, of. study of 31 dogs with PDH. S dog, with FAN .• nd 12 h... lthy dogs:" I. Aldosteron. oonctntmion. we...ignificontly lower in the PDH dog, than in the h~..!thy dog,. The authors suggc:ned that und .. chronic ACTH nimulation. some of T ab .. 11-'. _ " ' .. 00&< • • " " ; '. . f, .... ACnl ,,;......... toot. i. dar Al ~~,: 10"","", bypm."' _ _ tici .. • ......tu.u.J "' ......... v..,.. , I" n' ,,4 I" ~~ '" 120 J.jl ~ W;± llI 2ll ~ j<\ ~ III , """""" .. 0.- ." FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY th. aldon.ron._~ming cdls conv.rtal to ooniso]'KCming cdb as h.. oon d.""ikd in expuim.ntal.http and rat studies. 2 Aldostuon. OODctntr.u ioIU w... 'ignific;ontly grrarr, in Ih. FAN docs dUD in Ih. PDH dogs, but th. conctntr:u ions .'" prob.bly ,.laud to .. nin (angiot.",inogr_ n... ) coD".ntnrions via ch:ml:'" in blood volum. and dttlrolyt< CODctmnrions. E. Aldo,trron. to .. nin ratio l. Pwma .Jdosuronr oonctmrations are ,d.tM to renin concentrations via changes in blood volume or d,""trolyu oonctntrations. a. Aldoswonr production is stirnula!aI by angiorrmin II, which is prodUcffl by Ih • .. naJ ,d • ..., of,rnin in =pon .. to .. n:.! hypot.",ion or da:r] tubul ... b. R.nal ... po= to aldon.ron. result in retention of N.+ and ct-, which promote. plasma volume upaD,ion :md thm d=ra,aI stimulatioll of th. ""i".a"giot.,,,i" system. c. Aldose.roll. collomt"tiollS are a~.d to be g",atOdium. r<.I:crict.d diOdium.remict.d di with primary hY!""'drellocortici,m •• ldosr.ron. to ,,"i" ratio> w... coll5istently low.. (0.002- 0.08 pmollfmoU. [or 2-801,]) th.n the mio, ill 60 h.althy dogs {O.09- 1.6 pmollfmoll. [or 90-16001,]}" {note: the authors ..pon.d the rati", without ulli,,}. Ullits of the ratio. are rompla b.cau.. the author> musured ,,"i" a.ctivity (rath .. thall concentratioll) via the gen.ration of allgior.n,ill I; th. "tio .. pr=nt' the amount of .ldost. ron. {pmol} rdat.d to ","in activity (fmol of angiot.nsin I produced p.. S Thorn tm alld modifi.d Thorn tm"' .. A. 11I".ad of dir.aly m.a,urillg [conisol] aft .. all ACTH illjectioll. challges in [conisol] can be indirectly and roughly:assess.d via ch.nges ill blood [..,.inophil] (Thorn «>1) or chang.. illllrutrophil to lymphocyt. mio, (modifi.d Thorn tm). An [eo'illophil] is m.amr.d dir.aly. wh ....... the llrutrophil to lymphocyt. ratio i, calculat.d &om leukocyte diff... ntial COUllts determined 011 blood films. l. In. h...lthy dog. blood [eosinophil] is ap«t.d to decre;o .. alld the nrutrophil to lymphocyte ratio i. <x~.d to illcr.,... if blood [cortisol] inc",=>. 2 Failu.. to , .. a dec ..= in [wsillophil] or failure to..., .n illc..asaI n.... trophil to lymphocyte mio nt...dmininmioll of ACI"H sugge;ts the pr=lIC< of hypoplastic or atrophi.d ad",,,,,l gland •. IS/ ADRENOCORTICAL RJNCTION 027 B. lncr~a~ ~= of obtaining ""rum or plasma conisol con""ntrations and th~ir sup..ior diagnostic va.lu~ ha", mad~ th~ Thorn t~st and mooifi«l Thorn t~st nrarly obiOl.te. Howe""r. th~ rdationship be{v..""n blood [conisol) and blood leukocyt~ con""ntrations should bt ",membtr«l when int~rprffing routin~ romplet. blood count results. II. Con,entrations of oth.. steroid hormon~. A. Adrenal glands produce other ,uroid hormon~s. induding int.. mediates in the .ynth~tic pathwaY'. l. Inc",ased pro!:"st~rone com:entratiom han b.",n r~ported in dogs and a cot with FAN."'" 2. In a study of 53 dogs with confirmed hyperadr~nocorticism {including PDH and FAN <:aKs}, 69 % .... d an ~uwrated incru.. in [1 7-hydroIYprogest~ron~) in th~ ACTH stimulnion test, and 79 % had an exagg<'med incr..... in [coni50I)." 3. Anoth .. .rudy included 127 dogs with ''''peeted hyperadr~nocorticism. Aft.. ACTH stimulation, 59 {46 %} h ad an aaggemed increase in [conisol). Of those 59,42 (71 %) had an enggtm«l incrra.. in [1 7_hydroxypro~~rone).'" Also, an ""awrated inc~~ in [1 7_hydroxypro~.reron~) was found in 31 % of dogs that had na>plasms not rdated to pituitary or adr~nal glands. Th~,. findings emphasiR that adrenoconicol hyperplasia may occur =nd .. ily to non.drenal di ....... B. Mu.mr~m~nt of oth~r steroid hormone con""ntrations may hdp dff«t adrenal disord~", ... peeially if th~ synthetic pathwa)" a", defecti"". Some laboratories mrasu", conc~nt .. tions of the following ,t~roid hormon~s in pre_ACTH and post_ACTH samples: conisol, dihydroepiandrost~nedion~, estradiol, androstenedione, 17hydroxyproge;t~rone, proge;t~ron., and testost~rone. In some cas", steroid hormon .. oth.. than cortisol a", inc",ased.'" Refe re nces I. am"",, CB. G.oj.", VK. t986. U,.i,,J &.1""""'00 O/ c....,-;... An ......... Ph.iI.<\dphi", I.e.. 8.: Fd.iV'. 2. Dybdd NO. H_"" KM. Madip> IE. G.ibhk DH. ~ PC. Staknfdd, GH. t994. Dia&o>o'tic IN pttunuy p... in,,,,,,,,di. d"fuoctioo i.. 1.0."". I Am Y" Med Aa.oc 204>6Z7-<>J2. J. L.n«iool. to"[ tb,,,,xi .. , . ond h« thyroxin< «>o>« .. "atioo, in ..."'" and pi ...... of <10". J Am y" Mod A.oc 2t2,tS64-IS68. S. Pbilip Ell . M...,... SF. 1971. CdI ..... ....i.cion i .. tb, "P'"'" and m".boIian of rortiool by ani .. , ""tin, So""""',. c.",._."" 8. H,rtad Rl. lok_o .. SD. P.ot<mak DM. 1990. Effect. of ""'pI< k...dlins; 0 .. ad«DOo of b,p"'ado,DOOOrtici.... I Yrt 1m.... Med 10,1....,. 10. Fd.[", ... EC. Ndoon RW. 1994. Compo",;", .. poet, of Cuai.in,·, oyo>drom, in dor;, and ca". E..d.oai....J Mrtabol (]in N<>91. II. Duab 828 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY 14. M ...,. NT. IV. 1999. H"" to d i _ oq";"" pi...na.y 1"" . "..",odi. d,...<><J."" NM, 14S_ lol7. 15. BooM M, Griffin C. 1999. 1'""",,,,,,"0' """cio, ad.,n.l ro ... in • a' ..itIo. clinical .i&m of brP"'ad.,oororticiun. JAm Y" McdA_ 214,666-669. 16. Gala< S, KDoi.,,, HS, Vood.ou.t G, "'" "'" [D ~ TSGAM, MoIJA. no d,o IkJ 28,3}S-HIl. 17. Y.miDi B, V""o.oB.in.k PL. Rd'.J KR. 1997. 0....; ... m.oid cdJ tumo. -...hIio ~ l.tN", .....cl.t«! ..tth ~yp".d",><><<>rti.ci . m (Cwhio, ', di",...) in . doc- Y,,!'athol. 34057--60. 18. 1'..... "'0 ME. G.= Orth DN. 19S9. r.imuy b.rpood.""""",tician in un co", J Vrt Int"" Med },Sl--S8. 19. Co.'tilll. HoIf", ... AM. [?SIB. Ad.,o.J i.....rr.ci",<J' j., • """""tallOaJ. J Am Y" Med A_ 212,) S9t-I S96. 2(). o.nn K/. H ..... V ME. 1'!98 . Hrpooo.tiool ....... in . Ub..d.o............. J SmaIIAnim Pract J9,90--9}. 21. UI'ton 5J, Kine LG, z...b. co.. 1m. Gluoocorriooid d,fid,nt Ioyp"..d",.orortician in do,,, )8 'u , . (19S6--I'!9S) . JAm Y" MedA_ 21)9.2076-2081 . 22. I«>oi ... , HS, ru;ob..k A. van n. on «nOm «>nan"'';'"'' of akIo""""",. rooci",l. and"""';"'" in doop ... t!. pi .. ita.y-«ito! ,,,,,,tinin< ..tio " • tct=in ~ _ fo. ~yp " • oct=in, - . fo. kyp«>rticitm and ~yp win..., ",rti.roid/",,,cinin, .. cioo in.- ..it!. b,peo<_ tid."" 225 """'" (1979-19"9}) . I Am Y.. Med Aoooc 2OB,Bj....91. JI. Scorbini M. r....,..,i D. ~ M. c.......... M. 2004. Eq""" Cwbin ~ .lil.. oyndrooo" Dia&nooia and t!."'''P' in ..... "'.... Y .. R.. Coonm.o 2B(S.ppll ),J77- }S(I. J2. DonaIdooo MT. McDonodlSM. Scltanb.tdt<. BI . lamb SV. "kFadan. D. &.d.. I. 200S. Y..iation in 1'1...... od"""",rtiootropio ltonnon, rottCl odmini.ttation 00 ",..!t. of "",m bi.oclttroploic P''I''''tiooo in oonnal do, •. Au.t Yrt I 76.255- 2';7. 40. Ftank IA Otiv.. JW. 19"98. Compuioo .. of "'.... ",rtiool os. IS/ ADRENOCORTICAL RJNCTION 029 41. «,,"'ppaio,o RI, IId.....d EN. B.ocb KA. 2OOS. U" of rompoundod od.,~k """""'" (AcrH) /'0, ad"",a/ fuocoioa ,ati"ll: io «>«>rtici"" in d" ho,,,. Equio, Y" Ed"" 4'lJl- 13" 46. Eik. H, GobI, O. 0Ji.n I. 1979. A1tmal Pnd functi.oo in "" boot", [/foel> of ""'J""r<>pio (.,.,tl,...k}...d ro.-tkottopiD (Ba,.ni) otim..Jaoioa. Am I Y" R.. 40,724-726, .fl. br),., CA. Noduoi"" RF. 0..00'" RW. 0.11.". lB. 1987. Hyp<,.J«D<>OO. I Am Anim Hoop Aaooc 35,.(11-416. 53. Ecn CEo R..bin .... WF, H..nabI, CRR. 198J. Prim • .". &kIoot<>onian (Conn', oyndrout G. """ Slv.i;' FI, """ <><>ian in • ca. with "" .J..rud ",rtkal carcinom •. I Y ... 1m"" Med 19,JS5-358. 57. I",ad; s, Kooi"" HS. Mol JA. &." p, Bo.. WH, Rij""'d. A. 2003. PL."". &kIo .....", conc«>rtici.m. Y" Roc 15Jo521 - S25. 58. Oobaldi,,,,,, GW, c,...., T. 19~. E..timatinc;.dr.nal oortk.[ f.."<1 «>«noratio ... in do" ...it!. """odtmal """pI .. ia ...d doc, ...it!. RUp«tod 1oyp<<.d.,D<>OO.....J in "" doc...d ca•• s....;" Y" Mod 5~'I: (S .. alI Ani ..) 7,285-291. 67. Fd.d ...... EC, Ndooa RW, FdJm.n. MS. 1?96. U" of 10.._ • .,.J It.id>-Joo. d=m foot di~J.inc; pi...n~"" from od.maI ... """ byp<.odt«>rticiom in doc,. I Am Y" Mod Jw.oc 209,772-775. 68. KapI .. AI. P"",oon ME. «""'ppaio,n RJ. 1995. Effirta of di" ... on t!., ",.tit, of di."",otk "' .. , IN UK io d,,,,,,;"!: b1P"od.«>rtici.m iD do". I Am Y" Mod Jw.oc 207,«S-4S I. '0 "''';'''; A\doo"""""". u.. """"to Chapter 19 CAVITARY EFFU SION S General Concepts and Definitions .............. . ............................. Pathogeneses of Cavitary Effusions ........................................... I. Transudates ...................................................... II. Exudates ........................................................ III. Hemorrhagic Effusions............................................. IV. Lymphorrhagic (Lymphorrheal) Effusions ............................. V. Effusions Caused by the Rupture of a Hollow Organ or Other Tissue ...... VI. Effusions Caused by Multiple Processes .............................. Routine Analysis of Pleural and Peritoneal Fluid ................................ I. Sample Collection and Processing ................................... II. Ph~ical Analysis ................................................. III. Chemical Analysis ................................................ IV. [Erythrocyte] or Hematocrit (Hell .................................... V. [Total Nucleated Cell Concentration] (TNCCI .......................... VI. Microscopic Examination ........................................... Selected Analyses for Pleural and Peritoneal Effusions .......................... I. [Cholesterol] and [TG] ............................................. II. [Urea] and [Crt] .................................................. III. [Na]', [Cit, and [K]' ............................................... IV. [L-Iactate] ....................................................... V. [Glucose] ........................................................ VI. [Bilirubin]. ....................................................... VII. [Ammonium]. .................................................... VIII. Protein Concentrations............................................. IX. Enzyme Activity .................................................. X. Gram Staining .................................................... XI. Culturing for Microorganisms ....................................... Comments About Specific Effusions .......................................... I. Septic and Nonseptic Exudates ..................................... II. Differentiation of a Bacterial Exudate and Intestinal Contents ............ III. Effusions Associated with Neoplasms ................................ IV. Lymphocyte-Rich Effusions ......................................... V. Pericardial Effusions ............................................... VI. Amniotic Fluid .................................................... 832 837 841 843 844 845 846 847 847 848 849 850 851 851 851 856 856 857 858 858 859 860 861 861 862 862 862 863 863 863 864 864 865 865 831 B32 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY Table 19.I.Abb...,,,iatiom and symbols in {hi.. chapler [x] Con""mmion of x (x = .nalyte) ALP Chol: TG Albline phosphat..... Cholcswol to Irigly"",id. en Crntininc Ethylcnffliaminmuaac:Hic acid Fdinc inf~tjous ptritonitis Hematocrit Sp«ific gnvity TriglfC"ridc Total lIuel.alal cdl oonctmmion Total prouin Toul prouin d.urmin. lion by rCfr:lctoffietry EDTA FIF Hn SG TG TNCC TP TP_ GENERAL CONCEPTS AND DEFINITIONS I. body Clvilies ~cau .. of phys.iologic or p"thologic processes. Result. from the .rudY'is of the .ffu,ion .nd olh" clinic..! information ... <= k oollsultal for mor~ illformation .bout all:olytic:ol m..rhod. alld th~ micro,copic f....tures of reUs .nd oth~r SCructur~s ill dfu,ions.'-7 II. The pleurn .nd ptritoll .... l coviti..... lin..:! by a lay~r of m~sothdi.ol rell, (m ..othdium) with two surf.""s: th~ voc.r:ol surf."" rru.t conn vise",a (lullgs. immin~. HC.) alld th~ p .. i..ral surf. "", th . t m..:!i.o!linum and th~ pleural and ptriton~al w:olls. In mon .pteies• • small amoum of d~ar serous fluid ill the cavities provid.. a lubriamt alld malium for transport of dectrolytes and oth~r subnallces. Th~ covitie. a", c:oll..:! >trow kcause they com";l1 • dear waury fluid in h~.lth. Pleural alld ptriton.al fluids are form":! by simil.. processes that illvol"" th~ forces of Starlillg's law alld an.romic muctures. G~lIer:olly. a pt.srna filtrat~ I~.v<:s the copillaries •• mer.; imemitial SI""". and diffuses imo the serous coviti... from which it i...mo..-ed by the lymphatic 'pum alld return":! to plasma. Pku,,,{ fluid is form":! in h.. hh by diffusion of fluid through the I"rieul mesothe. lium to th~ plrural cavity (Fig. 19.1).' The fluid has a low [prot.in} alld low ""llularity. p"j"mu{ fluid is form":! simil.rly. with most of the fluid r~mov..:! by diaphragmatic lymphatic ==I.•.' 00""'" Ill. Comp<>!ition of pleural fluid .nd ptriton..al fluid ill h..alth. A. Th. ch.mic:ol comp<>!ition of a body covity fluid i. primarily determill":! by ptrme.bil. ity of capillari.. to H,O .nd solutes and. to a I..... extem. ptrme.bility of pleur:ol .nd ptritOIl.al mesothdium. l. Capill .. ies are ptrm~abl. to H,O . tlectrolyt .. (•. g.. Na+, K+, ct. c:..>+. bicorbonate. alld phosphates) and sm:ollllollprouin solutes (•. g.• glucose. u..a•• nd Cn). Other thall [prouin}. the imerscitial fluid adjacent to most copillaries has dectrolyte. u .... gluco ... alld Cn con""mr.tiom .imil .. to plasma. 19/ CAVITARY EFFUSIONS B33 p I Parietal Tissue e Inl erstHium C"'-'; , -- .. -, , Capillary :, , --t+ ,, , ~ 1--", -----_.' Lymphalic vessel Slomata - . ':. u r a I Pulmonary Tissue Interslilium C----'; , --- .. -, +-:i, Capillary , F I . ' 'd' U Lymphalic vessel i Fig. 19.1. PLeur:.l 8uid form. non md rem"""'. Forceo of St:uling'. low mov< prot B. C. D. E. 2. [nte",titial fluid is the SOUl""' of most pleural and J><'ritoneal fluid protrins. Varia_ tions in capillary J><'rmeability to pl.. ma proteins causes v. riations in in,.",tit;..l [TP]. For example. in pwple. the interstitial fluid [TP] i. near 1.5 gldL in skdrtal muscle, near 2.0 g/dL in SlIbcu,.n~u, ti .. ue. nrar 4 gldL in int ... ine, and near 6 g/dL in li""r.'· The [TP] of pleural fluid in health is n=- 1- 2 gldL." Pleural .nd peritonral fluids in healthy animals should not rontoin erythrocyt.. and should ha"" low ron""'ntratiom of nudratw ,,",II! {e.g.• mesothelial erlls. lymphocyt ... m.crophage •• and neutrophils}. How~er, thora~ntesis or alxlominocrnt .. is typicoJly damages small blood vessel.. and thus. f~ erythrocytes .nd "'" blood leukocyt...", not unurual in oollecrw fluids. Canine pleural and peritoneal fluid •. I. The .mount of pleural and peritoneal fluid. in healthy dogs is ""ry small. so it is difficult to rolJ= sufficient fluid for anal,...,i •. One "'urer .to,.. that healthy dogs ha"" 0-15 mL of prritoneal fluid . nd about 3 mL of pll ural fluid." 2. The", i. very little inform.. ion . bout the expectw fraru ... of thest fluid. in health. but data from multiple sources .uggest they should have thest feam"",: rolorless to slightly yellow, dear •• [TP...J < 2.5 gldL. a TNCC < 3.0 X 10J/IlL. and • mixture of mesothdial erUs. neutrophil., lymphocytes •• nd macrop• . " Typically. covital}' fluid it oolJrctM when there is an effusion • • nd the task i. determining thl cous<: of the effusion .nd not whether the fluid is .bnormal. Fdine pleural and peritoneal fluid" A> in dogs, the .mounts of pleural .nd peritoneal fluids in hralthy cots "e very small. '" it is difficult to 0011= .uflicient fluid for anal)"i •. [f fluid con be: ooJlrctw, the fluid probably rq>rr"'nts .n effusion. Equine pleural . nd J><'ritonral fluids: Healthy hors<:. do ha"" enough covitary fluid for ooJlrction and anal,...,i •. bur the rrpottW findings vary comiderably (f.ble 19.2). For Tabl. 19.1. IUponM r.. ...... f pl •• .1 aaoI. po,m-oI tha4 . .f ~Mhb.! 100.-' N....ba-""'-", Plno-d 0IWd' _.,,,.n,",,, " 1<>-100 "Lch<1- v,,",- ooIlort«l (mL) ~ ~ Y _ « ""-rdlClw"",,,y ~, n .. (cIdL) rnoc (x lo'II<1.l N ... «opI> . """""",. L""pbocr<" ~~ M ___ F";""I'IU M .. , «II Rod Wood ,dlo (x lo'¥1.l d.4' ( -dy < IS) 0.1-12.1 0 .... ,0.3 ' IO'II.<>.7 , ,00II.<>.2 , ,0'II •'<>-100 a... Li,bt rdlCku .. obthdr twbi Cl.1-H' U _l.s' n.I _ L~ .~, OS- I!).I ... , .. . ,. '. .16-7' .. _. I_II .. 1_17 .. .,. o.aO.ood o.,"",,"d U~ • O.l-~.' ,~ . I_I'" ,~ . 835 19/ CAVITARY EFFUSIONS Table 19.3. Repone.:! fcuut'eI of peritoneal fluids of hcal.lhr aide' P~rilon",,1 Numkr of allll~ TP.... (gldl) Fibrinogen .... (gldL) TNCC (x lo'lJ.lL) N.... lrophil. Lymphocytes Monocytes/mocrop. Eo!inophils " 0.1--4.6 fluid' P"ilon~.1 , fluid' P~rilon",,1 , 2.2--4.0 <3.0' 0.4--3.0 < 10.0 fluid" o. 1--{).4 5.0--30.0 12- 58 % 1- 28 % 1- 28 % 25- 72 % < 2.2 x lo'lJ.lL < 0.2 X 1001J.lL < 1.0 X 1001J.lL < 0.6 X 1001J.lL • • • • • Ewn thougb clinic:illy b..lthy. th. [TP",I > 2.0 gldL :md fn phs): Wilson .. aI." 'Sour«: An<\euon et .1." • So",", Kopdt. md Scbultu" 'Reponed .. [total "'~"' I (gldL) f o..cribed .. u".. lly. I : I ... io of neutrophiL. md mononocJeu ",lis. but rno up to 60 % "";nopbiL. bolh Ih~ pl.... ral :md pl is npcclw in h""lthy :mimol. of mon species. Thus, Ih. findings nuy nol lruly r~p ...~m data from heallhy ande. TNCC may k inc"".. w during Ih. first 2 wk poslpartUm." IV. o.,finilions A. Effolitm i. Ih. aa:umul.tion of fluid in • lxxIy 'P= or cavity. and .n ~ffo'ion is th~ fluid th aI ho. aa:umulated. B. Mcilr' is Ihe fluid .ccumulaled in a ..,rous cavily (Iypic:olly p"rilon",,1 cavily). II can be a u:mmdate, exudale. or oth" Iype of effusion (e.g., hemorrh:agic """il"). C. Tranrudation i. the p"""l:e of fluid or solute Ihrough a m~mbr:me bccau.. of chang.. in hydraulic or oncolic pressure gradi~nt •. D. A """",da" i. an efll"ion produced by chang.. in m~.nic:ol faciO .. SlIch :as oncolic or hydraulic pressu"" Wilhin capillary bcds. Such chang.. influ~nc. Ih. loss or resorplion of fluid. E. Hydroiftltic prruur~ i. Ih •• n~rgy (pressure) of. fluid al resl. F. Hyd,,,u/ir P"'''''' i. Ihe energy (pressure) of. fluid in mOlion. G. Exudation i. an exuding or oozing out Ihrough pores. H. An ~xuda" is :m .ffusion produced by incr""scd va.\Cular p"rme. bility 10 pl"'ma prolCins bccaust of inflammalion. I. Hmto"""g~ is th~ =p< or 10.. of blood from blood vessd. or hearl. J. LJmp/nrrl",K' i. the ..cap" or 10.. oflymph from lymph ves•.!.. K. A modifi'" tran",dau is • lransudale Ih al ru.. been modified by Ih. addilion of prolein andlor cdb. This classificalion is nol r«:omm.nd~d by the amho ... '" FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY l. P>thologic proc= that produ~ thest ~ffu,jolls ar~ V. riM and not consjn~ndy d.""iklr con um:dly k r..adily diningui,h«i from a typic;o[ exudat.; but /lUlly atrav.scuJ., fluids are modjfiffi transudate< or modjfiaJ exudate. with some chana.riniCJ of both, so r~ni_ tion of th. etiologic process may k difficult without a mo .. complete n:lminarion of the fluid."" Modifial transudate v,as Jistw :as on. tYP' of .ffu,ion in , 1977 ,,",urinary clinical pathology textbook." It is now" common l. btl or clowificat;on Nen though it moy not :K:CI1ratdy rrftrct the pathologic proa.. responsible for Ih • • ffu,ion. 2 Th. modified tranmdatr cl.ssifi"'tion is usually basN on J.bor.uory data r>th .. than the pathologic proass or pr<><='l<' m..t prodUcffl th. df."ion. S;"icoJJy. thi, cla"ificotion has bttn u...d for dft"ions th at had a [TP...J and/or TNCC in • grq ron. b.lWttn prot.in_poor t .. nmdot .. and typico.l exudotel. 3. A v.rigic. mylom. cong<sti"". n""plastic. and low1: ..d. inHammatory .!fll'liom. [nn"asN hyd .. ulic pressures contribu", to t ..nsudotion in congesti"" h.an f.ilu ... but t .. nsudation i, not th. prim.ry m.rnani,m ... ponsibl. for th. Huid that accumulo",. in h.morrhagic. most chylous. and moll n""plastic .ffusions. [n [hi, mapt....!fusion, a.. not das,ifi.d ., mooifia! t .. nmd.[el. b«a1lS< th. dassificotion d""" not d ... rly communicot. th. pathologic P"""""" that l.d to th••!fu,ions. and m any of th. ",-calla! ,,"odifid tramudam do not acrumula", b«au.. of t .. nsudo_ tion .nd mb..qu.nt modification. mot v. B",ic conc.pts of Starling·, law (Eq. 19.1 ) a.. nNda! to und..stand th. pathog.n...s of body cavitary .!fusions. [n th. context of most c>pillary b.ds. th. major variabl.. a.. illustr.u.d in Fig. 7.3. N {19. 1.} , =.. A. A majority of th. fluid that .nt.", th. interstitium from plasma return. to th. pl."". via the ppillaries (capillori.. that Op<1I into nllules). Th . .. mailld.. • nt.", th. lymphatic v. ssds and returns to the blood via the thoracic duct (most of th. body) or the right lymphatic duct {parts of h....d. neck •• nd thoraxl. B. Most copillory wooll, ha"" minimal ptrm ... bility to plasma prot.ins. but some prot.ins do .nt.. th. int. rstitium and th. resultant lymph. Th. pro",i", in the int.rstitial Huid c.... t. all ""travascul ar oncotic pressu.. (roJJoidal osmotic pressu..) {,.., Ch apt.. 7}. 19/ CAVITARY EFFUSIONS 037 I. Oncotic pressur~ in int~mitium of skd~uJ mu.d~ it about 30 % of th~ plasma oncotic pr~ssur~ in hnhh. 17 2. Oncotic pressu,"" in th~ hq.atic interstitium it n~arly «J.u..l to pla,ma oncotic pressur~ ba::au.. th~ w:oll, of h~p'lIic sinusoid,.,"" p Effu,ion, a.ccumulat~ in pleural and Jl<'riton~al covities wh~n one or more pathologic p~ (Tabl~ 19.4) cousr increasnl entry of fluid into the covity .nd/or d~crea..d removal of fluid from th~ covity. Th= proc.....' ..~ illustrattd in Fig. 19.2. Th~ oompo.tition of th~ effusion wiU provid~ ""id~n~ for th. type of pathologic process that ltd to th~ ~ffllSion (T .bl. 19.5). How"""r, fr«J.uently the rv:id~n"" is not di~no,tic of a diKlrder, and thus oth~r information Fig. 19.2. Scb.m. tic drawing of tl>< fin IJUjOl pathog<~ of pJ.ur:.l and pffitoneal .ffusioJlll. I. T nruudat .. form when m... i, inae""", vuruhr bydnulic prestwe .. itb or .. itbout dec",""", pI .."", oneotic preuw •. TIt. tJ.",oo. te form< 8uidt by tl>< lymphatic v""",1s .ither be-c ..... of incr"""! p........ ,..;thin the lymphatic """"lor b.a"", ~Ils ( •. g.. ....,pl..tic) .. e blodcing patbway>. Darruge to lymphatic """"It .nable. lymph to ""'I'" to c .. te . lymphocyt ... rich dTusion. If the lymph oonu.im dtylomicroru, tben a dtylous dJusion fornu. S. Darruge to enabl .. content! of thote ,tructu= to enter the body avity (e.g .. • uropffitoneum). TIt. content! ",Je...d from d:unaged :dimentuy. biliary, or urinary tissue• ..ill initiate an inJIamrrutory ..",tion and thut ",an I! jU Ii I 1 'I ° ,I 1 .iJIII ,111 Ii! If • ,I I ' I '! l. "! I'J '! '1! 'l '! I', i)~j~:. 1 1.'1 .'1 S 1 "f U . if! 1~il " 111 !~[ tH I1 1! • II'II r II ~ l" ' ~l 1 I . I j Ii : 1 l' [I ,. }'; O !,,!'!t' 1I I 1 iJ!I1 }1 fl fll }J ' 111;"11'1 . , 1 , It" lilt 1 l' i'J "1,1 j . it i j .Jll ,1 ~~. ~ ' • t,: :-;'lI1h! .ll 1 ~ i 1 !hilt!l ., i " Hil "1 i 'in ,11' I r! ° , 8. .~t" ·l :!lf~ .lil!l!j·!! i • ~ .. ·1 . 1 ° it ·11 ~ ,Iii ., I" ~' ;~ , iI ; ! e • e •• Tab .. , ..,. ,-. c -........... fofF"';"... -~ T""' ....... protria nm' ~,bo Phew f.-.ua' a. .... _ ]Nee (x ' oJ/eLl <" a. ..... do..dy -, ~ -- ~-, "0 "0 >M N......p.ilo ... ......... Bod no >\.0 N......p.ilo .. .......... no > ).0 Ncutropb.lt .. ,- "0 <M H..,. ,. do..dy "0 Ydlow, """"', K H..,. ,. do..dy "'C" H..,. .. do..dy H..,. '0 cIcnIy a. ..... ....,. Ydlow, ''''' "tom, """"', ~- ; R...arn m~ ' * ["., ..... "'r"" R...arn m~ ' * ["., ..... U< "'"_hoe<' ", o~ ", Ydlow, u.o, <=m, " "'C" ~,F!!" ..J",« M« Ydlow, u.o, <=m, " "'C" &..-., ","writ: ' .......;N·' • "I. < \.0 Ydlow, u.o, "" .... , fuo, d U. (r;ldll N......p.ilo ... ......... "'"_Iooc<' >M N......p.ilo ... ......... "'"_Iooc<. ~ lymr b I'''' .... -~ F.apo ...y _ "" 1.... <1 .... ........, !'JI~ pacaDp "'1, puuitt: ...y _ "" 1.... <1 .... ";""""'I'Y - , . "",uIu .... ",~, ..... ,;" """"' •. f"'h'l" ... ~ ~~- fib';" - H __, . df....., ""'~ ~2,0 > , •• ~2,0 > , •• -~ ~ ~2, o' < 10.0 Sao .. I)mpboq<, (OO ~ &- H __, . ... HUJ' to "I"'l'" ~ a.,Iouodf...... -~ ~mpboc)." (.... , HUJ'to .... '" <1..-";"') Mi=l~ (Iooc~) L,...,m"" HUJ'to oo..ty y"",,"pm, ,- df_' u.....,;_""" "'"tidy) Uropm-.... fe" 'm<)' '0:-0"_,, ' n" ................ .,. ...,. Sao .. I)mpboq< .. <2.0 u,;", aymoI, .. 'I""" ""'.....dy V,,; > u,;", "J"'I' "",.......u,- "'-' "'-I, ""' .......... V"i~ 1 <10.0 --.- S""""",,,bili< ... ..,..,.... fioid ",d ......... 1 . TI'.. _ ' " '" 1oIod,-;" L~ 19/ CAVITARY EFFUSIONS B41 (history, physical aamination, imaging finding>, or oth~r laboratory dan) is n=:lal to inwpm ,ignifiCOln", of .rudysis results or esublish th~ patho\:"n..is of:m dfusion, th~ L Transudat~s A, Pathogtnesi, L Transudau. typically accumulat~ in plau:d andJor I"'riton..J COIviti.. wh~n th~r~ is an a", .. diffusion of pla"na H,O from th~ vascular 'pa'" ba::.u"" of alterations in hydraulic and/or oncotic pressures, Th~y =y also .ccumubt~ wh~n lymphatic drainag~ from a COIvity is impai=1 ("'" F~, 7,3), 2, For most ti .. u~s, • transudate is prot~in poor ([TP..a < 2,0 gldL), but transudation from a liver COIn crnt~ a prot~in_rich peritone:d transud.t~ if normoprot~in~mia is pr=nt, [f. markal hypoprot~in~mia i. pr~.. nt, th~n th~ transud.t~ a",oci.ud with hq>atic transudation will ronuin a lo~r [TP] than if forma! wh~n th~ plasm. [TP] was not d«r~"""", 3, A tr.Insudativ~ pl~ural ~ffusion is COIU.ffl pri=rily by incr~aud hydraulic p.... u.. in the alveolar COIpiU.ries, Most of th~ pulmonary transudat~ r~mains in th~ lung {i. ~ " pulmon. ry a!~ma}, but it may flow into th~ pleural COIvity ba::.u .. of damaga! vi=ral pl~ura. Concurr.ntly, venou, coIIJ:<:Stion and th~ resuhant incr~• .ffl hydraulic pressur~ in th~ post~rior vena ",va ra!u"" lymphatic drainagt from th. pl.ural COIvity, 4, Port:d hypen~nsion is a fr'"'lu~nt rontributor to th~ formation of !"'riton.:d transu_ dates and occ:asion:dly pleural transudates, Po",,! hyptrtm;ion is a pathologic stat< in which tho hydraulic pressur~ in port:d blood vessds i, inc",asrd by oith~r incrnKd porul blood flow or, mor~ likdy, incr"",a! ..,isun", to pon.l blood flow, a, Blood ~nt.,.. th~ . bdomirud port:d venow syst<m from COIpillary brds in vis""ra (intestin~., sple~n, panc",.. , stoma.ch, . nd dinal.sophagus), tr.vels to th~ liv~r via the ponal vein and its branches in th~ port:d triad, flow. through the hepatic sinusoids (COIpilJari.. ) of th~ h~patic lobule to th~ ",ntral vein, aits tho liver via th~ hepatic vein, and .nt~" th~ vena COIva, u.ions within or adj=nt to th.", vessds or the h~.n may impair port:d blood flow and incrnle hydraulic pre,su.. within the porul.ymm, b, Ponal hyperronsive disord~" am be clas..ifial by overlapping .yst~m.,'~" The iI""",mic IJ'tmI is primarily bastd on grOSl anatomy (i,~" pr~hepatic, intra_ hepatic, or ponh~patic) and rd . tes to th~ flow of blood to, through, and from th~ liver, Th~ Vdscufilr IJ'trm id~ntifi~s the locnion of tho l.. ion rdativo to the hopatic sinusoid, (i,e" presinusoidal, sinusoid:d, or postsinusoidal), The disord ... that restrict port:d blood flow COIn be cla",ifia! .. lumirud (e,g" thrombosi.) and atralumin:d (e,g., hq>atic fibrosis), {I} Port:d hypen~nsion COlUSa! by right h... rt failure i, posthepatic .nd pominu_ soidal, B«au.. there is incrnla! hydraulic pm..u .. in th~ h~patic ,inusoids, the resulting effusion is protoin rich, {2} Port:d hypenension COlUSa! by hq>atic cirrhosis is intrahq>.tic but =y be .ith~r p... inusoidal or sinusoidal, Whon the incr~.sffl hydraulic pre .. u", is presinusoidal, th~ resulting ~ffusion is prot~in poor, Sinusoidal hYl"'rt~n,ion may COIU.. protein_poor transudation when hypoprouin.mia is marka!, B, Protein_poor transudares (Tabl.. 19A and 19,5) L Th= t B42 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY [albumin] <: 1.5 gldL. How,""". in mon disord"", hypoalbumin.mi. by itsdf will not must transudation. a. Analbumin.mi. occun in ~pJ. and is rn:oract.riud by ",rum [albumin] <: 0.1 gldL; {hu. pwpJ. typicolly do not han mnsudotj"" dlU,jons but may ha"" mild aI. ma. Com~nsatory process.. that pr.""nt transudatin dfusion! indud. incr~ 'ynth..i, of globulins, ,.ducal intr:IV'!CUw hydnulic pressurr, ..ducal im.,S{;{ial onootic pressurr, and inc..asallymphnic dninag<'. Th. pWIlliI oncotic pr<ssur. in .nalbumin.mic pn>pl. js .bout half of th at found in hralthy J'U'pl •. lO b. Th. J.ck of .ignificant tnnmduiv•• ffusion in ch• • b..,n"", of albumin .mph. _ s;u. thrtt oon~pts: {I} globulins oontribut. to onootic p..lSU.... {2} incrrasnl lymphatic drain,,!:' of. cavity may comp'nsau for increasN transudation, and {3} factors olh., than hypoolbumin.mi c. An acut~ on~ of mark«l hypoprot~in~mia (~.g .• mark«l blood lOS! folJow«l by intr:IVenom fluid th~rapyl =y l~d to transudation ba:.u,", th~ .. has bttn inmflici~nt tim~ for adjustm.nts in th~ oncotic pressure gudi.nt and lymph.tic drainag~. most common disord.rs that produce prot. in_poor tr'filUdates in dogs ..~ hq>.tic cirrhosis and prouin_losing n~phropathy (f.bl. 19.4). P.thologic prouin_ poor transudat .. a.. unrommon in cats. horse;. and canl~. [n both cirrhOli. and prot. in_IOling n.phropathi~s. tranmdates form baau,", of th""" two major factors:" a. D=~..al plasma oncotic pr<:S!ur~ (I) A:J th~ pl,uma [TP] f.lls. prot~ins (induding albumin) in the int~rstitial fluid a.. ..dimibutal to th~ vascular space. If th~ pw= [TP] and th~ int~rstitial fluid [TP] d.n• ..., by the sam. amount. th~n th~ oncotic P"'"SU" gradi~nt does not ch.nge. Thu •• th~", will not ~ .dditional H,O mov.m~nt from the v• ....,ls. Wh.n th ... is mark«l hypoprot~in~mia. 1= interstitial fluid protein is .vailabl~ for ..distribution to th~ vascular space. (2) Wh~n th~ onrotic pressu", in pl>s= da:..-..es mo", than th~ interstitial fluid. the onrotic p .... ur~ gradi~nt i. ralucM. and thus 1110'" fluid will .nt~r th~ interstitial space. However. a tranmdat. will not .ccumulat. if incrra...! lymphatic drainage .. mov.. the fluid. [n ptopl~. lymph flow can incrra.. by .t l... t a f.ctor of 10 to compenSsm •. " b. Inc",..al hydnuJic pressure gradient (I) [n cirrho.is and prot. in_IOling n.phrop.thies. abnormal regulation of blood ""lum. leads to "'t~ntion ofN.+ .nd H,O. Th. reuntion of H2 0 cau... an incrra...! plasma hydnuJic pressure and thu. a gr~ter rate of fluid l•• ving plasma and ~ntering interstitium." Ponal hypc:n.nsion .lso contributes to transudation in cirrhosis. (2) Th~ procc:...,. that l~d to th~ retention of Na+ .nd H,O in""l"" hormon.. and r~nal functions (= Chapters 8 and 9). 3. Occasionally. lymph nod~ di ........ prot~in_losing enteropathies . and idiopathic noncirrhotic ponal hypert.nsion cau.. th~ form.tion of prot.in_poor effusions. a. If. protein_poor .ffusion is associat«l with intrathoracic or intra_abdominal lymph node disrale. at Iran part of the mISOn for the ~ffusion is decreas«l lymph.tic drainage of fluid. that normally .nt~r the caviti~s. 2. Th~ 19/ CAVITARY EFFUSIONS B43 b. If a prot~in_poor ~ffusion is :associat~d with a prot~in_l05ing enuropathy, at 1..1St two mechani,nu Jruly b. contributing to the eHU,ion. If th~ ~nterop"thy has lod to a Jrulrkod hypoprouin~mia, th~n p"rt of pathogenesis includes a d""rea.o;od oncotic p",",ure gudi~nt. Aho, ,om~ prouin_l05ing enteropathies impair drain~~ of inte"inal lymph {e.g., lymphoJ\i:i=asi.j. c. Idiopathic noncirrhotic portal hy~rt~n,ion in dog> i, an uncommonly rNDg_ niud disord~r that Jruly b. caused by pon..! ""in hypoplasia. Dogs with this disorder hove protein_poor ~riton...J tran,udates and many oth.. labor:uory data sugge;tive of. port"'yst~mic shunt or hq.atic insuffici~ncy.>1 C. Prot~in_rich transudat~. {Tables 19.4 and 19.5} l. Prot~in_rich transudate, typically occur when th~ .. i, increa.o;od plasma hydraulic pressure in th~ liver or lunC' because of venous cong<'"ion. 2. Th~ most common disorders that produce protein_rich tramudates a.. congestive heart failure and portal venous hypert~n!ion {T.ble 19.4}. In both, transudation occurs b.alUse of incr ...,od plasma hydraulic pr~ssur~. a. In congestive hean failure, a romplex set of .vents d""r~a ... cardiac output, inc"","", hydrauli, p",",ure in ""in!, and incr~a ... Na+ and H,O .. untion (... Chapter 9). b. In portal ""nou, hypenension (eith~r po"hep"tic or posuinusoidall. the raU of plasJrul ~ntering the ,pace of Disse incre.... because of the inc==' hydraulic p.... ur~ in h~patic ,inusoid,. If there i, not a corre'ponding incre..., in lymphatic drainagt. th~ protein_rich Huid may move &om [he liver to th~ ~ritoneal cavity. c. In JlO'ple with th... disord~rs, a .. rum albumin_ascite, gradi~nt (.. rum [albumin] - .ffusion [albumin]) is used to hdp diff~rentiate effu,ion! cau,od by portal hypen~n!ion {• .g., cirrhosi, and congestive h~an failure} from other dfu,iom. Thi, gradient i, gr...ter in th... cong<:S{ive disorders than in exudative disorders."''' V~ry littl~ information about thi, gradient is av.ilable for dommic mammals. 3. Th. [TP] of. transudat. may incre..., wh~n an animal i, given a diuretic agtnt to reduce ed~ma or the quantity of.n effu,ion. 11. &udat.. {T.bl.. 19.4 .nd 19.5} A. An exud.t~ form, wh~n inflammation cause, incr....d vascular ~rmeability that allows plasma (and it, prot.in,) to ooze out of the blood. Th. exudation of prouin_rich Huid i, U1ually accompaniod by th~ migration of leukocytes (mostly neutrophil,) into the eHU,ion because of ch~motactic ,uNtance, in th~ Huid. l. Th. vascular permeability i, typically inaUKd by the eff=. of inHammatory modiato" {e.g., histarnin~, bradykinin, leukotri.nes, and suNtance P} that wer. rd~a ..d from inHamod tissue. The inflammatory modiato," Jruly .ho cause ,el""tive v;:uodiiation, so that more blood ~nters th~ inflamod tiosues and thu, ina• ...,' the hydraulic pr~ssu .. in th~ capillaries." When :wociatod with "P,i" secondary v:..cular dam~. increases the vascular perme.bility. 2. Th. inflamJrultion can directly involve blood v=d (vasculitis and phlebiti,), and then th~ vessel, becom~ very perm ... bl~ to pl"'ma proteins. B. Neutrophil, are th~ most common nucl..tod ceU in most exudat.. , but nucl~atod edl, in exudat.. wiU frequ~ntly b. prodominantly neutrophil, .nd ma.crophages or a minure of neutrophil" macrophages, and lymphocyte!. Occasionally, nucl... tod cell, of exudates wiU b. prodominantly ""inophih or possibly lymphocyte>. B44 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY C. Th~ l~~ of pro{~in' into th. interstitium rffiu~, the onrucie pr=u,", gndiem bttWttfl pla,JruI .nd interstitial fluid, :md rhm plasma h.., less ability to reuin H,O. The pl.mru, hydr>ulic pressure push.. a protein_rich fluid into the inurstitium. and Jess fluid rffurm in the nnom .nd of the capill:.ry W A pleuriti, or p",ilonili, moy dam"l:e mesothdium so that the protein_rich interstitial fluid . ..ily rno"". into the covities. D. Exudates are causffi by inf«tious and noninfa::tiou. "l:em •. The typt of audat. (i.e., infectious or noni"f=ioll'l) cannot'" rdiably determinal until the cou"";",, ~nt or """m Ita. bttn establimffl. I. Exudates "e &«\uently caUoN by.n infection with bacteria or other organisms. Some pwpl. consider >lptic and blle.friill to "" .ynonyms, but "p.is ,eftrs 10 .ny microbial infa:r;on (e.g. , ru.ctoriaJ, fungal, viral, or protozoal). To reduu th~ possibility of confusion, it may k ktt~r to ref.. to th~ inf«tiom exudm:s by the appropriat~ inf,""tious ~nt (e.g., b:ocurial nudate or fung:d audatd, when known. 2. Exudates ar~ :dso rommonly formed during noninf,""tious inflammatory responses. For enrnpl~, th~ inflamm>tory respo",e may bt du~ to n,""rotic tissue (~.g. , ~ndary to isch~mia or ntopl..,ia), th~ presonct of a st"ile for~ign body (e.g., surgical 'pong~ or barium), or th~ p ..,.nct of an irritating fluid (e.g., bile or urin~). E. When an ~ffusion i, protoin rich and Ita. a high [neutrophil]. it is not difficult to r'"""gni", th~ effusion as an nudal<. How~... it may k diffirult to det"min~ the c;ous.. of th~ aud.rion. l. [n b:ocurial .nd fung:d nudates, a microscopic oxamination of • fluid /lUy det«t bact~ria (rocci or bacilli) or fungal nructures (yea.tu or hypha~) in phagocytes (neutrophils or macrophages) or ntractllularly. However, if very few organisms ar~ in the fluid, th"}' m.y not bt det«ted during the microJCOpic n.:Imination, and th~ fluid should bt pla.ced in appropri.te rulture media to an~mpt to grow orgamsms. 2 With the excqJtion of the audato ofFlP, viral audat .. are unrommon. The types of proteins in the FIP exudate are very simil .. to the plasma protei", became the FIP ,""sculiti, enabl .. plasma proteins to oou through th~ inflamed ves .. l w:dls. Compo.red to most audat .. , the FIP nudate tends to have . low nucl~ated cellular_ ity btcm,. the inflammatory process is in the blood v.... ls and not in the body Glvity. MicroJCOpic n.:Imination of a st.ined preparation may reveal a pink granular background rdl'""tive of a high [TP]. 3. Protozoal audates (e.g., Lri>/",um;a sp.) a.. uncommon in the United Stat~'. A/lUStigote, GIn bt found in /lUcropbai:"S. Ill. Hemorrhagic effusions (fables 19.4 and 19.5) A. Wh~n the primary reason for .n effusion is hemorrhage, th~ fluid is a hrmorrhagk rjfo,ion. Rdativdy minor hemorrhage is • compon~nt of many nudates becau.. of th. vaocular damage a.tsociated with inflammation. Also, minor hemorrhage commonly occu'" during the collection of an ~ffi"ion; thi, is commonly GlUed traumIltk 19/ CAVITARY EFFUSIONS B. 845 Wh~n th~ ~ffusion forms b«:au.. of frequ~nt but SJIlaJl .mounts of h~morrh"l:~. th~ can ha"" a vari~ty of f~atures. l. [f coJl~ct~d 500n aft~r hemorrhagt. it might ha"" features of dilut~ blood 2. With p"..istent hemorrh.ge. the extravasation of plasllU prot~ifll di!fupts th~ oncotic pr=ur~ gradiem •• nd thu. H,O tends to diffu .. imo th~ cavity .nd dilut~ its coments. Concurr~mly, H,O .nd proteins .'" r~urning to plasma via lymphatic ~ ffusion -". 3. Some erythrocytes ..~ ~ngulf.d by macroph"l:es to form erythrophages, and some degrade h~moglobin to form hematoidin or hemosiderin. C. It i. difficult to set a da:ision th""hold for the cw..ification of h~morrhagic effusion b«:au.. many f""tors can alt~r the effusion', Hct (or hemoglobin or erythrocyte oon""mrations), and sometimes .n effusion devdop. became of h~morrhagt and oth~r pr"""s .... [f the effusion·. Hct i.:> 3 % , then h~morrhage is contributing to th~ effusion; for aample, an ~ffu,ion Hct of 5 % and. blood Hct of25 % suggests th at 20 % of the effusion·, volume is blood. D. Occasionally, an .bdominal .,mpl~ is coll~cted from an ~nlarged sple~n. Th~ bloody sample will ha"" a Hct :> 3 % , but it dO<::! not rqJresent a hemorrhagic effusion. A splenic origin ( aspirat~ or ruptu"'J ,hould be consid~red when h~matopoietic ""lh are in the rollecral sample but not th~ blood. [v. Lymphorrh"l:ic (Iymphorrh ....l) ~ffll'lion. cr.ble. 19A .nd 19.5) A. The", i. not a common term for the loss oflymph into a bOOy cavity. Two term. that occasionally are used a", Iymphorrhagt and Iymphorrh..... For this chapter, /ymplumnag~ wiU be used similarly to hnnorrhagr, that is,lymphorrhag~ is • P"""'ss that re",h, in lymph ,scaping from lymphatic vessel., just as hrmorrhagr i. a p""""" that results in blood escaping nom blood vessel.. I. L}mphorrhagr is som~im.. mal synonymou'!y with /ymphorrhra but is al", used to ,d'rr to • focal accumul ation oflymphocytes in tissu ... 2. L}mph~rrlua more commonly ",fen to the ate",alloss oflymph from dam"l:ed lymph atic """els. Occuionally, .utho", .ho u.. the term to "f.r to the pr"""ss that cam.. lymphedema or formation of a lymph-rich ,ffu.ion. B. Several p:uhologic sum IIUy cau.. Iymphorrh"l:ic effu,ioru. The pathogeneWi of these states can be divid.d illto two groups: tr,ullUtic and nomraumatic!' l. [n the mUllUtic group. ph)"ical dam~ to lymph atic ves.Jd. allows lymph to ,mer the body cavity. 2. [n th~ nontraumatic group, lymphorrhagic effusions occur when on, or more of the .. m.ro.nism. are present: {I } lymph stasis, (2) lymphatic hypertension, (3) defecri"" lymphatic val"" function bn:au.. of dilated lymphatic ""... h, and {4} increased perm .... bility of lymphatic ves ..ls.17 Aho, the persistene<: of. nontraumatic process might w.. ken a lymphatic ""sse! and cou.. lymphorrhagt when it ruptures. Clinicol findings suggest th.t many chylous pleural effusiom a", caused by obmuction of th, thoracic duct or cranial ""n. cav.; colltrast lymphangiography r~. aterui"" lymphangiectasia of mediastinal and pleural lymphatic vessel •. '" 3. Blockaj:~ oflymph atic ves.Jd. by neoplastic cdls con be a IIUjor "ason for effusions associated with malignanci ... However, ~nhanced vessel prolif~ration and increased v;u cular permeability may al"" contribute to th .... ,ffu,ions." C. Lymphorrh"l:ic .ffu,ions can be classified imo these two major groups based on the presen"" or absen"" of chylomicrons in the effu,ion: FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY '" l. Chylous ~ffu,ions a. A rhylour ,jfo,;on is produ=i when chylomicron_rim lymph l~"h from lym_ phatic nssd. and entn, Ih. pleural ",vicy to form a chylothorax or Ih. ptrito_ 1I.a! cavity to form a chyloalxlom.n (chyloptritonrum). Chylomicron, "r formal in intestinal mucosal ~ll" .nUr intestinal lymphatic '1."".1" and .nUr ptriph.ral blood vi. th. thoracic duct. Wh.n "chyloll'l . ffusion is p .... nt, it indict.. that lymphatic ve;sd, somewh.", ~""n Ih. ,moll immine .nd Ih. thoracic nil. ",va ... dam"l:ffl. b. Among th. domestic IIUnun.b, chylothorax j, most common in cots. It a m ~ causal by .\IN". l disorow; in am, dogs, .nd horst' (•. g., neoplasm., cordiomy_ op.thy, hra" failurr , trauma, lung Job. torsion, and infection.) but frequently i. idio!"'thic. ..... ' Th. l~ of chylomicron-=nuining lymph =1 '" di',""cly ,datrd to dam'4:M lymphatic vessel. but al", may ~ strondary to inc"""'M hydraulic pressu"" in the cranial or caudal ""na cava. Chyloalxlomen occurs rarely.... '" c. Chylomicron. may ~ abundant and thus c..ate a creamy white fluid. How",""r. thry may ~ p"""nt in low con""ntrations. requiring chemical a.. e.. mem of [TG) and [cholmerolJ to recognize their presence {= Sd.crM Analyses for Pleural and Peritoneal Effusion!. ""'t. I}. 2 Nonchylous lymphatic effiuions a. A nonchyfow Iymphlltir tjfo,;on i. produ=l when lymph without chylomicron! lrata from Iymph>tic ""... Is and enters the pleural G1vity or periron ... l cavity. The I""ion m ay involve lymphatic vessels that are not in the drain'4:e p:uh from intmine to thoracic duct and thus do not contain chyle or chylomicrons. How",""r, the a~n,,", of chylomicrons might indicate a la.ck of dietary intake of lipids (thus, the intminallymph did not contain chylomicron!) or that the chylomicrons (or TG ) did not persist in the .ffusion. b. These effusions do not have the unique f....tu,"" ,:reatM by chylomicron!, induding the chylou. pattern of the [TG) and [chol"""rol). Small lymphocyte, may ~ the predomi""fiI nudratM cell if the lymph originated from.n effe""nt lymph atic ves.sd. 3. rstudochylou. effusions a. P,fudochyfous tjfo,;om a"" fluids th>l grossly have a similar .ppearance to chylous effusions {e.g., white to cream rolorM, and doudy or turbid} but do not contain chylomicrons and do not have a high [TG). They have bttn r<'COgnized in people (rarely) but ..e not describffl in dommic mammals. Effusion! d e",ribnj as ' pseudochylous" in old.. veterinary literature had features of chylous effusions. b. Pseudochylom effusions typically have a high [chol""terol) th.c might result from the degradation of cell membrane. or the trapping of cholesterol. rich lipoproteins (e.g., low-density lipoproteins) in the body cavity. v. Effusion! caUsffl by the rupture of a hollow organ or other tissue (Tabl"" 19.4 and 19.51 A. t.....tmg., of urine from the uri""ry bladder, ureter, urethra, or kidney I. Uropmtomum occurs when urine ente .. the p"riton....J cavity subsequent to trauma, urolithiasis, or neopl ..i .. Attempts to expd urine forcefully from a distendM urinary bladder can ruptu"" it. 19/ CAVITARY EFFUSIONS B47 2. Initially, th~ periton~al fluid has f~ .tu ... of urin~ (5« Sd«tffl Analym for Pleural and P~ritoneal Effu,ions, sacts. II and Ill), but wh~n th~ pres~n'" of urin~ in th~ periton~~l cavity initi..t .. an inflammotory ... ponst, th~ dfiuion m~y han f ""mage ""mage VI. Effusion! coused by multiple proces.ses A. M.ny c.viury effu,ions .ccumulate bn:. .... of multipl~ concur",nt proa:s.ses. For example, an .bdominal neoplasm nuy ha"" necrotic areas that result in an inflammo_ tory r ROUTINE ANALYSIS OF PLEURAL AND PERITONEAL FLUID Analysis of pleural and periton..aJ fluid may yidd a cou ... for the effusion, but, more often, analpi' provides information about the fluid', compo,ition so that one or more possible explan._ tion! for its exa:s.s formation con ~ consid~rffl. The results of the fluid analpes ha"" been U'ffl to c",.t~ ~ffu,ion clas.ifications. Effusion da"ificotions ar~ not diagn~, jU 848 I. FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY Sample coll.crion and processing A. Sterile collection method. mould I>. used to enstm that bacteria or oth" org.niun, are not introducnl into the avity. The <:. placnl into two tul>..: one tul>. that conc.in, EDTA, which inhibits fibrin clot formation, and one sterile tul>. fur possible subntission for nticro_ biologic testing (e.g., culturing) or memi",1 arutlysi •. Tubes tha,.re not imm.di.tely .ubmitt":! to a laboratory should I>. kept oool.nd ddiver.d to the laboratory within 36 h. Concurrent mbmi .. ion of nain.d and unnain.d direct or eon""ntrat.d p ..""ra_ tiOfl[ made from an aliquot of the fresh fluid am I>. very helpful. Some EDTA tubes mayeontain .n additive that falsely increase, the [TP ...l .... He""rin may.lso I>. u,.d as an .nti~ulant, but it .Ite.. the st.ining prop<:ni.. of ""II, .nd thus may int.rfe.. with microscopic analysi,. C. Method, of p.. paring .p<:<:imefll for microscopic examination depend 5Om~hat on the ""lIularity of the fluid. When po .. ibl., th. cytoprepar.tions should I>. made .oon .ft" the ...."pl. is eollect":!, so th.t in vitro chang.. are Ie .. likely (•. g., ""II deterioration, organism prolifc:ration, or in vitro phagocytosi'). Th. fluid i, .ir..dri.d prior to sc.ining with routine stain,. For all of the film method., the f~th".d edj:e, which frequently contaim imporc.nt "",Us or structur.., .hould be in the stainable .rea of the slide. I. Dim! Im~ilr: Using the common technique fur making a blood film, a small drop of fluid i, .pr~d on a glass slide. Thi, method is usuaUy adequate for dfi"ions th.t have a T NCC;> 5.0 X 10'1J.lL, but an additional con""ntration method is d..irable for fluids with a TNCC of up to .t lean 20.0 X lO'IJ.lL 2. Lim p"pa'iltion: Thi, method is similar to the dir.cr .m~r, but the push slide is abruptly nopped and lift.d so that ""lis a", eon<:. us..:! for fluid, with a TNCC < 10.0 X lo'lJ.lL Howrvc:r, a high [TPl tends to make this ar ... too thick for a microscopic examination. Thi, can I>. partially rn:olv.d by adding. f1ow_b.ck step: when th •• pr.ooer slide is lift.d to form the terminal line, the slide is tipped up to allow the terminal lin. to flow b"",k and 'p..ad out .nol.Jl:h for I>.mr ""II visibility. 3. S,dimmr Im(an.re u•.d for dfu.ions with a low T NCC. The eon""'ntration method is not need..:! fur effu,ions that have. TNCC ;> 20.0 X lO'Ij.tL. a. Fluid is ""ntrifuged in a clinical "",ntrifugt by method •• imil.. to p..""ring urine s.diment. After .. moval of mo,t supernatant, the sediment is .....spend.d in a ,mall .mount of fluid, .nd th.n • direct sm ... r method i, used to distribute rell, on • gla .. .lide. b. Another method involves using special devires to allow gravity to form. s.diment .lowly. Thi, procedure mayor may not cre ate. monolayer of cdl. for micro,eopic examinations. 4. CytoCmtrifot' p' 'I"mltion: Comm"cial cytorentrifl.Jl:" are design..:! to con<:. pr..iict.d b...d on the transp"",ncy of the fluid. If the fluid i, cl ... r or hazy, the TNCC i, probably low, and thus a eon<: 19/ CAVITARY EFFUSIONS doubt, both types of cytoprq>. rations btu s1id~ for a.mill.tion, [I. 849 :u~ m>d~, alld th~ microscopin call sd~ct th~ Phy>ical .nalysis A, Color . nd trallsp" .. n (TP scale) .nd humall urille (SG ""l~) A r~fractom<'ter with a SG scale for dommic mammals is also availabl~ (s.., Chapter 8), To reduo. the eff~s of .mbient temptratu .. , temptrature_ comptllS3.ted .. fractom~t~'" ..~ recomm~nded, 2, The [TP] scale is calibrated for plasma [TP] .nd will approximate the tru~ [TP] in protein_rich Huids btalUS< th~ altered refrxti"" index is mostly due to proteins, E""n though the [TP...J m.y not bt .ccurat~, the estimated [TP] is. diffe .. ntial feature of dfu,ions, 3, Som~ clinical refr. ctometer> have. total wlid, (TS) ""Ie, 011 • gldL b..is, most di""lved solids .re proteills; other solids include da:trolyt .., urea, glu=, .nd other chemicol .nalyt.., 4, High con~ntratiolls of lipids (.. =n in chylous ~ffusiolls) wiU illcre""" th~ Huid's ",fracti"" index:md produ~ falsely illcr.... ed value. for [TP...il alld SG, High con~ntratiolls of solutes other than proteills {e,g" urea in uroptritolleum} will also fal ..ly incr ...... the TP.. value, 5, Susptnded cdl. typically do not int.. ~re with refractometric valu .., btcause the cd], do 1I0t alter ..fracti"" illdex significalltly (5« the Physical b.mination of Urine sectioll in Chapt~r 8), How""er, exmmdy high cdl conctntratiolls {> 100,0 X 100IJll} may, Whell"""r ill doubt, the values of Huid with suspended cdl. con bt romp. red to the Huid', suptmatant, 6, A vari<'ty of da:ision thresholds for [TP..] h.ve beell proposed for diffe .. ntiating effusions, Ho"",ver, several f.ctor> d<'t~rmine an effusion's [TP..], so mch da:ision thresholds should bt considered •• suggested guiddines, a, A lower [TP..] (usually < 2,0 gldl.nd frequ~ndy near 1,0 gldL) i, found prim>rily in prot~in_poor tr. nsudatr. and early in uroptriton~al effu,ions, b, A [TP...J > 2,0 gldl is found in most oth~r effusions, Typically, the greater the [TP ..] is, the gr .... ter is the protein ptrme.bility of blood ve=!s, 7, On. study de",ribed a falsely increased [TP...J {~,g" .. 1.5 gldL illcre..e} wh~1I ptritoneal Huid w;o. added to an EDTA tubt th . t also rolluined .dditive. to pr"".nt crystalli7..tion of th~ EDTA," Not aU commercial EDTA tubts conuin th~ additive, '50 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY Table 19.6. Conversion of refractive indi"". to (SG) rrr...J, (tolal.olid .], or specific gt'3Vity valu .... R~fractiv~ 1.3368 1.3376 1.3386 1.3396 1.3406 1.3416 1.3426 1.3436 1.3444 1.3454 1.3464 index Plasma [TP...] (&!dL) 0.0 0.5 '.0 '.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 [TP...I Pimna [TS] (/ddL) ,. , '.5 3. , 3.7 .., 4.' '.3 '.9 6.3 6.9 7.5 Ur;n~ SG 1.0 II 1.013 1.01 7 1.019 Plu m. SG" l.OlO 1.0 II 1.012 1.024 1.027 1.029 1.014 1.015 1.016 1.018 1.019 1.031 1.020 1.021 1.033 1.021 1.035 1.023 0.5 gidL interval.. Tn. ubI. provid..d by • T ho table ind»<J<, d.tI for from 1.0 to 6.0 gldl at AmericoD Optical b.. TP d . .. for .""h 0.1 gldL. Beca ..... th. ,el. tioJUbip' ar. """,[y ~ ....,. Olb., m.... can b. ... im. ted by inte,pobtion . • V:du .. orr rounded to the near .. ' thouundth ro ,hoy can mor ....ily ... compared ...ilk tho wi"" SG ..... and, by iudf, th. EDTA (n th. =mmrndffl oonctntr:llion) had minimal df.ct on Ih. fluid', ,di. ctivt index if Ih. EDTA tu~ v..as at [un on. _third full. Rdr:>c{om.try con b. Usffl to • ....,"" EDTA tu~s for th.ir poUntial to falsdy inc..... [TP...J values (i .•.• by asseuing the fluid ..fr:octometrically prior to .nd aft .. adding diff... nt volumes of it to EDTA tube;). C. R.f..ctometric mimates of ¥cific gravity (SG) I. Th~ SG scal. is calibrated for uriM and wiU approximate the tru~ SG of a protein_ poor .nd gluw..,_poor fluid b.cau.. most of the fluid·, ..fractive index is due to drctrolytes (the..fo ... limil", to urine refractive proJ><'nies). 2. Clinicl ",fracrometers do not have. scale for p"''''''' SG. but Leic. Am..icon Optical, and other ..fractom.ter manuf:octu .. rs provide ubles to conven p"""'" ..fr:octive index to SG." [n Table 19.6, not. that the diffe .. n"" in the SG ""lues (urine versus pl"'ma) krom .. gre.t.... th~ [TP",] incr......,s. Also not. that the tot:.! solids (TS) con""ntration is not equivalent to the [TP...J . 3. [f . laboratory .. ports. SG for a body fluid, the value might ~ a · urine SG" (if tak.n from ..f..ctometer sc:d~) or • "plasma SG" (if taken from. conversion ubI. ). Som. publi.hed m.thod. of d>.<SiJYing .ffusions use [TP...J and SG values. [f a SG of 1.017 or 1.019 is a decision th ...mold, the SG value w .. from the urine SG scale. A ..fractive index of 1.3386 converts to a urine SG of 1.01 7 and a plasma SG of 1.012 (Tabl. 19.6). 4. Using the ..fractometer to e'timate urine SG is very "",lUI b.ca"", [he SG valu.. typically ..fleet changes in urine osmolality (S« Chapt.. 8). Howev.., pl .. ma SG or osmolality values typicolly do not hdp with the int.rpretation of .ffusions. p"""'" Ill. Chemicl an.:dysis A. As d.1Cfibrd in the previous section, an effiuion·, [TP] is routindy estimated by m.amring its ",fr:octiv. index. A chemicl ..... y to mealU .. [TP] may ~ used, but additional expen.. IIU)' not ~ warr.nted. 19/ CAVITARY EFFUSIONS B. OSI Th~ con~lIIrations of ur~ .. Crt. cholesr.roJ. TG. da:trolytes • • nd oth~r mbnan~s "'" occasionally m~mra! to charaer~riR som~ effusions ( IV. [Erythrocyu] or h~matocrit (Herl A. Eith~r .n [~rythrocyt~] or a Her should be, dmrmina! wh~n th~ ~ffu5ion is pink to ra!. so cru.t thOM valu .. con be, comp:""'" with p.. iph~ral blood [~rythrocyt~] or Her. Th~ a.... lytic methods usa! for m V. [Total nucl~~tal cdl con~ntration] (TNCC) A. Ba::aU5t pleural .nd ~riton C. By it..lf. an ~ffu. 100.0 X 1001J.ll} . ", found in exudate> and neoplastic lymphoid ~ffusions. VI. Micro..::opic examination A. Wh~n cavitary effusions are d= and colorl~ .. (i .•.• look lik~ H,O .nd th ..efore ..~ prouin_poor and cdl_poor transudates) •• microscopic examiruotion of the fluid typically yidds very linl. useful information. For all othu ~ffusiom. the microJCOpic examin. tion of scainal cytoprepo.rations is frequently the most imponant p. n of the fluid analysis. Part of th. microocopic examin. tion is th~ aamination of ""n •• but examin.tion of oth.. structur.. and f.~tures is ~u.ntly imponant. I. Scains designa! for blood films ... fr~u.ntly used for the routin~ scaining of cytoprq>.rations. On~ major adv.nt"i:~ of using the blood st";m i, that people are f. mili.. with th~ staining of blood cdh and similar f•• tur~s ar. found in th. nud~ta! cdls of ~ffusions. 852 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY 2 Th~ microocopic examin ation should indud~ .11 pm. of on~ or mor~ cytoprep."'. lions ~"'" of th~ potenti'" un.nn distribution of ~Us :md ocher microscopic structur ... B. '\hjo. :up«:ts of th. ""amination .'" as follows: !'"'" l. Nucl~t«i cdl diff.",mial count is done to determine pe=nt"l:"" of ....ch nud.... <,<,U; the count is completed by eith.r ".ubi""';"" .s{imace or >II objective .numera_ lion. With" mea.u,ed TNCC and a nud.arM cdl diff... ntial coum. 'pproxiJrulte coII"'ntr>tions of ram cdl typ< can b. Gl.kulated and int.rpmed. For n.:Impl•. 60 % neutrophil, in a fluid with" TNCC of 1.0 X IO'fIlL is intupr.tn! differ.ntly than 60 % neutrophil. in a fluid with a TNCC of 100.0 X lo'llJL. If cd] ooll",lIlra· {ions are calculated from the perctnt"""., th<7 probably ,houJd b. consid.red crud. miJrultes kcaust of th. inaccuracies ofTNCC v:du .. and th. nud~~ttd cdl diff~ .. ntial counts. 2. Som~ pa>pl~ indud~ ntesothdial ""US in th~ diff,,~ntial cdl count, .nd othm do not. If th~:are not indudtd, it is mo .. difficult to interpret thelNCC, and con""ntr>tions of oth« nud... ttd cdl, cannot bt calculottd or cominendy esti_ m. ta!. Mesothdial ""lh ar~ oft~n in dust~rs, so th~ir indusion may l...d to incruKd vari.bility of the diff"ential munt, dep may ha"" microJCOpic fe. tur.. th.t ... simil.. to those .een in blood films; if so, th~ ~re called nondtgmml« ~"trtJphils (Pl.t. 14A and B) [for .ll plote., ..,., the color section of thi. book]. Th~ pm~nce of nondegenerate neutrophils in body fluids wuests • nonbacterial cau", of th~ effusion, but mch neutrophils can bt found in bacterial exudat... For example, neutrophils .dja",nt to mlonies of Amnomyur or Noca,dia may have dq;en~rat~ features, but oth" neutrophil. in the exudate =y not (Plate 14C). c. Deg~n.rat. neutrophil, ..~ ",ns that have .cquired ",ruin structural defects .fter they ha"" left the blood. Microscopic featu ... of the dassic deCC'nerate neutrophil indude. swollen and pal~_.uining nudeus, lock of nud... r chro=tin patterns, .nd variable degrees of cytopl .. mic v.cuolization (Plate 14C and D). {Il The.. a.. m. ny variations between the 5eVerdy degen".te and nondegon,,_ ate cdls, so it =y bt difficult to dassiJY the cdl, comi 19/ CAVITARY EFFUSIONS BS3 {3} Nondeg.n.rat. naltrophih can d.t ..iorat. within hour> of 5ampl. coUr<:tion. and thus th.y may acquire ftltures resembling deg.nerau naltrophih. The""fore. it may ~ difficult to diff.rentiate degtnerative change from in vitro deterioration if cytopreparations w... not mad. won after 5ample collection. d. Differentiating nond.gtnerate from degen.rate or det ..iorated neutrophils is 1= importont than ..arching for direct evidence of the cau.., of the in!L.mma. tion. becau.., degtn .. m·. appearing naltrophils may occur without ..",is and nondeg.n.rate neutrophils may occur with .. psis. How",,"r. the presence of d'gen.." e neutrophils sugg.m that the ..,arch for organisms should ~ more inten..,. 2. lymphocytes a. Th. lymphocyt.. in most .ffusioDl resembl. the ,mall lymphocytes t .... t are typically =n in ptriph..al blood films. Th • .., cells ....v. UDaU nuclei {with diameters < 10 Ilm in a""ao; where cells are well spread}. clumped chrom" in p>tt.rns. and .mall amounts ofblu. cytoplmn (Plates 14E and 15l). b. Wh.n they ...."" bttn stimulated. the lymphocytes may have features of ttlctive lymphocytes. plasm:>eytoid lymphocytes. or plasma cells {Plate 14F}. c. Neoplastic lymphocytes {Plau 14H and I} frequ.ntly have nuclei with diameters :?: 10 Ilm and !indy granular to homogeneous chromatin patterns. F""<J.uendy. the cells have mod .." e amounts of deeroph~ .. a. Outside of the vascular .yn.m. monocytes transform into macrophag .. or histiocyt ... Cdh with the 'ppearan"", of monocyt... m:>eroph~ ... or tran,ition pha.., occur in fluids. but they may aU ~ lumped into a m:>eroph~. category once the cell, have left the vasculature. b. Th. cytoplasms of m.croph:oges f""<J.uently contain vacuol ... lipid. or cellular d.bris. and might contoin .ngulfed celb {•. g.•• rythrocyt .. and leukocytes}. organi,m •• or foreign material (Plate 14M- 0). Ceu" organism,. or foreign mat..ial may und..go phagocytosis within 30 min in vitro (Plate 140). 854 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY {I} Erythrop/uge; ar. rdatively common in .[fusions wh.n blood comaminat" a fluid thor comains activaud macroph"l:" and wh.n cytopr'p"r>{;ons '" not mad. fOOlI aft., ... mpl. ooU«I;OIl. {2} Leukophag.,,; ... rdativdy common in .om. fluid" e:sp«iIlly in Ih. ptriton.al fluid of hOrK'. F~u.ntlr. Ih. J.ukoph'g<' contain nrutrophil, at v:lriou. nage. of degradation (Plat. 14B). 5. Mononuclrar cdh a. Th. Urm i. oft." u~ ooJJ«tivdy for cdh that ... not granulocyte; or mas! ~ll, (though they aho h."" only on. nuclau). Mon so-callal mononuclrar cdls in .!fusions . '" lymphocyte;, m:ocroph>g .. , or melOlhdiaJ cd]', and they can ~ categoriud as mch in mon ca .... b. In wdl_prqmal, wdl.slainM cytop''1'''fations, most small lymphocytes .nd ma",oph~ that have v:>cuolata! cytoplasm, or contain .ngulfnl m at"ia/ :u. =;Iy recogniud. How.ver, som. r~{ive lymphocyt •• and ,mailer monocytoid or hi,tiocytic cdh a", not ~asjJy difftr~ntialr~..,nt .rtifacts (~.g., membrane .picul~s, hypochromia, .nd r~fnctjJ~ m~mbranes) but may r~f1ffi: p.thologic stat~, {e.g., aamthocytes. polychromasia, and :wociata! organisms}. Findings .us~ta! to r~p .... nt • pathologic stat~ should be, confirma! by finding the "'m~ abnormality in a mined film of fresh blood. a. If th~ h~morrh"l:ic dfi"ion has oon pr=nt for at I.-as! a few hours, ~rythro. ph"l:es may be, found;" it may tak~ 2-4 d for .id"oph a~. to .p~r.""'" Also. hematoidin crystals may be, found in macrophag... b. Erythrophage., Jideroph"l:e., .nd oth" macrophag.. b.rom~ a mo .. prominent fe;otur~ of ~ffi"ion. the long~r th~ blood has oon in a covity. c. Wh~n th~ hemorrh"l:~ is cousoed by. nontraumatic ~""nt (e.g., h~morrhagic neoplasm), frequently th~", is ~ith" recurring h~morrhage or persin~nt low.grad~ hemorrhage. Thus, th~ ~ffusion may hav~ properti.. of both .n arut~ hemor. rhagic effusion (i.~., high Hct value) and a chronic h~morrhagic effusion {i.~., .id~ropha~}. d. If th~ making of a cytop.. paration .om~ case" th~n th~ was ddaya! for a few hours or less in p.... n~ of ~rythrophage. may .. present in vitro phagocyto.i •. D. Platde"ts con be, found in collffi:a!samples. Th~ pr... nce of platde"ts su~st. ongoiIli: (or ""I}' r=nt) h~morrh~ or contamination of fluid with periph~ral blood during collection. Clots indicote fibrinogen was in th~ ..mpl~, but the fibrinogen could be, present """'UK of audation, h~morrhage, or blood contamination. E. Nuclrate<\ cell. that ar~ I.... commonly found in pl~ural and periton~al fluids. l. Eosinophil. usually hav~ ftltu= similar to tho.., lttn in periph~ral blood {plate 15A}. 19/ CAVITARY EFFUSIONS OS5 Th~ pr"~I1"'" of rdativdy f..... man edb a.. not ullumal ill nudates .nd ar~ aho found ill oth~r ~ffusions (~.g.. h...rt failur~ ~ffusions). Unl~ .. th~ ""lls ha"" promi_ nent microscopic f~.tures of maligllancy. lleoplastic .nd nonllgrd bact~ria may stain. v.riety of 0010" from red to light grey or light purpl •. The shapts of the dead org.nisms may be, diff..~nt from tho,~ th at were live wh~1I the sample was preparrd for n.millation . •. Fungi may appear as yeasts (~.g .• B/m"'mym or Hii/upill"",,). hypha~ (~.g .• hfMrgillu, or MucDr). or y...sts alld pseudohypha. {CandkiJ.} (Pl " e 15G). Most yeasts will have a positi"" staining =ction with • Wright naill {i .•.• bl...,. or purpl..}. but hyph ae may have negali"" ...ctiom. Nonsuining hypha~ am be, detectrd as ddin... trd lin... r structures that ar~ displacing celh and oth~r suinrd 2. mat~rial. f. Lorva~ of Mmx"",wl 'P. may be, fuund in dogs with ptritoll... l cestoidia.sis." g. Lorge ciliatrd prot07.OO (~.g .• &iIlnridltJm sp .• Polymorph"iIl sp .• and Cyll1_ porth;um 'p.) may be, found in equine ptritoneal exudat.. or in int.. tillal taps {Pt.te 15H)'" Th~ protozoa typically a.. commensal inh.biunts of equille intmin.. bur may be, KCOlldary ill""d.rs of dis..srd intestillal mucosa. 2. Oth.. mat~rial th at am be, fuulld in exud. t.. and lipid ~ffu,ions indud.. urille crystals {Pl. te 151}. 'P"rm (Plat~ 151). bilirubin crysu ls. bil~ (Plate 15K). mucus. plant material. barium crystals {Plat~ 14N} •• nd lipid droplets (Plate 15L). Sudano_ philic dropl~ts em be, id~ntifird in th~ fluid and ill macrophages by using a SUdall stain (Plat~ 15L). To look fur Sudanophilic droplm. Sudan st ain i. pia=! on top of an .ir-drird cytoprep:mnion of th~ ~ffusion. and th~n a cov.rslip is pia=! on top of the liquid. Via microscopy. th~ Sudanophilic dropl~ts .ppear as round. orange to rrd structur.. that tend to float (jun und~r th~ coverslip) or ar~ within macrophag~s. 3. Th~ suining intensity of th~ spa"" be,tw..,11 edt. tellds to be, greater when the fluid has • high [TP). A drying artifact may cr ... t~ protein cr=nt, wh~1I th~ [TP) is increased {Plate 15NJ. ". FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY Table 19.7. The [TG]. (cholesterol ), and Chill, TG ratio. in chylou. and nonchylou. cffu.ion. of dog> and cal. Canine .AU,ions ChrJou, NonchrJom Fdin. dfusiom Chr!0us Fmsum nudy" Numb" in group Chol.nerol (mgldL) T riglJ'C"rid. (mgldL) Chol:TG ratio 6 37- 127 45 - 2,687 0.04--0.15 3 48--139 8- 33 3.88~.OO 4 38- 232 91--4,670 0.05--0.70 Nonrnr!0u, , 65-111 <>-59 1.88--110.00 W.ddl. slUdy' Numb" in group Cholm.rol (mgldL) Triglymid. (mgldL) Chol:TG ratio • So""" Foosum et oJ." 6 23- 70 110--610 0.04--0.64 " 3- ll9 3-'3 0.33- 27.33 " 18- 148 130--3490 0.01-0.64 " 16-107 23-93 0.23-7.64 • Sour«: W. ddI. :and Giger'" SELECTED A NALYSES FOR PLEURAL A N D PERITONFAL EFFUSIONS I. [ChoJ..I.rolj .nd [TG] A. If a diagnostician swp«ts an .AU,ion is chylous, but the routine anal)"'i' do.. nol provide enough rod.net for a firm conclusion, m~~mring the [cholesterol] and [TG ] of th~ ~ffusion and th~ anim.J·s ,.rum may provid~ d n a to diff,,~miat~ chylous and nonchylous dfillions. B. In. srudy involving dogs .nd em." .ffusions w~ .. ron,id~red to ~ chylous if th~re ~ microscopic appea .. n", of chylomicron. (i.e .• Sudanophilic dropl.uJ . the .<;;Impl. rould not ~ deared with ",mrifugation. or th~ fluid did d . .. . fter th. addition of eth~r. Effusions w". cl ... ified as nonchylous if they did not meet the chylous .ffusion crit.ria. Th. results of th~ study ... in T able 19.7. l. A> would ~ npected with th. pr... n", of Sudanophilic droplets in th. chylous . ffusions but not in th. nonchylous .ffusions, th~ triglyceride ron",mrations w... gr.~t" in th~ chylous than in th~ nonchylous. Also th~ Chol :TG ratios wtr~ much sm.Jl" in th. chylous .ffu,ions because of the g",.ter trigly",rid. con"'ntmiofl1. 2 Th. diagnoses for dog. with chylous .ffusiofl1 included thoracic lymphangiect"'i<>. inteo;tin.J lymphangiect"'i<> , .nd aortic lxxIy tumor. 3. The diagnoses for em with chylous .ffu,ions included thoracic lymph:mgiect:L!ia, diaphragmatic herni., cardiomyopathy, and metasutic pulmonary adenocarcinoma. C. In .nother study involving dog. and cau." .ffu,ions w". considered to ~ chylous .ffusions if lipopro .. in dectropho ..sis rev.aled a dininct chylomicron band at th. origin of th. dectrophoretic pattern. l. Th. major finding. in this srudy'_ laoorntory data "",re as follows (fable 19.7): a. Each chylous .ffusion had a [TG] ;.100 mgldL. and each nonchylou •• ffusiofl1 had a [TG ] < 100 mgldL b. Each chylous .ffusions had a Chol:T G ratio < I. wher.... most nonchylous .ffu.ions h ad Chol:TG "'tim;' l. 19/ CAVITARY EFFUSIONS '" c. Grossly. nnrly.ll chylou, ~ffi"ion' h.d whit~. milky appnrana:s ali" ~ntrifu_ gation. wh~reas non~ of th~ nonchylous dfusion, h.d that appnran~. 2. Th~ diagn<»es for dog. with chylou. ~ffusion indudn!lymphoIllil. nnom thrombo_ ,i •• cardiomyopathy. and idiop:nhic chylothoraL 3. Th~ di:agn°w; for cats with chylom dfu,ion indudn! cardiomyop>thy. diaphr>i:m>tic h~rnia. ~ricardial ~ffu,ion .• nd idiopathic chylothorax. D. Chol",~rol and TG ..... Y" trull ar~ designn! for ",rum ... mples (..., Chapt~r (6) typically cm k uK [I. [Ur~.] and [Crt] A. Wh~n a diagno,i, of uro~ritonalm i, king comid~rM. th~ m= .. m~nt of tho [uru] and [Crt] in tho ~ritonnl fluid md a tim~_m>tchM ",rum sampl~ may hdp esublish a diagno,is. It i, imponant m.t the dfusion con""ntrations k compar~d with r..ant ",rum oon~ntmion' ~""" of the rapid diffusion of ur ..... nd Cn from the caviury fluid to lymph and blood. B. U",• • nd Cn a.. t diffu", through capillary wall.; uru diffu.s.:s quichr than Cn. [n h~alth .nd most p.thologic sUtes that creat~ effusion>. the ",rum and ~riton~.l fluid oon~ntration' of ure> md Crt .r~ n~.rly th~ sam~. C. Wh~n th,,~ i, • r..ant .ddition of urine to ~riton~.t fluid. the r=.tlting fluid wiU h.n. gr~.t~r [u.....] and [Cn] thm pl.,IlliI con""ntrations ~= th~ [ur~.] .nd [Cn] in urine a.. typically much gre>t" thm pl"'ma or .. rum oon~nt .. tion'. With tim•• nd with diffusion of mol",ul .. du~ to con~ntration gradi~nts. the [u",.] .nd [Cn] in the extravascular . nd intnv;oscul.. fluid, will b«:om. similar :again • • nd both wiU k inc",asnl. I. Aft~r 45 h of ex~rim~ntally inducnl uro~ritoneum. the [Cn] in th~ ~ritonnl fluid woo. about twi~ the .. rum [Cn]; Th~ m~an ~ritone>l fluid [Crt] wa.< 11.6 mgldL (rml:". 5.5-15.6 mgldLI. wh"•• , the mun ",rum [Crt] was 5.2 mgldl (ran~. 1.2- 6. 7 mgldL). How"""r. th~ [u... ] in the effusion .nd ",rum at 45 h w~ .. nearly tho same. Th"", dau support the fact th>l u",•• nt.rs the blood fast" than does Cn. Azot.mia (.ith~r an incrn=\ [ure.] or [Crtll was det~ctabl~ in. f~ dog, within 5 h bur in :all II dogs by 2 I h after "ruptu ... " Th...rum [u",.] .nd [Crt] oontinun! to inc..... onr the 3 d study." 2. [n. retro'pectin study of 13 dog. with uro~ritoneum. th~ cr.atinine oon""ntn_ tion, in ~riton~al fluid w",e at l~.. t twi~ the "blood·· con""ntrations in II dog•. Th. durations of the sponunu>us rondition, w... not known. (Note; Th~ .uthon did not specifY wheth~r the "blood" c.....tinin. oon~ntmiom w~ .. ",rum or pl"'ma oon~ntntion .. )" The ratio. for .ight dog. with other ~ritonnl dfu,ion, were from 0. 7 to 1.2. 3. Ratim of ~ritonnl fluid [Crt] to .. rum [Cn] han ken r~ported for other anim:al, with uro~ritoneum; foal, (mun. 3.5; and ranI:". 2. 7-4.6)." horse. (;> 2.0)." and cats (m.... n. 2.0 md rang.. 0.8-2.4)." Th~ ratios can k u..d a,. guideline in the interpretation of data. but oth~r .. peets of the ca.. .should k carefully ron,id"ed. For enmpl •• a cat with a urinary tract ob,truction IlliIy ha"" bttn """"rely .7.0temic kfore th~ uro~ritonalm occurrM. Thm. the .. tio m.y not k;> 2 wh~n the cat is first enmined. 858 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY D. U""~ (or ur"" nitrog<'u ) and Crt '''''Y' that >r~ d~,jgnffl for strum sampl .. (Itt Chapt~r 8) typicaUy an b. U....:! (0 .ruolyu th. SlI~mat:m1 of ''''Jl«'M uropt,iton..,,] .ffusions. For some ...... Y'. the umple may ott coDctmrations within the :us;oy's analytico] rang.. Ill. [N.1 , let], :md [K'] A. N"" Cl- . and K' are f.edy diffusible through moS{ capiU . ry walls, and th", th.ir oonctntmiollS in plasma, intemitial fluids, .nd mOS! pleural and ptritoneal.ffi"ions .'" nearly th. sam • . Th. major exaption to this COnetpl occur.; in uroptritoneum. Comp""'" to plasma .nd most exmctUulu fluids, the urine [Na1 :md [en are las and th. urine [K+] .'" gr.... t.r. Thu., in Ih. early st"!:" of urop",;loneum, the ptrito_ ne.l fluid [N. 1 and [en will bt I... th,n ploUII" ron"'Dtmions, .nd Ih. ptritoneal fluid [K+] will bt gr....m than pla,ma concentrations. With tim~. diffiuion of d=ro_ lyt.. and H 2 0 will rrdu"" th~ diff~ .. n"" •. B. In a mrO!ptctiV<" study of 26 elK, of fdin~ urop..itoneum. th~ [K+] W>S m~a,urM in fiV<" ats. Th~ mios of th~ periton~al fluid [K+] to strum [K+] ranged from 0.9 to 204, with a m~an of 1.2." C. In. mro,ptcri"" study of 13 dogs with uroperitoneum, ratiO! of the peritoneal fluid [K+] to th~ "blood " [K+] ~re ~ 104 in all dog, (median, 2.7; and range, IA-4.9). (Note: The authors did not sptciJY whether the "blood" [K+] was a strum or pla,ma con""ntration. )" The ratios for eight dog. with oth~r periton...l effu,ions ranged from 0.7 to 1.2. D. In an experim~ntal production of uroperitoneum in dogs, hyponatremia and hypochJo_ r~mia were apparent betw«n 1 and 2 d and pronounad by 4 d. Hyperkal~mia was apparent in some dogs by 2 d and in nearly all dog. by 4 d." E. """"ys designed for strum [N.+] , [K+] , and [Cl-] (see Chapter 9) may not provide accu rate electrolyte concentrations in the supern>lant of avitary ~ffusiofll. Some ion_ .dective electrode methods require a proteinaCft>us fluid, 50 urine or a uroperitoneal effu,ion may not be accq>table. Anions oth.. than ct may rract with th~ d=rodes of the ct ass.y. For ""nul", .ampJes, measured [K1 might include K' rd=ed from cdls either in vim or in vitro. IV. [L-lactate] A. L-la.ctate i. the product of anaerobic glycolysis, and one reason for hyperlactatemia is tissue hypoxia (st<: Chapt~r 9). The L-Jacta te produad by""ns may ~nter venous blood to aust hyperlactatemia but also may ~nter int~mitial fluid. and diffust into pleural or periton~al fluid,. An increastd [L_lactate] in pleural and peritoneal fluids may be a marhr of incre..ed anaerobic glycolysis in the body cavities. B. An increastd [L-lactate] in peritoneal fluids has been found in animal, with nrangulatrd and nonstrangulated intestinal obmuctions, abdominal neopl ...ns, and bxt~rial exudates. I. Th~ [L_lactate] in periton~al fluid. w>s greater in colicky horses with intestinal ischemia secondary to strangulating obstruction (8.5 ± 5.5 mmoUL, mean ± standard deviation) comp",ed to colicky horses with nonstrangulating obstruction (2.1 ± 2.1 mmoUL) and healthy horses {0.6 ± 0.2 mmoUL)." Th~ [L_la.ctate] in "'Iuin~ peritoneal fluid i. u=i as an indirntor of the .everity of intestinal ischemia in rolid:y ho"" •. 2 The [L_lactate] in peritoneal ~ffusions was greater in dogs with intra_abdominal neoplasm. (h~mangio.arcoma, arcinomatosis, neur""ndocrin~ rumor, "...,tastatic 19/ CAVITARY EFFUSIONS os, ..d~lIocarcillom., and p.ncr""tic carcinom.} {3.8 ± 1.7 mmollL, m""n ± stand"d d~i.rioll} than in dogs th.r did not hav~ abdominalll b. In cots of on~ study, th~ [L-lactat~] was 6.2 (1.3- 1O.6) mmoJiL (maliall .nd rang~) in th~ bact~rial nudates (n = 5) .nd 1.4 {1.2- 1.6} mmoliL {malian .nd rang~} in th~ nonb.et~rial dlUsions {II = 2)." Similar data w~ .. found ill .noth~r lIin~ caU with bact.. ial ""udates, but a wid .. rang~ of valu .. was found in lIiM oth~r cats with 1I0llbact~rial ~ffusion'." c. Th~ diff~r~II""'s i>eCW,",,1I p"riton~al fluid [L-Iactatr] and plasma [L.lactat~] w~re usually greot.. in th~ ructer",l audat~ group than in th~ nonruet~rial group. A differ~n"'" gr""t~r than 2 mmollL was suggrnal as .n indicator of Jeptic p"ritoni . tis;" the 5ame criterion would h. "" incorrectly classifial th= of eight dog. alld five of nin~ cats in th~ second .rudy." The pathogrnesis of th~ increased [Llactat~] ill the bact~rial exudate. was not uplainal, but at least tv..o uplan.rions :U~ po"ibl~: {I} Bacteri. call produ"", both L-I.ctat~ .nd D.I.ctat~, and thu. bact~ri. could ~ a sauro:: of the L-lactat~ ill the bact~rial exudat ... {2} L-lactat~ could alsa ~ produced by I~ukocytes and ~rythrocytes in th~ audat~. Cdl con""'ntrations in th~ rueter",l effusions t~lIdal to ~ gr""t~r than thOJ~ in th~ 1I0llbact~rial ~ffusion •. 4. Th~ [L.l. ctatr] ill p"ritoneal fluid was incre:=d within 2 h of exp"rimental intesti· nal ",.ngulation ill dogs and continual to increase during th~ 24 h exp"rim~nt. Th~ plasma [L.lactate] changal ""'1 little in th~ survivor group, but inn~=d rapidly aft~r 8 h in the nonsurvivor group." C. As.5ays designal for .. rum or pl.. m. [L-laetat~] (see Chapt.. 9) typically can be u.aI to a""lyze the sup"r""tant of effusiolls. Int~rpretation of ~ffusion [L-lactate] mould also oonsid.. that erythrocyte> and leukocyte. ar~ a potent",l sauro:: of L.lactate. Aho, L· lactate could ~ produc:ffl by edls ill the effusion . fm sampl~ collection, and thus 5ampl.. n=:! to ~ processed and a""lyzed quickly. If samples canllot ~ . nalyzed quickly, th~ fluid oould ~ plac:ffl in .ooium fluoride tubes (see Chapm 9) or the edls r~moved from th~ ~ffusion. v. [Glu<:<>u] A. Because gluooJe in • p"riton~al ~ffusion rom .. from plasma, it is imponam to interpret the [glucose:] ill effusions with kllowledg~ of th~ plauna or .. rum [glucose]. B. Th~ glucose concentratiolls in p"ritontll effusiolls w~ .. Ie.. in dogs and caU with bact~rial exudat .. thall ill dogs .nd cots that had nonructerial p"ritonea.l effusions cowed by a variety of disord ...... ,OJ FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY l. [n dog., th~ glum .. con~mrations w,,~ 57 mmollL (ffiffli.n) in th~ b.ct~ri.J nudale:! (n = 7) and 137 (51 - 322) mmoUL (m 2. [n em. the glaco.. ronuntrations ~"" 100 (39-234) mmollL (meuri:d effusions (n = 5). 3. Th. glum .. oon"'niutions in bacterial exudu .. ~"" Jess than the corresponding blood g1uco.., rona.ntrations in :011 dog. and ne.rly.ll cats. Th. authors sugg<'u w". b3oCt~ria. C. Glucost assap that ar. d ..igna! for strum or plasma .. mples {s"" Ch apter 14} typically con bt usa! to analyu th~ "'~rnatant of effusions. Glu= will bt con,uma! by""H, in th~ ~ffusion, and thus <;;Impl.. should bt .nalyz.«! soon afw coHa:rion. [f sampl.. connot bt analy=l quickly, th~ Huid could bt pia=! in >Odium Huorid~ tubts (5« Ch apt~r 14) or th~ ""Hs r~mova! from th~ df'l!ion. VI. [Bilirubin] A. B«>Ust conjugata! bilirubin is diffu,ibl~ and pl.. m a proteins ar. pre.. nt in most nontranrudative effusions, th~ [bilirubin] in most ~ffu,ions should bt .imilar 10 the strum [bilirubin]. Th~ major exctplion to thi. con""pt is =n with bile p"ritonitis and th~ formation of a bilious exudate. When damage 10 bile dum or gallbladder r.. ults in th~ l~akage of bile into the p"ritoneal covity, ~rilonitis will devdop and the r.. ultant exudate will have an increasa! [bilirubin]. B. The", >re stveral potential COUstS of bile entering the p"ritoneal covity, induding blunt or p"netrating lrauma, bile duct rupture secondary to manual expression of the gaUbladder, necrotiz.ing cholecY"titi. or cholangitis, rnoldithiasis. and neoplasia of the biliary tissu ..." C. When the effusion d"",lop.'l .econdary to bil~ l~~, "rults of Huid anal,..,.i. reveal the p".. n"" of an exudat~ that typically h .. a [TP...] :> 2.5 gldL, a T NCC :> 5.0 X lo'lJ.lL, and:> 80 % n.... trophih." I. Microscopic ex;omination may deta:r a .mall to large amount of intra""Hular or extra""Hular amorphous yellow to brown bil~ pigment, or bilirubin crynal" extn_ ""Uularlyor in macrophages. 2 [f the bile l~ is secondary to a baclerial infa:rion. th. exudate may ronuin intracdlul >r or exlracdlul>r bacteria. 3. Typically, the [bilirubin] in the bile-rontaining effu.ion. will bt at le ..1 !Wi"" an animal". strum [bilirubin]."-" 4. When a gallbladder l.. ion ~nabl .. in mucoid secretion (coUa! whit~ bil~) to ent~r the ~rilon..aJ covity, the resultant effll'lion may contain light basophilic mucinom m aterial wh~n ,uina! with a Romanowsky stain." D. Rerults of blood and urine analy.is will depend on th~ COUst and duration of the biliary lracl leakaj:e; for example, if the animal had obstructive rnolesmis prior to bile leakage, hy~rbilirubinemia may h ave pr~ed th~ bil~ ~ritonitis. However, with trauma_ indu=! bil~ l~akagt, the hy~rbilirubinemia occurs aft~r the onstt of the bile p"ritoniti •. Likewi .., an inflammatory leukocytosis may bt Ihe result of a bacterial cholangiti. 19/ CAVITARY EFFUSIONS 86' that couses bil~ l~~, or an inHammatory l~ukocytosi, may occur aft~r th~ on.. t of arut< bil~ ptritonitis, E, Bilirubin assays that a.. design.d for .. rum or plasma ... mple. {."" Ch'ptu 13} typically em k ustd to analyu th~ .uptrnatant of ~ffusions. Bilirubin is d [Ammonium] A, Th~ [ammonium] in ptritoneal fluid was inc",a..d 24 h after ""pnt of dfusions, In vitro factors con alter the [ammo_ nium] in th~ oollectN .ample (= Chapt.. 13), VIII. Prot~in oon""ntration. A, With th~ JlO"ibl~ """qJtion of immunoglobulin!, th~ prot~ins d~tect.d in effusions repruent th05t that leak.d through copillary walk The gr..ter an,ount of protein and the larg.. prouins con p .... through th~ more prot~in_ptrmubl~ capillaria B, Protein dectrophor~.is I, Protein dectrophowis of pleural and ptriton~al Huid {.. peciaUy if pair.d with strum pror.in d~ctrophoresis} will provid~ infurmation .bout th~ ptrmeability of capillary bal., If th~ [TP] of the fluid is < 2,0 gldL, th~ fluid may need k oon""n_ trat.d kfu", electropho=i., 2, In FlP ""udaus, the electrophoretic patt~rn. of th~ cot's exudative ~ffusion .nd iu strum a", very .imil.. becau.. th~ disordds v:llculitis ~nables plasm. pror.ins to Opl~,. pleural effu.ion [TP] to .. rum [TP] ratio:> 0,5 i, indicotive of exudation if th~ ~ffusion does not ront.in blood, '" D, In pn>ple, ...rum albumin_asciu. gradi~nt (.. rum [albumin]- effusion [albumin]) h .. ~n us.d to help diff..~ntiau ptriton..l effusion! cousal by portal hyptrten1ion {~,g" cirrhosi. and oongestive he.n f.ilure} from effu1ions em..d by malignancies or inflam_ matory starr.; th~ gradi~nt i, grut~r in the portal hyp with tran""dat .. cou..d by hqJatic cirrhosis had a .. rum albumin-ascite. gradient :> I, I gldL, but so did about half of th~ dog> that had ~ffusions caus.d by n""plas"" pancr~atitis, oongtni"" hem f.ilur~, prouin_losing ~ nteropathy, and oth~r disord~rs," E, Con""ntrations of specific pror.ins con k measur.d in effusion., but the data availabl~ a", insufficient to establi.h th~ diagnostic value of such oon"'ntrations; for ex:ampl~, oon"'ntrations of « ,_.cid glyroprotein we .. grrat.. in plasma .nd ptritoneal fluid of cots with FI P than in h~althy cou," " B=UK CI.,_acid glyooprouin is • pOlitive acut~_ pha.. protein (= Chapter 7), similar findings would k ""p 862 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY IX. Ellzym~ :>ctivity A. Lip.....ctivity: P~ritoll~'" fluids from dogs with :>cute !",lIcreotitis h.d much gre>t" Ii""...ctivity than oth~r p"itoll~'" ~ffi"ioJlS and w~ .. at I""st doubl~ th~ animar • .. rum Ii""...ctivity." B. AlbJill~ phOlpru.t ....: III hon~ with colic or im",in.1 I~ioll'. ALP activity may illc""" ill th~ pctivity ill p X. Gram nailling A. Gram stain is us«! to diffe"'nt ooct"ia imo two large group': Gram_positive bact~ria and Gram_negativ. bact~ri .. Th. Gram stain illvolves two major step': (I) staining a bact~rium·. cdl wall with. dye {.ith~r methyl violet or crynal viol~} and iodille, and {2} .ttempting to decolorize the cell v..aU by using echanol or .ceton~ ",Iutioll. In theory. the dye_iodille complex becom.. fixw ill the Gram_positive orgallisms .nd thu, they rerain the d .. k purple stain. wh ....... the dy._iodill' complex i, di!.SOlvw out of th. cell walls of th~ Gram_neg.tiv. organisms to produce pink organISms. B. Th. Gram stain i, d..igllw for staining ofb.cteria .dectal ftom cololli.. grown in a vari~y of cultu.. mwi .. and it is diffimlt to apply the stain to cdlul.. ""mples or proteinaceous ~AU,ions uniformly .nd coJlSistently. Wh.n th~ srain is applial to air_ dri~d sm •• n or films of .AU,ioJlS. ""eral f.ctors .mould k COJlSid"w: I. D~.d Gram_positive org.nism. frequ.ntly have. Gram_lIeg.tive reactioll; tru.t is. th~ stain does not r~maill in the cell walls of d.ad Gram_positive b.cteria during decoloriz.ation. 2. Using too littl. ethallol or acttoll. may produce. faJ.. Gram_positive r""ctioll, whe .... using too much ~ru.nol or .ceton~ may produce. fal.. Gram-netarive ",.ction. 3. Cell debris, especially from nucl.i, has a Gram-netariv. ",action, and thus Gram_neg.tiv. bact~ria con k difficult to locat. amollg Gram_negative cdlul .. debris. 4. If usw diagllosticoUy, a .. t of known Gram_positive .nd Gram_n~tive dides prepared finm cellular .ffusiolll should bt stainal concur",ntly to .. rve as positive and negative collltol slides.. 5. Th~ use of Gram staiJlS is not n=ry to detect the pre..nce ofNct~ria. Routin. Rom.nowsky naillS are excdlelll ror this and offer much mo"" as well. Xl. Culturillg for microorgallisms A. Whellever.n infection is suspectw, fluid should bt submittw for possibl~ mlturing of orgalllSm •. B. UsuaUy, culturing con btn" detect org.nism, than con • mictoocopic examination of .ffusiolls, .. pecially wh~n very few org:mi,ms.re in th~ dF.uion. 19/ CAVITARY EFFUSIONS B63 COMMENTSABOur SPECIFIC EFFUSIONS I. S.ptic ~nd nonstptic nudates A. Th~ .. mlts of fluid ~nalY''' a... ometim~. classifi.d as eith.. stptic nudat.. or nonseptic nudat... I. The t.. m uptic in this context ~uendy i. u•.d synonymou.ly with bactnidl and is us.d when bact..i. w... found during the microJCOpic eumination of an nudate. Howrv... others u.. the term "pfir whenrv.. any microbe is fuund that it consid_ ...d th. cau.. of the inf'n:tious exudate. 2. The t.. m ncmtpfir in this contnt frequently is U,ffl synonymoudy with ~cnbllcfrrilll and is used when bact..ia we .. not found during the microscopic namination of a stained slide. Howe""r. the dfi"ion might ~ ~ stptic exud;o,.. a. Bacteria may havt bttn p .."'nt but not found via microscopy. Thi. i. not uncommon when vtry few org.nitms ... in • sample. b. Bacteria may havt bttn p .."'nt but not cuhu..d b«:a.us< of i""ppropri . te culm.. m.dia (• .g.• aerobic venus anaerobic organisms) or the aninul b.od bttn trut.d with antibioti", prior to collection of an effusion. c. The nud"ion is caused by. micro~ other than bacteria but was not found by microocopyor culturing. 3. Occasionally. bact..ia a.. found in an exud;ote but ... contominants. In the", ca .... the nud ate with bacteria might ~ a no~"ptir """"'fr. B. A classification of "pfir o:udi!fr is b..t .ppli.d to .n effusion when it is known that the exud"ion is caustd by an infectiou. ~nt. Convtrstly•• cl .... ification of nomtpfir (XU""fr is best . ppli.d to an effusion when it is known that the nudation i. not caus.d by.n infectious ~nt; this is typically not known" the time a fluid .. mpl. is "",lu_ at.d. so the term ncnuptic (xurldfr is not rrcommend.d fur the int.. p.. tation of fluid a.... ly.... When bacteri a a.. not found duriIll: the n;omination of an effiuion. it may ~ ~tt .. to classify it •• an ~dau of unknown clluu than to clas..ify it ... nc~uptic (XU""fr. [I. Differentiation of a bacteri.l nudate .nd intestinal contents A. ex:c..ionally. a collected sample will contain numerou. and pleomorphic bacteria that are consistent with intestinal flor .. The microJCOpist .hould consider that the umple might ~ eith.. a bacterial nudate KCOndary to intestinal leakage or that the sample inadvmendy WlL! collected from an intestine (called an inwtb",{ tap ). B. Both typ<s of "'mples may contain numerou. bacteria .nd evidence of ingest. (e.g.. f"'l:ments of plant or mu",le fi~ ... or granular debri.) and ha"" odors and ~ gross ap]l 2.0 gldl.., wh..... the int."i .... l fluid will not have many leukocytes •• nd n.... trophils. if p.... nt. will tend to ~ nondegenerate if the .. mple is processtd promptly. Rardy. th~ ptriton•• l fluid is collected within minutes of an intestinal rupture. and then it will not ~ pos.!ible to diff..entiate the ptritoneal fluid sample from int..,i .... l fluid. How"",r. the ..JUhant inflammatory r"lxmst will ~ evident in .. mpl .. collected. f..... hours lat... 2. Evtn though the int.. tinal b.ct"ia are not pathogen> within the int.. tine. they are recognized as fu ..ign structures in the ptritoneal fluid. Accordingly. they wiU k engulf.d by n.utrophil •• nd macroph:>gO'. Bacteria from an intestinal top m.y 864 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY und~rgo phagocytosi, in vitro within 15- 30 min af{~r oolla;tion, funh~r oomplic;;ot_ ing th~ djff~r~nt"'tion of a mixM p Effusiono associalffl with n""plasm, A. A n(opfmric rffoiion is a pleural or ptr;ton.al dlU,ion th. t romain, "..,plastic cdl,. Mon intmhoracic and imra-..bdomiruoJ neoplasm. that co"", .ffusions do not .lough ~ll, into the fluid; chq typically ClUst an .ffi"ion by othu process.. {. xudotion. transud ation, hemorrhage, or damagnllymphatic ,)'Sum)." Th. ~aru= of the .lfu,ions can be, ammdy variable, with only rare "..,plastic ""lis among many inft.mmatory cell, or with .n extr.rudy h;y. ron"",mration of neopJ:.s{ic cells. B. A critic! aamination of th. nucl.at to j.lm in diam~t~r and has nud~ar and cytoplasmic f~atur .. of h~h ~llular activity. Small~r neoplastic lymphocyt~•• r~ more difficult to r'""'gniu via mICroscopy. 2 Primary carcinoma! or ad~nocarcinoma. of alxlominal or thoracic organs may afoJjat~ cdt. into th~ dlUsion {Plat~ 15B J. Meustatic .,rooma! typiC;O/ly do not afoliat~ cdt. nor do th~y amst dlU,ions. 3. Met:lSIatic mast ~ll neopla'ia may dough ~Us into the effusion. 4. Hemangioma or hemangio",rrom. (malignant h~m:mgi""ndothelioma) of .plttn, li""r, lung, or he.n might rupture and causr • hemorrhagic effusion, but tardy wiU neoplastic endothelial cdh be found in the effusion. 5. Meustatic mdanoma! may damag~ tissue., damage lymphatic vesset., and nfoliau neoplastic cdl,. 6. Plalnl:md peritonul mesothdiomas cm produce effusions with numerou, anapl..,. tic ~lls (Plat~ 15C). However diagnonieolly, it may be difficult to differentiate neoplastic mesothelial cdl, from cU""inomatou. ~lls or proplastic mesothelial cdl, found in nonnwpl:lStic effusions. 7. Squamous ~pithdial ~lls in ~ffu,ion .,mples."" typiC;O/ly contaminants. However, occasionally they ""pre"'nt neopla!tic cells afoliatal from an alxlominal neoplasm; for example, gastric "Iuamom ~ll eorcinoma in hone•. Poorly differentiated squamous cdt. m.y be difficult to differenti.re from reacti"" mesothdial ~Us. In pregnant :mirnals, squamous cell. may be nom .mniotic fluid (Pl.re ISO). IV. Lymphocyt~.rich ~ffu.ioflS A. LJ",plnrylr.,kh iffotiom "'" those plalral and peritoneal effi"ion, in which th~ dominant nucleated cell is a lymphocyt~ (Plates 14E, G, and Hand 15L). Th. two moJi rommon causes of a lymphocyt~rich dfi"ion a.. lymphoid neoplasia (lymphoma) and :m :lCCIlmulation of lymph (Table 19.5). Occ:as.ionally, a low1:rade inflammatory process may produce a lymphocyt~rich fluid. B. Microscopic examination of the lymphocyte:! may enable a neoplastic lymphoid effusion to be diffe .. ntiated from a nonneopla.nic effusion. Th~ distinguishing futures includ. the following; l. Nuclear di.meten; If mo!! lymphocyte nudei a.. :> to J.lm in diameter, th~n the . ffusion is probably neoplmic. If mon lymphocyt~ nudei .re < to j.tm, lymphoid neoplasia m.y or may not be p.."'nt. 19/ CAVITARY EFFUSIONS 86' 2. Chrom.tin patt~tfll: Di'perK v. Poricardi.l ~ffusion' A. Th= a.. uncommon and mo,tly d~ectM in radiograph. and ultrasonogr.ms (echocar_ diogram.). Disord~rs associatM with pericardial ~ffusiom includ~ nalplaoi •• nd infection,. but they f=iu~ntly'''' idiopathic.'''''' B. Fr VI. Amniotic fluid A. Occasionally. amniotic fluid is inadvertently rollectM during para",nu,is. B. Unless th~ro i. a pathologic sUt~ involving a f.tu, or th~ amniotic sac. th~ amniotic fluid is cl.... r and watery. and. microscopic """mination rov~..t, numerous nudutM or anucl~ate oornifiM squamous epithdial ",II. (Pl.to 150). References t. R.. ..... R.E. M"", DJ. lOOt . Alto. .fC.,,;'" • ..d F.ii", C)JtO"o . Pbiladdpbia, WB Sa.od. ... 2. Co...!l RL. Ty\« RD. t992. CytolOCJ ",,,J H"""","o 'f'b. H"". Gal .... CkAmakao V s. P........ V. AJ.ah. RD. Rii. Re. 1979. C)JtO!.p of do. 0., _ ; Gst. So.t!. Btt>d. IN, Amt.ia.n Animal Hoopital Aaoociati.on. 6. Co...!l RL. Ty\« RD. 2002. [);.,...,.;, C)JtO!.p."J H.-.!.p of do. H.",. 2nd odition. St tn..l" CV M ~oby . ." FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY 7. VIlIi,n E. Bt.choood L. cd •. 2005. 11&1 VA M ....J ojc..;." ~"" F.ii_ C/;,,;..J P<Sth. !.rJ. 2nd editioo. GIou.c ..... , B.hl.k Snu.lI Aninu.I V«. B. M~ G. I '!97. PJ.y.iolo~ ...d patI.oph.pi.oIou,,( pkwal Bold tum'''.... E., Rtopi. J [0,219--225 . 9. Hooeood G, Saliob"'J' SK. c.... .... d1 HD. o.NkoIa DB. 19B9. [ntrap..itoncoJ elK .......... ...d dcainau in ...., .J.oc. y" 5"" 18,261_26iI . 10. G.""", AC. Hall JE. 2000. T,..... .f M.JKJ l'bJ«tati.on of ...,,,,,.. pt.... ric ....,pko. Comp<..J c.....rin Ed"" Pm' V.. lj,70}-7Q9. 14. Col .. EH 1967. V.......:.....,. Ch.K.J P_iorJ, I. edition. Pbiladdpbia. WB Saw.d .... IS. B.nj....in MM. 1961. Oodi.u o/V,,.,.;,, • ., 0;""'" P..h.l.o. 2nd «Iirioo. Am .., 10.... Sn", U..;....,.ity Po ... . 16. 0."".. JR. !'r.... KW. 1977. V........., Lsb......." M';;";"', h. editioo. Am .., ]"". Sat< U..;....,.ity P", .. . 17. Roo. BD. Po .. TW. 2001 . CO''';,,J PIryH. I.D .fA.iJ-EJ...."",J E""'-IJ" Dis."",-. 5t1. «Ii';"... N... Y",,1c McG",,,· HiIL 18. t-.idi. KN. AI...I.... ",-"".tIt PS. ZOOS. H<mat.,I", inl mal~ m • .,;o,nin& _ ..clt< •. Nat Oin Pract G ..""....".,I Hopatol 2, 1 1.l-11 6. 19. Molina E. R""'r KR. 2001 . N.,""""-k pottaI),yp<""no;""'. Qin u..... Dio 5,769- 787. 20. KDot BG. H""""" R. Po< DI. Naut. J. 2004 . c..",...n.J .....I1""..i•.......;'" Bioch<>nical ond dinia.I i"'pliati.o ... --A .-, "P'''' and li........, ........ Eo, I P,di.tr 16.H;c;'l~70 . 21. Sdu1 .. RW. E.«I.r T. 2001 . Gibb. """",~aI krtw,. VniIjOn,; kypotJ. .... of body fl..id .........., ~"i<>." lmplia-DCtpt in ...., di1l"=ntial d ~ io of • .cit .... Ano Int .... Mod 1l7.:!1j....220. PtobI Yrt Mod 25. Hooeood GI.. s.tiob"'J' SK. 1989. Patbo~1<>oa.x .. ...ci.tod ..ru. .i&\rt.oidcd hcut faiIur. in fivt <1<>. I Am Y .. Mod A.oc 2().j,B4-89. J}. Fo ....... lW. BiodoaodSI. l....bo RM. 1986. CIoylotbo... in.4 doc ~ I Am Y.. ModAuoc 188,I}Ij....I}IB . 34. Sdrum.d.", I. B.ui, R. Spano I. 1989. Chylodooo .. in "" An),i ... fill,. Eqoin, Y.. I 2101 }2-IH. J5. Willud MD. Con"" ID. 1985. o,yIotI.otax ....,cl.. od ..ith )'L,,"'rn1"'';' i... ~ I Am Y.. Mod Auoc 186,72-7}. 36. Got.. BR. s.., I. c...,...... II.. Ullntan SL 1994. a.yIouo ..cit.. in "'", Nin< "'''' (1978-I99J) . I Am Y.. Mod Aoox 20M 161 _ 1160. 37. M.... TS. Lod, YM. 1992. Ckylo.,..i"'''' ..... ....dated ..ru. _ .. .,.. of tbt I'T «>100. i .... J.on.. Y.. RL< lJb421 . JB. Nd.... AW. 1979. AnaIpi. of "loin, .,..ttonc..I fluid. Y.. Clio North Am Lu" Anim Pract ),267- Il4. J9. Wilooa AD. Hi..d. WM. 00I.0tn. AD. 19B5. AIxIooninoo<=tt.io in.-lt, T td...iqo, ond "it..;, 1"0. dia&-oio of pt~to";';' . Can Y.. I 26074-.80. 40. E.,ttF" IC. lopn. I. M.,...v..... R. Rodoipot.. M. A&uik .... T <j,,,, E. 2006. TI, inllu,,,,, of ... ticoa&uI.n" on ...., .................. of -..J P"'''''' <"'"i .. ....1 o..id. R.o Yrt Sci Bo,j....IO. sc. "'P"""" pt"'"i""""" "I""'" 86' 19/ CAVITARY EFFUSIONS 41. Ao...icaa 0ptiaJ. 1976. b.m_';.", fo "" .,.J , ... _/,1.. AQil TS "'''" (~ GoIJ"'T/l ..IN<<-.). Buffalo. NY, Ao...icaa 0ptiaJ. s.;,,,ci60 In"""""" OM"",,,. 42. M~, ..... SE. 2004. 1h< "",-t.d.i,J cdL Int I Bioch"" c.n Biol }609--16. 43. Ao""" VB. M<>bunmod KA. 1m. patl.oplt,.-.iolocy,,[ pkwal .poc< iokrtion •. s..ni" R...,o Infect 1409_ 17. .... Yom, S. Li FK. Chan TM. 2006. P";'o."."J meoo""'ti.1 «ll , ........ and bOoi.o&r. !'ni, Dial la' U;,162-171. 45. Kjdod.t-r; CR. Kni&\>' IA. 1986. &.& F!.UJs.. lAb ..""" Ex..,m,. .fl. Cu..., KI. I ....... MP. Fi,"" D. P.oboo RL. o...;"opb .. MM. ZOOJ. Cytolock dia&oo.ia,,[ pt<>nbi" KM. 0"", CM . 200 I. &aluati.on ,,[ ,bdo",in,1 o..id, P.. ip/tm.J bloc.l ",,,.tinin, and pota..; .... "tioo 1'0, di, ~. ,,[ ~""''''''' in dop I Y" E.n..r; c.it Cat, I t.27j....280. 53. Ridou,31 5- J24. 56. Lat"'" KM . N"0rt0 IE. lkld.oon.nioo RM. IR. 2005. &ahoation ,,[I"'~tonco.l fl..id!acta« ... mack.. of int...tinal iodoa.m.i, in oquin< «>lie. &toin, V.. I 37,J.(2-}46. 57. N._ DD. McC~ SM. Sd..dJ", DI. 2OOol. Biod.,,,,iaJanal,..ia of.....planit '''''''0' DoOOUt«>pl .. ti.< abdominal dfu.ion. in doc •. I Am Anim H~'I' A.ooc 40,372-375. 58. Booapuki JI. !...d";, Bartoo LI. Loa. A. P""" .... ME. 2003. Com"";"n ,,[ p<">"i""",1 o..id and p"ioooitia in d.oco and ""to. Y" S"'I: 32,)61_ 166. 59. ~ GM. Bona.puki JJ. !...d";, Bartoo 11. (..., AS. 2OOt.~« .. . di~ ,eot /"o' .... ti.< p<">"i.",,,,1 dfu.ion. in doc. and ca' •. I Am Anim Hoop A.ooc 4O..J64--Yll . 60. D.I... o"'" GA. C .......n RM. Jobn"'" RH I,. Sid"" IF. Baiod,a CR. M....I_p AR I,. 1989. I""",ood pntto.....t fl..id lactic odd ,.,j .... and p~ .......1 """",lation in "itooiti. in 24 d.oco .od 2 61. !...d";, ca'" A .......,pectt...nody (1987- 1994) . Y .. Swr; 2&90--98. 62. 0... ... SD. Goo..n R. MdlJ."""" MR. a...;' '''pJ." M.\1. SJ.,H, SM . 2003. Tht« "'... ,,[ "',,;.,., bit. pnttoniti, ";do """""'~ . "",«tial in ,bdomin,1 o..id .. tJ., rrooni""'" "iroo,al Iluid. Vrt Clin P,tho[ ,.,335- 341. 68. G..ij. "i"""al o..ido ,,[ k"""" ..ru. «>lie. Am I Y.. Rtt 42,888-891 . 70. 0...... /V. G.nin, EL. Goodb.om R. Mand ... ill, P. 1984. hpni"""tal iocb",ni.,,[ tl." a..,,,, and ""oemlra""'" ,,[ "'" .. ,.-ina[ i"""'P'" ,,[ albJin, pboop ........ in plaaaa and I"'dtonco.l fl..id. Eq";"" Y.. I 16021 j....217. do,. 0.......,. So""" u.. u.. u.. 868 FUNDAMENTALS OF VETERINARY CLINICAL PATHOLOGY 71. s...k.. MN. Cd.. CK. Tomq";', SJ. 20001. n.., di-", aod p~ va/u, of aLb.lio< pIooopb.tao< "',;,;ty in ........ aod 1"";""".1 o.id from .... "' • ..itI. 000" «>Ii<. J V ... IGum Med IS,W--S67. 72. Hom,,!, B. 2OOS. n. di.~• ...d pr<>&DOOtk .... "" of albJin., pho","'_ Ktivny in ........ ...d p..itoncd B..id from ....... ,.;th aoot< ",lie. J V.. I".,"., Mod 19,78.J.--784. 73. T .m"... Jr. K,; .... HJ, Moind ... AI!.. 2001 . r •....,~o..;. of mali&n_ udt<" Starlin, ' , Ia.. of «pill • .,. bm><>p<>iI>i .. AH. J""'" KA. 2OOJ. U.. of p«ia..d.i.llluid pH to dioti~.k brtov.", kliopaohk ...d nooplaotic diU';'",. J Yrt lnt<m Med 17,52j.-529. so. W.~ AE. ilc' '0 hcuu.toJou ~tion. ln, Kitch", H . K..~bicl JD. oda. Prw..Jm" ofth. F;", 1""",";....J Sp.."";_ of&p.m. H. - .I.p. 408-416. Gold",. co. ~D Aaoociation of Equin, I'..enli",,,,... 8}. Mo.1cy l'S, Dc.N"l'n M. 1992. Di~ . of ,"pturcd ... n...y bI.d.d" iD • ho.l by "'" id,oti6",tioo of a\ci"", ~"'" «r'"b in "'" """OOD'.1 fluid. J Am V.. Med Aa- 200, 151 j.-I 517. M. Andc.OOD DE. c.. ..... U D. Andc. .... l..S, 5'-J"D G. D",,,d.,,,, A. 199 5. Com ..... ,;'" ... .Jyr. of 1"';"",,,llluid from ~...d adul, attic. AmJ V.. R.o S6097J-976. ""&- do,. INDEX A.DO,. S« Arteri..l:o!veohr oxygen t< ... ion gr.odient AB syst<m, ]]9 kAte. S« Ac~oacrt;l .. roncenm.• ion Acrnthocyte, 143- 47. IHt Ac.runcy.37 Ac.t.minophon, H.in. body f",mation ""d. 186 Ac.toac<"'t< (AcAc) co"""D"",;on, 543---45 analytical ooJ>CCi, of, 584--91 Acid-b...d disaden, 562--65. 562f bLood gas d.ti in. 578. 578t cJ..sific"ion, 578, 579f compenu.ory duoges in, ""p«1«l, 578--1\] dilf... nces in definitions of, 585--&i mi=:!, 578 ump!., 578 Acimpenu.ory du"£",, in. 578. 581 t dilu,ional, 574--75 Hea,.. ond, 532 ino grouP' of. 574--75 bypercblo«rnic, 523 bypodtLonmi. and, 525, 526f phJ"iologic mmpeJUOtioa for. 575 normohlmi. rnd, 516 orgrnic, 509--10, 574 renal tubuhr, 532 '''pin,OJY. 575. 57S, SKU'OJy.574 ,CO' ..,d, 532 tim.tion:d,574 Aciduri., 456-57. 456t pu.odorical, 576 ACr..I« Activ:ot-23 mppr.,..jon ""'''', 81 '>-23 Adrenocorticotropic bo"""",,, (ACTH) conomtr1tion, 813_ 14, 814f cortisol, pt.rrruo ratio to, 814-15 "imut.tion test emu.., 818-21, 819f 96' 870 Alibrinog.nemi. INDEX ( BMBT md, 264 IT, prolonged rnd, 265 Agglutirution .JYlhrocytr. ]30. 136 late><, for foal .. rum, 400 AID. Sa An.rru. of inJI:omm>'OJY di ..... AlHA. 5« Autoimmun< "'"",lytic ..,.."i. Almi ... tranumirw>< (All), 6OW- 52 octivity, in=»«l ..... m, 651. 651. uulytical con""p". 651J.-51 psychoLogic process<s. 650 .. rum, 6'51. 651' ti ..... sour=<. 650 AJb.ndnoJ., tbrombocytopmi. md. 236 Albumin malytic:d principle. for to ..l, 372-79 C.'· md, 597- 98 <X>Domm.•ion, "'.,.lUring. 374 Crt to, ... io. 482 g..,.,ral ooncq>'" for tot:d, 370-72 pby.ioLogic pr0=s<5, 370- 71 ,onal """,..oon. 481 .ynth.. i" d.c", ... d, 383 urine ....Y'. qUIDti",,;,,,,, 478 AId_ ..o"" co=n.mion, 824_26 unm.. 824-26. 825. ~tion. 806, 807f AJluJ.mi.,561 hypioLogic procossel. 655 .. rum, inc,..."! .ctivity of, 65'>-58, 657. drugiborlROn< induction of, 6S6--57 >peCi<sIlx...,j differ..,,,,,, in, 65!!-59 AJluJinuri., 456<, 457 Allulose>, 585 comprnu.ory mati£"" in, 578 contuction, 531 hypochlor..ruc metabolic, 518 K·, .. rum and, 509- 10, 509f me.. bo~c, 510, 576 Cab urinary ""c...cion and, 60S bypodtlor<mi. and, 524-25 normohlmi. and, 516 ''''PintOl}', 523, 576-77 di....e./conditions c.wing, 577, 577t phy>ioJogic compenution .nd, 577 po. and, 620 AlJW",;", 561 Alop"', 697- 99 phy>ioJogic pro<J""e.r, 697, 698f S1lIlples, 699 effusion .rul}";" with, 861 inukel. bs<J=Se.J, 663-64 ,en.J acr, 21 "prodllcibi~ty of nndom, 22f .rources of, 37 W ... gard rules and detecting, 26-27 ArWytic.J pr<'Ci,ion, 21 _24 0 { and, 22_24 V1,i.tion and n...d fOr, 22 ArWytic.J .. mitivity, 25 An.pbylui.r, thrombocytopeni. :md, 243 A""I""'- teWrocyt<, 95 A""1'''''''''' 1"'1]1, 233 tbrombocytopmi. rnd, 242 A""I"""'" 'pp" infectious bemolytic """mi., 182 ''''lllXJ' A""1'''''''''''''''''''' leukocyte, 95, %t INDEX An.mia of in8ammatO<}' ialogic, 158-59. 159f «ticulocytosis ond. 151 oobabmin d.eficirncy ""d. 165 copprr drficirncy.oo. 165 .JYlhrocytr production. dra • ...d ""using. "" F. deficirncy ""d. 164--65, 16s-70. 169f fala'" deficirncy .00. 16'5 grnr ..1 infornution. 151 homolytic. 158, 170-93 All' g'io", aI. 170-76 ~uocytic. 187-68 rrytbrocyt< "".."irution rnd. 171 Heinz body. 186 idiop1tbic. 192 IMHA ... l n _79 PFKdeficirncy.189 PKddicirncy.188-89 porphytia producing. 190 .pbrrocytic. 192 homolytic, rrytluocytic mrubolic dd'oc •• 186-91 ""id..ivr damage and. 186-88 homolytic. immw>r. 176-81 disorder> of. 177--.'11 drug.ind"""". 179-80 pathogrnrsis of. 178f vacci",.induoed, 180 homolytic. inkctiow, 181 _86 A-p",,","'pp·.182 &b.';" .pp., 184 Cl4miti;um '1'1", 182-83 HAV.183_84 F.lV,I84 brmotropic mycopl .. m •• pecirs. 181_ 82 up''''P;''' .pp., 182 M}""plt""'" ",p .• 181 '" pathogrnr=. 181 TMkrU.'PP" 185 Tryp.u..-""", .pp.. 185-66 hyperchromic mocrocytic, 157 normocytic. 157 hypochromic mocrocytic, 155 miC7OCytic, 1!>6 normocytic, 157 of in8:urtm1'ory tivr. 151 - 53. 159--67 'pwi., pmr rod crU ""wing, 163-64 dirorde" cawing, 160-67, I60t rndocrinr diso,dr" cawing, 166 rrytbrocyt< prodoction and, 159 rrytbropoi •.u, inefJr<:tivr cru.sing, 163--67 g'. 535 ~...d, 538,538' ddinitioru.534-35 ina<=ocytosi.. 142 un INDEX Antibodies FDPs:md sp ,"'-"" thrombocytopeni. rnd, 239 Antiroagubn", . ndog<now AT 1S, 288-91 rnti_(phos pbo~pjd.pro"'in) ...tibodi.. ", 292-93 protein C 1S, 291 protein Z.., 291 - 92 [Up"', 292 Antidiumic hormone (ADH). i8S-67.!W ~"" Syndrom< of inappropri ... ADH _, .. ion Anti.{phOlpholipid_prot';n) rn,ibodi.., 292-93 Antithrombin 288-91. Thrombin_ :mtitbrombin compL.us :octivity d.c:r.ased, 269--90, 289, inc ..""!, 290 roagubtion ffizymeo inhibited by, 2H- 74, 275f conmmption, 290 Mpuin thenpy.oo comumption ai, 290 inte'Pretiv< comid. ...ioJU. 289 Joss, 290 plasm., 288 mbiJity, 289 units, 289 (An. Sa"". ApJ., .. , bo ... marrow, :u><mi. uu..d by, 162-63 lymphoid, pro..,u, synth..iJuuboJi,m ..,d, 389 lymphopenia of lymphoid, Mf. 85 pur. ",d all, >nerru. co= Apooonzymes. 640 ApoJipoprot ""~ ddinition. 83S hypoalbuminemia rnd formation of. 505 hypoprot';nomi. rnd formation of. 50S Asp ...... AST, S« Asp""'" tnn.amin>S< Asyncltronous nocJ..r lJU"'ration, 92 AT, S« Antitluombin ATP, S« Ad.nosi ... niphospJu.. Automtibodies, 794-9'5, S«1lls. ThyrogLobu~n . utoantibodi.. Autoimmun< b.molytic """mi. (A1HA), 179 Azot=>i" 427, 428f, 429- 33 in cm, 440 in "'ttl., 440 "m.., multi6.C1ori:ol, 432-33 cl..sific1tions, 429-32 Cn in .. rumlpl .. m. ""d, 437 differ.nti,tion, guide~,... for, 432- 33, 432t diwrd.l1, 429- 32 in dog, .. rum ch.mi,tty <=lit. in, .bnorm:ol routi ... , «<> UN:Cn ratio:tnd, 438, 43-St in ho..... , 477, 478t .. rum ch.mittty result. in, ,bnorrrW routine, «<> I""tr."uJ, 430t, 431 p",,,,n:ol,429-3O ru.ord.n rnd, 429, 430, ",tul v" 477, 478t ren.!, 430-31 pr ... n.! v" 477, 478t r.n'! di....... rnd, 433 ren.! f.oilw., .ru.. :and, 428 .. rum m.mistry =ul" in, mnorm:ol lOUtin<, ,,'-"'> UN in ......",Ipw.n.:and, 435 ureJCn wirucy acr.,ion :and, 429-31 Uf'" production, inc",....! rnd, 43O~ 431 _32 Bal",;.. spp" inf«tioUl hemolytic ," .nrmi., B:oct.,.iuri,,472 B... acrss in blood (BE,,), 567-6S B... aceu in ECF (BEla)' 567 B... ucetS in pw.n. (BE.), 567 B1tOf"'ni:o, 88 B1SOf>hil a>nomtr.,ions, mnomW, 88 kinetics, 59-60 pools, 59-60 B1SOf>hili., 88 diffusor cyhilic "ipp~ng, rry<1uocyt<, 138-A I, 140, B:oyer T rchnioon, III B& S" tl.lu.BiJirubin BE", S« B....,""'" in blood Brclanrn :on:olyzer, 528 BE IO , S« B... a= in ECF INDEX '" Bm"" jon<> pro,einuri., 385, 460-62 Bm.. thonium chLorid. :may, 478 Bmroc:Hn., H.in. body form1tion . nd, 186 BE" S" Bast "'''''. in pI .."", BHB, S" fi.HydroJ -'"' ditordet1/p>thogeneses:and, 531 - 33, 532' bypercbl",emi. :and, 533-34 iner.= .,.,,""'" din.!, 526-27 p=inuJ, 526, 527f qu:anrit1tion, umpLr for, 527_28 ren.! 10""" of, 532 ren.! tubulr fonction :and, 419 ~etion, 527, 528f Bicarbon>turi., 518 Bilt :ocid uulytic:d conc=>r>, 690-91, 690f . nt,ic cirrubrion of, 690 excr .. ion bilivy, 691, 692f dra.=ret.rion, 691 uri"", '0 COl .. rio, 485, 696-97, 697, Bilt 691 Bili..,. di ..... , 6n Bilirubin cholrnui. rnd, 6tH conc'" of, 683 phJ"iologic p"""'.... of, 681--.'13, 682f conjug.t«!, d.cre:ased rxcr .. ion of, 687--.'19 C,''', m"', rlJwion rn.!";' with, 861 hemolJ"it rnd, 684-85, 685' hrp>,ocyte up ..... of, 685-87, 685' byperbilirubinemi., 684--89, 684, icterus, hemolytic rnd, 684-85, 685' msun Bl.., ""U, 346 BWding di.ordrn, m.jor bWding p>tt:and p>thoge ...... of, 300-310 gend.r:and, 301 h<mot, .. it t<'" rnd, 302-A, 302, p.ttrm. in, 302--4, 303, p>'bog '" Blist B... a<J<St in blood: Complrt. blood ooun~ Nude.ted «d blood ",lis crUs,6 naining, 61--62 crosrm1tdting, 210 ditordrn, 30 I rrythrocyt,,", Ill, Ill, d ... di/fe",ncer . ad, 129- 30 fug"",nt1tion in, 191 gases, 559-91 ,cid_b... disorltd, 578, 578, rn:dytical ooncq>'" in, 565-73 calcul,,,d, 567- 70 comp" ....'ory cbang ... in, "'P""t Blood, 874 Blood ( dog, 119 erythrocytes and, 117_ 20, 208-10 bOl5<, 119 Hgb. 128-29 irnpedrn'" ceU counter, Jabontory ""Y", 7--11 J.ukocytes. 55 63--64 .bnornW concmtr:otion of. 70-90 ...Jwtion rnd. 60-61 lymphocytes, 58 processes influencing, 59 Dtr:otion of. 70--81 Left .hift ""d, 7!1-71 proo<sses influencing ""'.... n "'mp<"'''''' umpLes.5....6 rnticoaguhntl for. 6 Leukocyte ..mu. tion ..i'h. 60-61 ..rum, 7 'p<'Ci<. varutioJU in, 2](1_ 11 typing, 208_ 10 cat, 119 cattle. 120 dog, 119 ..., clotting ,i"", for, 279---80 gluros< conom'r1tion in. 706-23, 7l2. Blood ammonia, ~7 Blood films . nmin1tion of ",j... d, 61 pbteJ.t ....Ju.tion on, 227. 229--30 volmn.:and,247 Blood h<mocrit valufS, 49 _.. INDEX Blood lou :om"'. 167--61!. 16Sf chronic. 16&-70. 169f co ...... 167 hypoprot';nemio rnd. 385 put<J.t survinl. d.a ... "d md. 240 Blood tnosfusioru autologous. 131 blood groupo :md. 208-9 blood 'f"'Cies nri1tio", rnd. 210 c..1+. h.., coJ>CKOn<Wy to. 180-81 poottnosfusion purpuu. 2olO Blood """",Is (=dotb.li.J cells). 300 diwrd. ... 301 B_lympbocytes. 55 neopl ..i. hyp<rprot. :md. 3S 1...'15 lilioutoryidinic.J probJ.ms ...ith. 3S5 p>thog ..... is. 381...'12 BMBT. Sn Bocc:d mUCO<1l bleeding tim< Body 8uids bbontory 1S1>J'" for. 7...'1 umpte...7 ""~ c..". 596 _.. neopl ..i. in. 60 I Bo ... murow. 54-55. 323--65 1Jl1ly>i1. methods for. 328-34 ""I..tic. 338 clostific. tion md r"'P"n INDEX indi"",ions for. 327- 28 interpr .. ing remIts of. 357- 58 microscopic. 329-33 F•• 332- 33 /low cytometJy. 334 gr:umlopoi<sis , 00. 336--37 hema'opoi .. ic ""lis. 324-27, 329- 30 hema'opoi .. ic I"'f"'h,ions. 330-32. 33lf h.mic neopwu. 342-57 d , ,,ifi,,,,tion of. 342-46, 343' general romq>ts/ternu. 342-46, 344f hyperpl .. i•• 334-38. 335, bosophilic gr"'ulocytic, 337 ominophilic gr..,uIocytic. 337 gen monocytic. 337 hypopJ.,i., 338--40. 338, ..,.mi. cru..d by. 162-63 ..ytbroid. 339 gen...liUeytes, 332 lympbocytosis,340 ID1jor ronc '" nu,ririoruJ defici.ncies, 336 pos'mortem S1ffiples of. 334 reph""men'. 'brombocytopeni • ..,d. 237 umples.357- 58 Brodypneo. 561 Bromide. L-uc .. te m,,,.... ,,,,,,,,n' ..,d. 540 llucc:d murow bt...ding time (/1MB!) :m:dytic:d oonc '" pute!.t function 'est. 263--64 prolongM, 263 vurulu dUe ... .00. 264 vWD ,00. 263 Buffer base, 568 Bwrn.o>< kine"". hypoblemic mY"P .. by in. '" Burr cell. 147 Co'+. 5., C:dcium Cocbectic ....... protein oyn,hesi./",ubolism.oo. '"' C:dcitonin ronunmtion. 630-31 C:dciwn (Co'",). 593--631 ..,iom. dilfusible binding of. 608 ..,rioo>glll..,,, binding, 611 _ 12 bo .... 596 chloride. do'ting .00. 281 conces of. 594-96 pl1UIl1, 594-95 regul.tion of. 597- 98 .. rum. 594-95 diwr'terru for. 631. 631' e/Jwion rn:dJ"is with. 858-59 fr:ocnue he:iling .00 deposition of. 608 hyperc.Ja,mi, :md. 598--602. 599, incr....d pro,ein.bound. 602 mobili",tion of. 598-601 in, d«J,.. te.604--1! po. in'encOon ..ith. 596 reguutoJ}' hormo ..... 593--631 ren.! f.oilw./inmfliciency.oo. 601 r.n'! tubule. 596 ren.! tubule function ' nd. 422 .. rum. 594-95 fxto" determining. 595- 96, 595f uriJl1l}' acreOon of CO""""" 876 INDEX din"mp Canj"" '" Cast" uri"" .djm dm.. ",..,we UA • ..:42t. 445 USG. rdrxtomotric in. 450 D_xy\ooo .b.orption tell'" in. 754 Cant.. S" tWo H.ro..base '" "" .. rum, 535, 535. "rong. 586 c.. GGT in. 661 glocororticoid, in. ",usod by bypofellomi • • a,.PI, fKaJ in, 753 ACTH in, 813. 814f .ruenorortie>J function tens in, 821_ 22 ALP in, 659 azotemi. in. • bnorm:d routi ...... urn ch<minry biJ. acid cl"JLeng< test for, 693--96, 69'5, blood_group. ')""'10. 119 roba/:unin in, 747- 50 a>rcisoJ, uri... to en <11;0 in, 812-13. Sl3f EPI in, 740 F.LV in, ]64 infdrenocorticism in. 601 _ 2 inf'K1iow Mmolytic..,.",'" 181 J.ukogram p.... rns in, 90 m PU and, 668, 74S---46 porphyria, mng.niu! ~bropoifl:ic in. 191 lOan di...... in, 60 I ren.J f.oiJwe. bypok.J.mjc in. 519 r "" TSH in. 7 94 gluunldebydo "'ning of coJf ..,rum in. 400 b.moglobinwi •• p""p"rurient in. 189 h....diury b.nd 3 d.6ciency in J.p"""'" bl ..k, = hypoc.J""mi • • p:ururi.nt in. 6i:J6-7 " coJf oerum :md cbang .. of. 402 de6cimci .. in. 403 FPT of. 39l!-99 "",mllingl.. timating, 399---402 lNkogrom p atterns in. 90 Na,sO, for e>Jf sorum in. 401 _ 2 notltropeni. in. n - 79 potpbyri. , rongmiul orytbropoi.,ic in. 190-91 renoJ f.oilwe, 524 r 400-<" Cav:.li.. King Cl..,].,. sp:miel. thrombocytop'icudioJ.866 rupnu. -ausod. 847--48 .pecific, <X>JIlJDffi'" about. 863-66 St:oding·. t.w rnd. 836--37 INDEX '" Cb,R ddiciency. S« Cytochrome-b, nducus. d.efici.ncy CSc. S« Complete blood C.U counting. 63--64. 64f C.U (corpwcuhr) hemoglobin concmtr:otion mun (CHCM) ..,.",.. md inar...d. 157- 58 iJ>Cl....d. 157- 58 C.ntrifug. tion. wine. 455 Crph.Jospo,ins. immunr hemolytic """mi. induced by. 179....'10 C.rulopbsmin. 372 CHCM. S« C.U (ooq>wru.lar) hrmoglobin roncrntr:otion mrrn Chrdiak_Hig .. hi .yndromr. 99 Chemistry .... y>, 8 Chlor:unphenicol •• rytluoid hypopb.i. ""d. 339 Chlorid. (0·) o.limmucy tnet funetioJU pCivis with. 856-57. 857, hyp coun' m cr. S« Chlorid. Cirru.l. ting n Citr:o"'. 6 CK. Sn Cr • • ti ... kirw>< Oinical p. thology. 4-5 Clini"". S« Coppn<mi.. 182-83 Clotting ....Y'. ooaguhtion f:octo,. 287 calcium chlorid. md. 281 thrombop1tb" rnd. ~5 ..-hot. blood. times. 279 CNp. S« Circubting neutropbil pool Coagubtion. 268-93. S«tds. Gluur:old.ebyd. roaguhtion ACT. 279-80 :m.Jytic:d ron",p". 274-79 g< ... ro.l ' pp,,,,,cb to. 278 instrument>. 278 urnples.274-78 ....Y'.274 cosc::od.e .. rh. 263-69. 270f r ndog<now rntiooogulrn"'. 2118_92 AT ... 288-91 rnti_(ph"'pbo~pid_protein) antibodies .. . 292-93 protein C >s. 291 protein Z.., 291 - 92 r nzymrs. inhibitotl of. 273-74. 275f beto",. 269- 71. 272,. 273f .etiv:iti.. of. 286-.67 rntibodi .. . 292 dotting ....Y' for 'f'<"Cific. 2117 d.ficiencies in. 286-.67. 286, inhibition of. 310 inhibito"'. 292 """""';ynl1tic.269-7I fibrinogen rnd. 285--86 hypotbrnni •. homorrboge and. 278-79 OSPT.282-84 JUtb"'Y'.269 pby.iologic inhibito", of. 271 - 74 pby.iologicp~. 268_74 plVJ(A rnd. 287-118 PTT.280_.84 RYVr.288 "s'" ""'<'"' cimM.276-77 rolt.cting/hmdling.274-76 p=ing,...bility of. 277- 78 TF.282-83 ..-hot. blood dotting timrs md. 279-80 "8 INDEX Coogulopathy conmmpOY< b.mostotic test re",Jt~ .bno,rrW in, 309 =ognizing,30S---9 dilutional,309_ 10 Cobabmin {vi"min BtJ conam'<1tion, 74:7- 50. 7i8f .,..mi. ,"'-", uulytic:d <:<>11""1''", 407--.'1. i08f oLogic p""",..."Iooncepu rnd. 405 S,:or:Ii"ll" low, 405, 4061' Combs' test, 211, 2llf CompL.te blood count (COC). 60....62 bo ... marrow. 358. 360f compon .,..mi, ,,. Cortisol ACTH ,,,ti~ to pwm>, 814--15 conamtr .. ion. 808_ 12 CIt ",io to uri ... , 812-13. SUf hypercortisoLemi. and, 8OS-IO, 8091 hypocortiw!.mi. md, 810-12, SIlt ><ereOon, 806, S07f Gutin< kin ... (CK) uulytic:d con",pts and, 662 n .. rum, inc",ased activity of, 662-63, 662t Crutini ... (Crt), S« ,,"" Urinary protoin to " .. tini"" ..tio olbumin to, r:otio, 4S2 ....Y', 4:36--37 dur~ me, 438-39 a>n:isol, urine r:ono to, 812-13, SI3f effusion an:dY'"' with, 857- 58 pbyUologic p'o=sWconcepts reg:uding, 434f .enol ",but. function :md, 423 .. rum/pIa""" conom"ation of, 434f, 436-37 decreased, 437 in".... d, 437 UN oonuntr:otion v" 437- 38, H8t u.inary n",.tion of, 429-31 min< bit..ad to, mio, 485, 696--97, 697t Cros.m1tming, blood, 210 CRP, S« Co.rect«! .etirulocyte pen:pl .. i. cw.ifi",tion .ntb, 353, 353t Cytochrome-b, =lutt ... (Cb,R) defici.ncy, 199 Cytology, S bo,.. lIU.J<>W, 333-34 Cytometty, flow, 334 Cytopl:wn, foamy, 92 D:ocryocyte,147 DEA. S« Dog erytluocyte antigen D..:won limi.. , 20 D..:inon tbmbolds, 39---40, 40f bcton inlluencing e.ublidung, 48 .e/'eoma, limits v" 20 ROC 1tbologic .ute of, 500 types, 500 USG, refractometric ond, 450 o.ming method oompuison, 33-34, 33f INDEX Omdritic ""lis, 327 D._cion limi~ 25 D.'omidin<, hypergl}'<'<mia ond, 718 Duamrtl... on<, Sn Ills. High_do.. da>m m emin<, 81'5--18, 8161', 819, «p>in<, 822 f.li"",821 _22 Tll and, 743 Dextnn, biw=l :md, 520 prnc,.. tic, 716 types,71'5--18 Diognostic aCCll'><:y, 39 '" ""'. " ,-PI, fec:d in, 753 ocidosis in, 58lt ACTH in, 813, 814f .ruenorortical function tests in, 81'5--21, 816f .Jdos«ron< concentr:ocion in, 824-26, 825, ALP in, 658-59 ALT, .. rum, 651 :unmonium toIennce test in, 702-3, 703, azo«mi. in .bnorm:d routi ... oerum cbeminry , ..ul.. in, ." UN : Cn ,.tio ' 00, 438, 43-1!' bile .cid d"JLenge «n fo" 693-96, 694, 00"" marro.... dy.",ui. of poodLes, 100 CKin, 662 rob:d:unin in, 747- 50 a>rtisol, uri ... Cn l1tio in, 812-13, 8l3f p:miet., '0 '" el~ptocyt"'is ROC cwv< compariwn of, 45--46 Diognostic prope,ties <\dici.my in th.o,;., of, 44 bbol1tory ""Y, 38--45 Diognostic ....,.i,iviry, 38-39 Diognostic .(> in, 200-201 b.rediwy,200 en"roJ>1,by, protein_IOling in, 604 EPI in, 740 eIOC,in< prncr ...ic iruufficiemy in, 605--6 fot.« in, 7'50--52 GGT in, 661 H.in. body fo,m .. ion in, 186 hyper:odr<norortici,m in, 610 hyper:oldos«roru.m in, 824 hype'~pi DisrocytfS, 136 Dissemi ... t Pll md, 66/1, 745--46 pon"'yttomic ,bunts in, 20i pyometr:o in, 451 ,en.J di ...... in, 60 I 'ebrulocytOlis, 117 .iderobb nic ..,.",i. in, 202 SLE in, 240 Diognosticp~~,5 ", ., BOO INDEX Dog. (Nln';nud) "'~ .... ""d. 20] TAP in, 746-47 thrombocytopeni. in CavW., King Chuks 'p<'CtRn ddiciency in Dutch golden sp~,23S. 241--42 thyroid honno"" oo",",nttltiom in, 799. 799<, .~ thyroxin< md TSH in, 798 TLI in. 741-45. 743., 745' TSH in, 788, 794 UA, 442 •. H5-SS uroJith~ 488 USG, ",frxlomp«trin '" ~Cl.tion , .. n, 703---4 Dy.cr:ui. 1>0... rrurro..... ofpoo. 100 protein. 37] Dy.erythropoi..is, 348 (x>J>ll"nitid, in Herd'ord caJv.s, 167 in EngIidt spring .. ,!",n;'!., 167 Dydibrinog.... mia, 310 IT. prolonged rnd, 21!S Dy.bom1topoiesil, ocquir.d ><eoooary, 3'W Dy""pJ .. i., multi~"".g<, 347--48. Sa ali<> R.fnctory cytopmi • ...ith multi~ .... g< dY'Pw;:o: Refrxtory cytopmu ..itk multi~ ... ag< dJ"PJ.,i. :md ringed ud.robl .. ts Dy.pro",jnemi .. 371 hyp<rprot;' and, 507 normon.n.mi. in, 503-A. ~03. EDTA. &t EthyJen.di..ru"" .. r.. cotic 1-16 Ehrluhia, J.ukocyt •• 95 ElAV. S" £qui"" infoctioUl """"'.. virus Ekctrolyt.. conam.... ions :mAOrnW. 498 in .. 'P .... tion of. b .. ic concOnov~ •• 49'>-5~3 _:tk. 586 EUiptocyt<. 148 EUiptocytos;'.148 in dogs. 200-20 I horediury. in dogs. 200 Endoc> .".",i. ",used by, 166 hypopWi:o, erythroid c:w...d by, 339 Endomitriosis, 32~ Endotho~:d ",Us. 5« Blood """,Is Endotonmi. hypoffi1g ..... mi. :and. 625 noutropeni. c:wsed~. 78f. 79 thrombocytoponi. and. 242 Endotoxic .hod:. erytluocytos;, aused by. 1% Engli'h springor 'pan;'], dysorytbropoi"';, in. 167 PFK deficiency in. 189 Enteric di==. hypom..,.,....ru..:and. 624 Enteropathy. pro .. in_Iosing. 604 Envenoffi1tion. 192 thrombocytoponi. and. 241 Enzyo>eI. 63~9 1 -, hepatic. 680 .. rum. 645----17, 646f. 647. ddinition. 640 nomoncU.ruro.647_48 production ""nubr.644 ';""".644 rele ... of. incr • ...d. 643f <>en <=ap< :and. 642-43 ""nubr.643 "". f1 08' INDEX .ctivity, incre:ased, 6019--50, 650f ....)" of, 645---47, 646f, 647t m..,w.minophili., 86-1!8 co ..... , 86-S7, 87t 1Ul""'Pt.n;', 87--1\8 Eosinophilic Lruhmi., 88 Eosinophils conomtt .. ions, mnornul, 86-88 ki ... tics, 59 nomt1ining, gr=uLes of Epo, llO-ll Equine infectious anemi1 virut (BAY), 183_84 &ytluocyte<, 107_212, Sn alst> Dog ~brocytr rntigen, Winttobe', erytbrocyte indicet mnornulitiet, nonregenttOtiv< anemi. rn<"'i", vari1tiont rnd, 210-11 Cb,R defici=-51 color, 137- 38 compLem .t~ptocytotis in dogs rnd, 200-20 I fngmen"'tion of, in blood, 191 g.".ral f'e..tum, 136-37 ghost ""U, 137 H.in. hOOios, IiI hemolytic ..,.",i • .00 ex:omi ... tion of, 171 herediwy b.oo 3 d.ficioncy in J.pa ..... black cotd. rnd, 200 hypochromic, 137- 38 inclusions, ]38---42, I40t basophilic "ipp~ng, l38-41, liOt Hgb cry".ls .. , 141 Ho...n-JoUy bodios .. , 141 ",fuCliLe ..sinett ... 141--42 ,iderosic gnaw... 1S, 142 indi=, ..,.",.. d ... me>tion with, 153- 58, 154t kinetics, 112 megalobwtic . ... mi •• nd, 201 _2 met.bo~c defect in, hemolytic .... mi. rnd, 1s6-91 met.bolism of tn1rure, 115, 118f metbemoglobinemi. rnd, 198-99 morpbologic ' ¥:Im. tion, 120 morpbologic f""rureo of, 135- 51 orgmi",u, 138, 139t o"""Wity,I30-31 oxidrnt ..potwe ai, 198-99 patbogea..si,,13'>-51 pby.iologic pJOCelSe'l, 110_20 polyduom1topbi~c, 138 P=""",,", 110 p«>ction, anemi. rnd, 158-60 QBC mal)"is of, 127 roulem, 136 rubricytotit, 136-37 'k"!",, abnormal, IH- 51, l+it ,iderobl... ic ..,.",i. in '''"W BB2 INDEX &ythrocytosi.. 19}-98 IDsolut<.193 hypoxic, 1%-97 idiopathic, 198 I"'hogenesis. 19Sf pby.ioLogic, 196 prim..,... 19!!. 351 ,el .. i"" 193 > p.thog"""is of, 197 phJ"iologic pro<:<.... of, 197 =:on ,,', hemorooc Erythrognm,60, 120-23 Erythron. ]W_ 12 &ythroph.ge, 94 &ythropo;',is .ffi.cO"". bo ... JIUlfO'V ""d, 334 indJeco.... """mi. cruS«! by. 163--b7 bo ... murow md, 336 ESAlg. S« Erythrocyte uuf..c,e. . ..ocuted immunoglobulin Etwnol, hypogly<J<mi. and, 722 Ethyl .... diamine .. tr:ucetic:ocid (EDTA), 6 Ethyl .... g1yml hyperglycemia :md, 718 tnnoosi.,60S Euglobulin, 3n. Sntdi. $ .. . ugLobu~n t~t Enn', .yoorom<, 240 Exocri ... in... on<, 739--59 absorptive m.JfuJK1iOJlll of, 7oW-A I di ....... methods for .-mu1ting, 758. 759, Exocri ... panc:r .... 739--59 absorptive m.JfuJX1ions of, 7oW-A I Exocri ... pmc:r •• tic inruflici.ncy (EPI). 60S--6, ,,,,-,,, in c.ts, 740 in dog" 740 evaluating, m "'-ct F:oiJwe of P""'" trIDSfe, (FPll, 369 immunoglobin, 398---402 C:lWel of, 399 phpiologic co=pts of, 398-99 F.ts.. neg.ti", (FN) ,emIts, 3S F.ts.. po>i,i", (FP) ,esults, 36 ROC auv<.,.d me of, 45 F:onroni 'YndlO""', 620 FOP" S" Fibtin or fibri""ll"n dog,od.,ion prodIlc .. ~,S« Iron ~~ne IouUmi. virus (FeLV) erytbroid bypopl .. i. inducod by, 164 infoctioUl homolytic .".",i. mel, 184 ~LV, S« Felino leukemi. vi"" FE r:oti""'porc IT,394 Fibri""l!"noty.is, ina • ...,.], 298 FibrinolyUs,293-300 ....Y', 300 FDPS mel, 293-98 fibrin fugmon' D.dime,.oo, 299--300 in=...,.],297_98 pbpiologic p"""'...., 293 Fibrinolytic 'Y'trm, 293, 296, Fibrin or fibrinogen degr.oo:btion produru (FOP,), 293-98 :uulytic:d con«p", 2%-97 rntibody ,pecificity mel, 2%-97 cle:tr:oJ><:O, d.cn...d, 298 in=...,.], 297- 98, 298, intr'P«ti", roruid.mions, 297 INDEX 1._ . gglutirution immunoa ...ys for. 296 m< .... ring. 293 ump!'.293-96 uni'.297 Fight__jlight, 90 FN mults. Sn F:tIoe no... r~ml", Foamy Cl<=tic ID1U. d.=»es. hypoc.hmi. :ond. 609--10 G..,.,im distribution. 18. 19f G~iul 'nct hemonh>g •• 472 in8.rnm1tion.471 GFR. 5« Glomrrular filmoon n'" GGT. S" 1"Gluumyltnnsfrr.... Ghos, ceU. 137 Gibbs_Donn:on equilibrium. 405. 407f Globu~n rn:dytic:d princip!', for to ..l. 372-79 grnrr:d roncq>'" for tot:d. 370-72 pbysiologie pro=seo. 370- 71 toul. m..suring. 375 Glomuular filtntion. 417_ 18 conomtf1tionldilution of. 424-25 B83 Glom.rubr filtntion,.", (GFR). 417_ 18 po. :ond disordrn deau,ing. 618_ 19 G<-.,. hypergly=ni. :ond. 718 irnmuno".ctiv<. ro""",moon of. 730- 31 Glocororriroid ther:opy APP. positiv< conom'ntion incrr ... :and. 397 hypenlbumin<mi •• 390 hypotbyronmi. rnd. 792 Gluco .. .b.orprion ",n. in ho....., 755- 56. 756< roncrntf1tion. 708_23. 712' rn:dytic:d ooncq>'" for. 709- 13 pbysiologic P"""""" fOf. 708-9. 710f . /fusion rn:dysis with. 86i1--b1 hypergly=ni. :md. 713- 19 hJ'l'<'SIyc<mi. rnd. 719--23 i",u~n rnd. 722-23 immWXl",. etiw f1tio to. 729- 30 ren:d moo!. function :md. 422 in uri .... 462-64 rn:dytic:d roncq>'" of. 463 pbysiologic processe, of. 462-63 Gloco .. infwion. 620 GIOCOluri. dilorgUbtion 400 GIJ'C1",d bemoglobin. 723- 26 d.=as«l. 725- 26 incre~. 724-25. 724t Glyoogrnolysis. 720-21 Glycoouri •• disordrn. 463-64 GM , gongli"'ioo.il, 99 GM, g.ngli",ioo.i~ 99 GMD. Sn Glut:uJUtr debydrog.n ... Gold sat",. L\fT indu=l by. 23S Gr.." st:oining. effiuion ..,.Iyti, ...ith. 863 Grrnule~ toue. 92 test,. 8B4 INDEX Gnnulocym, S6 Grrnulopoios;" bon. murow rnd, 336--37 Griseofulvin. my.,[<»uppremv. effect. of, 236 Group , ... ting. 48_ 50 H,O dehydntion rnd, 500 ekrtrolyt< conan,mio", rnd, 498 hyp ,- N. ' Los. and, 502 pwe. 502 N.+.oo, 499, 502 reph"''''''n'' 502 "lifting of. 508 H,O H .... pr.apiunt m eJYlhrocytr, IiI f.lin<, II!] formation of, 186 h.molytic anemia, 186 Hmu' .. ' tlb&.t, 465 HenutOCli, (HClI, 120 conductivity, 131 d... nnin. tion of, 128 effusioru and, 851 _ 52 eJYlhrocytr d.", diJJ=nc~ and c:dcuhting, 129--30.129' HomatoLogy .,...lyl.e.. , ligbt...."." .. p attern! produe& by, 92 Hmu'oLogy....y>, 7-'" Hmu'opoi"," eydie. 80-S I, 86 rnnmedulhry,351 Hmu'opoi ..ic ""lb, 324--27 bo ... rrurro.... nud• • t«! ""U diff<,mo.] rount in healtb mel, 327 dmdritic ",lls.oo, 327 <'Y'brocyt< ~neog<, 325, 325' lympbocyt< li .....g., 326 m H_ .ynth"'i', porpbyru emS«! by pwu' 90- 92 bone nuno.., 342-57 clossific. tion of, 342--46, 343t g< ... r.l amer cbuact .. iring, 92 cWsifying, .... thods of, 352-57 cytochemic.J sum, 353, 353, immunophenotyping, 353- 56, 354, microoropic, 352-53 molecul:u ""d cyt"ll"netic studies for, 357 Wri&h' .uin, 352-53 tbrombocytOlis ""d, 24!-i5 H.moronom,ution, 193-96 hyp INDEX hemolytic, 174, 175f postp:orrun.n~ ina,de, IS9 H<m<>Iy.is,170 alloimmufl<, ISO-l! I bilirubin :ond, 68-t-S5, 6S5. .nvmoaution and, 192 ennnsruJar v, in'nV1K'UJar, 170.-71, 173. Mpllin.in 085 c.'+ sampl< :mtiC01guhtion .,jtlt, 611 _ 12 AT consumption .00, 290 eJYlhrocyte :agglutin1tion .00, 136 fibrinogen and, 285 hemolJ"is indu=! by, 191- 92 [MT inCl .... d'OO, 388 prot';n ')'Iltoois, t<>e<Jlut.r di ..... , 677 Hep>tocytes, bi~rubin "Puke by, 685-1!7, 685. Hep"ocytes, functioning, 680 H.","'uon am,"mnu"" leukocyte, 95- 97 H.","'"""n ",~is, leukocyte, 97 Hen\..bao.d testing for conle, 48-'50 abnorm.Jities in, 50 rn.Jyt~ "", .. in, 49-50 w.men' (HDOSn anin<, SI7_ IS feline,8lS Histiocytic neopt.,sio, 192 Histology, bon< m:orro"" 333-34 Histopathology, surgical, 8 Hi".p"""" ....psultst_ leukocyte, 97 Hiuclti rnalyzen, 52S_29 Hoto.nzyrnos,640 Honno .... , Sa altt> Ipmfr N"""~" c.", f..., coflC "" bile .cid iJ>Cl .... in, 693 blood.gro"P 'J"tem of, 119 HOi INDEX Hot... (=rinwd) roba/:unin in. 747- 50 ElAV.IH_-84 rJYlhrocytr :agglutination in, 136 g.. troint .. tinal ru.or<\thic hyper.. gmmt '. <1<6<;'00.. in, 403 foal .. rum . nd clung.. of. 402 FPT of, 396--99 "",.,wingl.. tim.ting. 399----402 bet""" toler.., .... "''' in, 7%-57 btu . gglutirution for fool .. rum in, 400 J.ulrogr>m p1t"rns in, 90 mJ>amtntion in. 757 USG, rd'nctom ,_>t, ,""-"" , How mw ," Hydrostatic pr~r<, 835 II-Hypni., 561 Hypercarbia, 561 Hyperchloremi., 522-23 di.orderslpath.ogoneses rnd, 522, 522' HCO,-, ., 808-10, 809t Hypem..oo, 166 Hyperferremia, 203, 203, Hyperferritinemi>., 207-.'1, 207, Hyperfibrinogenemi., 394-96 di.eues/oonditions awing, 394, 394, PP : F ratio, 39'5--96 IT, prolonged rnd, 285 Hyperglobu~nemi .. 392, 404 hyperprot., 731 Hypergly<J<mi., 713- 19 cl. .. ifica,ion, 713, 715' diili<"", meUi"" ."d, 713, 714, di.ordervcondirioru, 714-19, 715' pathologic,71'5--18 pbarmarologid,oxicol.ogic, 718-19 pbytio~c, 714_ 15 potrprrndi.J, 714 INDEX 887 ""roid""""....d, 714 u"""f'<'temi., disoro.r.lp1thogr""... cowing, 541 _-12 Hyper~p ... mia, lPS and, 667 Sa"". Hyper~pemia, n3-78 j, of, 501 - 3, 50lt H,O inuh.oo, 501 Hyperounob~ty, ooroo, gap and .. rum, 552-53 Hype'P'rathyroidiun frlint, refractomrtric USG.oo, 452 primuy, C. ".oo, 598 Hyperpbospb ....emi., 658 Hyperpbospb.temia, 618_ 19, 618t J"<000-, 619 Hyperplasia I=ophilic granulocytic, bo"" m:orro.. .oo, 337 bonr rrurrow, 334- 38, H5t g< ... ralizrd, 337- 38 rosinophilic gr:umlocytic, bo"" rrurrow ..,d, '" r J}'throid """mi., "'Il"nerativ< and, 152 bo ... murow, 334-36 granulocytic bosophilic, 337 bo ... murow and, 336-37 eminophilic, 337 lympb nnde, 362 mrgalmyocytic, bo ... murow and, 337 monocytic, bone m:urow and, 337 rr1<1iv<,362 Hyperprotrinrmia, 371, 379-85 di","";oonditions cousing, 379, 379, dy.protrinemu J>1ttem and, 382--1!3 hrmoronam'ration and, 379 hyp nroplasia, B_lymph<>ynW of, 83 N.+: K' ratio, docrrasrd.oo, 519 Hypoodrrnocroticism, rrfractometric USG and, 45 I Hypoalbuminemia, 391 - 92 albwnin syntbr.is, drar ... .oo, 383 ascit .. formation . nd, 505 di","";oonditions coming, 391, 391' hypoc:dcrmi., 602 in8. mm1tory, 391 p1tloog -'" hypoalbumi ... mic,602 11<1. tiorW,6iJ6-7 myopathies,610 nutritional, 607 1""«'" ';''' acu.. and, 606-9 J'1l'rurirnt,6iJ6-7 pbosphate e... mas ""d, 609 pregnoncy,6iJ6-7 prrputurirnt, &Y7 renal f.oilwr, .altr ..,d, 609 tumor ty.i, .88 INDEX Hypochlo«mi. (nnri~1mI) di.wes/patOOg<"""" :md, 524-25. 524, H,O ""cess group of. 525 hypo""".." .. and, 524_25 HypochoJ..tes and, 512-16, 513., 516, ,. 517. d.a.a=I .oul body ..,d, 516-19 inc ..""! ""cnnon and, 517- 19 myopathy rnd, in "''''. 519 ronal f.oiJw. :md, in eau, 519 Hypom>gn<><mi .. 604, 623--25, 624, hypoprot';nemi. and. 623 HypoJU,«mi .. 505- 9 dilutional,508 disordmlp. thogen<"'" ..,d, 505. 506. diuresil, prolo~.oo, 506-7 ECF. '507--.'1 H,O ""cess group. 507--11 hypoaldos'eronism. hyp<",,,ninomic and, 507 hypochlor..ru. and, 524-25 h'onun • ..,d, 507 N.· -7 Hypopu:othyroidism pseudo-,604 PTH .ctivity, :anemia caw«l ond, 409 di ......IconditioJU awing, JIl5, 386t FPT and, 389 Mmodilution . nd, 389- 90 byponug~mi. ond,623 pwm. lots . nd, 387-.'18 PLE, 387 PLN, JIlS-87 prot';n ""..bolism, increased mel, 388-89 prot';n syntberis, INDEX L-Ioct:lte production md, 54O-A I ">grunt, 584 byperl:ocutemi•• nd, HI ti<W,582--S3 Icterus hemolytic, 664-85, M5t in "'-', Idiopathic bypereosinophilic :yoorom<, 87 Iditol dehydrogerw>e (10), 6H Ig, Sn Immunoglobiru IMHA, Sn Immu ...·m«!i.ted bemolytic . ... mi. Immu ...·m«!i.t .W bbor1tory f'utum of, 179 Immu ...·m«!i.tpwu, 353--56, 3Ht lympborru, 364--<>5 089 Immunoructive PTH (iITH) ....Y', :oo:dytical concep" for, 626-27 mncentr1tion, 626-27 pwm., iDCl...&lao coU counte", 63-64 "'-tic principles, 63, 64f erythrocyte, 124-25 pt.reJ.t :oo:dy>is with, 230 IMT, Sn Immu ...·m.<Ji...d tluombocytopeni. InclusioJU, Sn.w DiibJ.'. indwion bodies, cytopbsmic erythrocyte, 138--42, 140t - .... b .",dituy di",rd." tbot b....." 99-100 mi"",UID<>COlc<mi. :and JJl1bbsorption, 741 po• • !>sorption in, 617 BOO INDEX In....m. (roMtinw.i) d.a,osed, 620 inc","""",619 [on diff.r=c<, 559- 91 uulytic:d conc0... rrurro..... 332- 33 <\dic;'ncy. :anemi. :md, 164-65. 16&-70. 169f J><'C<'" ... ns!'.rrin unu:otion and, 206 profile ,<suI... oompau.iv.. lOS, 209, .. rum, 202-5 malytical oo~ .. of. 202-3. 207_ 8 byp lsosth Isotonic fluid> d.hydr.otion and J.... of, '503---4 ..dem.:uxl n K'. s.. Pot"";um K.ppa 19rNm Kotamine, bypergly=ni:. and, 718 Koto:ocidosi.,465 Koto:unm..,7H_26 Kotog< .... is, Hi Kflonemi .. 5H---45 "'"- bodies, 543 con=>tr:oOon of. incn...d, 544-45 in uri.... 464....65 Kflonuri .. 464--65, 517 hypon>.trom" .00, 507 Kotosis, 465. 544-45 Kid""Y' albwnin ""a.cion by, 481 AMS and, 66S BHB nem;"" tbrough, 5H glomtr:ltion/diluOon and, m Hea,- Losses through. 532 K+ ",cr<..:ion and, 514--15 ine","""",517_ 18 LPS and, 667 po. d.",:..><,,,, by, 616-17 ure> ucr .. ion, disorde" incnrnng, 436 Loborotori.. in huaun bospiut., 36--37 Loborotory....yo, 7- 9, Saills. 'p«iP RiSal' og",."",nt KUJ'tobJ. d.gr .. of, 35 ...... ing, 32-34 bi.. plots and, 32, 32f D.ming mmp:uison of, 31 - 35 <x>ntrol ",mtiom and :assesling performan.., of, n en,436--37 0{,22-24 ",!.van"" of, 2}-24 d '0- b.p"tie,680 .. rum, 645----47, 646f, 647' fibrinolJ"is, 300 HCO,- , 528-29 bomatology, 7- 8 bopatic ffil:j'1D<, 680 immunologic, micro.tbuminwia, 481---.'12 iPTH, an:dytie>l oo""",!,'" fOr, 626-27 K+, 511 microscopy, 8 091 INDEX N.+, 500-501 Pi,617_ 18 pbtelet, IMT, 249 p~ctiv. v:;d"" of, 38---45 qu.Jjty ..... "''''''', 25-29 quantiutiv. urm. toul protein, 4:77- 78 nndom error, 37 rer. .. ne< inton':W, 16-20 ugnmcmt figwes in, 9-10 =mples of, lOt .. porting, 9 rules of, 9-10 mmt:u>ces intorfering ..ith, 24-25 'f'temic .. ror, 37 tCO',528-29 thyroid funcrion, 786-89, 787f UA, quantitotiv., 475-76 urea, -05 vit:unin D, 629 vWF,267 W.ng=[ rules for, 26-29 Lobor1toty methods rom]'1l'ing, 44--45 dilJ... ~in,29-31 .,.:oIu.ting, 44--45 ",>sons for, 36-37 implemtic inmflici.ncyldi ..... .00, 677--.'11, 678t, "" multiples test remit" 19_20 resulu rn:olytiC1l . in, 21, 22f cl .. tifi",tions of, 38 compmng, 31 diff.",nces in, 29- 31, JOf FN,38 IT, " mojo. m,,, n, " LoClote cJ..mic.t ltructurr, 539f conamtr1tion, 538--43 rn:olytiC1l ooncrp'" of, 540 phJ"iologic p"""'.... , 538, 539f [)'I""tote, 538---43 ilitorprion, 543 uulytiC1l roncrpu, 540 conamtr1tion, incr" ... d pl .. ma, 542--43 diilirtes meUitus ...d, 542-43 propylenr glycol ""d, 543 l-bctote, 538--43 uulytic::tl roncrp'", 540 conamtr1tion, inc", ... d pl .. ma, 540--42 ,lfurion rn:olJ"is with, 859-60 hyperbctotemio .00, ')41--i2, 54 It mr>suring, 540 pbsma, H0-41 Loctote dehydrogerw>e (1D), 653 octivity, incrr»rd orrum, 652t, 653 Loctotion hypom..."... mio.oo, 625 Loctooe toler.,,~ ttlt in h''''et, 756-57 Loctosuria, 517 lAD, Sn u..kocytr odhesion deficirncy u..r /low crU cytometer, 64-65, 64f rJYthrocyt., 125-27 pbtekt; :on:oIJ"is with, 230- 31 Lota >gglutirution imm=J", FDP, 2% lD, Sn lac"" clebydrogrn ... LDDST, S" lo....-do.e clenmetlu.onr mppression """'y, ~, LE, Sn lupus rryrherrutosus (lE) lre- White mrtbod, 279 lrft dtift bLood ..... trophil roncentntionr rnd, 70-71 cIDsmc1tion, blood neutropbil ronerntr1tioas ...d,70-71 clrgrnrntive, 71 nruuophili. rnd, 72, 73f r: F.li", lrukrmio virus ocu.., 346 :unbiguour li ....ll", 348 cminr,356 cluonic, 346 cluonic myrLoid, 76 drfinition, 346 ,lrrtron microooopy, 356 """inophilic, 88 myrLogenous, 326 myrLomonocyt:ic, duonic, 350 lrukrmoid r.. pont<, 74 lrulrocytr . dh .. ion de/icirncy (LAD), 100 892 INDEX L.u~, H _ lOl mnorm:ol morpbologic fe. ture> of, 92_ 100 >ggIutin.tionl"ll8feg1tion, 98-99 :uulytic:d principJ.. and m .bnornW concmtr:otion of, 70-90 umpl< for "",",,,,ring, 60-61 cmi"" din<JIlp<>' jnclusions in, 9'5 conam'<1t;oJlll, princip!.s of ~rnnining, ,'-'" bemocytometer method for, 62-63. 63f imp difr.renti.J, count, 62 microscopy. 67. 68. differen,i"ion, 55 disoro.r.. non""'pwtic, 100_ 102 .."',.... in uri"" 468 H,,,,,ouum """",a~~", in. 9'5--97 H,,,,,o,,,,,,, a~Ji in, '77 "","Iii...", His",!"""", in, 97 inclusions berediwy disorder> tbat b..... 99--100 mi"",Umeous con"i""" in, in, 97- 98 J.ukognm, 60, 62 MJ-98 9~95 u.ih",a~u, pby.iologic pr0cess<5 involving, 54---60 production, 55 SamKJfti, in. 98 'i..... , 56 T""",I"",,,, l!'~"jj in, 98 Trypa _ _ cut< in/lamm1tory. 84 morpbologic .""",. cion, 62 pat"""', 9{l-92. 9lt cat, 90 ca,d..90 dog, 90 bo,...90 , .... lts. ",btiv Levami",J.. immune b.molytic .... mi. indue .W t...v.y.Jenning, control dun. 27, 28f Light dwn identific. tion. n«>pl.,i • 3S4 Light dwn prot';nu,ia. 462 Lipase (iPS). &nrw P""ae. tic ~pa>< immunore""tMty :uulytic::tl oon«p" of. 666 prncr.. tic. 665-66 pby.iologic p~ 665--66 ,en.J ""aetion/in>ctiv:otion and, 667 .. rum. inc,......! ><1ivity of. 664t. 666-67 Lipids. 763-80 ...... m .oo. Li,", to Liver di ..... /inmf!iciency ..,.",i • 166-67 bbol1tory ,esults .00. .oo. tes, '" 6n~l. 67S,. Liver fWIl",.,efuctometric USG ""d. 451 Liver function. 675- 704 bilirubin oonomtution .00. -17, Sl6f feline,818 lPS. S" lip ... lAorbose intoxication. 190 lukes-Col~ru d...i/i",tion. 364 lupus :onticoagubnts. 292 fuels.oo. tes"'. INDEX lupus ~ho"",_ ... (LE). 94--9'5. Sn Ills. Sf>temic lupus erythem1ton" lympb. denitis. 363 lympb. denop1tby.358 lympb noo.... 58. 323--65 analysis. methods for. 3511-63 .. pir:ltion. 362 biopl)".358 m.jor fe. tures of. 359-62 colis in. of ho:dtby mom .... h. 362-63 clusification. 363--65 hyperpl .. i•• 362 lympbocyte< in. 59 ID1jor conc<J>",/tetrru. 358-63 noopwi •• 351. 363--65 re:octi"..363 umpJe.. cytologic. 359--62 lympboblm•. 363 lympbocytes. 56 blood. 58 processes infl.,."cing. 59 bone autro..... 332 conamtr1tions. mnorm:d. 81-35 effusioru md. 86'5 hem.topoietic cells rnd. 326 kin .pan. B93 lymphoprolifer:ltive disorders. 82- .83. 82f. 346 lymphorrbage. 835 effusioru md. 846-47 M. crocyte. 142 M. crocytosis. 142 M. gneorum (Mg"). 593--631 concentr:ltion. fr ... 625_26 .bnorm:d.625--26 rn:dytial oonc<J>'" in. 625 "",.. uring. 625 phJ"iologic process<> in. 625 concentr:ltion. total. 621 _25 .bnorm:d.625 rn:dytial ooncttoOon (MO-IQ ..,.",i. md in=..ro. 157- 58 Hgb tebtionsbip .. itb. 121 _22 incuaoed.157- 58 Mern ",U yomme (MCV). 131 Meon pbtelet componen' oonorntration {MPC}. m Meon pbtelet volume (MP\,). 228. 246 d<=..ro.247 increaoed.246-4 7 pbtelet :on:dyUs with. 231 _32 M. dofenunic :lcid. mJ"'losuppr ... i", effioct. of. 236 M~w"'.325 894 INDEX M~, 325. S«td", Dirbryocyt< immunof\""res<J<J>C< >Sl>J": Prorn.g.bJyocyt.. bo ... rrurro...., 330 M~;..i1,225 infKtioJU d.cuuiog, 236 M hyp ""d, 49--50 Methomoglobtin<mi., 19!!-99 ocquir.d, 19!!-99 di"llnosis. 199 f:unilial, in bolS<S, 199 Methomoglobinuri., 466 Methim1Z01<. ["'IT induced by, 239 Methionm., Hrin. body form"ion :and, 186 Meth.yIJbmninuri .. 480-83 uulytic:d oon",pts, 481--.'13 coni.,., 483 gen<'.J 00""'P'" 481J.-81 immunologic "'''Y''. 481--.'12 Microcyte, 143 Micro<:yt"'is, 143 ClUS<S of. 156-57 Microboma'oa;t, rrytltrocyt<. 124 Microorgrnisrru, culturing for, 863 Microscopy. 8 .krtron, J.uUmi. , 356 hemic nn~ diff...,nti.J, 67 1CCurxy of, 67. 6th lympborru.364 pLeunl.lperitonul fluid, 852-56 MNP. S« Mugi ..."", n Monocyt<>. 56 conomtmions. mnornuJ, 85--86 inflaaur..tion :and chaoges in. 94 kinetic., 59 pools. 59 Monocytop neopl .. tic. 8'5--96 , .. mid. 85 Morp/tin<. hyperglyc<mi • .oo. 7 19 MPC. Sa Morn pJ. .. let romponent oo~tr:otion M prot';",. 382 MPS. Sa Mucopolysoccbori<\o.i' MPV. Sn Mun pl1teJ.t volume Muoopolys:ocdl.1lidotis (MPS). 99_ 100 MJK"I....uriu",. J.eulrocyte. 98 MJK"p"''''''' 'pp .• inkctious b.molytic .... mi., 181 My.']iti~ 326 bo ... rrurro..... 341 MJ"'locym.326 MJ"'loclyspl .. tic oyndroID<5 (MOS.). 34!!-51 AMl ,., My.Jo6brosis bo ... aurro..... 341 chronic idiopathic. 351 MJ"'logenow J.ukomu. 326 chronic. 350 My.Joid colt.. 326 MJ"'lom., plasm. con. 351 MJ"'loneaooi,. thrombocytopJ>1tby hyp<><:>l",mi •• 610 hypohlmic. in 519 C,'''. N . +. St, Sodium N.,SOJ' Sa Sodium ",16.. N.·:K' mio. 519-20 N 1£'hth.t.n.. Hoin. body fOfm1tion . nd. 186 N 1tion.J I"'tim" of Sun<4rdt :and T ocbnology (N1Sl). II unit co"""ntio",. II. 12. N ocr<>tis bone aurro..... 341--42 ti ...... m ... i"". 513 NEFA conam .ntion. Sa No ...... ti6od fiotty . cid ooncontntiom N«>pwi. B_Iymph<> 895 INDEX bbontorylclinical probl..m ...ith, 385 pathog..,.,is, 3S 1--.'12 bon.,601 hi!tiocytic, 192,351 - 52 lPS and, 667 lympboid, 351, 363--65 lympbopro~ferativ<, 346 my lost of, 427, i28f Na' reabtorption in di"al, 499 proximal, HCO,- in, 526, 527f uri"" clung<" and, 425, 4261' Nephropatbies. Sn;rls~ Pro",in.to.ing di ...... and conditions cawing, 72, 72f in8aaurutory, 72-74 .atte, 72-74 , 73f chronic, 73f, 74- 75 ki ... ti"" 73f pbysiologic, 75, 76 ."'roid, 75 "'es>, 75 Neutropbils, S" Ciratbting neutrophil pool; Margiru"'" neutrophil pool blood, 57 abnormal concentration of, 70-81 Left .hili :and, 70-71 p=s>es in8.,."cing me .... r«l, 57 right dtift and, 71 - 72 "Is. giant, 92, 93 granulation, hereditary anomaly of, 99 hypeu"gmente6f rrurlOW, 57 pools, 56-58, 561' blood, 57 P.... do-P.lger.HoK't,IOI "ructural change> in, 92 ti.rue, 58 toxic, 92-93 Nicotinic :ocid, hyperglyc<mia and, 719 NIST, S« National Inttitu", of Sundard. and Technology Nitra'" poisoning, methemoglobinemia and, 198 Nitri"" in urine, 468 None>t Nonoeptic enl<:U"', S63--6i NormocbJoI<mi., 523 Normocytf5,I36 Normoblemi., 516 Normonatromi., 'W3--5 disorde" . nd p.thog< ...... of, 503---4 nRBCs, S« Nudeote an. ... ". INDEX o.moJ.~ty ((fm~'""",) plasm., N.+ and ngul.tion of. 499 .. rum, 547--48, 547. ...Jw';on of. 551. 55 If USG, 445--47. 446f O,mobrity. 545 Osmol<, 545 Osmom 545 f .....ing-point. H9 Osmom a,m",",545 o.motic pressu..,. 545 COP v., S-i6f OSPT. S" O ...... ug. prothrombin time O ..Jocyt<, 149 O ..Jocytooj.,14s Oub", •. 6 tnncity, 608 Osidmts, orythrocyte up<>5= to, 19l!-99 Osid.tivo ,"'-" Osim C, 571 - 72 blood rubstitut .. inwfering with, 572 Oxygen, 581--.'12. Sa "Is.. Anaw .mob, OXJ'E"n ",,,,ion gndit h<moglobin ..,ur."ion ...itk oxygen: PuIs. oxim r.ncn... 5., &<>crill< p.n'''.... Prnau,ic imuffic;'ncy. uocri"" rnd, 605--6 P:mcn1tic lira>< immunor~octivity (PU), 668 cots ""d. 668, 745--46 coJ>C P:m(n .. iti~ 741 Panhypoprotei ... mi.. 385 P:onneopwtic ..... trophilic kulrocytosi., 76 r ...protti""mi.. proLo~ IT.nd, 285 Poruiti,m, blood Joss aUS«! by. 167 P.. uitology. 8 r . utbyroid hormoJ>< (PTH). S" tW. lmmu""",,,,,ti,,, PTH: PTH.",bt prolong.d, 281t, 282 .borton.d, 282 P..,iog .00 Sililot. method oompariwn, Hf, 34 PCDW, S" Pbt immune Mmolytic ..,.",i. induc.d by, 17'9 IMT indu«<\ by, 239 P.mmt hemoglobin .. rumion with oxyg.n (SO,), %8-69, !>68f Peromt hemoglobin .. rumion with oxyg.n of arterial blood by pul.. oDmrtJy (Spo,), ~4 P..ant tnnsl'errin urumion, 206 Pericardial .ffusions, 866 p.riton PFK d.ficioncy, Su Pb"'phofructokin ... defici.ncy ,H bLood,559-91 :oruIytial oo~ .. in, 565-73 .. ronoow v.ru.. for, 573, 573, ph";ologic pro= ... of, 561-b5 .....ples for, 572-73 "'mp<mwr .00, 5]0, 570, urin<,455-57 Pb>goeytic .ytt<m, mononocl<1r, fi77 Pheruzopyridin<, Hein. body fonn1tion and, 186 Pbrnob.rbi,al thrnpy APP, positive oonc "7 INDEX ,.nn tubuk funetion :md, 422 dUh,617,619,6lO urinary e:nnnon, 61 !!-19 incr<:ased,619--20 Phosphofructoltin..< (PFfO <\<6c;'ncy, 189 Phosphorw (Pi), 593--631 m.Jytic:d coJ>CCC .W PIVKA. Sn Protrim induced byviumin K ...ugonism, .bsenor, or ddiciCC "" C." conc=tr..ion in, 594-9'5 compontn.tion in, 730-31 glocos< con=>tn.tion in, 708-23, 712t iJUulin, immunor.:octive, 726-30 iPTH, 627, 627t K+, reg»btion of, 510, 51H, 512f D.laet.te, 542- H LA:oc<:ote, 54O-A I Mg'·, 621 o>mOWity, . nd rrg»l .. ion of, 499 UN,477 uri"" ""nY" to, <1ti<>l, 476-77 PI ...... cytom., .. tnmedulluy, 351 PI ...... protein to 6brinogen (PP: F) ",tio, 395- 96 N.· PI ...kt componrnt coJ>C, 227 microoropic fut"'" of, 232-33 g. tr, 235 <,232 .il.<, 232 898 PI ...!... ("'n~·",,,J) 'plmic, 227 uuviv:ol. decr • ...d bl<><>mposition, 832-35. 833f .quin<, BH- 35, 834. microoropic nomination of, 852-56 routin< orWysU of, 848-56. 85 It Pleuritis, hypoprotrooutophiJ, 138 Polycytbemia, 193- 98 Polycytbemi. ""..,198,351 Polydipsia, 452. Sn """ Polyuria :ond polydipsi. Po]ym INDEX Posttnmi'umn I"'rpur>, 240 Pou•• ium (K"j. S« Na+K' ",tio olimenury I.... of. ~ 18-19 ....}".~1O- 11.511 concentmion. 509- 19 :orulyrical ooncep'" of, 510-11 ph}"iologic pro= .... ~09--10 term.runi", for. 5 I 0-11 aw "'--' hypolWemi. and, 516-19 totol body, ~16-19 depletion. ~08 .ffusion 'rW}"is with. 858- ~9 ucretion hypolWemi. and in=»«l. 517- 19 inc", ... d.51 7_ 19 hyperblemi. rnd. 511 _ 16 hypoblemia and. 516-19 incre»«l,513 totol body. ~ 1-l-15 winary ttoet obstructionllewg< and. 514_ 15 inul.e dea .... d.516-1 7 incrns1lBon :and. 42-i4. 43f interpr Propofol. H.in. body form1tion and. 186 INDEX 899 Propnnolol, bypergly=ni. mel, 718 Propy!.n. glycol Hein. body form .. ion . <>d, 186 D·uet.. e rnd, 543 Propylthiour:ocil immune homolytic mom.. inducod by, 179 IMT i<>doced by, 239 Prot: Cn. r1tio, S« Urinuy protein to c",.tini ... r1tio ",.Prote... inhibitor {a ,.PI}, feal, 753 Protein C octivity, decro...d, 291 coagul.tion inbibitodog.nous :anticoagulant, 291 Protein.losing dornutop .. by, 387 Protein.losing en.. rop .. by (PLE), 387 Protein.losing nopbropatlty (PlN ), 38'5--87 di"' .... rnd, 386 bbor1toty finding., m .jor of, 386-87 Proteins, 369--410, S« Acute pb..., protein: Hyporprot';nomi:o: H ypoproty«l '"'pon .. , 372 infl1JIUJl1tion :and, 380 dilOrom, 371 - 72 dy>i:o, 371 .!ectropboretic region contribution of .. rum, aw 3n,378t ent nh genor.! 00""'1'''' for tot:d, 370-72 hypd, 392 immunoet..:tropbOf";', 3n immunoglobim, 398--404 lOS! of PLE :and, 387 PlN:and, }s'5--87 from nscuur sp1O<, 385-S8 M,}S2 m.ubolism, liver function :and, 676 nophropathy . nd loss of, 460, 46lf pb)"iologic P"","""" 370- 71 pwm:o, 371 los. of, 387-S8 ren.! f.oilw. :and loIS of, 460, 46lf ren.! ruoot. function :and, 422 Si • • uglobulin ten, 3n- 79 'yntlt",i. d, 380--85 inc", ... d,380--85 tot:d, 372-74 biuret ro""rion for "",.. uring, 374 qu=tiuti"" urine, ....Y', 4n- 78 «fnctometty fOf mo .. uring, 372-74 tnn.oo. "" . <>d, 642-« in urine, 457-62 rn:d)"iul 00""'1'''' of, 458 ....Y', 4n- 78 miaoaloominuri.:and,482--83 proteinuri. rnd, 458-60, 459f Proteins indu",d by vit:unin K . nugonilm, . bs.mO<, or de/icimcy (PIVKA), 287--1!8 Proteinuri., 4 58-60, 459f Be""" Jo .... , 385, 460-62 cJa..ific1tions, 460 glomoruhr, 458---59 homonb"llic, 45~ infl:omm ..ory, 45~ light.cJu.in,462 p",nn.!,4SS tubuUr,459 Protein Z, .. endogenow :antiooagulant, 291 - 92 Protein Z-dependent pro..... inhibitor (PZI), 291 - 92 Protluombin ti"", (PT), 282-64, S« Thrombotest PT INR:and,284 prolongod, 283, 283t .bortenod,283 ...m:uin tl..rapy monitor aw 900 INDEX l'TH. &. P:u:othyroid bormoD< PTH·,.I1ted protein (PTHrp), 628 PTHrp. S" PTI-I_rebted pro.tin PTT. S" P:uti:d duombopwtin tim< P""C 00" hom1to]ogy 'f""" r J}'throcytr,l27 pbt<J.t :orWJ"is with, 231 RA. Sn R.fnctory onemia 1Udi:d immunodiffusion (RID), 399-----100 RAEJ!..1. Sn R.&a.:.ory ..,.",i • ...ith ""cess blos" I RA£Jl..2. Sn Rofnctory """mi. with """'" bl .. ts 2 IUpl"pon cw,i/ico';on. 364 RARS. 5« RdrXlory with ringed .i< in uri"" and, 463 hem. in wi ... and, W nitrito in wine and, 46-1! protrin in uri"", 45B USG .. tinution ond, 447 REAL 'l"tom. S« ~isod European! Arnericon Lympbo .... 'l""'m """mi. Receiver ope ..ting clu.. ctoristic (ROC) CIlrv.., "-Mi ....y oomparison, 45, 46f d.cision thresbold .... blisMd with, 4';-46, ." di>gnootic procedu« comparison by, 45--46 FP ..", in, 45 TP .. to in, 45 Red blood all (RHq, 120 apl";., 339 homocytome"'r method, 128 Red all distribution widtb (ROW), 122 R.fer..,,,,, di.tribution, 16 dot .. mining, 18 G • ....;on, 18, 19f .Uw.d, 18, 19f Refer..,,,,, individll.t, 16 .. t..:ring cri",ri. for, 17 R.ferenco intorvili, 16-20 ddinition of, 16 dot .. mining, 18 est:oblislunontof,17_ 18 Hp«",d finding. ond, 20 G • ....;on distributions :and, I B, 19f Malthy, 19 herd_ba..d "",ing, 50 bbor>tory mul", ond, 31 mo ..und v:ol.,.. ou",id. ai, 19 multipt. to" ",""l~ 19_20 .Uw.d distributions and, 18, 19f .ynonymous ",rms, 16 u .. of, 19-20 R.ferenco limi"', 16 d.cision thresbolds v., 20 dot .. mining, 18 Refer..,,,,, popuhtion, 16 R.fer..,,,, ron&<, t ...m, 16-17 R.ferenco umpt. group, 16 ost:oblisbing, 17_ 18 Refer..,,,,, val ... , 16 infornution on, 20 mo .. winglde"'rmining, 18 R.fnctil • ..,i6.cu, .rytluocyt<, 141-42 R.fnctom""y prot';ns, total mo1SUr«l with, 372- 74 USG, 443-45, 444f, H5f Refnctory anemia (RA), 349 Refnctory .... mi. with .. =s bl ..", I (RAER-I), ", ", ", R.fnctory :anemi. with ........ bl ..", 2 (RAER-2), R.fnctory :anemi. with ringed .iderobw", (RARS), INDEX ." Rd'nctory cytopcu... 427_29 1ZO«aW. and. 428 b~oemi . and. 609 urine voIum< and. 429 bovine, 524 ct+.oo. 601 chronic, 425- 27 c..". h.., concentr:otion.oo. 613 md.enc< of. 427 polyuri. in. 427 "",..l con",n't1tiag ""i~ty :md. 427 in horses. 60 I hypoblemic. in 519 N.+: K' t1tio, d.e<:r.....,j 520 pro,';n_loting, 460. 46lf R.n..l function f1ilur.Jinsufficiency. cbronic of. 425- 27 imp1ir.d. 425--26 pwm • ..wyt<. 416, 417' Rrn..l insufficiency .00, 60 I chronic. 425- 27 md.enc< of. 427 polyuri. in. 427 Rrn..l in' .... titrum. 424 R.n..l lou. bypon.tremi • .oo, 505- 7 R.n..l pl .. m. 80w (RPF). 423 Rrn..l ... but.. C.'+ .00, 596 functiont of. 418 :unino . cid • .00. 422 c.." rnd. 4ll Cr.oo.419 Cn . nd. 423 glomerular filtr:o« oonc <1". cr· .ad. HCO,- and. 419 K+.oo.419_21 m. jo• ..,Iuter rnd. 418-23. 420f Mg'· .00. 422 N. · rnd. 418-19 PO• .oo.4ll proteins 4ll u... .oo.423 gloco,ruri. rnd. 463 Rrticulocyte oo~tr:otion (RQ. 123 erythrocytr .,wyUs with. 131 Rrticulocyte prr""n'agr (RP). Sa tdi. CollKt.d rniruJocyt< prrcrnt::lg< rutoauted ,rdrniques for. 132 rrythrocytr ..w)"is with. 132-34 procggrrg .... 117 <::1,.11 7 cil<'llt.ting, ~f< '1' .... of. 115 erythrocytr :oppunnce and. 138 puner .... 117 dtih. 134 types. ]]5--17 R.ticulocytosis. 117, 138 1I>,ion and. 151 production of. 'f'<'Cies varution in. 152 RP. incrr= .ad. ru_ RomrnOWlllry . t::Ii.... 61 Roule ... lroule.ux. 136 RP. Sa Rnirulocytr prroenugr RPF. S" Rrrul pl .. m. 8""" RPL Sa Rrticulocytr production inda Rubricytoois :opproprio«. 137 r rythrocytr. 136-37 i01ppropru«. 137 Rumen overlo:od. 610 Ruminan... H';n. body forrruotion in. 186 902 INDEX Rw .. U', vip" vmom tim. (RVVI), 288 RVVT. Sn Ruu.U', vip<>' vonom.im. S:dintion, r = , 518_ 19 SampJ.s, 5- 7 ammonium ooncmtr:otion in plauru, 699 bLood, 5--6 rnti=guhntl for, 6 Leukocyte ..mu.tion ...irk, 60-61 bLood 11"'. 572-73 bLood pH, 572-73 bLood pl .. 6-7 body fluid, 7 bo"" rrurro..... 357- SS c..". coDC m., f,.., ooJlectinglhandJing,2]ol_76 p=ing,...biJity of. 277- 78 collecting. 21 r n,¥JD<, > FDr., 293-96 fibrin fngment [}.dimer, 299 handJing,21 Jabomory .. >min. cion of, 5 Jabomory , ..ul", ""d, 31 ...ron in, 21 L.vy.Jrnnings oontro] char. for monitoring, 27. '"' lymph nod. cytologic. 359--62 Pi, 617 ,.r...nce int..v:tls ..,d, 18 uri"",7 vit:unin D, 629 vWF, 266 SamKJiris, J.ukocytr, 98 SorOOIJU, histiocytic, 352 Schirocytr, 149--50 Seron ALP. incr.....d :octivity of, 65'5--58, 657. drug/holmo"" induction of, 656--57 ALT. increased octivity of. 651. 651 t mwcL. d :urt:lg< and, 651 AMS. incr.,...d . ctivity of, 664-65. 664. rnioo g1£>. 535 AST. incr • .....! .J>amtr1tion. inc",.....! in. 691 - 93. 692f.692' ci' i'xto" d.t .. mining. 595- 96. 59'5f IOu] ronconmtion. 59-l-95 ",tion. 535. 535, cltol"n .. ol roncenttotion in. 767- 71 CK. incr""'! octivity of. 662-63. 662, . k GMD.654 hyp [D. incre.O«l . ctivity of. 654 immunor..cOv.. 726-30 iPTH. 627. 627' P. 509_ 10. ~09f LD. incn...d 1Ctivityof. 652t. 653 low. 50 lPS. incr • ...d octivity of. 664t. 6li6--67 Mg'+. 621 i'xto" d.t ..mining. 621 _22 <>Imot..~ty. 547--48. 547' ....twtion of. 551. 55lf Pi. f.o"o" d.t ..mining. 616-17. 616f TG roncrnttotion of. nl- 72 Serum pro,';n dectropbomj, (SPE) g. mmop.thy. monoclorW. 383 pro,';n fractions detrnninr INDEX non..sl units. 10-12. lIt SI unit converuon of. 11 _ 12. 13t Six Sigma Q..wity M'll1gemtion :and. 499 conomt ... ions. 498-509 m:dytiC1l oo~t> of, 500-501 hypon. ts.mi. md. 505- 9 K' :and. 499 ph}"iologic proc=><s. 498--500 ",mu/ unit> fo" 500 tion md. 499 .lJwion m . l}"is with. 858-59 e = group, 502-3 H,O :and. 499 hYl"" ... oemi. md. 501 - 3 ion activity. 500 ,- H,O Jo.. . nd. 502 ,,,,,,.ring :and cut:meow. 507 third... p'''''' 507 nephron ",.bsorption of. 499 pwm. osmoWity regulation md. 499 qumtiution ... mpLe fol. 500 lenoJ tubule function :and. 418-19 mining. 501! ..... ting 111 CUW>eOUS Jo.. of. 507 Sod.iwn mUi", (N. ,SO,). 401 _2 Solu",. 5% Spectrin delici.ncy in Dutch golden reo~. 201 Spherocytes. 143. 150 ',,-, a>ntnction, erythrocytosis .:w....! by. 196 erythrocyt ... 111 _ 12 pl>",Lets in. 2IJ Spo:.. S« P.. cent hemogLobin ..",... ion .. ith ozygen of lIterioJ hLood by pulse o:xUnftty SSA. Sn Sulfuulicylic ocid Suining 1ZUrophi~c. 62 I=ophilic, 62 bLood. 61--62 """inophilic. 62 neutrophilic, 62 Surling" bw. 405, 406f aviwy .ffiuio... 1I1d. 836-37 Stem ",lis, 55 Steroids. hypeoglJ=mi' 1I1d. 714. 718 St....,t·, "",thod of ocid_b.se ' M}"is, 584-91 delinitioru.585-116 Stooutocyt<. 150 Stooutocytosi.. heredituy. 199--200 Strong ion dilfe",,,,,, (SID). ns mion g"P .nd. 589 equ.rions. 586-88 fonnul ... comparison. 588-S9 St....m·, mrtbod of .cid_"",,, . nalysis. 584_91 th.oty,591 yoJ ..... inte'P", ..tion of . hnornW. 589-90. 590t Sulf.oquinouli .... vitamin K 1I1tagonism . nd. 306 Sulfonamid ... L\fT induced hy. 23S Sulfosalicylic:ocid (SSA), 458 S..",ting equine. 507 hor... K' 10"' through. 519 K' Jo.. through. 519 N.· rut:meow lou through, 507 SynBiotics SCA2000 V_rin.". Coagulation AmJ)'.".r. 280 Syndrome of iJl1Wropri. te ADH SKUtion (SIADH). 507-.'1 SyU<mic luI"" .rytbenutoo", (S LE). 240 T.chypnea, 561 T :unm_HonfoJl protrin. 457 TAP. S« Trypsinogen ocriv:otion peptide TAT. S" lluombin-antiduombin oompl<=> tCO'. S« T otoJ • .mon dioxide TeT. Sn lluombin clotting tim< TF. S« r ... .,. f.oetOI TFPL Sn Ti.",. foctol p .. hc.y coagul.rion inhibitol TG. S« Tligly=ide T gAA. S" Thyroglobu~n mto1l1tibo 904 Tbrom~. INDEX 262-65 Tbrombocyth<mu .... nti.J. 351 primary, 244--45 Tbrombocytopeni., 233--43. S" ids. [mmun<_ """'-i. tthic. 241 _43 inf'K1ions and, 231'>-37. 242 irndi ..ion.oo,237 marrow "'ploc"mont and, 237 mn>t:ol alIoimmun<, 239 neopl .... md, 239-AO n«>pl .."",.oo, 242---43 pbt<J.t production, dKRn«! and. 23~37 drug. causing, 23'>-36 r"''''J.ts ..,d dinribution. obnornW of, 235 mrvinJ, <>d, 24~45 ">CIi¥<,245 pl ...J.t • .oo,24S---46 Tbrombogr>m, 60 p"'t<J.t,227_29 Tbrombopathi., 263. 264, dotting and, 264....65 Tbrombopo;"is,225 inf.ctioJU d.cr.:uing. 236 Thrombopoi dinicalsig .... 311 ddinition. 310 h<mo.utic . boorm.tities. 311 Thrombotest PT. 21>8 Thyroglobulin . utoantibodi.s ([gAA). 788-89. "'-" Thyroid function. 78}-800 :uulytic::d oon«p". 78S--89 ....Y'. 766-119. 787f hormorW rontrol of thyroid gl:onds and. 78485. 785f phy>iologie p~ 784-85. 785f r..po .... test,. 795--98 Ulppr~.. ion ""''''. 795--98 TgAA and. 794-95 thyroxin< and. 789- 93. 798 triiodothyronine a>J>omtr .. ions ..,d. 793--94 TSH con=>tr:otion and. 794. 798 Thyroid hormoD< oon=>tr:otio .... 799. 7991:. 800t Thyroiditis. mnctometrie USG and. 452 Thyroid p"roxid ... . utoantibodi.s. 795 Thyroid ... timubting hOfmOD< (ISH). 788 in e .... 794 conamtr .. ion. 794 in dog •• 794 in ho,"",. 794 r ..po .... test. 7~-96 thyroxin< and. 798 Thyrotropin...lming borrnon< (fRH). 796 Thyroxine ruto:ontibodios. a>J>omtr .. ion. 789-93 di.b."", .... Uin.. induc.d by. 717 f",~ . 793 hypergly=ni. :ond. 718 hyperthyronmi .. 789-90. 789t hypothyrournia. 790-93. 79h TSH ..,d. 798 TIBC, Sn T oul iron_binding capacity Ti...,., foetor (IF) roogubtion tert. 282-83 PT :ond. 282-83 ltrm< foetor pathwoy ro. gubtion inhibitor (IFP!). rn T"'- m lymphocytes in. 59 lymphoid. ~ DNtrophils. 58 Tn Sn T rypsjn_Iike immuDOr ..etivity T-lymphocytes. 55 TNCC. SH T oul nud..t.d ""J[ ronamtntion TN remltt. SH True n ." INDEX T ""icity, H6 Torocyte, 150-51 Total arbon dioxido (tCd) analytical ooJ>CCC1"" S« T .... positiv< r<sulu T r:uuferrin, 372 T r:uuuC K+ mention indued by, 5 15 myelosuppressive effern of, 236 T rimetboprim-sulf:unetbonzole hypotbyronmia rnd, 792 immu,,", hemolytic rnemu induced by, 180 T rimetboprim-wlforumide, mye""'uppressive effern of, 236 T .... negative (IN) remlu, 38 T .... positiv< (TP) r.ntl"" 38 ROC cwv< :md me of, 45 T .... v:.Jues, 24 T'1"",,""""" leukocyte, 98 T'1"",,""'''''' 'PP" infe.ctious hemolytic :memi., cnm: '"'-" T rypsjn_Iik< immunor..ctivity ( HI ) analytic cort"'ptl, 741 - 43 coJ>Ciologic P""'"'""', 741, 742f T rypsj""ll"n activ:.tion peptide (TAP), 746-47 TSH, S« Thyroid... timulating hormone IT, S« Thrombin time Tumor ly>is syndrome, . rute, 610 UA. S« Uri,UfU.ly>is UIBC. S« Unbouod iron_binding capacity UN. S« Ure. nitrog--6 Uniu, 10_ 16 abbreviations for ....jar, 12t unount~ ooncen~tion, 12-16 buic, of "",.. mem<s", 10, II t calruJ.tiom ond r.. ultontsignifiClflt figur .. , " me .... red v:alu<s, II metric 'Y'tem of, 10 NIST, conviologic pr""""",/coJ>C 906 INDEX Ur .. nitrogrn (UN ) ( d.a,osed, 435- 36. 435. inc","""",435 urin<,4n Ur..ruo. 4:29- 33 Urinmal";' (VA) in cau. 442., 455 in cattl.. 442, compo ... nu of. 441)_,11 in dog., 442•. 454- 55 in 00,.... H2. nujor co""",,, in. 440-41 proc , .... lts di",rde,slronditioJU ausing, 456, in"'rpr .. ing. 454--55 Urinary b1:od'om, 415-90 1ZOt<mi. rnd. 429- 33 .. rum ch.mistJy , .... lts in. ,bRO.rrW routi .... ,,'-"'> Crt ..rumlpJ .. m. <x>nc 416-25 ronal ooncm r-27 renn i..... llici.ncy, cbronic, 42'>-27 renn rubuJ. functions in, 418_23, 420f UA.oo, H41--41 qerumlphuru rona M u«mi. :md, 4:29-33 uru.. in, pbY'ical onmirution of, H I_52 uru.. ..dimont ",,,,min.tion rnd, %9- 75, 470t urolith rnnY'is, 487- 90 Urinory trxI epitb.lin ""Us in, 473- 74 in8. mm .. ion, urinary ..dimont "".."i ... tion :md,471 K', incr.... d and omtructionll..k"ll" from, 514-15 omtruction c.", h ... roncentrlltion and, 613 byp<><:ak<mi • .00, 609 N.+:K' ",tio, <J.cn...<J :md, 5241 Uru.. 24 h ""cmion .rudy of, 476 FE study v" 484 :malyte, to phuru ",tios, 476-77 ....Y', quantiutiv<: .tbu.min, 4:78 totn p.-in, 477- 78 bilirubin in, 466-67 C..'+ cona INDEX ." umpJ..,7 ",Iut< con""ntmion, 4H- 52 rnalytical ro~'" in, 443---47 phytiologic p~ for, 443 UN,477 urobilinogen in, 467-..6S USG:and,443---45 ..- . bnornul, 427, 428f ",nat bilu"" >cut< ond, 429 USG, rm.ctometric rnd, 454 Uri"" >ediment rnalysi,,8 "'cteri. in, 472 cost. in, 472-7 3 aystoh in, 474- 75 epith.lial in, 473-74 etythrocyt~ in, 47 1- 72 .nmin1tion, 469--75, 4 70, ""u. g..,.rn oo~"" ""9 J.ukocyte. in, 469- 71 orgrniuno in, 475 Uri"" >p«:ific gnvity (USG) ounoWity, 445---47, 446f re"il"nt "rip for e"im1ting, 447 r.fuctom in ho.... , 450 bypoudrmooonia,m :and, 450--51 byporal.do"tOP1"'thyroidiun rnd, 452 int<rpr .. ing val"", of, 447- 52, 449' liv ina....d,468 Urolith~ 487--88 rnalysit,487_9O con«p" in, 488-89 calcium " ..Lot<, 489 calcium phosph ... , 489 ani"",488 eytti"",49O fonn1tion facto" for, 489--90 pothog."..is of, 489 inorgrnic compotiaon of, 488, 488t .i~C1, 490 "JUvite, 489 ume, 490 unth;"., 490 Urope>'itoneum, 847 USG, S« Ur;'" .pecific gnvity V"",w.r di.tu.<, BMBT rnd, 264 V"",ulitit hypoprotes in, 628-29 endogenow, "'Ul"'" of, 600-601 nogenow, """"'" of, 600 fonn1tion, 628_29 hyporviumi""";" neopwm.... oc .. «d, 601 hypovit:uninooi., 604 rid"", rnd, 604--5 umple,629 Viumin K rntogonittt, 305-6 bt...ding dilOrden rnd rnugonism or deficiency of, 305--8, 307, deficiency, 306 hemonh>ge rnd, 306 hemotu,it rnd, :abnormal , .. ul", in, ,.m ,,'-' Vomiang, K· lots through,S 18-19 Von WiUebrrnd disen. (vWD), 265-66 ""quire 908 INDEX Von Wilkbrrnd di ..... (vWD) pbyUo~cp~, (RI~riud) 265 ."..ui6 Von Wilkb,rnd bctor (vWF). 265--68 uulytic:d con""p". 266-67 ....)" ..,d, 267 umpl< :md, 266 units rnd, 266-67 rntigen utio to, 267--68 d.a.osed, 267--68, 267' in"''Protiv. co.wde",ions fo, 268 g""',;c tests for d.t Whit< ",U im~c< count (WIQ. 65 Whit< ",U optic:d count (WOO, 65 Who!. blood mun' (WSO, 62 nRBC con<'Ction of, 68-69 WlC. Sa Whit. ",U imp..d:tne< count Wintrob.', erythr<> X_ny diffnction, 489 Xybzi .... hyp ns, W.nuin the ..py. PT monitoring of. 283 WBC. Sn Who\< blood count W,,,vro rule., 26-29 Zinc rulr..", (ZnSOJ, 400-401 ZnSO,. s.. Zinc rulr..", fib.._. . J'I.. I. """"'~ ofleuloocyw of m , ; ...... (d hIoo.I do Wrich' aiR) (' ~. bo.;" L Of>Pi A. T ..... "-I ..........p.iI ...'" 100..1 q>OPloo_ , ~ .. _ D6IaI. t.odi.., " ' -. B. Tooic .......,.t.a. ~ c. T.u: p ... ""'~ ,.;,., .oo..bI •• ",In. tnt! !Ollie boad """...p,a. .... D. Hyp<_"".........,w.""" 1!. !l<"';.. I rl! I". doc- _~, Go _tift ~ , h.-. H. ~ pl."""""" 1, ................. I. A. ""PIucr. .... "'do ........... ~ 1.. NNlmpIUI_ """'" boo m.l ... iW. at. • r .... 2. i'hooooU.o"'V'P/U of kukocyt< .boo.m.!"" (oIJ w,;p, ...u..d blood Iilmo wd. .. 0<1. ....... nat A. Mond. of ~ ....'.".. in ......ropIill. .J.oc. B. Mond" of ~"'" ,~. .. .. . """,.",bit, h.on, (from ASVCP .lid. «>0","""001 ..,. ).W. H.."".. I~J). C . MonJ. of EJ,,{KI,;,, ",.k in. ""'"'" Irmpl.oqt<, P...6boo RlpiO"n"bot o I!1J). E. G.....rocyt~, ~, . • .. ., :•... ,. •• ~ - ...... ~ • '\ • • rw. J. -..,;,~ of ,.,.u..ocy« ab..oaaal;,;', (oIJ w,;p-"';...d blood 01 ... wd. .. 0<1. ....... _«I) (5 ~m b.. ;" L ,!,pi;", to ,ad. fr ...,). A. RDu/,,,,,,, bon<. B. A.j:p.ti_ioo, - .f doc. C. Rubriqt<>.. (_orubricyt, ."" niliri rocyt<> of F, d.&;""J'. doc. F. ~, ,m.ruIocyt< •• "'~ ..rtI.yI A G ':• r .... ... -..,;,~ ... of ,.,.o""J« abooan.!;,;', (oIJ w,;p, ...u..d bIood!ilmo wd. .. 0<1........, nat<>triIutcd by D.C. B 19SIJ. C. M""""' ........,...i;, _ ,.R. do",".,d &om .- • cyt< •• coov (fromASVCr dOl. INJ). D. MY' I ( foIis, <8". f. a>Dtributod by E.G. Wdlo . .. d., ""'-""",.J..c. Eo M"",,,_ 1-_ n.ns,,;. ••!foli, ""', G. BaoopIillk H . Hdm W ... at. I . H,;", plum....., doc. m ....,....., bl .. nt>.I ....... <• • K. HowdI-J.IIy bodl ... ~. -W/io, of bod". _ J. H""""""'" ,.,.<>L ""', .Joe. L. HowdI.JoIly body, n. ~ ........ • o rw. s. -..,;,~ of ,.,.u..ocy« ab..oaaal;,;', (oIJ w,;p-"';...d blood 01 ... wd. .. 0<1. ....... _«I) (5 ~m b.. ;" L 'l'PI;., to ,ad. fr ...,). A. R <~~"' mKoocyt<> of • ~ ....... doc. F. P<>iUocyt" ;ad..dH.c on oIooptul """ (I..¥. ""IP~ ...d • cdI wid. . brood..,r-!oc< ("1,.. ...... >1. cot. G. ~", ""kai< h<~ .J.oc. H. c...Iocyo .. ,.J.oc. I. IIa<'T'" Cf<" .J.oc. J. ArtJ."oal do.'T""J"<>, doc. K. E !.on" L. Ed.o.ocy. ... Joe. • •• •• • r .... 6. -..,;,~ ... of ,.,.o""J« abooan.!;,;', (oIJ w,;p, ...u..d bIood!ilmo wd. .. 0<1........, nat ""c. II. l'<>Idod, kypochromi. mi"")'tic kptocyt< of F, JoG""""• .J.oc. C. Ov.locyt .. doc. D. Pioa=J edt.,.Joe. E. i')boqt< (4 o'd.od.). h-. F. Pybocyt", .... mrtJ.yI"" bI"" n..l ......, b.",. G. SdU-rt<>. <1<>,. H. Sdmocyt<. (ut"'''')' Difi;Qo* do,. I. Spl.oocym. ................!O.od ~ ......;,. .w.. Ik"",," .... 1992~ L. T..ocyt<. (utilia), Joe. rw. 7. I'hotomiaoppbo of ~ . in • •.tin. d~........, ' ... ....! platdrt . boo....Irt.,. in Wricb<.naID fu.b cit. .ed blood. doc- G. PLodot dmaty ....... do . .... m..! ?atdrt """"~, ...;.", doc- H . PLodot dump in iC.kn ....... i [,:pI ,j ,i f ~~1'<~q·l'lh·~ oo • '. . . 1. ir~t'[d[ l rJLi' r'te. !" ~r~, i' Ht,H''-j lrrr 9 1 ~!!~ l ··l~·,r, ,· Q t mltWt ~ ~ f iHHht b 1'" La s~"}'lH<' 8.. ro' U lL ~ ~"' g l L. ~ ; .~ d i h·~. f' '~t "H~"l a.~ . I"1~-.a..i iii i f'·i.< ~L hi H i I"I"!"-·. . .J'~1~~ . :::. . I . J 1 tmt l'l" ~ ~ . "-!! ~ P'l ~ 0. ~ i ~ff!'- _f;:~ f-: it~ ' ~' ~ ~ a. HI [im~~ ~ r ~ ; .t ~ p.i Hhmr~1 ~~~Hm!f II 0- ..".., J . 1", ~ ..., [ -~o- I rw. 9. ~boof cdl....d od.... ~ o..J. me' '" .......... oarnP" .....;.. « . ... "" _ _ ;, .,.<Wid«l (oIJ W.;P.-;...d lilio. of .............;..... unI.........;.,_cd) (ouk b.u .. 0 .ppI'" to oIJ fo ... " <= A. ~~.~. a M . ~, ~~~,~ •...Jb .."'..,"" ""~' ~ c. ~ '-...d' of "",1..«<\ trythroid .tri... doc. D. G .....locytK .tri.. from ,-, myd.oWam to """,ophd • .J.oc. Eo Hyptrcd.loI .. ........ ~ ..;th duldy ttaID ._«1 ~;." ... m ..;th """"""'" roIdtn ~ .J.oc. F. Mano.. fro_ ~ d.""....IIot ....<>p<>itric ,,!Io1 .. .,.. .J.oc. G. M. aopluop ...b "'~o.d n.baMo" ~.q...kd ct!I (/,ftl ~ po/yd..omotcphili< rubtKyt, (~,,~ __ iattd ...... u..m"" .. modiat«l """""""" ;.. ""...... doop. H. MydoIib.oo .... th. bw.dI .. of sbrotyt" ond <0«. h .... "...pm ond ....... Wn. .J.oc. I. Mo ~".ur.o.ioo of . .....~ ...d tbric rod.~ 1.1""",,1am. ~. J. U.,.j;f. fntn!;.-«I ...., ct!Io of " '," Jrukt ..... .J.oc. K. o,.opIut" ~~ edI. at. L DyopI •• " ,.,.du-oid cdlo (/,fi ~ ~,..,.,. at. M. O,. ...... tK ""p cell."" ......- ....p."" PI.<. II. """' .....".v~ of cdl. .... """" mK",","",", fu.d. U>c' .. 11"'1'10 ""'" "'I'int .. <>< «."'" fo. ' - ;. p....;.d.d lall W """run 1= <>< imprinoo woI_ odo.nri", . . «<11 «llilmo of I"",, bac .. N "I'pIi.. to oIJ fo ..... <=1" 0). A. P~1 •...!! .... ..,O" ... «!;'" lymp/>ocr<.. ap«to"O"i~ IymrJ-lm';'. <8-. F. HM.- """"'1''' JIm_ "","",," __ "''''''''''.", .. _ io • "'"'"",,"' v ...d a<nodJulady•• ;pof H),,,,,!.z, ... fuaco.llym plud J"'-. Buddinr; )" ... of ~ *"-t; •.'" .... ...", ."".... phi!. ;. diIIirmI o.aJ pi"". £Uncal Iympl.odmrt;" ""c. H. UM __ 'P...... . ic<><" ... """"'I'1Ulo .... ",,,,,,,,,-•• ~ m .........tta ...1. rod.~ .......1'1."•• .,-....... Iympb..l.ni<;,. doc. I. 10,,,. m,","", .... 1uJ:' ompI . ... lympioocr< ... Iympl.oou. ""c. J. CdI do.h of """"' . ... lymp!.ocr«. I d of", lbmp,-. Iympbo.. ~ dot;. K. SouII to DrojJI_i< lymphocyt ... lympl.oou. dot;. L SmolJ Cd........ doc- M . M,,_," ....., cdl •• "",u.."IUlo..... 11"'I'io.ocr< ... dot;. N. NmpI. ... myd.o;d ,.ti •• G. W'" ... , _Koid """mcdi.tc ... ......,;, mydcid ...... .,.;. I~ oucom. ). ""c. O. Soc",,,,], 'I';thd;o.l d . of ......hboJ .. aLTa<)' pod .... .. iod....... , of ,.,..«>qt" ;. th,......o.l .oIi.uy 11.01. •.&.-..y " .... "P"'" I"","",«l lrnopb ""'" "'I'int,). dot;. - , ..... 1 2 3 4 - ,,~ rw. ll. ~ of win< (..t) ...d photomK~ of """" ~ 6odinco (B-L). s.J;""", .... ~ =c .1Up.J.y .l.jo diIIiu., _ """...."" ...d ooIon cl""""."inr; D«K....... (I) ooIod. .. USG... _ l.OI4. oo moLolOy _ "IO~, (1) U[llrt rdI-. USG... _ I.OI4. ........t.u,. - Sll..-l.lkll [j) rdI-. USG... - I.OIJ, oo...t.lOy _ 192 ~ on ydloov. USG... _ 1.01.\ oomoIolny _ SSI.....,[J1.c- B. UuL.. C. ¥...cy«'. D. ~. ,10", .. d duot,,, of boa ..... oocr;' ... .,.;" (/,ft), ,ry 'l"''' ...d <>y ~. 'ct. .""roph;l. r-" ~~ IP .h",,- ~ . ,.''"- 2 f . , ,.. H,,,,,- H-H u~. I ...... ,....... ... • 1..... ~ a... U,,;a. Ctl ~IO'Y discn-. TP T u"" " pfIjI. ~ 0IiIb '-, u\lI. ''''IfO' u g'" ....... , _. I "". ... "-,~ ,I :'" i:= • rw. 12. s...... protrin d.rt"'l' ..... ,; • • " , - . , . toad",. ,.!k.l- "rut< ~ • ...d "N" protdn """",...ioo. Irom. d.op ...d R<"""" E..~. A. c... bcolth)" n.. B. C . . ~JP<'P<"';"'''''' n.. dmDtom".,. '"""'c ;, .. Obi.o «p«t C. b=oc_..oo.. ""' '" ~ c... ..a....-'Ydyop... ....... • •d.rt.... _ «Iotiody I... albumin ""'"pa Iiact.... 11., """DOd "'~ ...p... ;. probobly "'" .. Ux" .. cd """""'....... of I. .~ .. ",._roc\oI>uI'" (poGtW< """,.pI..", protrio.). lb, ;"'-""od 1'dobnJin "p.. ;, b....I.t... od .,.d ;. ,I.e. duo ... poIy.ili. ~ _ """'.. . ..1 ,Iwa oo..ld b. • no<>a<>dooo.l ~ . . . pc/rdoooal _ b y ..nh «.rirtcd ""em..... I. th;, c-. • p". . . . ." .... diac-- of fdio, ;olCroou. "',........ ...d "'" . b""", of B.lymploocyo, n«>pIao;. i.odkotod tho, d., J.rp.'I''''''.......... b)]><>al. bwaio.<m; • ...d ~ ..... du. duook ;o/bmm.oo... F. Doc. 10..1"", lb, &..; ........ " "'"" ; ......... aprocd ....... "" "'.Ithy d.op ..d ;, ~ ... ..fm,,,,,, potto"'" ....... ~ ;" .... J;~ of pn>trin 1Dct"'......Id b. bnd;. ""'" hoolthy dop. F. Doc, ;"&",m",,,!, bJP<'P<<><';""";" n.. d",oitomrt" """'c """'" • .dod;" r-um---<> "1'« ..... . p«><><>=«I poIyd.o...J """....".thy. I. ,IUO ""' . dind ';P' aod .....=cly h;p. tit" .. E,i,,{J,I,,,, ''''"' ;".d;,cotod thz tI., hrr<> ;. ""'"""" .......... "' ... aod.., ...odal .h.ou!d<,. 11., """. of ......... II,~ «p.. .,.d "" 'W""'" "«"m .. th, 1'~ """"ot"';"" ;, ;".d;,cot;.., of • m<>a<>do...1 """""",otioy of . """'~ ~ N. d.oc" b~ ;' .... duo to. mydoma aod th, ....... I~ """'."";",, ... ....blIy """",cd. H . Doc, ",okypop_do"";" n.. d. .. ~,,« ""'"'c ;. .. Obi.o _od "...to fo. • b..I,by '"" bu, ;, fouod ;" • kypoprotrio=>< .=pk. lb •• protrio """,",,_ ... ••",.. cd proportioo. t paa!oypoprotrio ..... Uxlo.d. "'~" blood 1".. ....J.LptiY< . od ...!aI-pt;.., d;o..d .... _ ..... "" ....... ond <>o. l1ll. ",~.,.n ;, ;".d;,cot;.., of p'-;"'Ioo;", ...p.ropotioy ;. wltKl. "" ~.,.,.] .. liI _ bun.. bo. boc..., ....., I"''''obk to pi ..... ~ b.ao..", of ~....Jo0'l'hrit;, 0' po ......... ~ •. I. _I. co. ........ ~ • «I •• ", ""', .. of th. u,.p.bul" ,.p.. ~ ", _m~ ~ to<> I.. ", tho""", ,10. filiczioo bun.. .... ...u., protdno '0 .nrio. '0 "'_ coo. Not< d,,, ..." """"" th.,., ;, !.ypoprotrio... ~. .,.d~ . d, ...... _ bypooIbomi.o""; • .ruIy . o.....u. '0 '0 bino,". J. ~_n.....; . boc.uo, tdmuia ;, • .d«........_;"..no .. ,h, ..... P""';" 1""''''' ;. _ "" """' .. "" d.oc" >O<' protrinuri. l. s...u.,., ,..do, ;, n.. d< ..;_rt<, "'hio "1""«<1 « dto ... . Idthy a«""- '" ""''''' .....~ Co_. of ponby. iod..& .... , blood Iou.~";"" and maJ.boo.pri.. d;oo.d .... ...,...;"", , .... ..; • ...d ,",co_ oionalIy t.;[uu. n.;, "'" hod """;"",0. Iym. phoma .. ;th Mott cdla ...d "" !.ypoprot,;.",, ~ .... prohobly d", to naoItipl, p""""" • 2. Uri.., Moot .."" P""""" "" .. th. ~.p.b..I" ,.p.., ""' •• ,., ";!h ""em .... of ~ ~p d.alno. n.. Btoe< }..... .............. p...i", thz ; •• ....... .."'"",•• , .... .... iaIly d.u, fo.m .K P"I""-"".... ....... P""';" P"''''''';' '''' th. """ .. "" d.oc' ......... a..p= r-- protrin ond ""'_." ... , typ< of. P"..".J protrinurio. (0« 8). N_,11., ............ p_ b;out ...d BCG....th<> lb, """" cakubtod by ..bo« •• ' '';1'' ...,., ocuuood, ,10. """"""""" ..... " .. tha, "" .Luk.. protrio baod .. th< .....p.. "",od th, ....... ~m d.&orn... of "" ,oadnt; ""'. H1P''I'rotrio=>< , ~ " ...,., d;[",od (mba I port """" to [ I"" ..I;., 0' ~ p..... ..u...) p< .. dort.opbo".;, ... '"'" th." ........ " Ii.o.co, ...., .. aalUp brt,.",. q....,"rof r_n. .. ,1, d....... b...d ...d th< ........ , of~ th" "" ... duo.cb ,t.. .np. n.. ....... «>tal protdn COOK . . .oat .... _ " .......,.od by th, Coom ..... b.&nt bI"" ....y. n.. ...., "",pi....." CODCttO".otod [G.o.td p...... .1," ...... _ .... ~ ""'ant ....ioo ...... • , ). , ..• • • ," ' o - .~ • , .. ' ...• , • 0 0 • 0 0 " Q .,l • ~ • 0 , . ~ ~ u.~nri<' diR -"" ........... fi_ L) _ 1py - " bot~ ......d (4 . .oJl~ .... ....,. ~ ;p..IIy ot.j«, .... (_ 0001....... 1Ot.!:..- .... "ob.~ .... . 10:< 01>;0<>;.. c.,....... A. Caka.. aut.o...c. 8. Cola ... ..,..!". 4~ ,.,.. ......... boa ..... C. c.kj""" ....... ~ •• .,...J. <.,...I. F. SuIIO..,.. .... Sc ...... ~ G. """ ....... ~ .. _ _ ~..,. 1.. Sf-m. """~ .. 1 Uri< .ad ,.,.. ..... H. y ..... I. _..,... .. .....nhyIo-bl.......... J.eq;a..;. ol 0.... S.~). K. L;p;d d""*,,. D • l'Iat. 1"- """'..... ~ of cdl. .... """" mK~ bad. Uoj:' ;m dimot .....n (C. G,J, ...d N) .. . ~ •. po<>oIlIui,j, Ioon.. C. n.",m. _. t-.Iod. -J baed; ...d b.d4 m. A.-I"'~ op. <>< 11«"";;" "1" (/,fi), mildly d.,.".... t< .""..p.;;. ~th. pb"""",,;ud b.cta-;. (..,..." pI.... ~ bornri.I =xI.t., doc. D. Dq;"",.", room< "'._ <>iniac ...... , ...... of roaoi, ........., "1""""" odl, p crn..., .I" ......; """"runt .. "m..." bc.nd."" (.".. .,.,..,. . .....u .""rop/UI. "'1' ' '"' ".........., - .. ~ ""...,j,,«1 ">crop., pImcaI ,ffo ...... .J.oc. G. Noop..;. " .. oI .. lympl.ocyt<. (!.:fi .... ~.,.~ •• ..,,1utK lymph.;.! 1"'';' """,.I .!h-.." at. H . ]""""odi", •..d Ia.v ....",1."" Iym. phocyt", ~"""' j~ pI .... ~ ~ .loco I . s.....u ""'" of """_....,;,,, .... ooh m>crop. pI"",,) Soid, ""'" L R .....J""'".,, ......... N . N...nopIUlo ...d .... aopluv roo<>iniac .......... fo.dp body .....;ro.,."I .....lat~ (oili. "''''''1 of J""')' n......., Mid.it; .. St><. u..m..ity). O. N"""""...,."" onrtrcplUlo ...d ">crop. . . .h ia ... .dlulac...d att. aiI .... put;..J"" octintoc &om a>IlmU>j: Ibd "",0 on Kri ............uu..c d.. ,..t., r", ,,;,... p~.J. pc>I Soid. c& co. do., ) "- PI.<. 13. PI.ot_;.,-""opb. of cdlo ...d oot>tk...........,. <><>t>ininr; t..dL). bornrioI aud ,, ~ .Joe. F. M..Jy _rophao (on< ~ morula._ . -nJa nup;&d in .... utod by <>< cyto=>trifu~ (.4, H. K-M ...d ....................... ,-.x..,......... cmwr D. W""/" aI., 1OO1~ G. ~'" of c..,/;,,/o .... and d.moc«I .......... cdl.. p mr«> ~ doc· H . l....w..l p......... (kfooad..,.." bootm..l p < _""..od" ......'" of . ~......, (ASVCP ...... ,....;"'-I by 1992~ K. B* ~" nrut...p,Jo, ...,j ____ p/>oe'P . ........... ~ ~ .. aod > P. M,W......... m"'~ ...d ~"""" ,...,;n, • .J.oc. N. M-=Puc'"' _rio """,.,p,.a., ........., r-- """ulu ",<>«in ...d ''''''''''', p«>t<"'"=uo ....d.t~ fd.io., ~ p

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