Forensic Diatomology and Drowning

FORENSIC

DIATOMOLOGY

AND

DROWNING

MICHAEL S. POLLANEN Department of Chemistry and

the Forensic Science Prog;ram,

Univ ersity of Toronto, Toronto, Canada

Consulting Scientist, Office of the Chief Coroner

Ontario, Canada

1998

ELSEVIER Amsterdam - Lausanne - New York - Oxford - Shannon - Singapore - Tokyo

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Preface

Odysseus bent his knees and sturdy arms, exhausted by his struggle with the sea. All his flesh was swollen and streams of brine gushed from his mouth and nostrils. Winded and speechless he lay there too weak to stir, overwhelmed by his terrible fatigue.

- The Odyessey, Calypso (Book V) Homer

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The postmortem analysis of drowning is a classical problem in the pathol­ ogy of sudden violent death, From a historical perspective, one of the most important issues in the study of drowning has been the search for a sensitive, specific, and easily applicable test for this cause of death. On this basis, the diatom test has emerged as the foremost laboratory proce­ dure for the detection of drowning. Although the history of the diatom test spans a century, no comprehensive monograph has been published to address the scientific and medicolegal basis of the diatom test. This mono­ graph is an attempt to fill this void in the English-language literature. The monograph is intended as a guide to forensic diatom analysis useful to the forensic pathologist, scientist, death investigator, criminalist, and police officer. The motivation and axiom for the diatom test are discussed in chapter 1 which also outlines the types of circumstances bodies may be recovered from water. In chapter 2, drowning is considered in the broad­ er context of rapid anoxial death and the postmortem findings of drowning v

are discussed. The medicolegal value and historical development of the diatom test are reviewed in chapter 3, which prepares the foundation for discussion of the author's research on diatoms and drowning using femoral bone marrow (chapter 4). In chapter 4, the utility and validity of the diatom test is analyzed on a quantitative basis. Chapter 5 describes the laboratory procedures for extraction diatoms from human tissues and pre­ sents guidelines for interpreting the diatom test in forensic casework. The various investigative applications of the diatom test are presented in chap­ ter 6, with specific reference to the role of the diatom test in homicidal drowning. The final chapter is a photomicrographic and descriptive guide to the freshwater diatoms that are frequently encountered in drowning cases. This chapter, along with chapter 5, will serve to guide forensic practitioners who are interested in performing forensic diatom analysis. The text is followed by a selected bibiliography which includes references that may not have been cited in the main text. Most of the cases used to develop this monograph are from the Office of the Chief Coroner for Ontario, Canada. I thank the professional and tecnhical staff of the Office of the Chief Coroner for Ontario for sup­ porting this project, and Drs. 1.T. Cairns and D.A. Chiasson for permission to use case material. Permission to publish the case study in chapter 6 sec­ tion 7 was kindly provided by the prosecutor of the case Assistant District Attorney E. Nneji of Ulster County, New York. The investigators in the case were Chief Daniel P. Miller, Captain George B. Turner and the Detective Division of the Ulster Police Department. I thank Captain Turner for providing crime scene photographs of this case. I am grateful to Drs. D. Noel McAuliffe, Helier 1. Robinson, and Raoul A. Robinson for reading an earlier version of the manuscript and clarifying ideas. I acknowledge Dr. R. Russell for providing access to the diatom collection of the National Museum of Natural History, Smithsonian Institution, Washington, D.C. This project would not have been possible without the dedication of the late Dr. 1. Hillsdon-Smith (former provincial forensic pathologist and forensic diatomist). It is to him that this monograph is dedicated.

vi

ment of the

Contents -;:: descriptive guide ~ ered in drowning . :-' i: ro guide forensic - I diatom analysis. -~ includes references - .}graph are from the k the professional -: :'or Ontario for sup­ n for permission . d' in chapter 6 sec­ ssistant District ~ in estigators in the ;: B. Turner and the -.:. I thank Captain ase. I am grateful .1 ul A. Robinson for larifying ideas. I :ie diatom collection -:t. onian Institution, " ~s ible without the ;::" rovincial forensic -.1: this monograph is

Preface ....... .. ...................... . ............. .. v

Contents ..... .. ... . ... ... .. .. .. ......... ............. vii

1. Bodies in water and the diatom test for drowning . . . . . . . . . . . . 1

1 Medicolegal investigation of bodies in water .... . ...... 1 2 What are diatoms? ......... .... ............ ... .. . 2

3 The diatom and drowning axiom .................... 3

4 Unnatural deaths and water ........ ... ...... ... .... 8

* * *

*

Accidental and suicidal drowning ... ..... .. . . .......... . 8

Homicidal drowning ... . ...... .... . .. ...... ... ... .... 9

Drowning in the bath ............... . ............... 12

* 5 References ..... ... .. ..... . .. . . .... ........... . 14

2. The forensic pathology of drowning . . ................ . .. 17

1 Pathophysiology of anoxia ........................ 18

*

*2

Mechanisms of anoxia .... . . . . .. .. . . .. . ..... ... . . . .. 18

Anoxic encephalopathy .............................. 19

The types of asphyxia ... ... ....... . .... . .... .. ... 21

Classification of asphyxial deaths . ........ ........ ..... 2]

*3

Pathology of asphyxia and rapid anoxial deaths ..... ... .. . 21

Fluidity of the blood . . ........ . . .......... .......... 26

Pathophysiology of drowning ...................... 26

VII

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Forensic Diatomology and Drowning

*4

Pathology of freshwater drowning .................. 28

Introduction ....... ........ .. ........... ........ .. 28

Peri mortem injury and events ........ . ........ . .. . . . . . 29

Water-related alterations .......... ... .... .. .... ... ... 30

Air passages ... ...... .. ... ... . . . ............ ... . .. 30

Lu ngs ....... .. .. . ....... . . .. . .... . .. ......... .. . 32

Pleural effusion .......... .. ... ...... .... ..... ..... 33

Hemorrhage in the petrous temporal bone ..... . .. ...... . . 36

Visce ral organs . . ... . .. . ............ . ... ..... ... . .. 36

* 5 Pink teeth ... .... ........ ..... ... .. ...... .. .... 36

*6

The history of pink teeth ........ ...... ... .. . .. ..... . . 36

The significance of pink teeth ..... ... ........ .. .. ..... 39

Aquatic decomposition ........................... 39

Aquatic taphonomic factors .... ................ ...... 39

Decomposition sequence in freshwater ..... .. . .... ... ... 40

Adipocere ... .............. ....... ...... . ......... 42

* 7 References .. . . .. ... ....... .. .. .. ... .... .. .. . .. 42

3. The medicolegal value of the diatom test for drowning .... ... 47

1 Analytical approaches to drowning ............. . ... . 47

*

Chemical tests for drowning . ............ .. ... ... ... .. 47

Micro-organism tests for drowning .. .. ... ..... . ..... . .. 49

* 2 The so called "War-on-Diatoms" .. .... .... . ........ 49

* 3 A brief history of the diatom test ................... 52

* 4 Are diatoms ubiquitous in tissue? ............ . .. ... . 54

* 5 Research and the diatom test ...................... 57

Experimental models ... ........... ...... ... ... . . .. . 57

Extraction of frustules .................. . .. ..... .. .. 58

Qualitative and quantitative diatom analysis .............. 58

Use of bone marrow ................................ 59

* 6 References ..... . ....... . ....... . ...... .. . ..... 60

4. Validity and utility of the diatom test using bone marrow . ..... 65

1 Utility . .. .. . ... . . . .... ........................ 66

*

Outcome ratios for the diatom test .. . .......... ... ..... 66

Seasonal and monthly variation of diatom test outcome ..... 66

Vlll

Contents

*2

· .. . ....... 28

Validity ..................................... .. 68

Concepts in support of the diatom test .................. 68

· . . ........ 28

... .. . . .... 29

Test outcome correlates with diatom content of water .... ... 70

•... ....... . 30

Frequency of concordance of frustules in

· .. ..... .. . . 30

bone marrow and water ........................... 72

· . . ........ 32

* 3 Sensitivity and specificity ...... ... ............. ... 77

* 4 References .................................... 82

Size and shape of drowning-associated diatoms ........... 76

· .. ........ 33

The diatom test and diagnostic indices .................. 78

· .. .. . . .. .. 36

.•. .. .... .. 36

· ... ....... 36

5. Laboratory procedure for the diatom test ......... ........ . 83

1 Collection of samples ............................ 83

· . . . ... . ... 36

*

· . ...... ... 39

· . . ..... ... 39

· ... ........ 39

· ... . ....... 40

Collection of samples at the scene ........ .... ... . . .. .. 83

Collection of biological samples ... . ............ . ...... 84

*2

•... ... ...... 42

· .. .. ...... 42

Chain of custody ........ . ....... ..... . ..... . .... . . 87

Laboratory method .............................. 87

Overview .... ................ .. ........... . .... .. 87

Materials ........................................ 88

- .... " .. . . . .. 47

... . . ... 47

••.. . ..... 47

*3

· .• _ ... ... 49

*4

· ... ..... · .... ...... · ... .. . . ... •• . . .. ....

49

52

54

57

· .. . . . .... •.. ...... · ... ...... • . .. ......

57

58

58

59

Procedure ............................... ....... .. 89

Laboratory method for inanimate objects . .... ... .. ... 90

Overview ......... . . ...... ..... .. ..... . .......... 90

Procedure ................................. .. ..... 91

Interpretation .. .. ..... .. .............. . ........ 91

Microscopic examination of bone marrow extracts ......... 91

Microscopic features of diatom frustules . .......... . ..... 96

Non-diatomaceous silica .... .................. . ...... 97

Significance of bone marrow diatoms ............. . ..... 98

* 5 Other methods ................................ 100

Electron and atomic force microscopy ................. 100

M olecular biologica l approach to the diatom test ......... 101

... . . . . ... 60

* 6 References .. . . . . ........ . .................... 103

·-::>oW... . .. 65

.... ... . .. 66

6. Investigative applications of the diatom test ............... 105

1 General applications ............................ 105

... .... 66

Indications for the diatom test. ....................... 105

*

Contra-indications for the diatom test ... . .. . ........... 106

lX

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Forensic Diatomology and Drowning

*2

Decomposition and the diatom test ............... .. 107

Aquatic decomposition and the diatom test ............. . 107

Skeletonized bodies and the diatom test ......... .. ... . . 107

* 3 Drowning in filtered water ................ .. ... . . 108

* 4 Dismembered body parts ..... . ................ .. 108

* 5 Diatoms as trace evidence .............. . . .. . . . . . . 112

* 6 Diatoms and homicide .. ... ............. . .. . .... 113

* 7 Case study: Murder in the Hudson river ...... ...... . 115

* 8 References ... .. ....... . . . ............ . .. .. ... 124

7. A guide to the freshwater diatoms of drowning ........... . 1 Centric diatoms ... . ... . ... . ................... 2 Naviculoid diatoms . . . . .... ... .. .... . ........... 3 Dorsiventral diatoms . . . .. .. .. . .................. 4 Epithemia ... . ..... . . . .... .................... 5 Nitzschia and Hantzschia . ..... . .. ............... 6 Asterionella and Tabellaria . ....... . .............. 7 Cocconeis ....... . .. . ... . ..................... 8 Heteropolar and other diatoms ... .... . ............ 9 Cuneate and other diatoms ... . .. ... . . . . .......... 10 Elongated diatoms . ............................ 11 Small pennate diatoms .. . ... . .. ... ... ... .. .... . .

* * * * * * * * * * *

125

126

128

130

132

134

136

138

140

142

144

146

8. Selected bibliography .. . . . . ... . ..... . . ... .. . . .... ... 149

Author index . . . . . . ...... .. ... . ........ .. .. .. ... .. . ... 155

Subject index .... . . .. .. ..... .. ....... ... ... . ... . . .. . . 157

x

· ... . . . ... 107

•• . . .... 107

•.•. ...... 107

· .... ..... • . . . ..... · .. . . . .... · . . .. . ... • •. . . . ... · ..... ...

108

108

112

113

115

124

· .... . ... · ....... .. .•... . .. · .... . . .. · .. ...... .. . . .... . .. · .. . . . ... · •.. .. . .. · ... ..... · .. . ..... · ... . . ... · ... . . ...

125

126

128

130

132

134

136

138

140

142

144

146

1. Bodies in water al1d the diatom test for drowning

A body recovered from water mayor may not have drowned. This leads to two basic problems: determining both the cause of death and manner of death. Both of these problems are difficult when confronted with a body from water, particularly if decomposition has altered the classical findings associated with drowning or another cause of death. In this chapter, the cir­ cumstances of death associated with water are introduced and the concep­ tual foundation of the diatom test for drowning is described. Although the diatom test may not, in most cases, contribute to understanding the man­ ner of death there is a role for the test in determining the cause of death in cases of bona fide drowning.

· ... . .... 149

· .•. . . .... 155

* 1. MEDICOLEGAL INVESTIGATION OF BODIES IN WATER

· ..... .... 157

Drowning is distinguished from many other violent causes of death since the pathology of drowning does not correlate with the manner of death. Unlike manual strangulation, multiple gunshot wounds, and multiple blows to the head, which are almost always homicidal, the postmortem findings in homidical and accidental drowning are indistinguishable. This matter is further complicated by considering the more general case of a dead body recovered from water. In principle, a body recovered from water need not have died in the water, and drowning may not be the cause

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Forensic Diatomology and Drowning

of death even if the death occurred in water (1). Therefore, the medicole­ gal investigation of a body recovered from water must consider the fo l­ lowing special questions: 1. Did death occur by drowning? Did drowning occur at the site of body recovery? 2. Did death occur by means other than drowning while in the water? 3. Was the body immersed in the water postmortem ? 4. Is the death due to a criminal act? All of these unique issues relate to the pathology of drowning and can, at least in part, be addressed by full postmortem examination. However, since the lesions of drowning are variable (chapter 2) there is an important role for ancillary tests for drowning include laboratory based analyses using tiss ues recovered at postmortem examination. The most important of these tests is the diatom test for drowning.

* 2. WHAT ARE DIATOMS? Diatoms are aquatic unicellular plants that represent a major taxonomic division of the phytoplankton (2, 3). The most dis tinctive feature of this unicellular organism is its extracellular coat or frustule, which is com­ posed of silica. The frustule of the diatom is a crystalline structure that is characterized by unique patterns of synunetry and microstructure. On this basis, the vast structural diversity of the frustule leads to a remarkable number of morphologically-distinctive varieties of diatoms. This vast degree of morphological variation gives rise to a large number of species. Recent estimates indicate that there are in excess of 10,000 different species of diatoms. Diatoms are most often encountered in naturally­ occurring bodies of water such as lakes, rivers, oceans, seas, ditches, and puddles. Some diatom species have preference for water of specific salin­ ity thus allowing general distinction between freshwater and marine diatom types . In addition, some diatom species are more frequently asso­ ciated with soil and puddles than lakes. Fossilized diatoms are another major source of frustules in the biosphere, although, these frus tules are derived from long dead diatoms. Such diatoms are mined for commercial 2

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Bodies in Water

_ :_e medicole­ - ..:le _ the fol-

use and maybe as forensically important as live freshwater contemporary diatoms. Mined fossilized diatoms are used in several commercial prod­ ucts including the fine abrasives in polishing compounds (4). One seldom emphasized feature of diatoms are their complex pop­ ulation dynamics and ecology. Diatom populations are constantly in fl ux and these fl uxes are the result of complex and poorly understood nu trient and aquatic cycles. The net res ult of these environmental variations is a monthly periodicity in the abundance of live aqu atic diatoms with blooms of diatom populations in the spring and autu mn (i.e. seasonal maxima) (5). In addition, there are temporal and spatial variations in diatom concentra­ tion in any body of water in response to local factors includi ng mineral content of water, temperature, water stratification, and acidity. A poorly understood aspect of diatom ecology is the variation in the species and genus distribution over seasons. These ecological characteristics of diatom populations have great, and under utilized, forensic significance. Among 11 the most important forensically relevant feature of diatom populations is the monthly variation in water concentration of diatom frustules that should, a priori, effect the outcome of the diatom test for drowning during vario us times of the year. The widespread distribution of diatoms in water and their morphological specificity makes the diatom an important foren­ sic marker of drowning.

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* 3. THE DIATOM AND DROWNING AXIOM

- __: ~ :e . On this .: _ remarkable - ~ -. This vast _ _ _ !" of species. .. . ~ 00 different ;;~:: ~ in naturally­ . di tches, and - ~ ~ -pecific salin­ ..:.:~ r and marine :':equently asso­ .-: _::: are another -'?:_f: frustules are -:'L ~-or commercial

The conceptual foundation of the diatom test for drowning is illustrated in fi gure 1.1. In the drowning process, water is inhaled and distends the alve­ oli. The diatoms that are present in the drowning medium perforate the alveolar-capillary barrier (Fig. 1.2 & 1.3) and enter the pulmonary venous circulation (6). Once the diatom-laden blood is returned to the left side of the heart, the diatoms embolize to organs and tissues such as the bone mar­ row. Therefore, the presence of diatoms in the femoral bone marrow is an indication of antemortem inhalation of water. If diatoms are detected in the bone marrow (a positive test) this implies that: 1. Drowning caused death or was a contributing factor to death 2. The individual was breathing upon entry into the water. 3

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Forensic Diatomo[ogy and Drowning

LIVE ENTRY

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PERCOLATION

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DISSEMINATIO N

1.1. The conceptual foundation of the diatom test for drowning using femoral bone marrow as the source to extract diatoms . If a dead body is immersed in water, diatoms may percolate into the lungs but cannot disseminate in the circu­ latory system. (Modified from Bernard Knight, Legal Aspects of Medical Practice, Churchill Livingston Publishers, 1972. Illustration by Jackie Robers).

4

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211

_::lg using femoral :- ' _ i immersed in '- te in the circu­ - ~_ I of Medical Ja b e Robers).

1.2. Diatom (Diatoma moniltformis) penetrating the wall of a distal airway in an experimental rodent model of drowning (Scanning electron microscopy, 6,000 X). Courtesy of Dr. Phillippe Lunetta, Department of Forensic Medicine, University of Helsinki. 1.3. Two diatoms (Phaeodactylum tricornutum) found in the lumen of an alveo­ lar capillary in an experimental rodent model of drowning (a = alveolus, i = inter­ stitium , c = capillary lumen, d = diatom) (Transmission electron microscopy, 6,600 X). Courtesy of Dr. Phillippe Lunetta, Department of Forensic Medicine, University of Helsinki .

5

Forensic Diatomology and Drowning

If a dead body is placed in water, diatoms may passively enter the tra­ cheobronchial tree and the alveoli by percolation (7). However, since the heart is not beating, diatoms cannot be dissemeniated to peripheral tissues. The silica-based frustule of the diatom allows for the easy extraction of diatoms from human tissues by digestion of tissues in agents such as strong acids. Although other methods for diatom extraction have been advocated, the acid digestion method and centrifugation has been the most consistently applied and gives highly reproducible results. Various tissues have been used as starting material for the extraction of diatom frus tules. Bone marrow removed from a femur is a popular choice. The advantage of the bone marrow extracted from an intact femur is that contamination can be minimized or eliminated since the marrow is extracted from a "closed" organ that can not be soiled by diatoms from some external source. For the test to be considered valid it is necessary, in most instances, to prove the origin of the diatoms by showing that the types of diatoms extracted from the bone marTOW are concordant with those in the drowning medium. This is accomplished by simple morphological comparison of taxa in the bone marrow and a sample of water from the site of drowning (Fig. 1.4). The forensic pathology community has been, historically, polar­ ized in its general acceptance of the diatom test as a definitive diagnostic test for drowning. The practical result of this polarization is that the medicolegal community in the United Kingdom, Japan, Europe, and South America (e.g. Uruguay) apply the test with greater frequency than in North America and elsewhere in the world. This is in part due to the origin of the test in continental Europe and because much of the research on the test is derived from laboratories on the continent. One of the main events in the historical debate on the diatom test was the demonstration, by some investigators, that diatoms may be present in the extracts of tissue prepared from non-drowned people. These inves­ tigators concluded that this finding limited the medicolegal utility of the test. However, this criticism of the diatom test has been successfully met by using the "criterion of concordance" which demands that the diatoms recovered from tissue be comparable to the diatoms in the putative drown­ ing medium (8, 9). This criterion makes the origin of the diatoms unam­ biguous and proves the diatoms originated from the drowning process and not some other non-relevant mechanism. 6

Bodies in Water ~='er

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1.4. Concordant freshwater diatoms [Stauroneis sp. (left set), and Asterionella formosa (right set)] extracted from the femoral bone marrow in a case of drown­ ing and the water in which drowning occurred.

7

Forensic Diatomology and Drowning

* 4. UNNATURAL DEATHS AND WATER Dead bodies that are found in water may have died in water or may have been relocated into a watery environment. Bodies in water are recongized to fall into four major categories: 1. Accidental and suicidal drowning. 2. Homicidal drowning. 3. The body intentionally placed in water posmortem ("the dumped

body").

4. Drowned bodies moved from the water.

Accidental and Suicidal Drowning Accidental drowning in open water during the Summer months accounts for the majority of all water-related deaths. The circumstances of acciden­ tal drowning are highly variable but commonly occur as follows: 1. Drowning de novo usually associated with water-related recreational

activity.

2. Drowning complicating natural disease (e.g. atherosclerotic coronary

artery disease).

3. Drowning complicating acute ethanol intoxication or other drug

intoxication.

Many victims of accidental drowning are intoxicated and this is an impor­ tant contributing factor in several water-related fatalities. The ability to swim is not an impOltant correlate of drowning since most victims of drowning are able to swim. Accidental drowning may also occur in mass disasters such as ferry accidents, and plane crashes. The role of hypother­ mia must be carefully evaluated in many cases of apparent drowning. Hypothermia may be a contributing factor or sole cause of death in some cases. A rare variant of accidental drowning is "micturation syncope" were a recreational boater may collapse unconscious into the water while uri­ nating over the edge of the boat. Suicidal drowning (10, 11) is infrequent and usually occurs in indi­ viduals with well a documented psychiatric history. Frequently, suicidal 8

Bodies in Water

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drowning will occur after discharge from a mental health facility or short­ ly after a psychiatric consultation. In exceptional circumstances, suicidal drowning may be the first manifestation of acute paranoia. Some victims of suicidal drowning will disrobe prior to entry into the water or will weigh themselves with down with stones or other heavy objects.

Homicidal Drowning mped

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Sir Sydney Smith reported that 5% of all drownings, in his experience in Egypt, were homicidal. This figure is consistent with experience in North America and underscores the need for careful consideration of the manner of death of all bodies found in water (12). In most cases of homicidal drowning, postmortem examination reveals other violent injuries, most commonly blunt force injuries of the head, or strangu­ lation. In such cases, drowning is usually a terminal event complicat­ ing the other inuries. However, homicidal drowning in the absence of other injuries can also occur and has many contexts . The most widely appreciated is the famous Brides in the Bath case . In December 1914, Margaret Loftus was drowned by her hus­ band, who called himself John Smith, while bathing (13 ) . Initial investigation led to a verdict of accidental drowning and the husbad collected an insurance settlement. S ubseq uent investigation revealed that several previous wives of Mr. Smith also died of drowning. The previous investigations suggested that the drownings were due to acci­ dent or the consequence of epilepsy. Smith was later convicted of murder and executed. The Brides in the Bath case illustrates difficul­ ties of assigning a manner of death in drowning. Determining if open water drowning is homidical may be con­ siderably difficult and often relies on careful postmortem examination to reveal injuries of assault or pressure on the neck. In addition, scene findings often provide important evidence for assigning manner of death, even if the scene is a deep lake. Bodies may be recovered with weights attached (14), or objects used to weigh down the body may be found at the bottom of the body of water if it becomes detached from the corpse (Fig . 1.5 & 1.6).

9

Forensic Diatomology and Drowning

1.5. Body recovered from a lake with rope attached to the belt-loops of the pants. A knot was found at the other end of the rope that was free-floating in the water. Autopsy revealed drowning , recent pressure on the neck, and diatoms were recov­ ered from the femoral bone marrow. (Photograph by Cst. R. S. MacLeod).

1.6. A patio stone found at the bottom of the lake near the site the body depict­ ed in figure 1.5 was recovered. The assailant rendered the decedent unconscious by throttling and drowned her by weighing her body down with the patio stone. (Photograph by Cst. R. Stiver).

10

Bodies in Water

1.7. Scalding injuries from bath water with subsequent drowning in the bathtub. This type of domestic drowning death is most often seen in the elderly with ath­ erosclerotic heart disease. 1.8. Old contrecoup contusions of the inferior frontal and inferior temporal lobes in a case of drowning in the bathtub. Old contusions may be epileptiform sites that led to a grand mal seizure disorder. Accidental drowning in bathtubs, and pools can occur as a complication of a seizure. (Photograph courtesy of Dr. J.R.N . Deck)

_=- . :.2~nI nconscious e patio stone.

.' 11

--1.

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Forensic Diatomology and Drowning

Drowning in the Bath Drowning in domestic water, such as in the bathtub, accounts for a large subset of immersion deaths (15, 16). Unlike open water drowning, the cir­ cumstances that are often associated with drowning in the bathtub are more limited and usually fall one of the following categories:

1. Accidental drowning complicating natural disease (e.g. coronary

artery disease, or epilepsy)

2. Accidental drowning complicating drug intoxication. 3. Homicidal drowning (e.g. Brides in the Bath) 4. Suicidal drowning (e.g. the frail elderly or infirm) The most common circumstance is accidental drowning that complicates acute collapse (e.g. grand mal seizure or cardiac arrythmia) due to natural disease. In the case of atherosclerotic heart disease, acute cardiac arryth­ mia may occur while preparing the bath and peri mortem scalding may occur when the skin is exposed to the hot water (Fig. 1.7). In these cases, significant hyperemia may be found in pharynx and air passages indicat­ ing aspiration of hot water and thermal injury to the mucosa. Features of drowning are usually, but, not always apparent. In the case of the latter, death may be related more to immersion and intercurrent natural disease than true "wet-drowning". An important preventable type of domestic drowning is drowning complicating an epileptic seizure (17) . In most cases, a clinical history of epilepy in known or anti-epileptic medication can be found at the scene. Several important observations can be made at the time of post­ mortem examination that may indicate an epileptic seizure-associated drowning including blunt injuries to the tongue (due to biting), and stig­ mata of the chronic use of phenytoin including gingival hypertrophy, cerebellar atrophy, hepatomegaly, and rarely interfollicular hyperplasia of lymph nodes. In a minority of cases, examination of the brain may reveal structural lesions to account for the seizure disorder. The most common lesion is remote contrecoup contusion of frontal and/or tempo­ ral lobes (Fig. 1.8). Other focal lesions that may be found infrequently include hippocampal sclerosis, polymicrogyri, neuronal heterotopias and gliomas. 12

Bodies in Water

One important variant of domestic drowning is spontaneous birth into the toilet (18) and infanticide by drowning of the newborn. These cases produce considerable practical difficulties since it is necessary to establish live birth. In the case of spontaneous birth into the toilet, the mother is often primiparious and has denied pregnancy until the time of birth. In principle, the diatom test for drowning can be valuable in estab­ lishing live birth; however, since domestic water usually lacks diatoms, positive test outcome are infrequent «10%).

The Concealed or Dumped Body

= ­ .:

~omplicates

- _ ral disease

- =:- drowning _ ,: . ·n · al history

-

~=

:'o und at the of post­ _ . ::":' _ ~e - ssociated . : :- . :.:.~g ) . and stig­ ~ - =_ :... nypertrophy, . ~ __ 3.[ hyperplasia _ .:. :- _ e brain may _ - ~ :-je r. The most - _- :, and/or tempo­ infrequently =-=-. :-.e .e otopias and _

--::- - ''"TI e

. :.:. -= "

Recovery of a body in water does not imply drowning. In some cases of accidental drug intoxication (e.g. heroin) the body in maybe concealed in water at some distance from the site of death by witnesses of the death that may also using drugs in a "shooting gallery". Homicide by means other than drowning followed by concealing the body in water also occurs. The corpse may be weighed down (see chapter 2, Fig. 2.26) to hamper efforts to locate the body. In the case of the dumped homicide victim or drug overdose, postmortem examination and toxicological studies are usually sufficient to establish a cause of death, unless the mechanism is asphyxi­ al. In a study of homicide victims disposed of in lakes of Missouri, U.S.A., Dix (14) found that all cases were weighed down with concrete blocks, barbell weights, wheels, or anchors. In all cases, significant aquat­ ic decomposition had occurred over the elapsed time since death and dis­ covery (4-10 months).

Drowning Deaths Moved from Water A small proportion of water-related deaths may be found remote of the water such that the scene does not imply drowning as a cause of death. Such circumstances, although rare, need to be considered particularly since cases of homicidal drowning may be moved to land and may be fur­ ther concealed by decomposition in an isolated location or burned post­ mortem. Some cases of accidental drowning in the bath may be removed to the bed to suggest a natural cause of death. 13

Forensic Diatomology and Drowning

Findings at postmortem examination may not be interpreted as drowning if the body is not recovered from water. However, the presence florid oedema of the lungs in the absence of congestive heart failure, and watery fluid in the stomach and sphenoid sinus maybe important indica­ tors of drowning in this circumstance. The diatom test for drowning may play an important role in determining the cause of death in concealed homicidal drowning. Therefore, some forensic pathologists will routinely harvest a femur for diatom studies in all cases of this type, even if the post­ mortem findings of drowning are not apparent at autopsy.

* 5. REFERENCES 1. Davis JR. Bodies found in water. American Journal of Forensic Medicine and Pathology 1986;7:291-297. 2. Round FE Crawford, R.M., Mann, D.G. The Diatoms: Biology, and morphology of the genera. Cambridge University Press, 1990 3. Cox EJ. Identification of freshwater diatoms from live material.New York: Chapman and Hall, 1996 4. Peabody AJ. Diatoms in Forensic Science. Journal of the Forensic Science Society 1977; 17: 81-87. 5. Hendey NI. The diagnostic value of diatoms in cases of drowning. Medicine, Science, and the Law 1973;l3(1):23-34. 6. Lunetta P Penttila, A. Scanning and transmission electron microscop­ ic evidence of the capacity of diatoms to penetrate the alveolar-capil­ lary barrier in drowning. International Journal of Legal Medicine 1998;in press. 7. Taylor n. Diatoms and drowning -- a cautionary case note. Med Sci Law 1994;34(1):78-79. 8. Timperman 1. Medico-legal problems in death by drowning: Its diag­ nosis by the diatom method. Journal of Forensic Medicine 1969; 16(2):45-75. 9. Hendey NI. Letter to the Editor, Diatoms and Drowning - A Review. Medicine, Science, and the Law 1980;20(4):289. 10. Copeland AR. Suicide by drowning. American Journal of Forensic Medicine and Pathology 1987;8: 18-22. 11. Auer A. Suicide by drowning in Uusima province in southern Finland. 14

Bodies in Water

::1terpreted as

=- . ~.. I1ant indica­ _ .

.

rung may .. : ~ :::h in concealed ~= will routinely -: ~ ::\"en if the post­ -:

. .:: ::

[0\\

-T­

_

~=5 :

Biology, and

Med Sci Law. 1990;30(2):l75-l79. 12. Copeland AR. Homicidal Drowning. Forensic Science International 1986;31 :247-252. 13. Kerr DJA. Asphyxia. In: Forensic Medicine. Sixth ed. London: Adam and Charles Black, 1957: 157-158. 14. Dix JD. Missouri's lakes and the disposal of homicide victims. J Forensic Sciences 1987;32(3):806-809. 15. Devos C Timperman, J., Piette, M. Deaths in the bath. Medicine, Science, and the Law 1985;25: 189-200. 16. Schmidt P Madea, B. Death in the bathtub involving children. Forensic Science International 1995;72: 147-155. 17. Ryan CA. Drowning deaths in people with epilepsy. Canadian Medical Association Journal 1993;148:781-784. 18. Mitchell EK Davis, J.H. Spontaneous births in toilets. Journal of Forensic Sciences 1984;29(2):591-596 .

. . e- -. 1990

- - note. Med Sci

ning - A Review.

15

2. Forensic pathology of drowning

Drowning is a member of a general class of sudden violent death due to the rapid exclusion of oxygen from mitochondrial oxidative phosphoryla­ tion. This class of violent death is known as the rapid anoxias and encom­ pass several distinct physical and biochemical modes of inhibiting oxygen entry, transport and utilization by the body. The rapid anoxial deaths have traditionally represented the most difficult of all deaths encountered by the forensic pathologist for many reasons including: 1. The lack of pathognomonic postmortem findings in rapid anoxial

death.

2. Widespread misconceptions regarding the sensitivity and specificity of the classical postmortem findings associated with rapid anoxia. 3. The numerous mechanisms that can ultimately lead to acute anoxia are diverse and many are not associated clear associated with post­ mortem lesions (e.g., suffocation of an unconscious indiviudal or one incapable of defense). While drowning is considered a rapid anoxial process due to obstruction of the airway with inhaled fluid, numerous other biochemical 17

Forensic Diatomology and Drowning

alterations occur simultaneouly due to hemodilution. On this basis, drown­ ing is anoxial but associated with acute electrolyte changes. However, many of the pathophysiological process and postmortem findings associ­ ated with drowning may be observed in the other rapid anoxial processes since the final common pathway to death is similar.

* 1. PATHO PHYSIOLOGY OF ANOXIA Mechanisms of Anoxia

All acute anoxial processes ultimately lead to a deficiency in molecular oxygen flow to aerobic metabolism. The four distinct mechanisms that leads to this oxygen depleted state are: 1. Anoxic anoxia which occurs due to the reduction of oxygen passage through the alveolar-capillary barrier leading to decreased oxygen saturation of hemoglobin (e.g., obstruction of any level of the respira­ tory system). 2. Anemic anoxia which occurs due to a reduction in the amount of hemoglobin able to carrier oxygen in the erythrocyte (e.g., carbon monoxide displaces oxygen from hemoglobin). 3. Ischemic anoxia due to the inability to delivery oxyhemoglobin to the brain due to reduced arterial perfusion (e.g., sudden carotid occlusion or acute cardiac arrthymia). 4. Histotoxic anoxia due to chemical uncoupling of the electron trans­ port chain (e.g., cyanide ion binding to mitochondrial cytochrome subunits and inhibiting the flow electrons used for energy produc­ tion). The anemic and histotoxic anoxias are sometimes collectively referred to as the chemical anoxias since both mechanism require an exo­ geneous toxin. Similarly, anoxic and ischemic anoxia are due to mechani­ cal forces and, in many circumstances, both anoxic and ischemic mecha­ nisms are coupled as in strangulation with compression of the airway and the neck vasculature. The net result of any rapid anoxial process is cere­ bral anoxia which ultimately leads to ventricular asystole or fibrillation. 18

Pathology of Drowning

:-.: basis , drown­ ~ .::-.g s. However, - ~ -= ::":1dings associ­ - ;: -c 0xial processes .3

-

=- ~. :n molecular '. Te banisms that ::: _.>::'ge n passage _. c" 'ed oxygen _~ e ~ of the respira­ - 3ffiount of ~

e . .~ carbon

-.=r::.o _lobin to the ;;- : ?--a:id occlusion

-.: electron trans­ _ ...;,~ ~y tochrome . .. ;: -Lergy produc­

'"":"':':"''Ues collectively _rn require an exo­ _ .:.Ie due to mechani­ - :1 i chemic mecha­ . -- of [he airway and - _. _.....: process is cere­ :t::'.e or fibrillation.

Anoxic Encephalopathy

The main pathophysiological consequence of any rapid anoxial process is cerebral anoxia. The morphological correlates of cerebral anoxia are mainly related to the duration of the anoxic insult and the interval of survival after the insult. Thus, in rapid anoxial deaths, no structural alteration can be found in the brain in most cases. However, if there is a limited survival interval (e.g., in hospital after resuscitation) after the anoxic insult subtle changes can be observed in the brain. The distribu­ tion and types of cellular change found related to the length of the sur­ vival interval such that a minimum of 4-6 hours is required to demon­ strate unequivocal cellular features of anoxia. The earliest indicator of cerebral anoxia in selective neuronal necrosis in areas of the brain that are preferentially vulnerable to reduced oxygen tension. These areas include the pyramidal neurons of the cerebral cortex, and Ammon's horn (Sommer's sector). Early neu­ ronal necrosis is typified by nuclear hyperchromasia and pyknosis and the perikaryon is shrunken and eosinophilic (Fig. 2.1). Oedema of the neuropil often accompanies the earliest evidence of neuronal lysis. Purkinje cells of the cerebellum may show early evidence of neuronal necrosis but may appear uninjured even when hippocampal neuronal necrosis is well developed. As the survival interval lengthens, the appearance of neuronal necrosis appears more widespread and this is accompanied by generalized cerebral swelling and herniation mass effects due to cerebral oedema. Although all neuronal populations ulti­ mately show features of necrosis, the neurons of the basis pontis, and the cranial nerve nuclei may appear resistant after many hours of sur­ vival in the brain dead state. The aftermath of selective neuronal necrosis of the cerebral cor­ tex is a conspicuous pattern of laminar necrosis (Fig. 2.2). This corre­ sponds to local phagocytosis of the necrotic pyramidal cell layer, and often adjacent neuronal laminae, and may show microscopic evidence of astrogliosis and capillary proliferation. Laminar necrosis of the neo­ cortex is observed only after a prolonged survival interval, usually of several weeks, if somatic death does not occur in the period of maximal cerebral swelling. 19

Forensic Diatomology and Drowning

2.1. Necrotic neurons with pyknotic nuclei and oedema of the neuropil in hypox­ ic cerebral injury. Neuronal necrosis and oedema are the earliest microscopic lesions of cerebral anoxia and take hours to develop. No microscopic changes in the brain are found in rapid anoxial death. (Hematoxylin-eosin, 400 X) 2.2. Laminar necrosis of the cerebral cortex in a case of throttling with a survival interval of several weeks in hospital. The zonal loss of selectively vulnerable cor­ tical neurons gives a well demarcated line of necrosis .

20

Pathology of Drowning

* 2. THE TYPES OF ASPHYXIA Classification of Asphyxial Deaths

Asphyxial deaths represent a specific subset of the rapid anoxias and are defined as deaths due either to anoxic anoxia or ischemic anoxia or a com­ bination of the two mechanisms. The main categories of asphyxia are clas­ sified, thus: 1. Suffocation due either to obstruction of the mouth and/or nares, or

inhalation of oxygen-deficient gas.

2. Choking due to physical obstruction of the airway. 3. Pressure on the neck due to manual or ligature strangulation, hanging, or other modes of neck compression. 4. Positional asphyxia due to restriction of chest expansion during ven­

tialtion. This includes asphyxia of restraint and traumatic or crush

asphyxia.

5. Drowning. Pathology of Asphyxia and Rapid Anoxial Death

The lesions of asphyxia and rapid anoxia can be divided into three types of pathological changes : 1. Local physical injuries. 2. Lesions related to increased venous pressure. 3. The signs of rapid anoxial death (the "soft signs" of asphyxia).

:: :".::- j e. t microscopic _. .: opic changes in ' - ~ L -lOa X) -- : " ~D g

with a survival

-. dy \'ulnerable cor-

The local physical injuries, if present, are usually associated with the mechanical effects of producing the anoxic insult. The injuries in the later category are violent and usually relate to pressure on the neck, or suffoca­ tion. The paradigm violent asphyxial process is manual strangulation (1-3) that can be used to survey the lesions of asphyxia and pressure on the neck (Fig. 2.3-2.13). Manual pressure on the neck may produce superficial abrasion and/or contusion to the ventral aspect of the neck (Fig. 2.3). These superficial injuries can range from absent or minimal to florid, par­ ticularly if the victim's clothing is caught by the strangling hands and 21

Forensic Diatomology and Drowning

2.3-2.6. The Pathology of Manual Strangulation. 2.3 Multiple punctate abra­ sions of the ventral neck. 2.4. Fresh bruising of the strap muscles. 2.5 Submucosal laryngeal hemorrhages. 2.6 Bilaterally symmetric fractures of the greater horns of the hyoid bone.

22

Pathology of Drowning

: .Jhiple punctate abra­ "rrap muscles. 2.5 ccri fractures of the

• Z.9

2.7-2.10. Occult Laryngeal Injury in Strangulation. 2.7 & 2.S. Parasagittal sec­ tions of the superior cornu and associated cartilages of the larynx from a 21-year old female victim of manual strangulation (right) and a control case of a 21-year old female victim of an accidental heroin intoxication (left). There is focal hem­ orrhage (arrows) within the cartilage of the superior cornu in the victim of man­ ual strangulation. 2.9. Acute perivascular hemorrhage in the laryngeal cartilage. 2.10. Acute microfracture of the laryngeal cartilage. 23

Forensic Diatomology and Drowning

abrade the skin. Curvilinear abrasions may be present indicating fingernail marks, and rarely, digital pad pressure (mostly from a thumb) can lead to punctate contusions. Hemorrhage may occur in the soft tissues of the neck including the subcutis and platysema, strap muscles (mostly the ster­ nomastoid and stenohyoid), and about the carotid sheath (Fig. 2.4). Horizontal tears of the carotid artery intima may be found due to carotid manipulation and stretching. Due to the exposed location, the hyoid bone and larynx are sus­ ceptible to a range of injuries. Among these injuries is fracture which can be determined to be antemortem in the fresh body by the presence of adja­ cent hemorrhage. In some cases of manual strangulation, the hyoid bone will be symmetrically fractured and in other cases, one horn will be frac­ tured, usually on the side of the neck with maximal soft tissue injury. A remarkable range of local and remote indirect hemorrhages occur due to pressure on the neck. The mechanisms of these hemorrhages were investigated by Camps & Hunt in their landmark paper (1) "Pressure on the Neck". Pressure of the neck leads to two main types of indirect hemorrhages: local submucosal hemorrhages in the larynx (Fig. 2.5), pos­ terior pharynx, and pyriform sinuses; and remote petechieal hemorrhages of the cheeks, eyelids, conjunctivae, and serosal surfaces of the lungs and heart (Fig. 2.11-2.13). Local submucosal hemorrhages of the neck organs are perivenous and due to occlusion and rupture of the delicate laryn­ geopharyngeal venous plexus during strangulation. Petechieal hemor­ rhages of the conjunctivae, likely relate to increased jugular venous pres­ sure and subsequent rupture of the capillary bed. However, direct anoxia and release of endogenous mediators are also a possible contributing fac­ tor (4). The classical site of fracture to the larynx is the superior cornuae of the thyroid . cartilage. T his pattern of fracture is most commonly observed in manual strangulation and may be found in pressure of the neck from a carotid sleeper hold. Many victims of strangulation are you ng females with laryngeal cartilages that are not calcified and are, therefore, pliable and deformable in the process of manual strangulation. This leads to intracartilaginous laryngeal hemorrhages that the same location in the superior cornuae as fracture in older victims of strangulation (Fig. 2.7­ 2.10). Gross examination of the base of the superior cornuae may often 24

Pathology of Drowning _~ _

_

::ng fingernail can lead to

__""": I

- . .> :-tyoid bone

_:;: .\ ill be frac ­ .: e lOJury. _ - -: ' emorrhages .>-e'hemorrhages - _- -:- ( 1) "Pressure _: ;-es of indirect - _ Fig. 2.5), pos­ hemorrhages _ -=- , :' [be lungs and o

~

2.11-2.13. Petechial hemorrhages in Asphyxia. 2.11. Eyelid. 2.12. Conjunctiva. 2.13. Epicardium.

_; __" venous pres­ =- '~:-. direct anoxia , : ontributing fac­ • :: :Jperior cornuae :n t commonly :'. --ure of the neck

~.e

location in the ; , rion (Fig. 2.7­ - : ::mae may often 25

Forensic Diatomology and Drowning

show acute hemorrhage upon sectioning. Microscopic examination con­ firms the presence of acute perivascular hemorrhage that dissects between collagen fibres in the chrondroid matrix. Feature of strangulation or compression of the neck may be found in many cases of homicidal drowning. In cases of this type, drowning may occur after neck compression and death may due to the combined asphyx­ ial effects of drowning and strangulation.

Fluidity of the Blood Rapid anoxial death, such as strangulation and drowning, is often associ­ ated with a highly reproducible but poorly understood series of non-spe­ cific postmortem findings. These include postmortem fluidity of the blood, perimortem urinary and/or fecal incontinence, and in the decomposed body, the presence of controversial pink teeth. Persistent fluidity of the blood likely relates to inhibition of the coagulation process due to some unknown mechanism. Initial studies on the biochemical basis of the persistent fluidity of postmortem blood in rapid anoxia revealed endogeneous fibrinolysin aci­ tivity (5, 6). This has led to the proposal that the blood rapidly clots post­ mortem, and the fibrin is subsequently lysed producing fluid blood. However, using Western blotting for the detection of fibrinogen in the fluid blood of vicitms of drowning and strangulation, we have found that the fibrinogen is the blood is not depleted (Pollanen & Boyle, unpublished observations). This indicates that the persistent fluidity of asphyxial blood is due to failure of the blood to coagulate rather than the lysis of clotted blood.

* 3. PATHOPHYSIOLOGY OF DROWNING The physiological consequences of the inhalation of water have been stud­ ied in animal models (dogs, sheeps, and rabbits) of drowning and using meta-analysis of reports of drowning and near-drowning in humans (7). The physiological changes in drowning can be addressed in four major areas: 1. Blood gases and acid-base changes. 2. Ventilatory effects. 26

Pathology of Drowning

oination con­ ts between

_ . _. Crowning may - ~ c~b ined asphyx­

- :: brinolysin aci­ _ __ idly clots post­ __': :'1g fluid blood. - : ..ugen in the fluid 'e found that the o--:.le, unpublished •. phyxial blood ~e lysis of clotted

NG ,:-;?!' have been stud­ ___ wning and using :- -::g in humans (7). ':: _ d ·in four major

3. Changes in blood volume. 4. Changes in serum electrolytes. Experimental studies indicate that the lethal physiological changes in drowning is anoxia due to airway obstruction. Although some investiga­ tors have emphasized rapid alteration in serum electrolytes, this effect seems less important since profound disregualation in ion homeostasis is not an important feature of cases of near-drowning with a survival in hos­ pital. Analysis of oxygen tension in experimental models of drowning and near-drowning indicate that arterial hypoxemia (oxygen content and hemoglobin saturation) is the most rapid and physiologically important change after the air passages fill with water. In several different species (man, dog, sheep, and rabbits), profound changes in oxygen tension occur with inhalation of 1-3 mLlkg (7) . Hypercapnia occurs simultaneously and may lead to hyperventilation if the anoxia does not lead to a rapid anoxial death. Both metabolic and respiratory acidosis occur with the respiratory element predominating early and metabolic acidosis occuring with a sur­ vival interval. The principal intra-pulmonary consequences of drowning, which ultimately lead to hypoxemia, are airway obstruction and intrapulmonary shunting (V/Q mismatch) due to perfusion of non-ventilated parenchyma. This has been likened to a "space occupying lesion in the alveoli ". If sur­ vival occurs in a near-drowning, there may be delayed lung injury (e.g., diffuse alveolar injury, and bacterial pneumonia) that further comprimises lung function and can lead to ongoing respiratory acidosis, hypoxemia, and delayed death due to respiratory failure and sepsis. Massive and rapid shifts may occur in blood volume due the inhalation of a fluid medium into the alveoli since there is a highly per­ meable barrier between the alveolus and the capillary. Such shift in vol­ ume status can be monitored by alterations in otherwise stable electrolyte concentrations. In the case of freshwater drowning there is an increase in the systemic blood volume due to the influx of a hypotonic fluid (8). Similarly, in saltwater drowning there is an decrease in the systemic blood volume due to the influx of a hypertonic fluid. Such shifts can be moni­ tored and measured postmortem and this has been the rationale of the 27

Forensic Diatomology and Drowning

chemical based tests for drowning (discussed in chapter 3). In the case of drowning in a hypertonic medium (e.g., sea water) the reduction in the liq­ uid component of the blood results in increased osmolality and viscosity that may impair pulmonic perfusion and contribute to the VIQ mismatch that occurs due to alveolar distension with fluid. The magnitude of the change in the electrolyte concentrations that occur in sea water drowning (hypematremia and hyperchloremia) and freshwater drowning (hypona­ tremia, and hypochloremia) are usually not lethal.

* 4. PATHO LOGY OF FRESHWATER DROWNING Introd uction The pathology of freshwater drowning is complex and still poorly under­ stood. The main difficulty is that, in many cases, the pathological features of drowning are non-specific or grossly unimpressive. However, the descriptive pathology of drow ning has been possible because some cases of drowning show numerous distinctive and florid lesions. Why some cases of drowning have extensive gross changes (e.g., massive pulmonary oedema, frothy air passages, mastoid air cell hemorrhages) and others cases show minimal oedema of the lungs and no other features of drown­ ing is unknown. It is true that, arguably, all the morphological features of drowning are non-specific and can be potentially explained by other processes (i.e. oedema of the lungs is not specific for drowning). In the daily practice of forensic pathology, this is usually not problematic since circumstantial evidence provides additional information that may con­ tribute to the weight of the evidence for determining that death occured by drowning. However, since the lesions of drowning are problematic, ancil­ lary tests such as the diatom test have an important role in the medicole­ gal assessment of putative victims of drowning. The problem associated with the determination of death by drown­ ing is eloquently illustrated by famous case of The Man Who Never Was in which the tenuous nature of the morphological features of drowning are paramount. In 1942, intelligence officers Lieutenant-Commander Ewen Montagu and Flight-Lieutenant Charles Cholmondeley developed Operation Mincemeat to facilitate the Allied invasion of Scilly (9). Their plan was to plant a dead body ("Major Martin") into the Mediterranean

28

Pathology of Drowning ~~

.

:: . In the case of

~~_ ::jon

in the liq­ d viscosity _ -;: ·IQ mismatch I;ater drowning . :ning (hypona-

ING

_

. poorly under­ ~" _ .)gic al featu res

...:..:, -iYe pulmonary -:- ~e ) and others :e-.::ures of drown­ . . __ical features of ~ :-. 'ned by other ~:- .::-owning). In the _. "'-ublematic since ::-. (bat may con­ ~ I:..eath occured by .. :-:- . lematic, ancil­ . .. the medicole­ - _:-death by drown­ ,' }10 Ne ver Was in ~ _ f drowning are . -" .:' ~nm ander Ewen

~ - == :ey developed - .: : Scilly (9). Their :..-::: Mediterranean

Sea complete with documents indicating Allied plans for an invasion of Sardinia, contradictory to the real plan to infiltrate Scilly. The body was to land on the Spanish shore and the bogus document would bo transmitted to the Germans. The main diffculty according to Montagu was: "What would a postmortem reveal?". Montagu consulted Sir Bernard Spilsbury who indicated that there were no major pathological barriers to the plan, presumbly due to the difficulty in excluding drowning as a cause of death in a body recovered from the open sea. Furthermore, when Montagu obtained a suitable corpse to become "Major Martin" he learned that the individual had probably died of pneumonia. Sir Bernard indicated that .. .the pneumonia was a help, for there would tend to be some liquid in the lungs, as might well be the case if the man had died while floating in the rough sea. If a postmortem examination was made by someone who had formed the preconceived idea that the death was probably due to drown­ ing there was little likelihood that the difference between this liquid, in the lungs that had started to decompose, and sea water would be noticed.

Indeed, a postmortem examination in Spain concluded that death was due to drowning and the bogus documents were accepted as authentic. Postmortem findings in freshwater drowning include the lesions of drowning as well as the alterations that occur due to immersion and decomposition in water. Special importance can be placed on perimortem injuries that may indicate the events immediately preceding death.

Perimortem Injury and Events Several important perimortem injuries and changes can be observed in freshwater drowning . Some of these observations such as cutis anserina, ("gooseflesh" ) are no longer believed to have diagnositic utility. Among the most important posmortem indicators of live entry into water is cadav­ eric spasm in which the decedent may grasp at vegetation or clothing dur­ ing drowning and the articles may be tightly bound in the hand due to "instantaneous" rigor mortis. Abrasions of the face and nose may be found if there was a terminal collapse into the water. Other injuries may suggest homicidal drowning (e.g., evidence of neck injury or blunt force head injury).

29

Forensic Diatomology and Drowning

An important type of injury that infrequenly occurs in open water drowning is avulsion and hemorrhage into the muscles of the shoulder gir­ dle (Fig. 2.14). This is due to a violent struggie that may ensue in the drowning process were the decedent thrashes and forcibly extends the arms against clothing or stable objects in the water. The violent contrac­ tion results in local tearing of the muscle from the periosteum of the scapu­ la. The injuries must not confused with blunt impact injuries of the back that may occur in ligature strangulation using a posterior assault.

Water-related Alterations The main water-related change that occurs after several min utes of immer­ sion (approximately 20-30 minutes) is pallor and wrinkling of the skin of the soles of the feet and palms, so-called "washerwoman skin" (Fig. 2. 15). This change can also be found in the skin in other parts of the body but in the early stages is less conspicious. Water immersion may alter the appear­ ance of recent wounds of the skin including lacerations and incised wounds, resulting in pallor of the wound margin. In this case, local dis­ section of the wound may be necessary to determine if local subcutaneous hemorrhage is present indicating an antemortem inj ury. Similarly, post­ mortem wounding of a body floating in rocky water may occur and dis­ section will fail to reveal local hemorrhage. The distribution of lividity will be effected by the position in which the body floats . In some circumstances, lividity may be concentrated in the head and neck region, particularly if the body floats in the face down posi­ tion for several days. This position, along with aquatic decomposition, may result in boggy red discolouration of the subcutaneous and subgaleal tissues that must be differentiated from recent hemorrhage.

Air Passages The main lesion of freshwater drowning is found in the air passages. The classical finding is the presence of abundant stable white foam that emerges from the nose in a mushroom-like fashion (Fig. 2.16). Similar foam can be found in the mouth, and tracheobronchial tree. The foam is moderately resistant to decomposition, which may stain the foam a red­ brown colour. The stability of the foam is due to the presence of alveolar 30

Pathology of Dro wning

_ - _. in open water _ .e houlder gir­ ~ . ensue in the . ~-: ~ \' extends the

2.14. Stable foam emerging from the nose in freshwater drowning. Per­ imortem blunt force injuries to the bridge of the nose, lower lip, and chin are due to terminal collapse. 2.15. "Washerwomen feet" due to water immersion. The skin of the soles of the feet and palms most frequently show this effect of water.

~

he body but in _ alrer the appear­ ~ ns and incised . . - case, local dis­

: --0.

::~

2.14

2.16. Avulsive injury and fresh hemor­ rhage in shoulder girdle muscles in a case of suicidal freshwater drowning. Injuries of the shoulder girdle muscles are due to violent thrashing while drowning. (Case of Dr. D.A. Chiasson).

ition in which entrated in the ecomposition, - and subgaleal

-e . passages. The e white foam that F~ . 2.16). Similar ::ree. The foam is :-. me foam a red­ ~=::- ence of alveolar 31

Forensic Diatomology and Drowning

surfactant, which along with the drowning medium and transudative oede­ ma fluid, comprise the foam. The air passages may also contain water if large amounts of water has been inhaled in the drowning process. If the water contains particulate matter, this may be found in the fluid including fragments of vegetation, and gravel. However, water and particulate matter may enter the trachea of a dead body immersed in water particularly if decomposition is advancing in an aquatic enviroment. Therefore, the presence of water and and foreign matter in the decomposed body from water may not indicate drowning. It is often helpful to open the sphenoid sinuses in cases of putative drowning since the presence of fluid in this site may occur in the absence of fluid elsewhere in the respiratory tract. Watery-fluid from this site can also be sampled for diatom analysis if samples of the putative drowning medium are unavailable for comparison with diatoms extracted from tis­ sues. The stomach may contain large amounts of water since swallowing water often occurs in drowning prior to inhalation of water. However, the utility of watery-fluid in the stomach as a sole sign of drowning is prob­ lematic since the water may have been recently ingested some time prior to immersion. The diatoms of the fluid in the stomach may be useful in discriminating, if tap-water or freshwater was ingested, since the former usually lacks diatoms (10).

Lungs In the classical circumstance, the lungs in drowning are hyperinflated and may show indentation from ribs, and the medial edges of the lungs may meet a the centre of the mediastinium ("kissing lungs"). The main gross lesion of the lungs in freshwater drowning is oedema. The pulmonary oedema of drowning has been studied mostly by deterntining the weight of the lungs in case of drowning. In cases of freshwater drowning 90% of cases have lung weights between 500 and 1, 000 grams which is greater than lungs from non-drowning death that have hlllgS weights typically between 400 and 500 grams (11). UnfOltunately, there is overlap between the lung weight of the approximately 15% of drowning cases when com­ pared with non-drowning cases. In addition, the nonspecificity of pul­ monary oedema make the weight of the lung, as a sole indicator of drown­ ing, of lintited value. However, in cases of drowning with foam in the air

32

Pathology of Drowning

" - of water : - ~, - particulate " f vegetation, _--"'!' :he trachea of .: ;:: i5 advancing _- ~ j and foreign __-c: Jrow ning. ;: of putative ~.~

:hi s site can _..::: e drowning '-..:. :,ced from tis­ -. :e swallowing ii=

the former

-_ :--:. rerinflated and .__ ,~ [he lungs may ~_ . The main gross The pulmonary : ::-:- ' -. ing the weight -;: ':':0 \'ning 90% of ,- ,,-ruch is greater = ,,:;:' ghts typically '_ - :werlap between - ~ ;: ' -es when com­ -- __ ci ficity of pul­ e rr: '::cator of drown­ ,::--. 'oam in the air

passage the magnitude of the lung weights may be an important consider­ ation in determining if drowning occured. Clearly, the positive predictive value of pulmonary oedema increases with the weight of the lungs in the appropriate investigative setting. The microscopic fingings is freshwater drowning include emphysema aquaosum and oedema aquaosum (Fig. 2.17-2.20) (10). Emphysema aquao­ sum refers to the local or regional expansion of alveolar spaces and oedema aquaosum refers to proteinaceous-fluid in the alveoli. Both these findings are non-specific but may be extensive in some cases of drowning, usually the cases with impressive gross evidence of drowning and diatoms in the femoral bone marrow. It may be possible to find diatoms in routine tissue sections pre­ pared in cases of freshwater drowning (Fig. 2.21-2.24). Despite the rapid death, alveolar macrophages can occasionally be found to contain phagocy­ tosed diatoms (Fig. 2.20). There is some evidence that alveolar macrophages may be "washed out" of the lung in drowning, although this does not appear to be an important microscopic feature of the lung in drowning (12). A non-specific finding in many cases of drowning in stagnant pools of water, and "hot-tub" baths is advanced postmortem bacterial overgrowth in the terminal bronchioles and alveolar ducts. Bacteria-laden water innocu­ lates the distal airways and the bacterial proliferate in the early postmortem interval. Microscopic examination will reveal preferential bacterial over­ growth in the distal airways and little or no growth of bacteria elsewhere in the lung or in other organs. If an individual survives an episode of near-drowning only to suc­ cumb later in hospital, the lungs may show diffuse alveolar damage andlor bacterial pneumonia. The diffuse alveolar injury is typified by hyaline mem­ brane formation, and type II pneumocyte proliferation and may show exten­ sive organization and interstital fibrosis if the survival interval is prolonged. In this circumstance, there is usually accompanying anoxic encephalopathy. The bacterial pneumonia is likely due to bacteria introduced into the lung by drowning. The pattern is usually diffuse rather than preferentially in the lower lobes.

Pleural effusion The presence of fluid in the chest cavities is variable in freshwater drown­ ing, and is likely due to diffusion of inhaled fluid from the lungs. In the 33

Forensic Diatomology and Drowning

2.17-2.20. Microscopic Features of Freshwater Drowning. 2.17. Emphysema aquao­ sum (250 X). 2.18. Oedema aquaosum (250 X). 2.19. Localized bacterial over growth in lung (400 X). 2.20 Alveolar macrophage containing a centric diatom-like particle (1,000 X) (Haematoxlyin-eosin).

34

Pathology of Drowning

·r .

2.24

:="'l1pbysema aquao­ .: :- ~:erial over growth ~ ~ ::'~:llOm-like particle

'{y :',;

2.21-2.24. Diatoms in Lung. 2.21-2.23. Diatom frustules in alveolar air spaces (Haematoxlyin-eosin, 1,000 X). 2.24. Diatom frustule at alveolar wall (Toluidine blue, 1,000 X).

35

Forensic Diatomology and Drowning

fresh state, the fluid is serous or slightly serosanginous. H owever, as decomposition precedes, decompositional fluid may appear in the chest cavities that may be confused with the pleural effusion of drowning. Hemorrhage in the Petrous Temporal Bone

Often the air cells in the petrous portion of the temporal bone will show extensive congestion and hemorrhage in cases offreshwater drowning ( 13) (Fig. 2.25). When the lesion is present it is conspicious although there are no studies on the sensitivity and specificity of the lesion for drowning. Florid congestion and hemorrhage into the temporal bone can also be observed in ligature strangulation presumably due to jugular venous obstruction. However, a similar appearance of the temporal bone may also be observed in non-violent deaths particuarly if the position of the head causes lividity at the skull base. The diagnostic significance of this find­ ing, although associated with drowning is uncertain at present.

Visceral Organs The main parenchymal organs that have been studed in drowning are the heart and spleen. No specific lesions of the myocardium or the spleen have been found in drowning. However, there may be contraction banding in cardiac myocytes in cases of drowning with significant atherosclerotic coronary artery disease (14). The spleen in freshwater drowning may be slightly reduced in size, although the significance of this observation is not known (15).

* 5. PINK TEETH The History of Pink Teeth

The subject of postmortem pink or red colouration of the teeth has been long investigated in the scientific literature and was first described by Bell in 1829 (16): I have frequently examined the teeth of persons whose death has been occasioned by hanging or drowning, and invariably found the whole of the osseus part coloured with a dull deep red, which could not possibly have been the case if these structures were devoid of a vascular system.

36

Pathology of Drowning

_

. ;.!owever, as in the chest

, 3r

- ::>!1e will show .;'c" Jrow ning (13) _ -'")r drowning.

also be .. .:ular venous :' ne may also _""",",""'n_,n .. of the head .-e of this fi nd­ _ -tC ~an

_~0\\'n ing

are the c :: :he spleen have _:on banding in

2.25 Mastoid air cell hemorrhage in freshwater drowning.

_ -ervation is not

~ =-.e

teeth has been : ':e cribed by Bell

- . ossibly have

37

Forensic Diatomology and Drowning

Since the observations of Bell, several reseachers have studied the mech­ anism and frequency of pink teeth in bodies that undergo medicolegal postmortem examination (17-23). The most famous series of cases were the victims in the Christie case of 1953 (19,23,24). During the investiga­ tion of the case, after the accused confessed to the murders , the bodies of two of his victims were exhumed. At the original postmortem examination (about 3 years prior to exhumation), the cause of death for both victim was ligature strangulation without mention of pink teeth. At exuhrnation, pink teeth were found in one vicitm. No carboxyhemoglobin was detected and spectroscopic and and the benzidine reaction suggested that the pigment was derived from hemoglobin. The Christie case initiated debate over the sensitivity and specificity of pink teeth for asphyxial modes of death. The chemical composition of the pink pigment is now well characterized and likely originates from hemogloblin or proteolytic fragments of hemoglo­ bin. This has been confirmed by direct biochemical characterization of the pigment using spectrophometry, histochemistry, and thin layer paper chro­ matography (25-28) . Although postmortem pink teeth have been described in hanging, barbituate toxicity, carbon monoxide toxicity, and strangulation, the most consistent association occurs with drowning (24). In addition, pink teeth are not associated with freshly drowned bodies but usually occur after 1-2 weeks of aquatic decomposition. A critical examination of the forensic lit­ erature on postmortem pink death indicates that there are two possible and non-mutually exclusive mechanisms to form pink teeth. The prerequisites for the genesis of pink teeth include: 1. Congestion and extravestation of blood in the tooth pulp. 2. Decomposition - particularly in a moist or aquatic environment. The first prerequisite occurs in two major circumstances, either through a congestive mode of death such as occurs in strangulation (mostly ligature strangulation as in the Chrisite case) or when lividity accumulates in the head due to positioning of the body (as in aquatic decomposition) . In the circumstance of strangulation, the blood vessels in the pulp presumably rupture and spill blood pigments that subsequently stain the tooth after decomposition. In the case of lividity of the tooth, decomposition and 38

Pathology of Drowning ~c:ed

the mech­

. --:e:::: examination =,m victim was _bnation, pink - detected and

- - =...Jr'on, the most _22 :.on pink teeth occur after 1-2 - _.:: ~: e forensic lit­ ': _,' 0 possible and ..e prerequisites

hemolysis may give the same net effect without initial antemortem con­ gestion. On this basis, the presence of pink teeth is less an indicator of the cause of death but rather, in some cases, the mechanism of death and the effects of decomposition on the congested tooth pulp.

The Significance of Pink Teeth Pink teeth are not pathognomonic for a specific cause of the death. Although initial studies showed a consistent association with unnatural death and drowning, it is now appearant that other causes of death (e.g., gun shot wounds, explosion, natural causes) can also be associated with the pink teeth if decomposition occurs in the appropriate environment. However, the initiating event of pink teeth formation in an individual case may be related to a congestive mode of death such as strangulation. In such as circumstance, postmortem pink teeth relate to the mechanism of death despite the fact that no definitive conclusion can be made about the presence of pink colouration of the teeth. Pink teeth in drowning likely relates more to aquatic decomposi­ tion with congestion of the tooth pulp associated with drowning or float­ ing in the head down position (i.e. lividity). Therefore, pink teeth are not definitive for drowning but, rather, are part of the spectrum of changes that occur in aquatic decomposition.

* 6. AQUATI C DECOMPOSITION Aqu atic Ta phonomic Factors

·!Unment. __ . either through a . _. mostly ligature .:.:' 1ffiulates in the '::~. sition). In the :: ul p presumably 2~. he tooth after ':c' mposition and

The progressive dissolution of the body in water is due to the action of sev­ eral independent degradative forces , termed taphonomic factors (29-33) . The factors include both intrinsic properties of the body itself and envi­ ronmental factors such as water temperature, the presence of aquatic scav­ engers , the action of currents, tides, and rocks in the water, and depth of the water. Aquatic decomposition is a dynamic process that does not fol­ low a specific timetable and can be slowed or arrested by changes in water temperature that occur with seasonal variation or with depth of submer­ sion. Since there is an inverse correlation of depth with water temperature, bodies exposed to cold deep water for several years may show only mod­ erate decomposition. 39

Forensic Diatomology and Drowning

Putrefaction in water may lead to dismemberment of the body due to the action of currents, boat propellors, and canal locks. Unlike terrestri­ al decomposition, animal scavenging is not a major taphonomic factor for aquatic putrefaction since most marine saprophytes do not dismember. However, the action of crabs, shrimps, and other crustaceans may produce localized marks of the fleshy parts of the face that must be distinguished from antemortem injury. It is not uncommon that decomposed body in water is fragmented or artefactually disrupted upon recovery from the water. Such postmortem injuries must be distinguished from antemortem wounds. One pruticularly common artefact that occurs on bodies recovered from water is the impres­ sion of metal mesh of the stretchers that rescuers may use to transport the body to the mortuary. This mesh leaves a distinctive impression on the skin especailly on the face if the body is prone. These postmortem pressure marks must not be confused with antemortem injuries. Decomposition Sequence in Freshwater

Aquatic decomposition, like decomposition on land, progresses through a well-defines series of stages (30-32) (Fig. 2.26-2.29). However, the rate of progression through the various stages is too variable to be used as a defin­ itive guide to determine the time interval between deathlimmerison and discovery of the body. Several variables control the rate of progression through the decompositional sequence, including, but not limited to, water temperature, body composition, whether the body is floating or complete­ ly submerged, and the influence of natural forces such as tides. In general the sequence progresses from a fresh body to fragmented skeletonized body over a period of months but may take several years. An "average" decomposition sequence starting with a fresh body commences with rigor, and livor mortis, and early discolouration of skin (0-2 days). Early decompsition (2-7 days), results in extensive discoloura­ tion, bloating, and early putrefaction of organs. After 1 week to 2 month there is advanced decomposition with the onset of adipocere formation, fragmentation, and extensive putrefaction. From months to years the body may become skeletonized with exposure of bones, and encasement by adipocere.

40

Pathology of Drowning

: L:- the body due . '__ nlike terrestri­

dismember. _"'.:..:)- may produce - .~ distinguished

_ . ..ut

_::.c~:

the impres­ .;: :) Iransport the _:: _-ion on the skin

.:_~:' unmerison and

_ . _:~ of progression

- _: imited to, water

- : _:..ing or complete­ · - _. -ides. In general

· ~~. :ed skeletonized

~~

. :th a fresh body · _. _.ouration of skin :en ive discoloura­ - _ ·.\·eek to 2 month ere formation , - .0 years the body - ~ encasement by

2.26-2.29. Stages of decomposition in freshwater. 2.26. Hair loss, skin slippage, bloating, and green-white discolouration of skin. 2.27. Early water-related changes of the skin with slippage and pre-adipocere fOImation. 2.28. Extensive water denudation of muscle and soft tissue with exposure of femur. 2.29. Adipocere fonnation of the entire body (inset, close-up of adipocere). 41

Forensic Diatomology and Drowning

Adipocere Adipocere ("grave-wax") is a white-waxy substance that is the product of the hydolysis and hydrogenation of fatty acid (e.g., oleic acid) in the adi­ pose tissue of dead bodies (34) (Fig. 2.29, inset). Adipocere fonus during decomposition in an aquatic or moist humid environment and is remark­ ably stable once formed. In the early stages, the hydrolysed adipose tissue of the subcutis may herniate through adnexal pores in the skin and hard­ ens as the chemical process of adipocere formation continues. The result is that the skin is encased in a waxy substance that may harden as a solid protective layer for the long bones and organs. Adipocere may preserve a corpse for years in the water. The presence of a protective layer of adipocere on the leg makes the femoral bone marrow an excellent choice for the starting material to extract diatoms in decomposed bodies found in water. The mechanism and temporal seqence of adipocere formation has been the topic of extensive study (35-39). Adipocere formation usually takes about 2 months, but is highly variable and cannot be used as a reli­ able indicator of the time of submersion. Water temperature seems to be a key factor in adipocere formation, and immersion in a cool medium may prolong adipocere formation for one year or longer (35). The biochemical pathway to adipocere from lipid is complex but appears to involve the action of bacterial catalysis by both anaerobic or aerobic microbes (36) (e.g., Micrococcus luteus) .

* 7. REFERENCES 1. Camps FE Hunt, A.c. Pressure on the Neck. Journal of Forensic Medicine 1959;6:116-135. 2. Gonzales TA. Manual strangulation . Archives of Pathology 1933;15 :55-66. 3. Luke JL. Strangulation as a method of homicide. Archives of Pathology 1967 ;83 :64-70. 4. Jaffe FA. Petechial hemorrhages: A review of pathogenesis. American Journal of Foresnic Medicine and Pathology 1994;15(3):203-207. 5. Takeichi S Wakasugi, c., Shitkata, 1. Fluidity of cadaveric blood after 42

Pathology of Drowning

~':e

,orms during ~e::.: and is remark­ . .:. c adipose tissue - ~ skin and hard­

:001 medium may _- :-he biochemical ..:.:~ to involve the - _: - microbes (36)

= _:TI

e.:.

I of Forensic of Pathology

,... ~: ': e . Archives of

.e ~~e' : . American

- _: : :_03-207. _~. eric blood after

sudden death: Part II. Am J Forensic Med Pathol 1985;6:25-29. 6. Mizutani Y Yamada, T., Sugie, 1. Studies on the fluidity of cadaveric blood in sudden death: Fluid blood in the cadaver from the view point of clotting function. Jpn J Legal Med 1978;32:277-284. 7. Modell l H. The pathophysiology and tretment of drowning and near drowning. Springfield, IL: Charles C. Thomas Publishers, 1971 8. Gettler AO. A method for the determination of death by drowning. 1. Amer. Med. Assoc. 1921 ;77: 1650-1652. 9. Montagu E. The Man Who Never Was. (2nd edition (first edition, 1953) ed.) Great Britian: Oxford University Press, 1996 10. Polson CJ Gee, DJ., Knight, B. Drowning. In: The Essentials of Forensic Medicine. Oxford: Pergamon Press, 1985: 11. Copeland AR Assessment of lungs weights in drowning cases. American Journal of Forensic Medicine and Pathology 1985;6:301­ 304. 12. Betz P Nerlich, A., Penning, R., Eisenmenger, W. Alveolar macrophages and the diagnosis of drowning. Forensic Science International 1993;62:217-224. 13. Mueller WF. Pathology of temporal bone hemonhage in drowning. Jounral of Forensic Sciences 1969;14(3):327-336. 14. Lunt DWR Rose, A.G. Pathology of the human heart in drowning . Archives of Pathology and Laboratory Medicine 1987; 111 :939-942. 15. Haffner HT Graw, M., Erdeikamp, J. Spleen findings in drowning. Forensic Science International 1994;66:95-104. 16. Bell T. Anatomy, physiology and disease of teeth.London: Stewart and Company, 1829:9-17. ; vol 1829). 17. Beeley JA. Pink teeth appearing as a post-mortem phenomenon. Journal of the Forensic Science Society 1973;13:297-305. 18. Kirkham WR Andrews, E.E., Snow,C.c., Grape, P.M., Synder, L.. Postmortem pink teeth. J Forensic Sci 1977;22: 119-13l. 19. Whittaker DK Thomas, v.c., Thomas, R1.M. Post-mortem pigmenta­ tion of teeth. Br Dent J 1976;3:100-102. 20. Brondum N Simonsen, J. Postmortem red coloration of teeth. Forensic Odontology 1987;8(2):127-130. 2l. Miles AEW Fearnhead, RW. Post-mortem color changes in teeth. Journal of Dental Research 1954;33:735. 43

Forensic Diatomology and Drowning

22. Brondum N Simonsen, l Postmortem red coloration of teeth. A retro­ spective investigation of 26 c ases . A m J Forensic M ed Pathol 1987;8: 127- 130. 23. Borrman H Du C hesne, A., Brinkmann, B. M edico-legal aspects of postmortem pi nk teeth. Int 1 Leg M ed 1994 ;106:225-231. 24. Brondum N Simonsen , l Postmortem red colouration of teeth . American 10urnal of Forensic M edicine an d Pathology 1987;8(2): 127­ 130. 25. Van Wyk CWo Postmorte m pink teeth : in vitro production. J Oral Pathol Med 1988; 17 :56,8-572. 26. Sainio P Syrjanen, S., Kellala, l, Parvianen, A. Postmortem pink teeth phenomenon : an experimental study and survey of the literature. Proc Finn Dent Soc 1990;86:29-35. 27. lk:eda N H arada, A, Takahashi, H ., Suzuki, T. Experimental formation of pink teeth and their analysis. 1pn 1 Legal M ed 1988;42: 179- 183. 28. Van Wyk CWo Postmortem pink teeth: H istochemical identification of the causative pigment. Am J Forensic Med Pathol 1989;10:1 34-139. 29. N awrocki SP Pless, lE., Hawley, D.A. , Wagner, S.A Fluid transport of h uman crania. In: H aglund WD Sorg, M.H ., ed. Forensic Taphonomy: T he postmortem fate of human remains. New York: C RC Press, 1997: 529-552. 30. B rooks S Brooks, R.H . The taphonomic effects of flood waters on bone. In: Haglun WD Sorg, M .H. , ed. Forensic Taphonomy:The post­ mortem fate of human remains. New York: e Re Press, 1997: 553-558 . 31. B oyle S Galloway, A , M ason, R. T. Human aquatic taphonomy in the M onterey Bay area. In: Haglund W D Sorg, M .H ., ed. Forensic Taphonomy: The postmortem fate of human remains. New York: e Re Press, 1997: 605-613. 32. Sorg MH Dearborn, l H ., Monahan, E.I., Ryan, H.P., Sweeney,K.G., David, E. Forensic taphonomy in marine contexts. In: Haglund W D Sorg, M .H., ed. Forensic Taphonomy:The postmortem fate of human remains. New York: CRC Press, 1997: 567-604. 33. O'Brien TG. Movement of bodies into Lake Ontario. In : Haglung WD Sorg, M .H., ed. Forensic Taphonomy:The postmortem fate of human remains. New York: eRe Press, 1997: 559-565. 34. Mant AK Furbank, R. Adipocere - A review. 10urnal of Forensic 44

Pathology of Drowning

:- :eeth. A retro­ :: \1ed Pathot

.'- ~ m pink teeth . ~: [erature. Proc - ..ental formation .. :.+2:179-183. . _ _dentification of :: 9: 10:134- 139. .:: .A. Fluid transport R .. d . Forensic - - _-ew York: eRe

Medicine 1957;4(1):18-35. 35. Mellen PFM Lowry, M.A., Micozzi, M.S. Experimental observations on adipocere formation. J Forensic Sci 1993;38:91-93. 36. Takatori T Gotouda, H. , Terazawa,K., Mizukami,K., Nagao, M. The mechanism of experimental adipocere formation: substrate specificity on microbial production of hydroxy and oxo fatty acids. Forensic Sciences International 1987;35:277-281. 37. Gotouda A Takatori, T. , Terazawa, K., Nagao, M. Tarao, H. Experimental adipocere formation: Hydration and dehydration in microbial synthesis of hydroxy- and oxo- fatty acids. Forensic Science International 1988;37:249-255 . 38. Takatori T Yamaoka, A. The mechanism of adipocere formation: II Separation and identification of oxo fatty acids in adipocere. Forensic Science 1977;10:117-125. 39. Takatori T Yamaoka, A. The mechanism of adipocere formation. I Identification and chemical properties of hydroy fatty acid in adipocere. Forensic Sciences 1977;9:63-73 .

~:-

flood waters on - _:- ..onomy:The post­ 1997: 553-558 . . - :aphonomy in the _.H.. ed. Forensic :: '\"ew York: eRe

-

=--.

...: ?.. Sweeney,K.G., . In: Haglund WD _~e!TI

fate of human

..:-:; b : Haglung WD ~ -:-:=:::-. fate of human ~ _ ~al

of Forensic 45

3. The medicolegal value of the diatom test for drowning

* 1. ANALYTICAL APPROACHES TO DROWNING Laboratory approaches to the postmortem diagnosis of drowning can be divided into two basic procedures: the chemical detection of a change in blood volume status due to drowning or the detection of aquatic micro­ organisms in the greater circulation or organs. The chemical tests depend on the flu id component of drowning medium, whereas the other method depends on particulate components. The common theme in both approach­ es is to examine a parameter in the body that changes after drowning medi­ um enters the lung and blood. The chemical tests for drowning are largely historical and not widely practiced. The principle reason for this is the large variability in postmortem electrolyte concentration that occurs as a function of the postmortem interval. While the micro-organism based tests are more widely used, the aquatic micro-organsim of choice is the diatom, rather than other aquatic organisms such as protozoa, dinoflagellates, or invertebrates. The preference for diatoms relates to technical considera­ tions and the postmortem stability of the diatom in human tissue. Chemical Tests for Drowning The basis of most chemical tests for drowning is that fluid in the drown­ ing medium passes into the left sided heart blood via the alveolar-capillary

47

Forensic Diatomology and Drowning

barrier resulting in hemodilution if the fluid is hypotonic (freshwater) or hemoconcentration if the fluid is hypertonic (salt-water). Most investiga­ tors have studied hemodilution due to freshwater drowning. In principle, drowning-associated hemodilution can be measured by the concentration of a solute in the left heart blood as compared with the right heart blood, thereby determining the magnitude of hemodilution using the equation, (SR - SL) / SR' where SR is the solute concentration in the right heart blood and SL is the solute concentration in the left heart blood. Most investiga­ tors have directly measured electrolyte concentration and calculated the hemodilution factor or used freezing-point determination of the osmolali­ ty of the plasma (1-4) . Popular variations on the hemodilution test are to assay the electrolyte concentration of other fluid compartments that change with blood volume and electrolyte alterations including the cere­ brospinal fluid and the vitreous humor. The first ion used to assay hemod­ ilution was chloride as described by Gettler in 1921 (5). Unlike potassium and other plasma ions, the chloride concentration of postmortem blood is relatively stable and can, in some cases, be used as a index of hemodilu­ tion in freshwater drowning. In addition, when drowning occurs in a hypertonic medium such as salt-water, hemoconcentration of the left heart blood may increase the chloride concentration. Similarly, hypertonicity of the drowning medium may lead to pleural effusion that has a chloride con­ centration greater than that of normal plasma (6). Unfortunately, the post­ mortem concentration of all plasma ions changes with postmortrem inter­ val such that the values obtained in drowning and non-drowning cases overlap too extensively to be useful in those cases of drowning that have values in the borderzone. An important and promising variant of the hemodilution test is the quantification of blood strontium levels in cases of drowning (7-9). Strontium is a trace metal that is found in relative abundance in the crust of the earth. On this basis, strontium is widespread in sea-water and, to a lesser extent, other naturally-occurring bodies of water and even domestic water. Since strontium has a low concentration in the plasma, the addition of minute amounts of strontium into the blood during drowning may lead to relatively large increases in the strontium concentration in the blood. The level of strontium in drowning cases is dependent on the strontium content of the water; salt-water drownings have higher strontium levels 48

Medicolegal Value of the Diatom Test

~

, ~ o- t

investiga­ - "' ~ . In principle,

Micro-organism Tests for Drowning

_::on test are to _ :: . " artments that - ':,JJing the cere­ _ ~ _:0 say hemod­ : ~':-. :ik:e potassium :- .:: .:: ·~ ortem blood is _ - ':eX of hemodilu­ ~,-=--1] g occurs in a .:: ­ of the left heart __ . . !:lypertonicity of :::. a chloride con­ ~ :, _,Eately, the post­ [[u ortrem inter­ ~ ~ -drowning cases - ~(" w ning that have ~

than freshwater drownings. Therefore, strontium analysis may be useful for the diagnosis of drowning particularly in the salt-water environment. One of the limitiations of the test is that the detectable strontium is corre­ lated with the length of time of aquatic decomposition, making the test of questionable value in decomposed bodies recovered from water.

:- . .j it tion test is the

_~ . owning (7-9). __-:.:a.nce in the crust - _'"_-\ ater and, to a ~ ... -=d even domestic :.. . _ -ma, the addition =- .: ~ 'Ding may lead _', n in the blood. _ - r ::':1 the strontium _ ~: 5 ~ rontium levels

The introduction of drowning medium into the pulmonary capillary bed allows both the fluid and particulate components of the medium to enter the circulatory system. This particulate matter ranges from micro-organ­ sims to amorp hous inorganic debris (e.g. microscopic sand grains). The presence of micro-organisms in the lung parenchyma, tracheobronchial tree, and circulation and its organs has been extensively studied, mostly in the context of diatoms. However, diatoms are not the only aquatic organ­ isms detected in the tissues of the the drowned. Other important organisms include chlorophyta, dinoflagellates, invertebrates such as Daphnia, and the protozoans including the ciliate Tetrahymena (1 0-18). In the case of Tetrahymena, the ciliate can be propagated in culture indicating that the organism entered the body alive during the drowning process (19). In one study, the presence of diatoms in the left atrial blood was also correlated with the presence of alveolar macrophages with anthracotic pigment indi­ cating the point of origin of the diatoms (18). In theory, the presence of any aquatic micro-organism derived from the drowning medium in the tissues of a drowning victim is sufficient to prove drowning. However, diatoms are the preferred organism for detection in the laboratory. This relates to several important features of the diatom including the stablity of the siliconiferous exoskeleton (frustule) and the abundance of diatoms in bodies of water. T he stability of the diatom allows relatively easy extraction from postmortem tissues. Other organisms such as the ciliates, and dinoflagellates have delicate cell membranes that cannot resist the harsh extraction procedures usually employed.

* 2. THE SO-CALLED "WA R-ON-DIATOMS" Spitz has referred to the debate on the validity of the diatom test for drowning as the "war-on-diatoms" (20). Before discussion of the scientif­ 49

Forensic DiatomoLogy and Drowning

ic aspects of research on diatoms and drowning, it is useful to understand the nature of the debate and how this debate emerged. The "war-on­ diatoms" appears to be that medicolegal facilities in Europe more fre­ quently utilize the test than in North America. This may be due, in part, to the origination of the test in Europe and the relatively minor contribution of American researchers to the scientific aspects of diatoms and drowning. In addition, many medical examiner's facilities in the United States lack appropriate laboratory facilities to extract diatom frustules from tissue, whereas many European departments of forensic pathology are housed in institutes of legal medicine or Universities. Despite the lack of the widespread use of the diatom test in the United States, the current state-of-the art assessment on the medicolegal significance of the diatom test for drowning is best summarized by the American forensic pathologists DiMaio and D iMaio in their standard text­ book on forensic pathology : The material , such as bone marrow, is digested in concentrated acid. The deposit is examined with a standard microscope for the presence of the diatoms. The water in which the individual has allegedly drowned is sam­ pled to see what type of diatoms are present and a comparison is made between that in the water and that foun d in the body. While a positive com­ parison is helpful, a negative result does not rule out drown ing. Even a completely negative analysis for diatoms does not rule out drowni ng ... Di Maio DJ, Di Maio VJM. Forensic Pathology, Elsevier, New York, pg 363-364, 2nd edition, 1993. The view of DiMaio and DiMaio is not unique among authorities in foren­ sic pathology; many classical texts of forensic medicine state similar views on the di atom test. M any of these books are considered the classical textbooks of forensic medicine. Professor Keith Simpson, of the Home Office in the U nited Kingdom, supported the use the diatom test in cases of drowning and advocated the use of bone marrow or brain as the starting material for extraction. In "Taylors Principles and Practice of Medical Jurisprudence", he wrote: Only the live body with a circulation could transport diatoms from the lung to the brain or marrow. Acid digestion does, in fact, reveal a remark­ 50

Medicolegal Value of the Diatom Test :- ~: 0

understand . The "war-on­ - _rope more fre­ _ ~ ue, in part, to rn.:..::or contribution _-- - and drowning. '_'::::ired States lack __:es from tissue, - ;. =. - are housed in r'-'"

Eight years later, after further scientific research was published, he sum­ marized his views on the test as follows:

~

atom test in the the medicolegal ... ~arized by the :.:,.cir standard text­

:.=nrrated acid . The -~ presence of the . _fow ned is sam­ • _ T"'. arison is made e positive com­

O\-vning. Even a

:: __ _drowning ...

~ . New York, pg

= _.J :borities in foren­ '::.ne state similar ,... :..ered the classical

in the United _ of drowning and r:ing material for : _ -:1 Ju risprudence",

-::

able collection of most delicate and beautiful diatomaceous bodies in brain, marrow and other specimens from cases of drowning--as distinct from immersion when dead, and the finding, if not absolutely diagnostic is of the greatest value. The balance of probabilities is, in our experience strongly weighted in favour of immersion in life when diatoms are found in the brain and bone marrow. Simpson K. Taylors Principles and Practice of Medical Jurisprudence, 12th edition, J & A Churchill Ltd., pg 377, 1965.

~ ro ms fro m the . =-e\eal a remark-

... microscopic diatomaceous matter rapidly enters the circulation in a live victim of drowning. Microscopy of spun acid digests of brain and bone marrow tissues will often provide this laboratory proof of immersion alive. A dead body thrown into the river shows no such changes, though water may run into the dead throat or upper air passages ... Simpson K. Forensic Medicine, 6th edition Edward Arnold publishers, pg 92, 1972.

Professor Francis E. Camps was also generally supportive of the reliabili­ ty of the diatom test: Whilst the tests are not necessarily conclusive, they provide reliable sup­

portive evidence. Tests are made on all enclosed organs, skeletal muscle

and femoral bone marrow.

Camps FE. Gradwohls Legal Medicine, 3rd edition, Bristol: John Wright

& Sons Ltd., pg 353, 1976.

In one of the most influencial texts in forensic medicine, Polson, Gee and Knight expressed the view that the diatom test had particular value as a confirmatory test for drowning: The demonstration of plankton and, more especially, diatoms in the sub­ merging fluid and in the body of the victim, is of particular value as a con­ firmatory test. The test material is treated by acid digestion as a prelimi­ nary step in diatom analysis. In the case of decomposed bodies, the demonstration of diatoms in enclosed organs and especially in the bone marrow, due care being taken to exclude contamination, is strong if not conclusive evidence of death by drowning . 51

Forensic Diatomology and Drowning

ic aspects of research on diatoms and drowning, it is useful to understand the nature of the debate and how this debate emerged. The "war-on­ diatoms" appears to be that medicolegal facilities in Europe more fre­ quently utilize the test than in North America. This may be due, in part, to the origination of the test in Europe and the relatively minor contribution of American researchers to the scientific aspects of diatoms and drowning. In addition, many medical examiner's facilities in the United States lack appropriate laboratory facilities to extract diatom frustules from tissue, whereas many European departments of forensic pathology are housed in institutes of legal medicine or Universities. Despite the lack of the widespread use of the diatom test in the United States, the current state-of-the art assessment on the medicolegal signific ance of the diatom test for drow ning is best summarized by the American forensic pathologists D iMaio and D iM aio in their standard te xt­ book on forensic pathology: The material , such as bone marrow, is digested in concentrated acid. The deposit is examined with a standard microscope for the presence of the diatoms. The water in which the individual has allegedly drowned is sam­ pled to see what type of diatoms are present and a comparison is made between that in the water and that found in the body. While a positive com­ parison is helpful, a negative result does not rule out drowning. Even a completely negative analysis for diatoms does not rule out drowning ... Di Maio DJ, Di Maio VJM . Forensic Pathology, Elsevier, New York, pg 363-364, 2nd edition, 1993. T he view of DiM aio and D iM aio is not unique among authorities in foren­ sic pathology ; many classical texts of forensic medicine state similar views on the diatom test. Many of these books are considered the classical textbooks of forensic medicine. Professor Keith Simpson, of the Home Office in the United Ki ngdom, supported the use the diatom test in cases of drowning and advocated the use of bone marrow or brain as the starting material for extraction. In "Taylors Principles and Practice of M edical Jurisprudence", he wrote : Only the live body with a circulation could transport diatoms from the lung to the brain or marrow. Acid digestion does, in fact , reveal a remark­ 50

Medicolegal Value of the Diatom Test

efu l to understand _e rg d. The "war-on­ ~~ in Europe more fre­ . may be due, in part, to .- eol\" m inor contribution '- ~ '::atoms and drowning. -: :b.c United States lack J

able collection of most delicate and beautiful diatomaceous bodies in brain, marrow and other specimens from cases of drowning--as distinct from immersion when dead, and the finding, if not absolutely diagnostic is of the greatest value. The balance of probabilities is, in our experience strongly weighted in favour of immersion in life when diatoms are found in the brain and bone marrow. Simpson K. Taylors Principles and Practice of Medical Jurisprudence. 12th edition, J & A Churchill Ltd., pg 377, 1965.

Eight years later, after further scientific research was published, he marized his views on the test as follows:

--

~'0r

the presence of the . gedl. drowned is sam­ ~ _ omparison is made '- -...... Wh ile a positive com­ ~_ ;: out drowning. E ven a -, : rule out drowning ... _ • E:se\'ier, New York, pg

=,ng authorities in foren­ . -:. ~ dicine state similar ...I:: 2 0n idered the classical -" Office in the United - , es of drowning and :~.e tarting material for _: _ edical Jurisprudence",

-;:)rt diatoms from the

_ . fac t, reveal a remark-

u~ ­

...microscopic diatomaceous matter rapidly enters the circulation in a Ii\"t~

victim of drowning. Microscopy of spun acid digests of brain and bon

man'ow tissues will often provide this laboratory proof of immersion ali ve.

A dead body thrown into the river shows no such changes, though water

may run into the dead throat or upper air passages ...

Si mpson K. Forensic Medicine, 6th edition Edward Arnold publishers, pg

92, 1972.

Professor Francis E. Camps was also generally supportive of the reliabiE ­ ty of the diatom test: Whilst the tests are not necessarily conclusive, they provide reliable up­

portive evidence. Tests are made on all enclosed organs, skeletal muscle

and femoral bone marrow.

Camps FE. Gradwohls Legal Medicine , 3rd edition, Bri stol: John Wri ght

& Sons Ltd ., pg 353, 1976.

In one of the most influencial texts in forensic medicine, Polson, Gee an . Knight expressed the view that the diatom test had particular value a ., confirmatory test for drowning: The demonstration of plankton and , more especially, diatoms in the sub­ merging fluid and in the body of the victim, is of particular value as a con­ firmatory test. The test material is treated by acid digestion as a preli mi­ nary step in diatom analysis. In the case of decomposed bodies, the demon stration of diatoms in enclosed organs and especially in the bone marrow, due care being taken to exclude contamination, is strong if n conclusive evidence of death by drowning. 51

j

Forensic Diatomology and Drowning

Polson CJ, Gee DJ, Knight B . The Essentials of Forensic Medicine, 4th ed ition, Pergamon press, pg 434, 1985.

We now discuss some of the historically important reseach on the diatom test for drowning with specific reference to early investigations, recent studies on the reliability of the test and developments in empirical meth­ ods fo r isolating frustules from tissue.

* 3. A BRIEF HISTORY OF TH E DIATOM TEST An impressive volume of scientific investigation has been published on the issue of diatoms and drowning. The literature is chiefly devoted to the his­ tory of the test, the best choice of tissue for diatom detection, and indirect and direct studies of the specificty of the test for drowning. In the last decade, investigators have focu s ed attention on procedural modific ations to the classical methods of the test. Much of the classical literature on diatoms and drowning has been reviewed by Peabody (21). It may have been Revenstorf who first noticed diatoms in the lungs of the drowned in 1904 (22). However, Revenstorf credits Hofman with priority for this discovery eight years prior to his own work. However, there seems little doubt that Revenstorf conceived what is now generally held to be the important concept behind the diatom test; that diatoms could enter the systemic circulation through the lungs during drowning. The modem era of forensic diatom analysis was initiated in the 1940's by the work of the Hungarian investigators, Incze (12, 13, 23 -25) and Tamaska (26, 27). Incze demonstrated that the lungs, blood, and blood-filtering organs of rabbits drowned in suspensions of diatoms con­ tained diatoms. The specificity of parenchymatous diatoms was shown by immersing dead rabbits in a similar suspension and demonstrating that the peripheral organs lacked diatoms whereas some diatoms could be found in the lungs, presumably by passive infiltration in the postmortem period. Mueller and Gorgs (28-30) carrier out similar in vivo investigations inde­ pendent of Incze. Tamaska extended these observations by detecting diatoms in the tissues of witnessed drownings. Furthermore, Tamaska failed to demonstrate diatoms in the tissues of several non-drowning deaths that were recovered from the Danube river. 52

Medicolegal Value of the Diatom Test

. . 1edicine, 4th

_-_::en on the diatom c tigations, recent :-: empirical meth-

TEST published on the deyoted to the his­ ~::'~~:ion and indirect ~~ 'Ding. In the last ;:~ Jral modific ations :';;c

jrowning has been " ,::"who first noticed .:' .:eyer, Revenstorf . ~...... prior to his own :-onceived what is . e diatom test; that _=' .he lungs during _ ... as initiated in the - ~?e (1 2, 13, 23-25) - ~·:g s . blood, and . 2f diatoms cor.­ _ :":':_ was sbo n b. _ -.., . :raling that £b:

c::

.:-~~ ... .d

be foun

:r.

It was not until after World War Two that diatoms and drowning became a major research focus in forensic medicine. In the 1960's and early 1970's investigators in Belgium led by Timpennan developed a comprehen­ sive research prorgam on the the practical application of the diatom test to death investigation (31-37). In 1969, Timpennan published the classic paper on diatoms and drowning, "Medico-legal Problems in Death by Drowning: Its Diagnosis by the Diatom Method" describing a decade of research (37). During the same period other investigators in Japan and the United States published similar data in general agreement with the work of Timpennan. For the first time, using a large series of drowning cases, Timpennan provided evidence for the validity of the diatom test by demonstrating: 1. Diatoms could be reproducibly recovered from human tissues in

freshwater drowning.

2. The acid-extraction method gave consistent results when applied to

several different tissues including bone marrow.

3. Quantitative analysis of the lung diatom burden may be useful in dif­ ferentiating live and postmortem immersion in water. 4. The concordance of the types of diatom in various organs and the

drowning medium was independent evidence for the validity of the

diatom test.

Studies on the diatom test for drowning in the last three decades have extended and strengthened these conclusions. H owever) at the time of the work in Belgium, some investigators suggested that the rationale for the diatom test may have been based on an erroneous postulate: that diatoms are absent in the tissues of the non-drowned. Spitz wrote: ... in 1963 we questioned the validity of this method by the recovery of diatoms in the liver and in other organs of individuals who had died of causes other than drowning. Spitz WU, Fisher RS . Medicolegal Investigation of Death, 2nd edition, Charles C. Thomas Publishers, pg 570, 1993. This led to the study of the specificity of the diatom test for drowning as determined by the ability to extract diatoms from the tissues of the non­ drowned.

53

Forensic Diatomology and Drowning

* 4 . AR E DIATOMS UBIQUITOUS IN TISSUE? The main evidence against the validity of the diatom test for drowning is that some studies have found fru stules in tissue-extracts of cases of non­ drowning deaths (37-45) (Table 3.1). This finding has been interpreted to mean that the non-drowned, indeed anyone, may have diatoms trapped in tissues and that these diatoms did not enter the body by drowning (46). However, an equally logical explanation is that some, if not all, of these problematic diatoms originated in the laboratory rather than the tissues since studies show that there is no universal agreement on the presence of diatoms in the non-drowned. The two issues that have medicolegal impor­ tance and need consideration are: 1. Is there a plausible mechanism for entry of diatoms into the non­

drowned?

2. What is the significance of the presence of spurious diatoms on the

diatom test for drowning?

A priori, diatoms can enter only enter the body by two routes: the respira­

tory tract and the gastrointestinal tract. The fact that diatoms have been found in the air and diatomaceous pneumoconiosis (47) is known to occur indicates that diatoms may enter and reside in the lung. There is no empir­ ical evidence that extensive hematogenous dissemination of diatoms inhaled in the course of tidal ventilation reaches the parenchymal organs or bone marrow. It is also uncertain by what mechanism diatom that are resident in the lung could breach the alveolar-capillary barrier and evade local lung defenses (e.g. cilia, mucus, alveolar macrophages). However, it must be conceded that a massive pulmonary burden of diatoms may lead to disruption of lung homeostasis that may lead to diatoms escaping into the capillary bed. Evidence in support of this is that asbestos and berylli­ um may be observed in the peripheral organs of some individuals with asbestosis and berylliosis. On this basis, the empirical evidence would suggest that the diatom test for drowning could be confounded by the presence of spurious diatoms in individuals with diatomaceous pneumo­ coniosis. The empirical evidence for the entry of diatoms through the gas­ trointestinal mucosa is that liver may contain diatoms and diatom-like par­ 54

Medicolegal Value of the Diatom Test

.

~e - t

for drowning is .~ ': - of cases of non­ ..:.:; been interpreted to ." diatoms trapped in .' by drowning (46). ~. if not all, of these ~ .er than the tissues

_ '1': on the presence of

e medicolegal impor-

TABLE 3.1 Specificity of the Diatom Test Using Various Organs t

# of Cases§

+

Study

Lung

5 0 7 6 4

5 18 28 15 13 16 29

0.22 0.96 0.84 1.00 0.65 0.72 0.88

Otto, 1961

Porowski, 1966 Koseki, 1968 Neidhart & Greendyke, 1967 Schneider, 1969 Timperrnan, 1969 Calder, 1984

21 I 6 6

29 1 30 14 12

0.97 0.05 0.97 0.70 0.67

Mueller, 1963 Spitz & Schneider, 1964 Koseki, 1968 Schneider, 1969 Ti mperrnan, 1969

18 21 14

0.95 0 .88 0.88

Porowski, 1966 Neidhart & Greendyke, 1967 Calder, 1984

18 1 12

0.95 0.06 0.40

Porowski, 1966 Schellman & Sperl, 1979 Calder, 1984

23

into the non­ diatoms on the

'- routes: the respira­ i diatoms have been - - is known to occur - g. There is no empir­ ~~ati on of diatoms . ?arenchymal organs , m diatom that are ~ . barrier and evade :-,hages). However, it - .:-:' diatoms may lead :::....1.roms escaping into stas and berylli­ ~~,e individuals with ~ al evidence would :: -. onfounded by the __:L!llilCeOUS pneumo­ • . 2",- {hrough the gas­ . -:-. diatom-like par­

Specificity

Liver

Kidney I 3 2 Bone Marrow

15 18

t Only studies with> 15 cases, all cases are non-drowning deaths §

+ = false positives, -

= true negatives

55

Forensic Diatomology and Drowning

ticles (37) indicating that the hepatoportal circulation may contain diatoms due to the products of digestion. It is uncertain how particulate matter could entire the hepatoportal circulation which usually accepts soluble by simple diffusion or facilitate transport across cell membranes. However, for diatoms in the gastrointestinal tract to be a relevant source of diatoms in peripheral tissues requires a fantastic journey in the bloodstream. Diatoms in the hepatoportal circulation must progress through hepatic sinusoids and enter the inferior vena cava, progress to the heart, pass through the capillary bed in the lung, re-enter the heart, disseminate into the arterial system and become enlodged in a peripheral capillary bed. Throughout the enter process, the diatom must pass undetected by phago­ cytes, in the sinusoids (e.g. Kupffer cells), the pulmonary capillary bed, and a terminal capillary bed. If we accept that some diatoms may reach distant sites, such as the bone marrow, what is the significance of this for the diatom test? This question was anticipated by Timperman who indicated that the medicole­ gally significant diatoms in a tissue-extract were concordant with diatoms from the drowning medium. This concept was further articulated by Hendey (48): If the confusion and uncertainty under which the diatom test has laboured is to be dispelled it should be regarded in the following light. The diatom test for death by drowning must be able to show that the diatoms recov­ ered from the deceased person are part of the diatom flora taken at the site where the subject was lying or where the subject entered the water or was thought to have entered the water. On this basis, if diatoms ar~ occasionally found in the tissues of the non­ drowned it is of no relevance to the diatom test for drowning if the crite­ rion ofconcordance is rigidly applied. In fact, once this crtierion is applied the specificity and positive predictive value of the diatom test for drown­ ing using femoral bone marrow is similar that of routine diagnostic tests in clinical medicine (chapter 4). In summary, the possibility of the presence of diatoms in the tis­ sues of non-drowned people is the root of much of the criticism of the diatom test. This criticism is based on an imperfect understanding of the basic principles of the test and misconceptions about the significance of 56

Medicolegal Value of the Diatom Test

- _..:l:. e . However, _:-::e of diatoms _ bloodstream.

- .:: ~seminate into _ _ capillary bed . .:..'"'=- ~te d by phago­ apiUary bed,

_ -. with diatoms - - ~ articulated by

; - - T he diatom . ~ :::jruo ms reeov­ - __ ~3.:'e n at the site

-.:: water or was

frustules in human tissue. On this basis, it is important to realize that a medicolegaUy important diatom test result not only demonstrates the pres­ ence of diatoms in tissue but also the same diatoms at the site of drown­ mg.

* 5. RESEARCH AND THE DIATOM TEST Since the classical studies, most research on diatoms and drowning has emphasized three main areas: 1. Studies with experimental models to test the validity of the diatom

test for drowning.

2. Development of novel methods to extract frustules from human tis­

sue.

3. Development of different approaches to the quantitative and qualitia­

tive analysis of diatoms in drowning.

Research with experimenta) animal models of drowning has facilitated further understanding of the fundamental events that occur in drowning, and represents the logical continuation of the original work of Incze. The search for alternative procedures for the acid digestion of tissue for frus­ tule extraction underlies most new laboratory methods. Finding different and meaningful ways of interpreting forensic diatom analysis is largely based on determining the significance of pulmonary diatoms in bodies recovered from water.

Experimental Models ~

_.: g if the crite­ :-:erion is applied .": : st for drown­ . - ;: diagnostic tests _ : roms in the tis­ .,. ~ri[icism of the _ .:=_ randing of the -i gnificance of

Two experimental models have developed to study the validity of the diatom test for drowning. Ranner et al have re-examined the fundamental principle of the diatom test for drowning using an animal model of drown­ ing to show that diatoms from the drowning medium are an important source of diatoms in the body (49). In an ingenious experiment, these investigators drowned rabbits in diatomaceous water after amputating one leg and ligating one renal artery. Quantitative analysis revealed that the diatom burden of the bone marrow and kidney increased after drowning indicating the hematogenous distribution of diatoms from the lungs . In a 57

Forensic Diatomology and Drowning

similar model, Lunetta et al have demonstrated that diatoms in the drown­ ing medium perforate the alveolar-capillary barrier and enter the systemic circulation (50). These experiments show, for the first time, that drowning is the direct causal link between diatoms in drowning medium and those recovered from postmortem tissues.

Extraction of Frustules Several procedures have been developed to replace the original acid diges­ tion method for extracting frustules from human tissues. Most of the meth­ ods rely on alternative chemical or physicochemical (e.g. ultrasonic radia­ tion (51)) means to solubilize tissue. Methods that have been particularly useful are enzymatic digestion of tissues with proteases (10, 52) such as proteinase K. Strong anionic detergents such as sodium dodecyl sulphate and nonspecific tissue solubilizers (51) have also been shown to be effec­ tive for frustule extraction. Simple centrifugation is usually used to isolate diatoms from extracted tissue-suspensions. However, membrane filtration (53), and gradient centrifugation (11) have also been advocated. Although many methods have been suggested as alternatives to acid digestion there is no evidence for improved yield of frustules since few studies have directly compared methods. Little is known about the centrifugational properties of frustules, in particular, the centrifugal forces that gives optimal quantitative isolation. This may be relevant since small pennate diatoms may be more difficult to isolate, using differential cen­ trifugation, than larger genera such as Navicula.

Quantitative and Qualitative Diatom Analysis A new concept is emerging in forensic diatom analysis due, in large mea­ sure, to the research of Auer (54, 55). Until recently, the diatom test for drowning has been based mostly on qualitative characteristics including the presence and concordance of diatoms in tissues. Little attention has been given to the magnitude of the diatom-burden of tissues, or the signif­ icance of the inter-organ variance of the diatom load. This is particularly important in the context of diatoms recovered from lung since diatoms in this site may not, in all cases, originate from the drowning process. This has resulted in the concept of combined quantitative and qualitative diatom analysis in an effort to establish valid laboratory criteria for a positive

58

Medicolegal Value of the Diatom Test

the drown­ .~. :he ystemic _ ±at drowning J ill and those lD

. J.:

~

~ ~ . . . g. -

acid diges­ . -,:-: of the meth­ _ . ~ onic radia­ \..-:1 particularly _' . -2) such as

ed to isolate ane filtration - : . ated. rernatives to .- :ru tules since --: ~\\ n about the • ~r ~rifu gal forces ,"r since small . z'erential cen­ _

n

diatom test. The basic concept underlying this approach to the diatom test is that diatoms recovered from tissues must be ecologically consisent with the environment of drowning (i.e. the qualitative factor; a variant of the criterion of concordance) , and that the diatom load of the lung and organs must be consistent with previous empirical experience (i.e. the quantitative factor). A corollary to the qualitative factor in the analysis is that it may be possible to identify the site of drowning provided that the diatom popula­ tion is not too homogeneous. Pachar & Cameron (56) substantially agree with this concept of combined quantitative and qualitative diatom analysis. In a recent study, these investigators found that a potential threshold level for a significant lung diatom burden may be - 15 diatoms per 100 gram of lung. In agree­ ment with Auer, this study found a correlation between the presence of diatoms in the pelipheral organs and in the lung, suggesting that the lung was the portal of entry for the diatoms (i.e., drow ning). Ludes (57, 58) has advocated further development of the ecologi­ cal aspects of the diatom by emphasizing the role of diatom community structure in the diagnosis of drowning. In a recent study, Ludes reported the results of comparing the diatom flora from continuous samples of rivers in France and drowning deaths in the same areas. There was a sig­ nificant correlation between the prevalence of specific genera in the river samples and tissue-extracts indicating the utility taxonomic inventories of diatoms. Unfortunately, in many large countries with extensive waterways the continuous monitoring of diatom flora is not feasible.

Use of Bone Marrow . . ~ . in large mea­ -:: _:arom test for .. ::c including

- ~ " rocess. This __ali tative diatom .:.! for a positive

Many different organs have been adovcated as starting material for frus­ tule extraction. The most obvious choice of organ to use as starting mate­ rial for simple qualitative diatom anlaysis is the lung. However, there are several problems with the lung as an index source for diatoms that have entered the body from drowning. It is theoreticaly possible for diatoms to enter the lung passively by percolation after death. In addition, the diatom burden may be quite large if the local fl ora is highly concentrated. This can lead to false positives if the burden exceeds an arbitrarily set threshold value. On this basis, the best tissue for frustule extraction would have the two characteristics: 59

Forensic Diatomology and Drowning

1. Be perfused by blood that could, in principle, have just passed

through the pulmonary capillary bed.

2. Be immune to spurious postmortem contamination of diatoms from

the drowning medium.

The two tissues that fulfill each of these criteria are the blood in the pul­ monary viens or left heart if harvested with care in fresh bodies, or the bone marrow of an intact long bone (e.g. femur). Based on this reasoning, we have studied the utility of the femoral bone malTOW as a source of tis­ sue for the diatom test for drowning. This research in discussed in detail " in the next chapter.

* 6. REFERENCE 1.Schwar TG. Drowning. Its chemical diagnosis. A review. Forensic Sci 1972;1:411-417. 2. Foroughi E. Serum changes in drowning. Journal of Forensic Sciences 1971; 16(3):269-282. 3. Moritz AR. Chemical methods for the determination of death by drowning. Physiol Rev 1944;24:70-88. 4. Coutselinis A Boukis, D. The estimation of Mg2+ concentration in cerebrospinal fluid (C.S.F.) as a method of drowning diagnosis in sea water. Forensic Sciences 1976;7:109-11l. 5. Gettler AO. A method for the determination of death by drowning. J. Amer. Med. Assoc. 1921;77:1650-1652. 6. Kerr DJA Asphyxia. IIi: Forensic Medicine. Sixth ed. London: Adam and Charles Black, 1957: 157-158. 7. Piette M Timperman, 1., Parisis, N. Serum strontium estimation as a medico-legal diagnostic indicator of drowning. Med Sci Law 1989;29(2): 162-171 . 8. Azparren J de la Rosa, 1., Sancho, M. Biventricular measurement of blood strontium in real cases of drowning. Forensic Science International 1994;69: 139-148. 9. Amin MA Samia, AH., Kabil, M.A et al. Serum strontium estimation as a diagnostic criterion of the type of drowning in water. Forensic Science International 1985;28:47-52.

60

Medicolegal Value of the Diatom Test

e blood in the pul­ .~ -h bodies, or the .) this reasoning, source of tis­ ~ ~u ssed in detail

--i e\ . Forensic Sci

_:: n of death by

r

- :; ncentration in ~ .iiagnosis in sea

: .. --. by drowning. J.

e=!. London: Adam

estimation as a \led Sci Law ~

:neasurement of :::orensic Science _':')Jtium estimation _ ::-. water. Forensic

10. Kobayashi M Yamada, Y, Zhang, W-D., Itakura, Y, Nagao, M., Takatori, T. Novel detection of plankton from lung tisue by enzymatic digestion method. Forensic Science International1993;60:81-90. 11. Terazawa K Takatori, T. Isolation of intact plankton from drowning lung tissue by centrifugation in a colloidal silica gradient. Forensic Science International 1980; 16:63-66 . 12. Incze G Tamaska, I., Ggyongyosi, J. A vizbefulas bizonyitasa aver es a szervek planktonvizsgalataval. Orv Hetil 1951 ;7: 1003. 13. Incze G Tamaska, L., Ggyongyosi, J. Zur blutplanktonfrage beim tod durch ertrinken. Dtsch Z Gasamt~ Gerichtl Med 1955;43:517-523. 14. Mikarni Y Kanda, M., Kamimura, 0., Okuyama, M. Experimental study and practice on the detection of vegetative planktons in the bone marrow of the drowned dead body. Acta Med Okayama 1959;13:259­ 268. 15. Golaeff DA. Use of phyto-plankton method for differentiation between drowned persons and bodies thrown into water. Odessky Med J 1928;3:425-430. 16. Merkel H. Plankton findings in lungs of drowned persons and their practical significance. Deutsche Ztschr.f.d.ges. Gerichtl. Med. 1939;31:211-212. 17. Tomonaga T. Demostration of plankton in organs of drowned persons. Jap.1. Med.Sci., VII, Social Med.and Hyg. 1940;3:197-198. 18. Karkola K Neittaanmaki, H. Diagnosis of drowning by investigation of left heart blood. Forensic Sciences International 1981; 18: 149-153. 19. Chardez D Lambert, 1. Protozoaires cilies et thanatologie. Forensic Science International 1985;28:83-101. 20. Spitz WU. Drowning. In: Spitz WU Fisher, R.S., ed. Medicolegal Investigation of Death, Guidelines for the Application of Pathology to Crime Investigation. Springfield, Illinois: c.c. Thomas, 1973: 21. Peabody AJ. Diatoms and Drowning - A Review. Medicine, Science, and the Law 1980;20(4):254-261. 22. Revenstorf V. Der nachweis der aspirierten ertrankungs flussigkeit als kriterium des todes durch ertrinken. Vierteljahresschs Geritchtl Med Off Sanitaetswes 1904;28:274-279 _ 23. Incze G Gyongyosi, 1. Wirkung des wasserdrucks auf die postmortale flussigkeitstromung in die luftwege. Acta Morphol Acad Sci Hung 61

Forensic Diatomology and Drowning

1955;5:349-353. 24. Incze G . [Phytoplankton resorption in drowning] . Acta M orphol Hung 1951; 1:421-430. 25. Incze G. Fremdkorper in blutkreislauf ertrunkener. Zentralbl AUg Pathol Anat 1942;79: 176. 26. Tamaska L. [Diatom content of bone marrow in corpses in water]. Orv Hetil 1949;16:509-511. 27. Tamaska L. (On diatom demonstration in the bone marrow of drowned dead bodies]. Deutsche Z. Ges. Geric htl. Med. 1961 ;51 :398-403 . 28. Mueller B. [On the problems of the occurence of diatoms in the organs of cadavers not having lain in water] . Deutsche Z. Ges. Geric htl. Med. 1963 ;54:267 -272. 29. Mueller B. Zur frage des vorkommens von diatomeen in organen von leichen, die nicht in wasser gelegen haben. Dtsch Z Gesamte Gerichtl Med 1963 ;54:267-272. 30. Mueller B Gorgs, D. Studien uber das eindringen von corpiscularen wasserbestandteilen aus den lungenalveolen in des kreislauf wahrend des ertrinkungsvorganges. Dtsch Z Gesamte Gerichtl Med 1949;39:715-725. 3l. Thomas F Van Hecke, W., Timperman, 1. The detection of diatoms in the bone marrow as evidence of death by drowning. Journal of Forensic M edicine 1961 ;8(3): 142-144. 32. Thomas F Hecke, W ., Timperman, J. Death by drowning - a problem of forensic medicine. Ciba Sympos 1961;9:154- 16l. 33. Thomas F Van Hecke, W., Timperman, 1. The medicolegal diagnosis of death by drowning. Journal of Forensic Science 1962;7(1): 1- 14. 34. Thomas F Heeke, W., Timperman, 1. Diagnostic medi co-legal de la mort par submersion par la mise en evidence de diatomees dans la moelle des os longs. Ann. Med. Leg. 1962;42:369-373. 35. Timperrnan 1. The detection of diatoms in the maITOW of the sternum. Journal of Forensic Medicine 1962;9(4): 134-136. 36. Timperman 1. Observations on the diatom question. Dtech Gesamte Gerichtl Med. 1968;63(2): 127-128. 37. Timperman 1. Medico-legal problems in death by drowning: Its diag­ nosis by the diatom method . Journal of Forensic Medicine 1969; 16(2):45-75.

62

Medicolegal Value of the Diatom Test

\lorphol Hung

""s in water]. Orv ~ow of drowned . : ~ :398-403. ~ - in the organs . Gerichtl. Med.

--. ' n organen von G;:-amte Gerichtl

02

\.2 n corpiscularen cei lauf wahrend Gerichtl Med

n of diatoms in

ng. Journal of

...

~~egal

diagnosis of __ :- 1):1-14. di o-legal de la J[omees dans la

-= : of the sternum. ~ . _.

Dtech Gesamte

ning: Its diag­ : _:-e ic Medicine _ _ - l,

38. Calder YM . An evaluation of the diatom test in deaths of professional divers. Med Sci Lwa. 1984;24(1):41-46. 39. Koseki T. Investigations on the bone marrow as a material in the diatom method of diagnosing death from drowning. Acta Med BioI 1969; 16(2):85-90. 40. Neidhart DA Greendyke, RM. The significance of the diatom demon­ stration in the diagnosis of death by drowning. American Journal of Clinical Pathology 1967;18(4):377-382 . 41. Schneider V. Versuch einer wertung der diatmeenprobe. Dtsch Z Gesamte Gericht Med 1969;26:92-96 . 42. Schneider Yea. [On the determination of radioactively labeled diatoms in the organs]. Beitr Gerichtl Med 1969;25:158-164. 43. Schneider V. Evaluation of the diatom test. Beltr Gerichtz Med 1969;26:92-99. 44. Schellman B Speri, W. Diatmeen-nashweis im knochenmark (femur) nichtertru nkener. Z Rechtsmed 1979;83:319-324. 45. Porawski R Investigati~n on the occurrence of diatoms in organs in deaths from various causes. Journal of Forensic Medicine 1966; 13(4): 134-137. 46. Foged N. Diatoms and drowning - Once more. Forensic Science International 1983;21:153-159. 47. Dayan AD Morgan, R.J.T., Trefty, RT., Paddock, T.B.B. Naturally occurring diatomaceous pneumoconiosis in sub-human primates. J Comp Pathol 1978;88:321-325 . 48. Hendey NI. Letter to the Editor, Diatoms and Drowning - A Review. Medicine, Science, and the Law 1980;20(4):289. 49. Ranner G Juan, R , Udermann, R [On the evidential value of diatoms in cases of death by drowning]. Z Rechtsmed 1982;88(1-2):57-65. 50. Lunetta P Penttila, A. Scanning and transmission electron microscopic evidence of the capacity of diatoms to penetrate the alveolar-capillary barrier in drowning. International Journal of Legal Medicine 1998;in press. 51. Fukui Y Takahashi, S., Matsubara, K. A new method for detecting diatoms in human organs. Forensic Science International 1980; 16:67­ 74. 52. Ludes B Quantin , B., Doste, M ., Mangin, P. Application of a simple 63

Forensic Diatomology and Drowning

enzymatic digestion method for diatom detection in the diagnosis of drowning in putrified corpses by diatom analysis. lnt J Legal Med 1994; 107( 1):37-41. 53. Funayama M Aoki, Y, Sebetan, I.M., Sagisaka, K. Detection of diatoms in blood by a combination of membrane filtration and chemi­ cal digestion. Forensic Science lnternational1987;34:175-182. 54. Auer A. Qualitative diatom analysis as a tool to diagnose drowning. American Journal of Forensic Medicine and Pathology 1991; 12(3):213-218. 55. Auer A Mottonen,M. Diatoms and drowning. Z Rechtsmed 1988; 10 1(2):87 -98. 56. Pachar JV Cameron, J .M. The Diagnosis of Drowning by the Quantitative and Qualitative Analysis of Diatoms. Medicine, Science, and the Law 1993;33(4):291-299. 57. Ludes B Coste, M . D.iatomees et medecine legale.Paris: Tec & Doc Lavoisier Editions Medicales lnternationale, 1996 58. Ludes B Coste, M. Tracqui, A., Mangin, P. Continuous river monitor­ ing of the diatoms in the diagnosis of drowning. Journal of Forensic Science 1996;41 (3):425-428.

64

.~

diagnosis of

1:- : J Legal Med

~-.

Detection of -~.:. jon and chemi­ , ~ 75-182. ~3 se drowning . .:..!1d Pathology Z Rechtsmed wning by the

;; river monitor­ ;: _mal of Forensic

4 . Validity and utility of the diatom test using bone marrow The quantitative aspects of the utility and validity of the diatom test for drowning using femoral bone marrow are now described. The basis for the present description is the quantitative analysis of cases (n = 771) processed for the diatom test for drowning at the Office of the Chief Coroner for Ontario between 1977-1993. Most cases were drownings recovered from water in Ontario, Canada"; therefore, the results can be reasonably extrap­ olated to most temperate freshwater environs. In the analysis that follows, the outcome of the diatom test for drowning is correlated with the site, sea­ son, and month of drowning to induce ecological factors that influence test outcome. Bone marrow from intact femora (or rarely, an intact kidney) was used for the extraction of diatoms using the method of Timperman with minor modifications as described in the next chapter. Full description of the studies summarized in this chapter can be found elsewhere (1, 2). The data presented is based on the belief that use of femoral bone marrow as a staring material for the extract of diatoms is the most reliable. Reasons for this have been described in the previous chapter, but it is important to reiterate some of the basic characteristics that make bone marrow a scientifically preferred specimen for the extraction of diatoms. The main advantage of bone marrow over, for example, lung tissue is that the diatoms in the marrow must have derived from the hematogenous dis­ semination of particles from the lung requiring a functioning heart. Lung 65

Forensic Diatomology and Drowning

tissue may become laden with diatoms by passive percolation in the post­ mortem period if the body is submerged in water. Of course, this does not exclude the possibility that some of the diatoms recovered from lung tis­ sue may have originated from drowning.

* 1. UTILITY OF THE DIATOM TEST Outcome Ratios for the Diatom Test One way of determi ning the medicolegal value of the diatom test for drowning is to show, in a relevant population of cases (e.g. a group of puta­ tive drowning cases), that this test may be helpful as an ancillary method to determine if drowning was the cause of death. We can describe this quantitatively as the utility of the test, defined as the ratio of the nu mber of all positive test results to the sum of all positive and negative test out­ comes. This ratio represents the frequency of a positive test outcome in the relevant forensic population. The utility of the diatom test for drowning using femoral bone marrow was calculated in a series of 771 cases of drowning that occurred during the summer, late spring and early autumn (Fig. 4.1 & Table 4. 1) in Ontario, Canada. The diatom test for drowning was positive in 205 cases (28 %) of the fres hwater drowning cases but was rarely positive if drown­ ing occurred in water from a domestic source that lacked diatoms due to water filtration and processing (Table 4.2). Only four cases (12%) of domestic drownings had a positive diatom test. Drowning medium was available for analysis in two of these cases, and diatoms were found in both cases. The diatoms were derived from an abrasive cleaning agent in one case and from gravel added into the water in the other case.

Seasonal and Monthly Variation of Diatom Test Outcome There was a conspicuous monthly variation in the incidence of a positive outcome of the diatom test (Fig. 4.2). The diatom test was most likely to be positive in April (-40%), July (-40%), and November (-30%) and least likely to be positive in the Winter months. However, the pattern correlated with the well documented monthly variation in diatom population due to cyclic diatom blooms. The principal diatom blooms occur in early spring and autumn with a sharp decline in the live diatom population between the

66

Validity and Utility of Diatom Test

: _:!on in the post­ . this does not -: ~ from lung tis-

diato m test for ;: ;. ..1 group of puta­ ~_ ciliary method ~ ~..n describe this of the number - ~ ~=gat ive test out­ • _~ - . outcome in the

TABLE4 . J Outcome of th e Diorom Testfor DrOlV/zillg by Seasoll

Season

Number of Drownings J

Number of Positive Tests 2

Winter

13 8177 1 ( 18 ';()

26/138 ( J9 ';()

Spring

1721771 (22';()

431172 (257(')

Summer

2931771 (387()

93/293 (32o/()

Autumn

168177 J (227()

431168 (257c)

J

",. = femoral bone

- . . . g that occurred

- . _ - Table 4.1) in

.:...ve in 205 cases

sitive if drown­

-=.:! diatoms due to -:- ..z ::ases (12%) of ng medium was were found in

eaning agent in -:-". -" ~ case.

Indica tes the number and proportion of drowning compared with the total series

2 Indica tes the number and proportion of positive diatom tests for alJ cases in the season

16 ,-------------------------------------------~

-;j!.

en en 12 c

c

3:

e c '0 c 0

- ,::. • Outcome

8

"€

0

~

e of a positive - most likely to - ~ - 30%) and least ::: ~ ttero correlated - _ pulation due to ;:: _!" in early spring ~ _ .::.tiOD between the

0~

Q.

Jan Feb Mar Apr Ma y Jun Jul Aug Sep Oct Nov Dec Month 4.1. Relative proportion of drowning cases in various months for 771 cases of drowning in Ontario. Canada between 1977 and J993. 67

Forensic Diatomology and Drowning

blooms. The high incidence of positive diatom tests in the summer months was presumably due to persistence of the insoluble frustules of the dead diatoms in water. Although the monthly variation of test incidence corre­ lated with diatom blooms, a basal concentration of diatoms in freshwater must have been maintained since seasonal variations deviated by only 13% from Winter (-19%) to Summer (-32%) (Table 4.1). The appearant monthly variation in the utility of the test has important implications for the validity of the test (section 2).

* 2. VALIDITY OF THE DIATOM TEST Concepts in Support of the Diatom Test The relationship of test outcome with diatom population dynamics is important corroborating evidence for the validity of the test since it is pre­ dictable that the diatom content of the water would influence test outcome. The correspondence of the high incidence of positive test outcomes with peak months of diatom blooms is consistent with the water concentration of diatoms being a key factor in test outcome. However, since diatom frus­ tules persist is freshwater throughout the year, drownings in months with suboptimal water concentration of diatoms still provide occasional posi­ tive test outcome, although at a lower overall rate. Other data support the reliability of the diatom test for drowning. For example, (i) the high false negative rate indicates that postive results, and therefore, false positives are infrequent; (ii) domestic water drownings had negative diatom tests unless the water contained diatoms derived from some external source (e.g. abrasive cleansers, contamination of the bath with diatoms from the out-of-doors environment); (iii) drowning in water with no or few diatoms (e.g. some samples of Winter water) gives a nega­ tive test result; and (iv) paired drownings in the same body of water had the same diatom species in the bone marrow. Some additional features (discussed below) also support the valid­ ity of th diatom test. This includes th fact that diatoms extracted from femoral bone marrow in cases of drowning represent a distinct subset of freshwater diatoms that conform to certain size and shape criteria . Evaluated together these data provide evidence in support of the utility and reliability of the diatom test for drowning. The data suggest that a positive

68

Validity and Utility of Diatom Test

. --e ~ u mmer months ~_ -_ le s of the dead :: :!1 idence corre­

in freshwater

_ ( d by only 13% - ~ . The appearant ::: :m plications for

TABLE 4.2 Outcome of the Diotol11 Test for Drowning by Site ofDrolVning Outcome Site of drowning

Total

Positive

Negative

Naturally-occuring body of water!

738

205 (28%)

533 (72%)

33

4 (12%)

29 (889C)

771

209 (279C)

562 (73%)

Bathtub. pool. or toilet

__..;.:~o n dynamics is -e :e-[ since it is pre­ :- _c:: ~ e test outcome. :=: :;:~t outcomes with _cr concentration :<-: -:nce diatom frus­ - ~ in months with ::c occasional posi­ ~

:e : for drowning. :~:.i: postive results, ~ water drownings _"_"vms derived from -.arion of the bath irowning in water ~ er) gives a neg ae ::-ody of water had

•

. 0

_: :upport the valid­ _~ extracted from . _ di stinct subset of - -l shape criteria " __ :1 of the utility and 5~es t that a positive

All locations ! Lake, river, ditch. or pond

40 ,-----------------------------------------~

.....0 e~~ li/ ­

30

-eCil eI) UI-

._

Eel)

->-.~ ... '='­ _CII

cO

oc.

20

~

10

Jan

Feb Mar Apr May Jun

Jut Aug Sep

Oct Nov Dec

Month 4.2. Monthly incidence of positive diatom tests in 771 cases of drowning in Ontario. Canada between 1977 and 1993. Reported values are the proportion of drownings in a given month that had a positive diatom test.

69

Forensic Diatomology and Drowning

test outcome is useful in determining if drowning was the cause of death or a contributing factor in death. Conversely, a negative outcome of the test does not exclude drowning as a cause of death since many factors deter­ mine test outcome including the concentration of diatoms in the drowning medium. It is important to elaborate further on three of these key issues: 1. The relationship between test outcome and the diatom content of the

drowning medium.

2. The presence of concordant diatom types in bone marrow and the drowning medium. 3. The size and shape of drowning-associated diatoms. Test Outcome Correlates with Diatom Content of Water The diatom content of lake and river water submitted in the course of the investigation of the 771 cases of drowning used for the present analysis was investigated. Microscopic examination of the water samples for the presence of diatom frustules revealed that December freshwater was large­ ly devoid of diatoms (Fig. 4.3). In contrast, freshwater samples collected in the summer and autumn months usually contained diatoms. Most sam­ ples of freshwater contained diatom genera that were typically associated with drowning (Fig. 4.4). The reduced rate of a positive outcome for the diatom test roughly correlated with the lack of detectable diatoms in the putative drowning medium (Fig. 4.2 & 4.3, c.f. June and December). The fact that a small proportion of diatom tests are positive in the Winter months, and that some samples of Winter water contain diatoms, indicates that some of the genera that are usually associated with drown­ ing are found in freshwater despite the season. Figure 4.4 show the over­ all frequency of the presence of some genera of diatoms in all water sam­ ples examined in the study period. The genera analysed represent some of the most common freshwater diatoms founds in cases of drowning. The high frequency of these genera, over all months of the calender, indicate that the diatoms associated with freshwater drowning are ubiquitous in both time and space. Although there is some constancy in the presence of freshwater diatoms, diatoms are not static in freshwater. Changes in abundance as well as genus diversification occur III freshwater (Fig. 4.5-4.7). New 70

Validif), alld Uti/iJ.r of Diato/ll reSf

-:; .:au e of death .,,:arne of the test factors deter­ .c. the drowning ~ - e key issues:

60

50 QJ

>

-=.,, --.

40

-

30

g' ~

Z fIj

....011)

QJ

C l­ 0

...

'­1: ... QJ

~~

20

E

a..

10

e-~

of Water

-'- :; ourse of the -': :-,resent analysis :: -:unples for the _ ___ .. ter was large­

Jan

Feb M ar Apr May Jun

Jul

Aug

Sep

Oct Nov Dec

Month

4.3 . Monthly frequency of fre shwater samples of putative drowning medium devoid of diatoms .

~ -':~

les collected . :: . 2ms. Most sarn­ _ ~ :.:illy associated ~ ..:-utcome for the :; iatoms in the - .:. Jecember). _~ p sitive in the -- .:.ontain diatoms, _._.':ed with drown­ - - -how the over­ 1water sam­ ~ -;;-D[esent some of ~:- drowning. The :: --': ender, indicate = :.:-e ubiquitous in

100 ,--------------------------,

Cocconeis

Cymbella

Navicula

Genus

_ ":-::e of freshwater :!l bundance as ;:: _ -1-.5-4.7). New

4.4. Proportion of fre shwater samples of putative drowning medium with repre­ sentative dia tom genera. CoccoJleis. CYl11he//o . and N(II'iculo. that are often asso­ ciated with fre shwater diaLOms.

71

Forensic Diatomology and Drowning

approaches to the diatom test may take advantage of the important eco­ logical properties of diatom communities. For example, Ludes has pro­ posed that continuous monitoring of freshwater sites and comprehensive species-level inventories of diatom flora at these sites may be useful in the medicolegal investigation of drowning deaths (3).

Frequency of Concordance of Frustules in

Bone Marrow and Water

The foundation of the diatom test for drowning is that the diatoms extract­ ed from tissues are derived from the drowning medium and not a medicolegally ilTelevant source. On this basis, the diatoms present in tis­ sue-extracts of the drowned should be concordant with those diatoms in the putative drowning medium. Such a concordance would be evidence for the validity of the diatom test, a validity not solely dependent of the mere presence of diatoms in tissues. We have studied a large series of cases of drowning in which both a femur and a sample of the putative drowning medium were available for analysis. Table 4.3 shows the distribution of cases on the basis of the diatom content of the drowning medium and the presence of frustules in the bone marrow. In a total of 52 cases, the bone marrow and drowning medium contained diatoms. FOlty-seven (90%) of these cases had diatom types in the bone marrow that were also represented in putative drowning medium. In five cases (10%), the type(s) of frustules found in the bone marrow differed from those in the putative drowning medium. In addition, in 34 cases of drowning that occUlTed in water that was devoid of diatoms, 29 cases (85 %) lacked demonstrable diatoms in the bone marrow. The monthly incidence of positive diatom tests with concordance of diatoms in the bone malTOW and putative drowning medium (Fig. 4.8) indicated that concordant tests were most frequent in April, July and August and lowest in February and December (no cases were available for analysis in January). Similarly, there was great seasonal variation in the frequency of positive diatom tests with concordance of diatoms in the bone marrow and putative drowning medium (c.f. Fig. 4.2 & 4.8). The one major weakness with the present analysis is the practical constraints placed on the concordance criterion used to determine the ori­ gin of the marrow diatoms. The series contained 5 cases that had marrow 72

Vulidity und Utili'.\' of Diutolll Test

portant eco­ L d s has pro­ -:: '- m prehensive . ~ u eful in the 30

,-----------------------------------~

•

Diversity Score

0

Temperature

25

15

25

~

'0

~

01

'':::

.....~

-.. ~

~"\

e diatoms in

~

evidence for

..:

5

20

U

~

::s

t! Q,)

-5

15

Q.

E ~

to­

10 4-~----~----~----~----~----~~ -1 5

and drowning ~.!SeS had diatom -; _: tive drowning - , ~ d in the bone ~ m. In addition, -_ .-aid of diatoms,

Sept

Oct

Nov

Dec

Jan

Feb

Month

4.5. Relationship between dia tom diversity (a rbitrary scale) and temperature in the Credit River, Mississauga, Ontario.

~arrow.

~ concordance ::-.edium (Fig. 4.8) - April, July and ere available for . variation in the :­ .atoms in the - _ & 4.8) . . - is the practical _ :. :ermine the ori­ . at had marrow

73

Forensic Diatomology and Drowning

4.6 & 4.7. Diatom genus diversity and the predominant species in water changes with the season. 4.6. Diatoms from October water from the Credit River, Mississauga, Ontario. 4.7. Diatoms from December water from the Credit River, Mississauga, Ontario . There is a distinct change in the genera present in the water samples (samples collected by R . Kashni, B.Sc.).

74

Vulidil), und Ulilil), of DiulOIll resl

60

-...

,-----------------------------------------------~

c

I II

5'J

'tJ 0

u

C

o ~

U~

40

O~

~~

c

lIl

~E

30

;'0 E c

20

.-u_ 0 C to

.. 0

::E Feb

lIilar

Apr

May

Jun

Jul

Aug

Scp

Oct

Nov

Dec

Month

4.8. Monthly proportions of freshwater drownings cases with bone marrow diatoms that match those in sample of putative drowning medium.

_- in water changes ~ ~~

Credit River,

__the Credit River,

~=r ~ ra

present in the

Number of Diatom Species in Bone Marrow

4.9 . Multiplicity of diatom species recovered from bone marrow in 37 positive diatom tests in Ontario. Canada.

75

Forensic Diatomology and Drowning

diatoms that were not represented in the drowning medium, and 5 cases that had marrow diatoms but the samples of putative drowning medi-um were devoid of diatoms. Although these cases comprise less than 4% of the series, the discrepancy must be explain if the quantitative data is to be use­ ful as evidence of the validity of the test. There are two possible explana­ tions: (i) diatoms in the bone marrow were present before drowning; or (ii) the samples of the drowning medium submitted to the laboratory are not representative of the fluid inhaled during the course of drowning. In this study, it is likely that the samples of water analysed were not representa­ tive of the drowning medium. In many of the 10 cases with anomalous results, the bodies were decomposed and found at sites that may have been remote of the site of initial immersion (e.g. "floaters"). In addition, in many bodies of water the local diatom flora can change both in abundance of species predominancy due to short-acting variables such as depth and light. These cases can also be used to calculate classical indices that are commonly used to evaluate the diatom test as a diagnostic test (section 3).

Size and Shape of Drowning-associated Diatoms The remarkable diversity of diatoms has led to a complex taxonomy and forms the basis of much of the botanical research into diatoms. However, the diatoms extracted from the bone malTOW of cases of drowning are not as diverse as the flora of freshwater. The diatoms extracted from the bone marrow represent a distinctive subset of freshwater diatoms characterized by size and shape. In most cases, no more than three distinctive species of diatom are found in any bone marrow extract (Fig. 4.9), frustules are usually <30 flm in size, and less than ten frustules per femoral extract are typically observed. The these characteristics are highly reproducible and the num­ ber of frustules rarely exceeds ten. The later criterion is an important prac­ tical feature of the diatom test since in cases of inadvertant laboratory con­ tamination of a bone malTOW sample dozens to hundreds of frustules are usually found in the extracts. Both pennate and centric frustules are routinely encountered in bone marrow extracts, but, pennate-type diatoms predominate in most cases (see Chapter 7). The ratio of the longest and shortest dimension of most drowning associated-pennate diatoms (aspect ratio) range from 1:-2

76

Validity and Utility of Diatom Test

.0. ,:, m . and 5 cases -= .:.:-uwning medium _c- - than 4% of the

~ : boratory are not : . . owning. In this :=-:e not representa­ with anomalous . may have been . In addition, in __ c tb in abundance :: _ :::uch as depth and . -.;:] indices that are _ :~ test (section 3).

.:: J iatoms ~:, _ e:x

taxonomy and : ::.a oms. However, - ~~ drowning are not ~ ed from the bone _:~m.s characterized

~

pe.:-ies of diatom are .::.Ie usually <30 /lm

_ ::__ t are typically _ .:-! ~ Ie and the num­ - III important prac­ "' - . t laboratory con­ of frustules are

• - ;: - ly encountered in ::":-;::iorninate in most _ .00 t dimension of ~ range from 1:-2

to 1:-5 and is seldom less than 1:5. In addition to the size of the diatom, this characteristic is the most important common feature of the frustules extracted from bone marrow. These diatoms are typically represented by of the genus , Cocconeis, Navicula , and Cymbella. For example, for the species of Cocconeis that are frequently found in bone marrow, the breadth, length and aspect ratio are approximately 18 /lm, 26 /lm, and 0.7, respectively. For the species of Cymbella that are found in bone marrow extracts, the breadth, length and aspect ratio are approximately 10 /lm, 33 11m and 0.3, respectively. In most cases, the number of distinctive types of diatoms in the water samples vastly exceeded the number of species in the bone marrow. On the basis of qualitative analysis, the diatom species found in the bone marrow, usually, represented the smallest diatoms in the putative drown­ ing medium. When more than one distinctive type of diatom is present in the bone marrow, the relative abundance of the different types of diatoms in the bone marrow was similar to that in the putative drowning medium. In addition, most of the water samples contained diatom types that were greatly in excess of 30 /lm in size (e.g. many Pinnularia spp.) and these diatoms are never, or rarely, found in extracted bone marrow. However, on occasion, small «30 /lm) fragments of typically large diatoms are recov­ ered in bone marrow extracts and can be found in whole and fragmented forms in the putative drowning medium. In these circumstances the frus­ tule fragments were usually near elliptical in shape with the axial ratio of 1: - 5. In some instances small numbers of Synedra frustules in excess of 30 /lm in maximal dimension can be observed in some bone marrow extracts. Such frustules were always fragmented and had an axial ratio of 1:4 to 1:8.

* 3. SENSITIVITY AND SPECIFICITY The epidemiological interpretation of a laboraotry test relies on numerical indices that relate to the ability of the test to detect the presence or absence of a particular disease or cause of death. The distinction between and "good" test and a "poor" test can, in part, be made by assessing several quantitative parameters including the sensitivity, specificity, and the posi­ tive predictive value of the test. Application of these concepts to the 77

Forensic Diatomology and Drowning

diatom test for drowning is problematic since it is not possible to calculate the relevant indices without making potentially prejudicial assumptions about the case population. Despite this, reasonable assumptions can be used to facilitate the calculation of some of the important indices such as sensitivity, specificity, and positive predictive value. T he Diatom Test and Diagnostic Indices

The commonly used test indices are defined in Table 4.4 and can be used to calculate various quantitative parameters using a set of assumptions and the data from Table 4.3. To calculate the relevant indices for the diatom test it is necessary to clarify the meaning, for the purpose of the present analysis, of true positive and negative, and false positive and negative. In the usually sense, a false positive is an erroneous test outcome in a population that is devoid of the disease that the test is designed to detect. In the case of the diatom test, this would correspond to the presence of diatoms in the femoral bone marrow in a non-drowning death. Since we have no information on the presence of spurious diatoms in the bone marrow of the non-drowned, this value is not known. However, this value can be estimated by assessing the number of individuals, in a series of freshwater drowning cases, that have diatoms in the femoral bone, and lack diatoms in the putative drowning medium. Similar defintions for true positive, true negative, and false nega­ tive can be deduced based upon the relationship between the diatoms in the bone marrow and the putative drowning medium (Table 4.5). Table 4.6 shows the results of calculating the various indices list­ ed in Table 4.4 using data from Table 4.3 and the assumptions described in Table 4.5. These traditional measures of diagnostic test accuracy and efficacy indicate that the diatom test is relatively insensitive, and highly specific. The high positive predictive value indicates that a positive indi­ vidual is statistically significance and reliable. The sensitivity and low negative predictive value indicates that a negative test can not be used to exclude drowning as a cause of death. These data imply that the diatom test using femoral bone marrow may not be the best screening tests for drowning but may provide useful and important information in individual cases. The definition of sensitivity used for the present analysis is similar to utility (section 1), in both concept and magnitude. On this basis, it is

78

Validity and Utility of Diatom Test

i

assumptions _~:n tions can be -! inclices such as

TABLE 4.3

Relationship Between Presence of Diatoms in Bon e Marrow and the Presence of Diatoms in Putative Drowning Medium

: ;..€ s

- .:s. can be used to . -.:: mptions and the . -- e diatom test it is .... , sent analysis, of = . In the usually - _ ?Opulation that is in the case of the

- ~ .. 'ormation on the .- ~ r:on-drowned, this . . _3 by assessing the - g cases, that have :,utative drowning . . c. and false nega­ ="'~ me diatoms in the

~

Diatoms in Bone Marrow I

Positive

Negative

Positive

47/52 3 (90%)

134/202

Negative

5/34 ( 15 %)

29/34 (85%)

(66%)

Diatom s in putative drowning medium 2

I

Numerator

2 Denominator 3 Concordant diatom types; True positives

.ai us indices list­ _:::lptions described .: est accuracy and c ":. iove, and highly :.:". .1 a positive inru­ .::.:. ~ -jtivity and low '- n not be used to ;:' '. ' that the diatom reening tests for _ . n in individual

~ -- ::malysis is similar ..,. . this basis, it is

79

Forensic Diatomology and Drowning

TABLE 4.4 Definition of Commonly Used Indicies in Descriptive Epidemiology

Test index

Definition

Meaning

Sensitivity

a/(a+c)

Ability rule-out disease

Specificity

d/(b+d)

Ability to rule-in disease

False-positive rate

b/(b+d)

Frequency of a positive test in disease-free patients

False-negative rate

c/(a+c)

Frequency of a negative test in patients with the disease

Positive predictive value

a/(a+b)

Proportion of true positives among all positive tests

Negative predictive value

d/(c+d)

Proportion of true negatives among all negative tests

a = true positive b = false positive c = false negative d = true negative

80

Validity and Utility of Diatom Test

TABLE 4.5 Estimates of Standard Param eters Used to Calculate Diagnostic Indices for the

Diatom Test for Drowning Using Femoral Bone Marrow

.-dem iology Concordant Diatoms Parameter

-

. ~:

di ease

Bone Marrow

Water

+

+

True positive True negative

-: ~

itive test in =':' :;cnts

+

False positive

+

False positive

TABLE 4.6 Diagnostic Indices for the Diatom Test for Drowning Using Femoral Bone -

- ~!Ue

positives _::i\'e tests

~ ~-

:rue negatives .... =;r ive tests

Test

Value

Sensitivity

0.26

Specificity

0.85

False-positive rate

0.15

False-negative rate

0.74

Positive predictive value

0.92

Negative predictive value

0.18

81

ForellSic Diotom% gy and Drowning

apparent that sensitivity of the diatom test changes wi th the month of drow ni ng. This is analagous to the increasing the prior odds of the test and increasing the posttest probability of a positive test outcome. It should also be apparent that procedural variants of the diatom test for drowning, such as the use of diatom-burden in the lung with a threshold concentration for a positive test,would most likely give a higher false positive rate thus decreasing the specificity of the test. However, it is also likely that a test using lung ti ssue should, in principle, produce a lower false negative rate thus increasing the sensi tivity of the test. It is unclear if this putative increase in sensitivity would be medico legally significant if the specifici­ ty of the test were reduced to give reasonable doubt about the tests signif­ Icance.

* 4 . REFERENC ES I. Pollanen MS Cheung, L., Chaisson, D.A. The Diagnostic Value of the Diatom Test for Drowning. 1. Utility. : A Retrospective Analysis of 77 1 cases of Drowning in Ontario, Canada. Journal of Forensic Science 1997;42(2):28 1-285. 2. Pollanen MS. The Diagnostic Value of the D iato m Test for Drowni ng II. Validity. : Analysis of diatoms in the bone marrow and drowni ng medium. Journal of Forensic Science 1997;42(2):286-290. 3. Ludes B Coste, M. Tracqui, A., Mangin P. Continuou s river monitor­ ing of the diatoms in the diagnosis of drowning. Journal of Forensic Science 1996;41 (3 ):425-428.

82

: irh [h e month of :- c d of the test and ":Dme. It should also :0, drowning, such .. ~ 'oncentration for .,;: JO itive rate thus - 0 likely that a test ;:r :alse negative rate : ~ ar if this putative [ if the specific i­ :.It the tests signif­

_?ostic Value of the -'.\"e Analysis of 771 -:' ~ Forensic Science Te. t fo r Drowni ng and drowning .: "6-_90. lOU river monitor­ "'Ium al of Forensic ~ow

5. Laboratory procedure

for the diatom test

* 1. COLLECTION OF SAMPLES To initiate the diatom test for drowning the preparation and collection of appropriate samples commences at the scene of body recovery. There are three important considerations for the collection of samples for forensic diatom analysis: 1. Collection of samples of the putative drowning medium at the scene

of body recovery.

2. Collection of biological samples at postmortem examination. 3. Maintaining appropriate chain of custody of samples for submission

to the laboratory.

Collection of Samples at the Scene

Samples of the putative drowning medium should be obtained fro m the scene of body recovery as soon as possible after the body is discovered. Water samples can be placed in clean containers, sealed with appropriate evidentiary tags and labeled with the decedent's name, location of sam­ pling site, along with the date, time and name of the individual sampling the water. Since the diatom content of freshwater varies with temperature, time of day, and season , approximately 500 mL to 1,000 mL of water is

83

Forensic Diatomology alUi Drowning

indicated in a typical case. In many cases, it is possible to extract diatoms from a far less volume of water, particularly if the concentration of diatoms in the water is high. If the body is recovered form a deep locale, it may be necessary to sample water at several depths to obtain an adequately representative sam­ ple of diatoms. This is best accomplished using police marine units equipped with investigative divers or search personnel. In some instances, the corpse may be found in a location that is remote from the actual site of drowning due to the action of natural currents in the water. On this basis, if investigative evidence suggests a particular site of entry into the water, it is recommended that water samples of the putative drowning medium be obtained closest to the site of entry into the water. However, in the case of a grossly decomposed body it may not be possible to locate the site of entry. In this circumstance, postmortem samples may assist in obtaining a representative sample of drowning medium.

Collection of Biological Samples There are two types of samples from the postmortem examination that are useful as starting materials for the diatom test for drowning: 1. Tissue(s) from which potential drowning-associated diatoms can be

chemically extracted.

2. Samples of fluids from the body that may be representative of the putative drowning medium.

Postmortem Tissue Samples Although many types of tissues have been advocated for the diatom test for drowning, the most useful for general application to all putative drown­ ing deaths is the femoral bone marrow. The main reason for preferential use of the femoral bone is that the marrow cavity is protected from decom­ position and postmortem contamination by extraneous diatoms that are present on the external surface of the body. In addition, in the badly decomposed body when the parenchymal organs have putrefied the femur will still be available as a source for diatoms. The main reason to prefer the femoral bone marrow for starting material for the diatom test is the 84

Procedure f or Diatom Test

extract diatoms ~oncentration of

;: :.J .=.

:- _y be necessary to r~ re-sentative sam­ : :' ~: e marine units _ I::: some instances, :::'::1 the actual site of -. er. On this basis, ;;' ..rry into the water, .~~. 'ning medium be ;?': r, in the case of : ,:- ~ ~ate the site of ..:.~ ;; is( in obtaining a

~

- mination that are :--~g :

~_

i toms can be

_: .:X : utative dr W:: ­ _ _:-.

~-or

prefe re - -

extensive scientific investigation that has validated the use of the femoral bone malTOW as a site to extracted diatoms from postmortem tissues. In the author's experience, a satisfactory alternative to the femoral bone, in the fresh body, is the blood from the left side of the heart (pulmonary veins or left atrium). Blood from the left side of the heart may contain diatoms that have just ruptured through the alveolar-capillary membrane and entered the systemic arterial circulation. Samples of left heart blood should be harvested as soon as the chest is opened. The pericardium is incised and blood is aspirated from the left atrium with a syringe. The blood can be placed in a tube with no preservatives and frozen or refrigerated prior to submission to the labora­ tory.

Removal of the Femur Postmortem removal of the femur can be easily achieved using standard autopsy equipment (Figs. 5.1-5.4). The main priorities for removal to the femur is that the femur be intact (e.g., free of fracture) and that the possi­ bility of contamination of the bone surface with exogenous diatoms is eliminated. The latter can be achieved by extensive washing of the surface of the body, and in particular, the legs prior to dissection. It is also impor­ tant for the prosector to extensively wash the gloves used while removing the femur if the gloved hands have been contaminated with diatoms that might be present in the stomach contents or on the skin. To remove a femur, the skin of the knee is incised just medial to the patella and the inci­ sion is continued along the anterior patellar border, and along the lateral a pect of the leg to a point just proximal to the greater trochanter. The muscle layers and fascia are dissected to free the bone, and the joint cap­ -ule of the femoral head and knee are incised. The femoral condyles can freed fi rst and the entire bone can be maneuvered and rotated to free the ~ moral head from the acetabulum. The femur can be frozen in a plastic g prior to submission to the laboratory.

stmortem Body Fluid Samples '--.I

_roms from samples of fluids from the body may be indicative of the 'om content of the drowning medium. In particular, in the drowning 85

Forensic Diatomology and Drowning

5.1-5.4. Procedure for the diatom test for drowning. 5.1. An intact femur is dis­ sected from the thigh after washing of the body. 5.2. The femur is stored frozen in the laboratory until use. 5.3 The femur is cut longitudinally using an electric saw. 5.4. The femoral bone marrow is removed from the medullary cavity and used for acid digestion. (Photographs by Jeff Arnold) .

86

~:-_ intact femur is dis­ : ::C':TIur is stored frozen ~ -. Jly using an electric -:; ~ .edu llary cavity and

Procedure for Diatom Test

process (usually of conscious victims), large volumes of water can be swallowed and distend the stomach. As well, water may be found in the sphenoid sinus and tracheobronchial tree. Water from both these sites may be useful as a basis for comparison with diatoms recovered from the femoral bone marrow. However, since water may passively enter the body in the postm0l1em period, particularly in rough water, the presence diatoms in the stomach contents, sinuses, or tracheobronchial tree cannot be used as evidence of drowning, in most cases. The use of diatoms extracted fro m stomach contents or the sphe­ noid sinus fluid can be used as a reference to compare with diatoms in the femoral bone marrow. This is of particular value if samples of the putative drowning medium are unavailable or if the site of drowning is not known. Chain of Custody

It is important that all samples from the scene and derived from the post­ mortem examination be appropriately retrieved, and labeled. If the sam­ ples are to be analysed in a referral laboratory, the chain of custody must be documented to show continuity of the physical evidence. Analytical results of evidence that has not been well doc umented can lead to exclu­ sion of the evidence in a court of law. On this basis, each individual labo­ ratory that performs the diatom test should have a standard routing proce­ dure to track specimens through processing to reporting. Prior to processing of the water samples from the scene, the water can be stored at 4 degrees centigrade (standard refrigerator temperature) for several days to inhibit extensive growth of microbes. Alternatively, a diatom-free formalin solution can be added to the water (1 mL 2% forma­ lin to 500 mL of water) to inhibit microbe growth indefinitely and the sam­ ple can then be stored at room temperature. Material collected at the post­ mortem examination hould be kept fro zen prior to processing.

* 2. LABORATORY METHOD Overview

The extraction of diatom frustules from samples of water, fluids, and femoral bone marrow is achieved through chemical or enzymatic digestion 87

Forensic Diatomology and Drowning

and centrifugation (Fig. 5.1-5.4). Although several extractive methods have been reported, nitric acid remains the best tissue solubilizing agent, particular for femoral bone marrow since the marrow contains trabecular bone. A major concern in the extraction process is the elimination of possible contamination of the evidentiary samples with exogenous diatoms or cross-contamination of the samples with diatoms from other samples. To avoid contamination, several elementary principles must be considered: 1. Use reagents that are free of diatoms as confirmed by microscopic examination. This includes the soap and distilled water used to clean instruments and glassware. 2. Minimize the number of glass surfaces and vessels with which the

acid-extract comes into contact.

3. If possible, process the femoral bone marrow prior to water samples to reduce the likelihood of cross contamination in the laboratory. 4. Use disposal pipettes and change pipettes frequently to avoid crosscon tamination. 5. Clean all equipment with large volumes of diatom-free water. 6. Examine reagents on a periodic basis to ensure diatom-free status. 7. Keep laboratory records of sequential positive test results and investi­ gate possible laboratory source contamination if several positive test results occur in sequence.

Materials 1. Electric band saw. 2. Instrument to extract bone marrow (spatula). 3.250 mL beaker and vented fume hood. 4. Nitric acid and distilled water (diatom-free). 5. Pasteur pipettes. 6. Centrifuge and glass conical centrifuge tubes. 7. Oven and refrigerator. 8. Mounting medium, slides, and coverslips. 9. Cleaning materials.

88

Procedure for Diatom Test

tractive methods _ubilizing agent, ;: _tains trabecular

, :.::e elimination of : _ .\ ith exogenous i.", ms from other ri _ciples m ust be

_ microscopic _' r used to clean ::h which the L'

-,\-ater samples

-e ~ aboratory. : ") avoid cross-

e water. . :,m-free status. -".::Wts and investi­ ~ e_ positive test

Procedure Extraction oj Femoral Bone Marrow The initial stages for the preparation of bone marrow extracts is best per­ formed by two people. One individual cuts the bone and the other isolates the bone marrow th us minimizing the possibility of contaminating the bone marrow by transferring potential contaminants from the outer surface of the bone. Alternatively, one individual can cut the bone and remove the bone marrow provided that gloves are changed between the two tasks. As a general rule, it is desirable to rinse gloves in distilled water prior to use. 1. Partially thaw the fem ur, typically for 30 mi nutes if the femur has

been stored at -20 degrees centigrade.

2. Hemi-section the fem ur along the long axis using the band saw. 3. Isolate the bone marrow using a spatula and pLace the marrow directly into the 250 mL beaker. Isolate both the yellow (fatty) and red (hematopoetic) bone marrow and much of the trabecular bone at the epiphyseal ends of the bone. Although the yield may vary, approxi­ mately 50 g of marrow is usually harvested from an adult femur. 4. Add approximately lO mL of concentrated nitric acid to the bone marrow in the 250 mL beaker and place onto a hot plate in a fume hood_ S. Once the bone marrow has turned a semi-fl uid brown-black colour (indicating hemolysis) added an additional 50 mL of nitric acid and si mmer on the hot plate. 6. Incubate on the hot plate at approximately 30-40 degrees centigrade and avoid boiling the acid-suspension. Digestion times are variable but may take up to 3 days using 8 hour cycles of simmering and 16 hours of incubation at room temperature. 7. Replenish nitric acid as the acid fumes from the 250 mL beaker. 8. Once the suspension has turned a translucent pale yellow colour in a final volume of about 50 mL, cool to room temperature_ 9. Depending on the initial amount of fatty marrow in the extract, a lipid layer may separate on the surface of the acid suspension_ If the lipid layer is minimal, aspirate the lipid from the surface and collect the acid suspension. If the lipid layer is extensive, the beaker can be

89

Forensic Dia{om%gv {fnd Drowning

refrigerated to solidify the lipid and the acid-suspension can then be pipetted from beneath the lipid. 10. The lipid-free acid suspension is placed into the appropriate number of glass centrifuge tubes, diluted with distilled water and centrifuged for 20 min (300g - 500g). The supernatant is aspirated, and discard­ ed. The barely visible or indiscernible pellet and the last 5 mL of the conical tube is retained. To the remnants of the acid-suspension, addi­ tional distilled water is added to fill the tubes. 11. The washing and centrifugation steps are repeated 2-3 times. On the final centrifugation, the last 1-2 mL in the conical tube and the indis­ cernible pellet are aspirated and placed onto a glass slide. The slide is dried in an oven, and mounted. The slide is permanent and can be stored indefinitely. Extraction of Fluid or Water Samples

The procedure for extraction of diatom frustules from fluid or water sam­ ples is similar that used for tissue. The samples are first centrifuged to pel­ let particulate matter (including diatoms) and the supernatant is discarded. The last 5 mL of supernatant in the conical tube and the pellet are retained and approximately 25 mL of nitric acid is added, and the suspension is incubated at room temperature for I hour prior to re-centrifugation, sequential washing, and mounting as for femoral bone marrow extracts. If water samples are to be processed the initial volume of water to use for diatom isolation is dependent on the concentration of diatoms in the water. On this basis, a small amount of the water can be examined microscopically using a water preparation to determine the optimal vol­ ume to use for diatom extraction. If numerous diatoms are visible as little as 20 mL of water may be sufficient to extract a representative population of diatom species in the water. In selected circumstances, it may be nec­ essary to concentrate in excess of 500 mL of water, however, this is rare.

* 3. LABORATORY METHOD FOR INANIMAT E OBJECTS Overview It some circumstances, it may be necessary to extract diatom frustule from the surface of an inanimate object. This analysis is an investigati e

90

Procedure f or Diatom Test r

an then be

m priate number ~ and centrifuged ~: e . and discard­ last 5 mL of the --u pension, addi­ _- 3 times. On the . b and the indis­

tool that uses the diatom frustule as a tracer that may indicate if an article of physical evidence has been exposed to a particular water source. It may be possible to identify and enumerate specific diatom types that are shared between an article of evidence and, for example, a specific river or lake. Such a concordance of diatoms can indicate that the diatoms on the article of evidence could have originated from a relevant water source. The extraction of diatoms from physical evidence is a simple mod­ ification of the other methods for forensic diatom analysis. Suspensions of distilled water washings of the physical evidence items are used for diatom extraction. Reference water samples from the body of water of interest are prepared separately.

Procedure

:~

id or water sam­ to pel­ ~ tant is discarded. .c pellet are retained the suspension is re-centrifugation, ~ marrow extracts. . ol ume of water to ~~.lon of diatoms in ~r an be examined -e t e optimal volMe visible as little "cntative population . ~c . it may be nec­ ~ever, this is rare. .

~en trifuged

IE OBJECTS

..:: diatom frustules _ : - an investigative

In addition, to the equipment needed for routine diatom extraction, several water-tight plastic bags are used to prepare washings of the articles of evidence. Each plastic bag used for preparation of a wash­ ing should first be extensively rinsed with large volumes of distilled water to remove any possible contaminating diatom frustules. The articles of physical evidence are individually washed in plastic bags using an arbitrary volume of diatom-free distilled water. As a general rule, enough distilled water to liberall y cover the evi­ dence is satisfactory. The washings are produced by agitating the evi­ dence and the resultant suspensions are prepared as any fluid starting material.

* 4. INTERPRETATION Microscopic Examination of Bone Marrow Extracts Approach to the Analysis of Ex tracts

The acid-resistant extracted material isolated from the femoral bone mar­ row must be examined microscopically to detect the silica-based frustules of the putative diatoms . Several characteristics of the extracted material need to be examined and considered in interpreting the significance of test: I. Recognition of diatom frustules , if present. 91

Forensic Diatomology and Drowning

5.5 & 5.6. Basic frustule shapes. 5.5. Diatom with radial symmetry. 5.6. Diatom with two axes of symmetry orthogonal to each other.

92

Procedure for Diatom Test

5.7-5.10. Morphological variation in frustule shape. 5.7. Large pennate diatom with prominent raphe. 5.8. Oval pennate diatom with peripheral punctate mark­ ings. 5.9. Pennate diatom with prominent raphe and rib-like structures. 5.10. Pennate diatoms with the typical naviculoid shape.

ra ial symmetry.

93

Forensic Diatomology and Drowning

5. 11-5.14. Colony formation in freshwater diatoms. 5.11. Lateral assembly of diatoms into rectangular arrays attached at corners. 5.12. Radial spoke-like arrays shaped into stars . 5.13 & 5.14. Points of attachment to form colony aggre­ gates of diatoms . 94

Procedure for Diatom Test

~ __eral

assembly of

.:. Rad ial spoke-like : " nu colony aggre-

5.15-5 .18 . Morphological characteristics of acid-insoluble material in bone mar­ row that co-purify with diatom frustules. 5.15. Spicules of crystalline material. 5.16. Spicules arranged into star-like clusters. 5.17 . Short branching spicules. 5.18. Crystalline material with polygonal shapes.

95

Forensic Diatomology and Drowning

2. Awareness of the physical features of diatoms usually associated with drowning. 3. Awareness of acid-resistant bodies that may superficially resemble

frustules.

4. Comparison of marrow frustules with diatom frustules from reference material.

Microscopic Features of Diatom Frustules The frustules of diatoms have several characteristic microscopic features under interference or phase contrast microscopy. These highly character­ istic morphological feature make an extensive discussion of diatom taxon­ omy and structure inessential for the forensic scientist using the diatom test for drowning. The cell wall or frustule of most diatoms conforms to one of two body plans: 1. Centric shape characterized by circular configuration (or rarely,

polygonal) with radial symmetry (Fig. 5.5)

2. Pennate shape characterized by one or two axes of symmetry (Fig.

5.6). The most common axis is the sagittal or longitudinal axis and

the other major axis is transverse (orthogonal to the sagittal plane).

Diatomist also recognise other forms of frustule symmetry; however, in diagnostic preparations from human tissue, most frustules are have an en fasse orientation making the other modes of symmetry less important for frustule recognition. Since, a microscopic particle with centric or pennate shape may not be a diatom frustule, other important features (Fig. 5.7­ 5.10) include: l. Substructure of the frustule surface. Many pennate diatoms have a central line along the sagittal axis known as the raphe and most frus­ tules have periodic striae or pores on the surface. Parallel rib-like structures may radiate from the raphe of pennate diatoms and radial lines may emerge from a featureless hyaline central zone in centric diatoms. Araphid diatoms will have similar substructure. 2. The frustule is refractile. The frustule scatters light particularly at the edges.

96

Procedure for Diatom Test

ociated with

conforms to

r rarely,

_ inal axis and plane).

~ ~.l.::>i ttal ~.3-

however, in are have an en important for .:-e~ rric or pennate : .1IU r eS (Fig. 5.7­ . ';

·Ld~· -

:

,:' _:oms have a - .e ::.nd most frus­ . -:::.... el rib-like '"' : ":ns and radial ZO:1e in centric ::rre.

ularly at the

3. Colony fonnation. In water samples, diatoms may associate into aggregates that have a specific, usually periodic, supra-structure. Individual diatom frustu1es of the same type that form a colony in the water sample are sometimes extracted from postmortem tissue (Fig. 5.11-5.14). In addition, to these three basic features, other subtle morphologi­ cal characteristics can be used to differentiate between pennate taxa. Recognition of these features will assist in detetcting frustules in tissue­ extracts. The two main characteristics are the overall shape of the frustule and the shape of the apices. The shape of the pennate frustule varies con­ siderably ranging from a narrowly linear shape to dorsiventral (i.e. two facing margins have a different slope of curvature). Further complexity is formed by variation of the apices which may be blunted and featureless, or have subtle variations on a capitiate shape. As well, most diatoms are homopolar (e.g. symmetrical identical apices), but, some important fresh­ water taxa are heteropolar. A guide to the morphological range of frustu­ lar anatomy is illustrated in chapter 7. In water samples, diatom frustules have a wide range of size. Small diatoms are typically about 10 flm in length and large species can exceed several hundred micrometres in length. However, diatoms extracted from the bone marrow that are associated with drowning, represent a small sub­ set of the total diatom population. These so-called "drowning associated diatoms" have dimensions that allow embolization in the arterial system and eventual deposition into the peripheral tissues such as the bone mar­ row (Chapter 4 section 2). Typically, these diatoms are only a few times the size of an erythrocyte (10 - 40 flm) and are seldom longer than 50 flm.

Non-diatomaceous Silica Many geometrically or regularly shaped microscopic particles in bone marrow extracts can resemble a diatom frustule (Fig. 5.15-5.18). These particles are crystals of inorganic silica that are found in varying amounts in normal human bone marrow extracts. Since the crystals are composed of silica, like the diatom frustule , these particles resist digestion with nitric acid. In some bone marrow extracts, the crystalline material may be the dominant material in the acid-resistant extract and may obscure the detec­ 97

Forensic Diatomo!ogy and Drowning

tion of diatoms. Although some individual silica crystals may have an overall configuration and size similar to a pennate diatom, several mor­ phological features can be used to differentiate this crystalline material from diatom frustules : l. Each crystal has a regular geometric structure with a crystal face . 2. The crystal has a continuous surface with no fine structure (e.g. raphe, pits, striae) 3. Numerous crystals with identical morphological properties will be present in the extract. 4. The crystals may aggregate into radiating arrays surrounding a nucle­ us of debris.

Although the amount of crystalline material in bone marrow extracts is highly variable, most extracts have very little or none of this interfering substance. The bone marrow burden of this siliceous material may be related to occupational or environmental exposure during life. Other inert and insoluble microscopic particles can sometimes be found in acid-extracts. Most of these insoluble particles are amorphous and may be incompletely digested collagen and elastin fibres. On occa­ sion, asbestos filaments can be found in acid-extracted bone marrow. The Significance of Bone Marrow Diatoms

Diagnostic Criteria of the Diatom Test Once diatom frustules have been found in acid-extracts several criteria must be fulfi lled before the medicolegal significance can be interpreted: 1. Frustules extracted fro m tissue must be intact and whole, or 2. A fragmented frustule should be cleaved along a line of symmetry; thus allowing the observer to deduce the overall configuration of the intact frustule. 3. The number, size, and shape of the frustules extracted from the bone marrow must confonn with the routine standards of the diatom test. 4. The frustules in the tissue extract should be concordant with diatoms in the drowning medium.

98

---------------

-

-

----

-

---

Procedure for Diatom Test

may have an . ~m . several mor­ -talline material

.::;. stal face. ...rure (e.g. raphe,

~ ..:~din g

11:" .:..:TOW

a nucle­

extracts is

c " {his interfering ::naterial may be :: life.

· e are amorphous - ii res. On occa­ · - ne marrow.

n

Determining if the diatom frustules in the bone marrow are concordant with those in the drowning medium can be accomplished with either a direct morphological comparison of the frustules (e.g. photomicrography) or by taxonomy. In the case of a taxonomic analysis, diatom frustules can be classified to genera and species by an expert diatomist and the types of diatoms from each of the samples compared, Taxonomic classification, however, is not necessary since simple morphological comparison is suf­ ficient to establish the identity of the diatom types in different samples. Frustules in acid-extracts of the femoral bone marrow, in principle, may be derived from laboratory contamination if strict attention to detail is not observed in specimen preparation. However, such false positive test results can be easily differentiated from a legitimate test. The main factors that differentiate false positive tests from true positive tests are:

everal criteria be interpreted:

or ·c of symmetry; :-'0le ,

,;;." from the bone · ' . e diatom test.

::...c.r with diatoms

1. False positive test will have vast numbers of frustules compared with the typical <10 frustule per marrow extract in true positive tests, 2. The frustules in false positive tests involve a broad range of sizes and shapes, whereas the true positives represent a limited subset of "drowning-associated" diatoms. 3. A source of contamination may be apparent. No meaningful conclusion can be drawn if the number and size of the diatom frustules in the bone marrow exceed that of a typical test. In such cases, the diatom test should be reported as noncontributory or incon­ clusive. In selected cases, it may not be possible to obtain samples of the puta­ tive drowning medium. In this circumstance, fluids obtained at postmortem examination can greatly assist in the interpretation of frustules in the femoral bone marrow. In particular, frustules in the marrow can be compared with those in the sinus fluid and stomach contents. When fluids from the sinuses or stomach are not available and only a femur is used for analysis, the signifi­ cance of frustules in the bone marrow is more difficult to asceltain. If the diatom frustules in the bone marrow conform to the criteria it is virtually cer­ tain that the diatoms originated in the drowning process. However, the best approach to the diatom test is an analysis of all the relevant samples including the femoral bone marrow and a sample of the putative drowning medium.

99

Forensic Diatomo[ogy and Drowning

Evidentiary Value of the Diatom Test The rules that govern the evidentary standards of science in the courtroon are evolving (1). In the United States of America, two main sets of rules of evidence are used to determine the admissibility of scientific evidence in a court of law: the Frye Test and the Federal Rules of Evidence. In the Frye test, the scientific evidence must be "generally accepted" as deter­ mined by meeting three criteria: 1. The evidence must be based on a fundamental scientific principle or

discovery.

2. The technique used for appyling the fundamental scientific or discov­ ery must be valid. 3. The technique's specific application on which the expert testimony is

to be based must be valid.

The Federal Rules of Evidence are less directive in providing criteria for assessing the admissibility of evidence and allows the trier of fact to deter­ mine if the expert testimony is well informed based on education, experi­ ence, skill, or special knowledge. At this time, the diatom test for drowning has not been challenged under the Frye rules of evidence but the test has been ruled admissible as sci­ entific evidence in several states in the United States of America, and provinces in Canada. Clearly, the diatom test meets the criteria of Frye rules 1 and 2. The diatom test is based on sound scientific principles regarding the penetration of diatoms through the alveolar-capillary barrier, and is logically consistent (Frye rule 1). As well, the procedure used to extract diatoms from bone marrow is a standard and generally accepted method in many countries over many years and can be quantitatively assessed (Frye rule 2). Frye rule 3 must be assessed relative to an individual case; however, if the methods outlined in this chapter are used with proper care, the criterion of this rule would be satisifed.

* 5. OTHER METHODS Electron and Atomic Force Microscopy

In most laboratories that perform the diatom test for drowning, acid digests are examined by light microscopy using phase contrast optics. 100

Procedure for Diatom Test

-;;:_ the courtroon ~ .n sets of rules . ':-entific evidence :.: E ·idence. In the ~ ' epted" as deter-

[

T: ::... principle or .~:-.ti fic

or discov­

;:--e:1 testimony is

(' idi ng criteria for ..c:: of fact to deter­ __ ~ du cation, experi­

- : been challenged dmissible as sci­ . ' a, and provinces - e :ules I and 2. The ,;:~ the penetration of :_~. - consistent (Frye . . ~ bone marrow is a :- . 0 ·er many years -: ~ must be assessed .... cd in this chapter :Je - tisifed.

:CDY

- -,. drowning, acid contrast optics.

However, some investigators advocate the use of scanning electron microscopy for the detection of diatom frustules (2) . The scanning elec­ tron microscope gives a higher resolution image of the surface features of the frustule and greater magnification can be used to compare the individ­ ual morphological features of frustules extracted from samples of putative drowning medium and the bone marrow. However, for most applications the degree of resolution afforded by phase contrast microscopy is satisfac­ tory for forensic diatom analysis. In addition, most laboratories are equipped with light microscopes whereas many forensic pathology labo­ ratories and mortuaries lack scanning electron microscopes. Since the advent of electron microscopy several new imaging tech­ niques have been invented and are now routinely applied in biological research. One of the most promising methods developed recently are the scanning probe microscopes which include the atomic force microscope. The atomic force microscope is a non-optical method of imaging the sur­ face structure of microscopic and mesoscopic structures. This imaging method produces a computer-generated topographical map of the sample surface and has routine magnification and resolution at par with, or beyond, routine scanning electron microscopy. The atomic force micro­ scope produces images of diatom frustules at similar resolution to scan­ ning electron microscopy and may eventually be useful for routine foren­ sic applications (Fig. 5.19) .

Molecular Biological Approach to the Diatom Test Traditional analytical procedures to detect diatoms in human tissues have relied on the physio-chemical extraction of the diatom frustules from sam­ ples. However, recent developments in forensic molecular biology indicate that it may be possible in the near future to detect minute quantities of diatom DNA in human tissues from victims of drowning. This possibility is made likely by the ability of the polymerase chain reaction to amplify small amounts of DNA from postmortem samples. Theoretically, it may ultimate­ ly be possible to extract DNA from bone marrow in a case of putative drowning and to demonstrate diatom DNA by polymerase amplification. Initial studies using minute quantities of ribosomal RNA from picobacteri­ um added to human tissues in vitro indicate that microbial nucleic acids can be easily amplified in the laboratory under forensic conditions (3). 101

to

o

-

5.19. Atomic force microscopy (A-C) and scan ning electon microscopy (D-E) of diatoms. The same type of diatoms are showed in the corresponding images A & 0, B & E, and C & F. (Technical assi stance pro­ vided by E. Sild and P. Markiewicz).

~.

~

Cl

a

I:)..

;os

~

'<

Oo

c;-

Cl

;:l

Ci

aIS·

'"Ci·

~ ;os

6J

Procedure for Diatom Test

The use of diatom DNA for development of a molecular biological test for drow ning is supported by important characteristics of diatom DNA. Diatom DNA is an ideal candidate for amplification from human tissues for three main reasons: 1. Limited homology between human and plant DNA. 2. High copy number of non-nuclear (plastid) genes in plant cells. 3. The presence of highly conserved genes in the plant kingdom, includ­ ing the genes that encode the enzymes for photosynthesis.

The main candidate gene for potential amplifiation is the plastid gene encoding the major photosynthetic enyzme ribulose 5-phosphate decarboxylase. This enzyme regulates a key regulatory step in the inter­ mediary metabolism of plants and is well studied. Similarly, since diatoms have a characteristic frustule that is a feature unique to diatoms, genes encoding frustule-associated proteins (e.g. frustulin) are also important targets for amplification.

* 6. REFERENCES 1. Bohan TL Heels, EJ. The case against Daubert: The new scientific evi­ dence "standard" and the standards of the several states. Journal of Forensic Sciences 1995 ;40: 1030-1 044. 2. Pachar JV Cameron , I.M. Scanning electron microscopy : Application to th identific ation of diatoms in cases of drowning. 10urnal of Forensic Science 1992;37:860-866. 3. Kane M Fuk unaga, T, Maeda, R., Nishi, K. The detection of picoplank­ ton 16S rDNA in cases of drowning. Int J Legal Med 1996;108(6): 323­ 326.

103

6 . Investigative applications of the diatom test

* 1. GENERAL APPLICATIONS OF THE THE DIATOM TEST Indications for the Diatom Test The decision to perform the diatom test for drowning is made by either the investigating officer or the forensic pathologist when there is a suspicion of drowning or if there is no anatomical cause of death after a complete postmortem examination in a criminal case. In the latter circumstance, it should be remembered that even if a body is found out of water (i.e., on land) death may have been due to drowning. This occurs in at least two cir­ cumstances : 1. Accidental or suicidal drowning with postmortem drying and dressing of the body. The deceased is usually found in bed. 2. Homicidal drowning with the body moved from water and concealed. The deceased may be decomposed, skeletonized, or burned post­ mortem.

In more usual investigative circumstances, the diatom test will be used as an ancillary procedure to confirm drowning as a cause of death as indicat­ 105

Forensic Diatomology and Drowning

ed by postmortem findings and the circumstances surrounding the death. For routine application, the diatom test for drowning is usually applied to the following: 1. Bodies recovered from water with investigative or postmortem evi­ dence of drowning. The diatom test can be used to confirm drowning (e.g. scuba diving fatalities, boating mishaps, suicidal drowning). 2. Bodies recovered from water with li ttle investigative information or

postmortem evidence of drowning (e.g. a dead body placed into the

water as in a drug overdose and a dumped body). The presence of

diatoms in the bone marrow will discriminate live entry into the

water.

3. Decomposed bodies in water. 4. Dismembered body parts in water.

Contra-i ndications for the Diatom Test There are important contra-indications for the diatom test for drowning using femoral bone marrow. These contra-indications related to the possi­ ble contamination of the femoral bone marrow with exogeneous diatom frustules that are not related to the drowning process. This occurs in two main circumstances. 1. Fracture of the femur as may occur with postmortem damage and dis­ memberment due to the action of propellors, and tidal forces coupled with decomposition. 2. Selected cases of prolonged immersion in water at great depths . However, in many circumstances of prolonged immersion in water the analysis of the femoral bone may still be a reliable tool to assess drown­ ing. In several cases, of prolonged immersion postmortem the femoral bone marrow will not contain diatom frustules indicating that diatoms do not inevitably enter the decomposing femur. This may be due, in selected cases, to adipocere which may encapsulate the femur and pro­ tect it from exposure to water despite prolonged immersion. On occa­ sion , diatom frustules can be recovered from cases of prolonged immer­ sion and the size, shape, and quantity of frustu1es is typical of a true positive test result. 106

Applications of Diatom Test

..... _:mding the death. -u lly applied to

* 2. DECOMPOSITION AN D THE DIATOM TEST

J

Aquatic Decomposition and the Diatom Test :::-rmortem evi­ ~'""'~ drowning . ~ drowning).

-=-::..~

presence of __ ~~. into the

:c [ for drowning ted to the possi­ :: -:. :)geneous diatom ~m occurs in two

:-~ _

amage and dis­ forces coupled grear depths. ;-:- -ion in water the . "' __ 0 ssess drown­ -"::em the femoral . ~: : __ g that diatoms may be due, in ::.e fe mur and pro­ .

~!,')lo nged immer­ _ ..: pical of a true

The lesions of freshwater drowning are non-specific and in a decomposed body the changes may be indistinguishable from the effects of decompo­ sition. In particular, in a decomposed body recovered from water, heavy boggy lungs, the presence watery-fluid in the airways, and pleural effusion are consistent with drowning but may also be due to decomposition. On this basis , gross postmortem findings are seldom useful in the diagnosis of most decomposed drowning deaths . The diatom test for drowning has a special value in these cases, as this may be the only positive factual evi­ dence that corroborates evidence of drowning revealed by police investi­ gation. The diatom test may be successfully applied to bodies in water for many years . In cases of immersion for 2-12 years, acid-extracts from femoral bone marrow have revealed diatoms that conform to the charac­ teristics of a typical positive test (Chapter 4 & 5), indicating that the diatom did not enter the marrow postmortem. In addition, adipocere may act as a protective layer. Besides this empirical evidence, some experi­ mental evidence suggests that aquatic putrefaction does not deposit diatoms in the marrow of long bones. The medullary cavities of long bones immersed in particulate dye solutions do not become discoloured, indicat­ ing that the cortical bone is a barrier to diatom impregnation. Timperman also describes the absence of diatoms in the marrow of sea burials that have been exposed to rough sea water for extended periods of time.

Skeletonized Bodies and the Diatom Test A useful, albeit rare, circumstance for the application of the diatom test is the skeletonized body found on land. Although the maxim "a body in water may not have drowned" is occasionally true, it is also apparent that "a body on land may have drowned". Although the latter is true, most death investigator and pathologists will not entertain drowning as a cause of death for a body found on land. However, the diatom test for drowning applied to cases of this type may provide surprising results that indicate drowning is the cause of death despite the circumstances to the contrary 107

Forensic Diatomology and Drowning

(see section 8). Some forensic pathologists use the diatom test in all sus­ picious or homicidal deaths with no apparent anatomical cause of death. A drowning death that is found on land is almost always a homici­ dal death that is concealed in a remote location. Bodies are usually gross­ ly deomposed or skeletonized at the time of recovery (Fig. 6.1 - 6.3). The diatoms test may be easily applied to the cases of this type since the long bones are intact and may be partly protected by mummified soft tissues. However, it is usually necessary to take great care in processing the femur in the laboratory to eliminate the possibility to transferring exogeneous soil diatoms on the outer cortical surface of the bone into the medullary cavity. Extensive washing of the periosteum may be helpful in removing extraneous diatoms.

* 3. DROWNIN G IN FILTERED WATER Drowning in the bathtub, and other circumstances in which the drowning medium in water derived from a filtered source will usually give a nega­ tive diatom test. This is due to the absence or very low concentration of frustules in the drowning medium. Frustules can be extracted from the bone marrow in about 10% of drowning fatalities in filtered water. In these cases, the frustules do not originate from the water but are derived from a contaminant present in the water (see section 6, case 4). For example, gravel, diatom-laden soil, and some commerical products may leave a par­ ticulate residue on the surfaces of bathtubs (Fig. 6.4). As a consequence, the filtered water is contaminated with a suspension of frustules. The diatoms that originate from commerical or industrial products may contain diatoms from mined miocene fossil diatom deposits . These diatoms are structurally dissimilar to the freshwater diatoms that are more typically observed in routine forensic casework. In most instances, these mined diatoms are highly fragmented and are usually recognized as ireg­ ular portions of centric diatoms (Fig. 6.4) or marine taxa.

* 4. DISMEMBERED BODY PARTS Dismembered body parts may be found floating in lakes or stream and, on occasion, are dredged from deeper waters by fishermen (Fig. 6.5). The main mechanisms that result in isolated body parts in water include: 108

Applications of Diatom Test

test in all sus­

.: pe since the long .£led soft tissues. g the femur

",-,,~, ~ ., c·

'tJall give a nega­ oncentration of ~x[[acted from the

~

e derived from a -t . For example,

_s: instances, these ~~~ ognized as ireg­

0[

6.1 - 6.3. Homicidal drowning in a seasonal puddle. 6. L Skeletonized remains found in a wooded area near a large seasonal puddle. Postmortem examination reveals sharp force injuries of ribs. Diatoms were found in the femoral bone mar­ row (6 .2) that are concordant with those in a puddle near the body (6.3). (Case of Dr. John Hillsdon-Smith).

stream and, on ig. 6.5). The 109

Forensic Diatomology and Drowning

6.4. Typical fragment of a large centric fossil diatom that may be fou nd in bath­ tub residue and in some commercial or industrial products.

110

Applications of Diatom Tes t

6.5 & 6.6. Drow ning and aquatic dismemberment. 6.5. An intact whole leg recovered in a lake. 6.6. Margins of the dismemberment showing postmortem chopping wounds consistent with propeller marks . The presence of diatom frus­ tules in the femoral bone marrow is an important ancillary investigation in such cases.

ttl

Forensic DiatomoLogy and Drowning

1. Homicide by means other than drowning, dismemberment, and dis­

posal of the body parts in a lake.

2. Aquatic decompositional dismemberment of a drowning fatality. The diatom test can complement postmortem and anthropological exami­ nation of isolated limbs. Since the diatom test has a high positive predic­ ti ve value, the presence of diatoms in the marrow of a long bone is good evidence for drowning (1). Unfortunately since the negative predictive value of the test is low, the diatom test is not useful as a method to exclude drowning as a cause of death in the case of intentional dismemberment. However, traditional postmortem examination may be sufficient to dis­ criminate between purposeful dismemberment (e.g. intentional disarticu­ lation) from the effects of decomposition or propellor action on the limb (Fig. 6.6).

* 5. DIATOMS AS TRACE EVIDENCE Diatoms can be used as trace material that may imply the recent history of an article of clothing or other inanimate object (see section 7, for further details). The literature, mostly from Peabody of the Home Office Forensic Science Laboratory (2) , contains several important examples of the use of diatom frustules as trace evidence including: 1. Three men allegedly broke into a safe with ballast consisting of

diatomaceous earth . The frustules were transferred to the clothing

and was similar to the frustules found in the ballast. The scientific

evidence corroborated the confession of two of the accused men. All

three men were convicted.

2. Car polishes containing diatoms have been used to link the hub caps to particular cars. 3. Metal polishes that contain diatoms and are used for silverware have been used to link stolen articles to certain households from which they originated. Other investigators have used diatoms from pond sediment to link accused assailants to a particular crime scene (3). In a case in Connecticut, 112

Applications oj Diatom Test

_ ent, and dis­ ng fatality_ ~

logical exami­ positive predic­ Lng bone is good p~ative predictive ..,., [hod to exclude _ . memberme nt. -ufficient to dis­ ;:.. tional disarticu­ :..~!ion on the limb

CE

:e recen t history of _ .on 7 , for further :le Office Forensic u_ples of the use of

:-::sisting of .;:' the clothing . The scientific ... cused men. All

pond sediment from the shoes of a suspect was analysed for microflora and compared with the microflora of the victim's shoes. A comparative analysis of the biota from both sites revealed a similarity in the diversity of diatom species. In addition, the relative abundance of three species of Eunotia, a common freshwater diatom, was similar in extracts from the shoes and a sample from the pond. On the basis of this analysis , it was pos­ sible to conclude a common origin for the diatoms, and this implicated the pond as the source for the frustu les fo und on the clothing of both the vic­ tim and the accused.

* 6. DIATOMS AND HOMICIDE The most important application of the diatom test for drowning is in cases of homicide and suspicious deaths. Homicidal drowning usually conforms to one of two major patterns: primary or secondary drowning. In the case of primary drowning, drowning is the sale cause of death and postmortem examination fails to reveal other life-threatening inj uries or inj uries that may relate to physical restraint in the drowning process. Primary homici­ dal drownings usually involve children or drow ning of an adult that has been rapidly incapacitated in the water. T he more frequent pattern of homicidal drownin g is the terminal inhalation of water complicating other grave injuries (e.g., blunt force head inj ury), or incapacitation by asphyx­ iation (e.g. , press ure on the neck). These cases are more readily apparent as homicidal drownings, and drowning is usually the "final common path­ way" although the other injuries may be suffic ient to cause death or, at least, threaten survival. The diatom test can be used in both forms of homi­ cidal drowning as indicated by the following series of brief representative cases:

k the hub caps

4-

ilverware have from which

d ediment to link -e in Connecticut,

Case 1. Primary drowning The body of a male child (-5 years old) was found submerged in a lake. At autopsy, there were no specific anatomic findings . A nitric acid extract of femoral bone marrow revealed the presence of two distinctive types of diatom frustules. The same two types of diatom were found in samples from the lake from which the body was recovered. Death was attributed to drowning. The father later admitted to drowning the child in the lake. 113

Forensic Diatomology and Drowning

Case 2. Drowning and strangulation The body of a 56 year old woman was found face down floating in a river. At autopsy, there was recent hemorrhage into the right sternomastoid mus­ cle with fracture of the right greater cornu of the hyoid bone with local hemorrhage and recent bruising involving the tip of the tongue. There was bilateral pulmonary oedema (right lung- 450 g, left lung- 400g) and 300 mL of watery fluid in the stomach. Death was attributed to the combined asphyxial effects of strangulation and drowning. A nitric acid extract of femoral bone marrow revealed the presence of five distinctive types of diatom frustules. The same five types of diatom were found in samples from the river from which the body was recovered. Case 3. Drowning and head injury The body of a 25 year old woman was found face down floating in a river. At autopsy, there were extensive recent blunt force injuries of the head with mul­ tiple scalp lacerations with diffuse subgaleal hemorrhage. Multiple recent abra­ sions and contusions were found involving the nose, lower lip, chin, and ven­ tral neck with recent hemorrhage into the left sternohyoid muscle. The hyoid bone and laryngeal and cricoid cartilage were unfractured. In addition, multi­ ple recent abrasions and contusion of the extremities, left breast and left hip were observed. Recent ligature marks were found along the circumference of both wlists. There was froth in the nares, bilateral pulmonary oedema (right lung- 350 g, left lung- 310) and 100 mL of watery fluid in the stomach. Death was attributed to the combined asphyxial effects of strangulation and drowning. A ni tric acid extract of femoral bone marrow revealed the presence of six distinctive types of diatom frustules. The same six types of diatom were found in samples from the fiver from which the body was recovered. Case 4. Bathtub drowning and strangulation A 38 year old male transvestite was foun d dead submerged face down in a bathtub full of soapy water. Cat litter gravel had been added in to the bathtub water. At autopsy, there was recent hemorrhage into the ster­ nomastoid muscles and deeper strap muscle. There was extensive recent bilateral fractures of the thyroid cartilage. The hyoid bone was unfrac­ tured. There was bilateral pulmonary oedema (right lung- 530 g, left lung­ 540). Death was attributed to the combined asphyxial effects of strangula­ tion and drowning. 114

Applications of Diatom Test

ling in a river. r!J omastoid mus­ - . bone with local e :.. ng ue. There was _~ ~ - 400g) and 300 ~ o the combined

""

. cd the presence - e types of diatom . was recovered.

_ ring in a river. At ~!e head with mul­ . _ -= ~riple recent abra­ ~ lip, chin, and ven­ . • P.luscle. The hyoid In addition, multi­ =.:- reast and left hip the ircumference of . . oedema (right the stomach. Death .on and drowning. .~ ' ~ :lled the presence ~ 'types of diatom _iJdy was recovered.

..=ed face down in t added into the - extensive recent ne was unfrac­ ~ ::- - 530 g, left lung­ ~:':-e ts of strangula-

A nitric acid extract of femoral bone marrow revealed the presence of four distinctive types of diatom frustules. The same four types of diatom were found in samples from the bathtub water from which the body was recovered .

Case 5. Drowning concealed by fire The largely burned remains of a teenage girl was found in a suitcase in a parking lot near a dumpster (Fig. 6.7 & 6.8). There was accelerant at the scene. At autopsy, there were no injuries. However, 5 mL of watery fluid was found in the right maxillary sinus. The lungs (right- 290 g; left- 250 g) were mildly oedematous and congested. No anatomical or toxicological cause of death was established. Nitric acid extracts from the fluid from the maxillary sinus revealed in excess of ten distinctive types of diatom frustules. The right and left femoral bone marrow contained four distinctive types of diatom frustules that matched frustules present in the maxillary sinus fluid (Fig. 6.9-6.11). The cause of death was determined to be drowning . In cases 1-4, the diatom test corroborated scene and postmortem findings indicating that drowning was the cause of death. In case 5, the diatom test provided the only evidence that death had occurred by fres h­ water drowning. This underscores the importance of the use of the diatom test in some unusually suspicious deaths were the body is fo und on land. The diatom test for drowning, and trace evidence investigations using diatoms, can provide important information that may not be forthcoming with other investigative measures. For example, in somes case of homicidal drown­ ing, it may be possible to deduce the location of drowning or to discriminate between potential sites of drowning and link an alleged assaliant to a specific body or water. These features of the diatom test can be illustrated with a case of homicidal drowning in which forensic diatom analysis played a critical role .

* 7. CASE STUDY: MURDER IN TH E HUDSON RIVER Synopsis In May, 1996 the body of woman was found in the prone position overly­ ing a wooden bulkhead in the Hudson river in the Ulster County, New York 115

Forensic Diatomology and Drowning

6.7 & 6 .8. Homicidal drowning in a burned body. 6.7. Site of body recovery near a dumpster in an indu strial car park. The body was found in a suitcase that was burning. 6.8 . Appearance of the body at postmortem examination showing postmortem charring of skin.

116

Applications of Diatom Test

6.9

6.9-6.11. Homicidal drowning in a burned body. Frustules recovered from the femoral bone marrow showing typical freshwater genera Cocconeis, Cymbella, and Navicula.

~

of body recovery in a suitcase that

117

Forensic Diatomology and Drowning

(Fig. 6.12 & 6. 13). The body was identified as a 58 year old resident of the city that bordered on the river. Postmortem examination revealed superical lacerations of the buccal mucosa, laceration/contusion of the lower lip, and scattered fresh contusion of the limbs. There was local focal hemorrhage of the soft tissues of the neck and a fracture of the left greater cornu of the hyoid bone. The right lung weighed 1,050 grams, and the left lung weighed 900 grams and the cut surface exuded frothy fluid. The right femur was retained. The cause of death was given as asphyxia due to neck compression and drowning and the manner of death was determined to be homicide. Toxicological analysis of postmortem blood revealed diphenhy­ dramine (1 ,100 ng/rnL) at a concentration greatly exceeding the therapeu­ tic level. Police submitted the femur, and samples of water from the Hudson river and decedent's bathtub for diatom analysis. Nitric acid extracts of the femoral bone marrow revealed two dis­ tinctive types of diatom frustules representing species of Diploneis and NitzschialHantzchia. The sample of water from the Hudson river revealed abundant diatoms representing in excess of 30 species of diatoms. The river water also included the species of Diploneis and NitzschialHantzchia that were found in the bone marrow (Fig. 6.1 4). The sample of the bathtub water revealed at least three species of diatoms but none of the species were found in the decedent's bone marrow. The presence of concordant freshwater diatoms in the femoral bone marrow and the river water sup­ ported the conclusion that drowning had occurred in the Hudson river and not in the decedent's residence. Police investigation indicated that the 63-year old male live-in companion of the decedent may have played a role in the death. He became the chief suspect shortly after the onset of the investigation. After the results of the diatom test for drowning were communicated to the police, the nephew of the suspect was interviewed by investigators. At that time, under the agreement of immunity to prosecution he recounted eye­ witness testimony about the events surrounding the death. He indicated that he and the suspect drugged the decedent in her home. The decedent was placed in a car and drive to a bank of the Hudson river. Later in court, the eyewitness testified that the body seemed "lifeless". Upon arrival at the river's edge, the body was removed from the car and dispatched into the Hudson river. The apparently "lifeless " body regained consciousness and 118

Applications of Diatom Test

.a due to neck to be e ed diphenhy­

~ .3~ermined

:;:- Diploneis and n river revealed _ diatoms. The " -- chiaIHantzchia -': .e of the bathtub of the species -'- ~e of concordant

__

male live-in ~_. the death. He ., '-= ·tigation. After n-: _nicated to the ~ :igators. At that .c recounted eye­ _ ~ _~ . He indicated j

began swimming. According to the eye witness, the chief suspect then waded into the river and drowned the decedent. The live-in-companion was charged with second-degree murder. It was later discovered that the accused had served a prison term for conspiring to kill a former wife. On further interviewing by police, the eyewitness indicated that that some articles of clothing worn by the accused had been concealed after the alleged murder. Articles including wrist watches, and a wallet were recovered in a sealed pouch in a outhouse belonging to the nephew of the acc used. T he shoes of the accused were recovered from the side of a road where the eyewitness said the shoes were discarded after the alleged m urder. Each shoe was found separately in the same general vicinity but in distinct locations. These articles were submitted for diatom an alysis and comparison with the diatom frus tules recovered from the decedent's femo ral bone marrow and Hudson river (Fig. 6.15 & 6. 16). In the laboratory, distilled water washings of the articles were pre­ pared, and diatom frustules were extracted using nitric acid digestion and centrifugation (Chapter 5, section §3). Washings prepared from the sur­ faces of the wallet, and both of the shoe contained species of freshwater diatoms foun d in the Hudson river. In addition, the species of NitzschialHantzchia found in the femoral bone marrow of the decedent was also recovered from washings of the wallet and left shoe (Fig. 6.17). This finding was consistent with a common source of origin for the diatoms found on the property allegedly belonging to the accused and the tissue of the decedent. The origin source of origin was most likely the Hudson river. The accused was convicted of second-degree murder and fourth degree criminal solicitation and was sentenced to 25 years to life in prison. Discussion This case illustrates several important uses of forensic diatom analysis beyond the confirmation of drowning as a cause of death. Several issues become important in the course of the police investigation including the site of death, and the independent scientific verification of eyewitness tes­ timony. In this case, the main contributions of forensic diatom analysis include deducing that: 119

Forensic Diatomology and Drowning

6.12 & 6.13. Murder in the Hudson river. 6.12. Decedent found laying over a bulkhead in the river. 6.13. Decedent's face with the ab sence of foam exuding from the mouth or nose.

120

Applications of Diatom Test

6.14. Murder in the Hudson river. (A & B), Diatoms frustules extracted from the femoral bone marrow. (C & D) . Concordant frustules extracted from a sample of water obtained at the time of body recovery (Top row - Diploneis; Bottom row - NitzschialHantzchia) .

. ~~ laying over a _ f foam exuding

121

Forensic Diatomology and Drowning

6.15 & 6.16. Murder in the Hudson river. Items allegedly belonging to the accused charged with homicidal drowning. 6.15. Wallet and watches. 6.1 6. Shoes. The items were allegedly worn while the accused drown ed the decedent shown in 6.12 and 6.13 .

122

Applications of Diatom Test

6.17. Murder in the Hudson river. NitzschialHantzchia frustules extracted from the surface of the left shoe (left), Hudson river (centre), and femoral bone mar­ row of decedent (right). There is a concordance of the diatom type found in all three specimens consistent with a common origin.

Forensic Di(Jfomo!ogy and Drowning

I. The site of death was the Hudson river not the decedent's bathtub. 2. Despite the high level of diphenhydramine the decedent was alive at

the time of immersion in the Hudson river.

3. Concordant diatoms in the decedent's femoral marrow, on articles allegedly belonging to the accused, and in the Hudson river material­ ly linked the accused to the decedent. Furthermore, the presence of the same types of diatoms on the articles of physical evidence despite their recovery in different locations is strong evidence for a common origin of the diatoms. The presence of the single species of NitzschialHantzchia in the decedent's bone marrow, and the left shoe, and the wallet of the accused are further evidence in support this proposal. This case of homicidal freshwater drowning also illustrates the logical consistency of the rationale of the diatom test for drowning.

* 7. REFERENCES l.Thomas F, Van Hecke, W., Timperman, 1. The detection of diatoms in the bone marrow as evidence of death by drowning. Journal of Forensic Medicine 1961 ;8(3): 142-144. 2. Peabody AJ. Diatoms in Forensic Science. Journal of the Forensic Science Society 1977;17:81-87. 3. Siver PA Lord, W.D ., McCarthy, D.1. Forensic limnology: The use of freshwater algal community ecology to link suspects to an aquatic crime scene in southern New England. J Forensic Sci 1994;39(3):847­ 853.

124

~-o : ' _

°

bathtub. was alive at

. on articles ': ri\Oer material-

the articles of _ ..t ions is strong c:-,\..c of the single ...r.ow, and the left in support this _0 ill ustrates the r drowning. C' •

:-:on of diatoms in Ii g. Journal of of the Forensic

oogy : The use of e t to an aquatic ~ . 1994;39(3):847­

7. A guide to the freshwater diatoms of drowning

The atlas of diatoms presented depicts the range of diatoms that common­ ly occur in freshwater drowning in North America. Many of the photomi­ crographs have been produced from bone marrow extracts while others have beem made from samples of putative drowning medium from foren­ sic cases. The diatoms presented are not comphrensive representations of the diatoms of freshwater; just the diatoms that have the size and shape features that allow entry into the human body during the drowning process. More comphrensive guides to freshwater diatoms can be found in standard texts and atlases of diatoms. However, it is useful for the foren­ sic practitioners to have reference photomicrographs of common diatoms that occur in forensic diatom analyses to aid in diatom detection. All pho­ tomicrographs were made using direct inference contrast microscopy using a magnification of 1,000 x with an oil immersion lens. Most of the indiviudal plates are organized to include diatoms of similar general morphological feature and size. Therefore, in some instances, only one genus of diatom may be represented on one plate. However, since there is morphological overlap between taxa some plates show different genera with similar structural features or size. The diatom images are not meant as a key to diatom identification but rather a primer to assist in the detection of diatoms during microscopic examination of case material. Those interested in diatom taxonomy are referred to texts on the subject for identification keys. Cox (1996) has produced a practical and useful text, particularly valuable for casework. 125

Forensic Diatomology and Drowning

PLATE 1 CENTRIC DIATOMS

In human tissues of freshwater drowning centric diatoms are recognized into two basic forms: enfasse views (1.1-1.7) and short chains (1.8-1.11). Figures 1.1-1 .7 show the morphological features common to most centric freshwater diatom genera (e.g., Melosira, Cyclotella, and Stephanodiscus) with a circular profile and radial spoke-like features . Some centric diatoms such as Melosira spp. may be alTanged into short chains composed of a small number of sequentially aligned frustules . In freshwater, these colonies may attain great lengths; however, when found in human tissues the chains are seldom longer than 2-4 units.

126

Atlas of Diatoms

recognized :­ : S (1.8-1.11). : _ most centric _;eplzanodiscus) ':cntric diatoms ~o mposed of a ~:-e-h water, these .2"

127

Forensic Diatomology and Drowning

PLATE 2 NAVICULOID DIATOMS

One of the most common, and easily identified, freshwater diatoms are the naviculoid (boat-like) diatoms typified by the genus Navicula (2.1 -2.4). These frustules are pennate, isopolar, and have a prominent raphe and del­ icate rib-like structures aligned orthogonal to the raphe. The apices of many speci s of Navicula are blunted, may show capitate or rostrate struc­ ture and are always symmetical about the long and short axis of the frus ­ tule (unlike heteropolar diatoms, c.f. plate Vill). Although the typical nav­ iculoid pennate diatom is represented by NavicuLa, other genera have a similar frustular structures (e.g., Stauroneis [c.f. Chapter 1, Fig. 1.4]) and maybe found in femoral bone marrow extracts if the species in small enough to pas throught the alveolar-capillary barrier and disseminate in the circulatory system. This excludes many species in the naviculoid genus PinnuLaria. Some naviculoid diatoms with rostrate apices (2.5) may be difficult to distinguish from other genera such as Cymbella.

128

Atlas of Diatoms

e_ diatoms are the ·cula (2.1 -2.4) .

. raphe and del­ . The apices of ~r rostrate struc­ .....xis of the fru s­ :he typical nav­ _ genera have a _ L Fig. 1.4]) and

r:ayjculoid genus ~ e- (2.5) may be

129

Forensic Diatomology and Drowning

PLATE 3 DORSIVENTRAL DIATOMS

The dorsiventral diatoms are those diatoms (e.g., Cymbella , and Amphora) with biconvex surfaces, and symmetry about a horizontal equator through the midpoint of the diatom (3.1 -3.4). The morphological features are oth­ erwise rather similar to Navicula. The most common dorsiventral diatom in femoral bone marrow extracts from cases of freshwater drowning is Cymbella (e.g., 3.1). However, in acid-extracted material it may be diffi­ cult to distinguish Cymbella from Amphora (see 3.3) since the latter appears similar to Cymbella in some orientations.

130

Atlas of Diatoms

dA mphora) uator through

-iYcntral cli atom drowning is i ~ may be diffi -

_. ~r

131

..

"

Forensic Diatomology and Drowning

PLATE 4 EPITHEMIA The diatoms of the genus Epithemia are highly characteristic and easily recognizable due to their unique morphological features (4.1-4.4). This dorsiventral diatom is isopolar with prominent en fasse detail that can be seen at different focal lengths (c.f. 4.3 & 4.4, showing the same diatom at different focal lengths). There are prominent transverse costae (4.3), and the raphe curves toward the centre of the frustule forming a conspicious V­ shaped structure on a reticular background.

132

Atlas of Diatoms

~:

ri tic and easily ~~ c-t l-4.4)_ This

- e ~

~ ame

diatom at 0 tae (4_3), and conspicious V-

133

Forensic Diatomology and Drowning

PLATE 5 NITZSCHIA AND HANTZSCHIA Nitzschia is one of the most diverse genera of freshwater diatoms which commonly undergoes taxonomic nomenclature revision (some species formly assigned to this genus are know classified as Hantzschia). Since Nitzschia spp.and Hantzschia spp. are common and abundant, these frus­ tules are frequently encountered in cases of freshwater drowning. Frustules are hnear to fusiform (5.1, 5.3-5.8) and may be sigmoid (5 .2) or gently arcuate (5.8). The raphe is found along one border of the frustule, and there are usually conspicious striae along the other border. The apices are highly variable and range from capitate (5.8) to acutely angled. There is a great variability in the length of the frustules; however, in femoral bone marrow the length is seldom longer than approximately 50 flm.

134

Atlas of Diatoms

diatoms which -orne species .- chia). Since , these frus­

135

Forensic Diatomology and Drowning

PLATE 6 ASTERIONELLA AND TABELLARIA

Two highly stereotypical and common types of freshwater diatoms found in the femoral bone marrow in drowning are Tabellaria spp. (6.1-6.4) and Asterionella formosa (6.5-6.8). Tabellaria spp. are linear frustules that are inflated at the midpoint and have isopolar capitate apices. Often these frus­ tules have fine striae that may be difficult to resolve. Colonies of Tabelleria form by lateral assembly and can be recognized by the conflu­ ence of the midpoint inflations that form a lacuna-like area. Asterionella formosa is are elongated pennate diatom with bulb-like ends that resem­ bles the shape of a long bone. Colony formation may be apparent in fresh­ water samples (c.f. Chapter 5, Fig. 5.12 & 5.13) but not in acid-digests of bone marrow.

136

Atlas of Diatoms

·e. jiatoms found

" :- ~. (6.1-6.4) and

~ ~s rules that are

Of:en these frus­

. 'f:. Colonies of

cd y the conflu­

_"""e . Asterionella . ;:nds that resem­ .:.:: arent in fresh­ :-. acid-digests of

137

Forensic DiatomoLogy and Drowning

PLATE 7 COCCON EIS Diatoms of the genus Cocconeis, like Navicula, and Cymbella, are among the most common diatoms to be extracted from bone marrow. This is largely due to the widespread distribution of this genus in running water (e.g., rivers), as well as their size and shape that permits ready passage into the alveolar capillary and peripheral arterial tree. Frustules are mostly broadly ellipitical with a prominent corona of scattered light. Frustules are monoraphid, with a raphe that is readily visible (7.2, 7.4, & 7.5). One valve displays conspicious rows of colinear pores (7.1, & 7.3).

138

Atlas of Diatoms

running water _Jy passage into . Je s are mostly :. Frustules are One

139

Forensic Diatomology and Drowning

PLATE 8

HETEROPOLAR AND OTHER DIATOMS A great variety of pennate and heterpolar diatoms exisit that are occasion­ ally observed in freshwater drowning (8.1-8.9). Many of the genera have characteristic morphological features such as the rounded apices and transverse striae found in Diatoma (8.5), the complex raphe found in Diploenis (8.4), and the highly asymmetrical apices of the raphid diatom Gomphonema (8.6 & 8.7). The araphid diatoms of genus Flagilaria (many species previously assigned to Synedra) may be found in femoral bone marrow (e.g., 8.9) but are usually limited to the small pennate species of this genus (c.f. plate 11).

140

Atlas of Diatoms

...:.: are occasion­ - he genera have ed apices and _ pbe found in e raphid diatom F.agilaria (many :J. femoral bone

141

Forensic Diatomology and Drowning

PLATE 9 CUNEATE AND OTHER DIATOMS

Cuneate diatoms (e.g., some Gomphonema, 9.5-9.8) have heteropolar apices and in some orientations have a characteristic tapering shape with prominent striae (e.g. , 9.5) and may have a pair of apical lucencies (e.g., 9.7). Some varieties of pennate diatoms have a rectangular shape (9.1 -9.4) and nonspecific profiles with marginal punctata (e.g. , 9.1 ). These diatoms are infrequently fo und in cases of freshwater drowning but may be present. This likely relates to the aquatic distribution of these genera since Navicula, and Cocconeis are relatively more abundant.

142

Atlas of Diatoms

~

e heteropolar • ng shape with . J encies (e.g., r.:h pe (9. 1-9.4)

since

143

Forensic Diatomology and Drowning

PLATE 10 ELONGATED DIATOMS

Several types of elongated or filamentous diatoms are occasionally found in cases of freshwater drowning. These diatoms are not as frequently encountered as the naviculoid or dorsiventral diatoms, presumably due to their length. These diatoms range from needle shaped (e.g., Synedra and Flagilaria) (e.g., 10.6 & 10.7) to rectangular (e.g., 10.5) to sigmoid (e.g., 10.1) and may form lateral assemblies (e.g., 10.2 & 10.3).

144

Atlas of Diatoms

: ·-onally found - frequently - ~ -u mably due to e g.. Synedra and : :J sigmoid (e.g., .J:

145

Forensic Diatomology and Drowning

PLATE 11 SMALL PENNATE DIATOMS

Small pennate diatoms (11.1- 11 .19) are often difficult to recognize in acid-extracted material due to their size and admixture with insoluble debris. However, several types of the small pennate diatoms have suffi­ ciently characteristic morphology to allow detection. This group is taxo­ nomically complex and many species are araphid (e.g., Achnanthes). The frustular structure is usually simply geometrical ranging from rhombodial (e.g., 11.12) to ovate (e.g., 11. 14) with blunted (e.g., 11.15) to capitate apices (e.g., 11.13). Many species have prominent transverse striae (e.g., 11.16). Some small diatoms (e.g., Achnanthidium, 11.6- 11.8) must be dif­ ferentiated from inorganic crystalline material that may co-purifiy frus­ tules in bone marrow digests.

146

Atlas of Diatoms

:

:~

recognize in with insoluble

0

- group is taxo­ • :1. chnanthes). The

rse striae (e.g., .1 .8) must be dif­ o-purifiy frus­

-l;

147

8. Selected bibliography

Adamo, M. (1949). Plankton in the lungs of near drownin gs. Zacchia, 12, 46 . An gelini Rota, M., Gualdi, G., Macc hiarelli, L. (1983). Analysis for diatoms in the diagnosis of drowning. Boll Soc Ital BioI Sper., 59( 12), 1973- I 979. Antonenko, N. E. , Ferris J.AJ. (1987) . Diatom analysis in the determination of death by drownin g. Journal of the Canadian Society of Forensic Science, 1(I ­ II ).

Auer, A., Mottonen,M. (1988). Diatom s and drowning . Z Rechtsmed, 10 I (2),87­ 98. Auer, A. ( 1991 ). Qualitative di atom analysis as a tool to diagnose drownin g. American Journal of Forensic Medicine and Pathology, 12(3),2 13-218 . Buhtz, B., W. ( 1938). Determ ining place where drowning occurred by measuring amount of diatoms in the lung. Deutsc he Ztsc hr.f.d.ges. gerichtl. med. , 29, 469-484. Burger, E. (1968). [On the problems of corroborative significance of finding diatoms in the greater circulatory system]. Deutsch Z. Ge sGerichtl med, 64, 21-28 . Calder, Y. M. (1984). An evaluation of the diatom test in deaths of professional divers. Med Sci Lwa ., 24( 1), 41-46. Cox, E. J. (1996). Identification of freshwater diatoms from live material. New York : Chapman and Hall. Dayan, A. D., Morgan, RJ.T. , Trefty, R.T., Paddock, T.B .B . (1978). Naturally occurring diatomaceous pneumoconiosis in sub-human primates. J Comp Pathol, 88, 321-325. E"idiin, L. M. ( 1968) . [The value of plankton in the di agnosis of drownin g (review)]. Sudebnomed Ekspert, 11, 18-21. Einbrodt, H. (1957). Phase microscopy of diatomaceous earth in lungs. Deutsche Z Ges Gerichtl Med, 46, 2-. Foged, N. (1983). Diatoms and drowning - Once more. Forensic Science International, 21, 153-159. 149

Selected Bibliograpln

Fukui, Y , Takahashi, S., Matsubara, K. ( 1980). A new method for detecting diatoms in human organs. Forensic Science International, 16,67-74. Funayama, M ., Aoki, Y, Sebetan, I.M., Sagisaka, K. (1987). Detection of diatoms in blood by a combination of membrane filtration and chemical diges­ tion. Forensic Science International, 34, 175- 182. Furuno, J., Yamaski, T., M oriya, H. (1963). [On the distribution of diatoms in the bones and teeth of the drowned cadaver. Nagasaki Med J, 38, 82-86. G eissler, U., Gerloff, J. ( 1966). Das vorkommen von diatomeen in menschlichen organen und in der luft. Nova Hedwigia, 10, 565-577. Giri, B. B. , Tripathi, C. B ., Chowdary, YB. (1993). Characterization of drowning by diatom test. Indian J M ed Res, 98,40-43. Golaeff. D . A. (1928). Use of phyto-plankton method for differenti ation between drowned persons and bodies thrown into water. Odessky Med J, 3, 425-430 . G ualdi, G. (1968a). [Diatoms in the diagnosis of drowning: I Biology of diatoms]. Zacchia, 4, 31-74. Gualdi, G. (1968b). [Diatoms in the diagnosis of drowning: II The medico-legal problems]. Zacchia, 4, 187-244. Gy lseth, B., Mowe, G. (1979). Diatoms in lung tissue . Lancet, 29( 1375). Hendey, N. I. (1973). The diagnostic value of diatoms in cases of drowning. Medicine, Science, and the Law, 13(1), 23-34. Hendey, N. I. ( 1980). Letter to the Editor, D iatoms and Drowning - A Review. Medicine, Science, and the Law, 20(4), 289. Incze, G., Tamaska, I. , Ggyongyosi, J. (1951 a). A vizbefu las bizonyitasa aver es a szervek planktonvizsgalataval. Orv He til, 7, 1003. Incze, G . (1951 b). [P hytoplankton resorption in drowning]. Acta Morphol Hung, 1,421-430. Incze, G., Tamaska, L. , Ggyongyosi, J . (1955) . Zur blutplanktonfrage beim tod durch ertrinken. Dtsch Z Gasamte Gerichtl Med, 43, 517-523. Isaev, L. (1991). T he forensic medical evaluation of the results of determining diatomaceous plankton in the diagnosi s of drowning . Bud Med Exspert., 34(2), 27-29. Jaaskelalnen, A. J. (1967). D iatom findings in bodies found in water. A new method for quantative measurement of diatoms in the body. Dtech Gesamte Gerichtl Med., 61(2), 41-47. Jaaskeli:ilnen, A. J. (1968). Influence of blood alcohol on diatom findings in drowned bodies. Dtech Gesmate Gerichtl M ed, 64( 1), 29-32. Jarosch, K. (1961). [The effect of industrial waste waters on so-called demon­ stration of plankton]. Deutsch Z . Ges. Gerichtl. Med., 51 , 409-420. Jianping, Q., Enshou , W. (1992). A study on the diagnosis of drowning by exam-

ISO

Forensic Diatomo!ogy and Drowning

fo r detecting

. ­ ---­t - - . Detection of

.- diatoms in the

1- 6. n menschlichen

- =r~~riarion between .:.-d 1. , 425-430. g: I Biology of

: he medico-legal :'9( 1375). of drowning.

~_ ~

rung - A Review.

- _- \ lo rphol Hung,

", . nfrage beim tod ,- - ,,

:_ of determinin g _ _-' \ led Exspelt.,

-

.. water. A new Ore h Gesamte m finding s in lied demon­ .. in e by exam-

ination of lung chlorophyll(a) of P lanktons with a spectrofluorophotometer. Forensic Sci Int, 53,149-155. Kanda, M . (1963). [D iagnosis of death by drowning, especially practical appli­ cation ofdetection of diatoms on di agnosis of death by drowning]. J Kumamoto Med soc, 37, 71-81. Kane , M., Fukunaga, T, Maeda , R., Nishi, K. (1996). The detection of picoplank­ ton 16S rONA in cases of drowning. l nt J Legal Med, 108(6),323-326. Kasparek, B . (1936). Diagnosis of death from drowning established by presence of plankton organisms in lungs and duodenum. Deutsche Ztschr.f.d.ges. gerichl. Med., 27, 132-142. Kasparek, B . (1937). Bei trage zur diagnose des ertrinkungstodes durch den nach­ weis von planktonorgani smen in lunge und duodenum. Deutch Z Gerichtl M ed, 27 ,1 32- 142. Khat tah, M. (1975). D iagnosis of death by drowning - a quick method for detec­ tion of diatoms in bone marrow and different organs. J Egypt Med Assoc, 58, 537-540. Kobayashi, M ., Yamada, Y, Zhang, W-O ., Itakura, y , Nagao, M ., Takatori , T. (1993). Novel detection of pl ankton from lung tisue by enzymatic digestion method . Forensic Science International, 60, 81 -90. Koseki, T. ( 1968). F undamental examination of experimental materials an con­ trol animals on the diagnosis of death from drowning by the diatom method . Acta Med B ioi, 15,207-2 19. Koseki, T. ( 1969) . Investigations on the bone marrow as a material in the diatom method of diagnosing death from drowning. Acta Med Bioi, 16(2), 85-90. L udes , B., Q uantin, B., Doste, M ., M angin, P. (1994). Application of a simple enzymatic digestio n method for diatom detection in the diagnosis of drowning in putrified corpses by diatom analysis. l nt J L egal Med, 107(1), 37-41. Ludes, B ., Coste, M. Tracqui, A. , M angin, P. (1996a). Continuous river monitor­ ing of the diatom s in the diagnosis of drowning. Journal of Forensic Science, 41 (3), 425-428. Ludes, B. , Coste, M. (I 996b). Diatomees er medecine legale. Paris: Tec & D oc Lavoisier Editions Medicales In ternationale . Lunetta, P., Penttiia, A. (1998). Scanning and transmission electron microscopic evidence of rhe capacity of diatoms to penetrate the alveolar-capillary barrier in drowning. International Journal of Legal Medicine, in press . Macchiarelli, L. e. a. (1964). [O n the presence of diatom s in pleural exudates and transudates in living subjects]. Zacchia, 27, 454-458 . Matusumato, H., Fukui, Y (1993). A simple method for diatom detection In drowning . Forensic Science International, 60, 91-95.

151

Selected Bibliography

Merkel, H. (1939). Plankton findings in lungs of drowned persons and their prac­ tical significance . Deutsche Ztschr.f.d.ges. Gerichtl. Med., 31, 211-212. Merli, S., Ronchi , U. (1966) . Experimentelle untersuchungen minch im Kern­ Reaktor bestrahlter diatomeen Uber das entrinken. Arch Kriminol, J 38, J 31­ 136. Mikami , Y., Kanda , M ., Kamimura, 0 ., Okuyama, M . (1959). Experimental study and practice on the detection of vegetative p)anktons in the bone marrow of the drowned dead body. Acta Med Okayama, 13, 259-268. Mueller, B. (1963a). Zur frage des vorkommens von diatomeen in organen von leichen, die nicht in wasser gelegen haben. Dtsch Z Gesamte Gerichtl Med, 54, 267-272 . Mueller, B. (l963b). [On the problems of the occurence of diatoms in the organs of cadavers not having lain in water]. Deutsche Z. Ges. Gerichtl. Med., 54, 267-272. Naeve, W. (1956). Zur praktischen gerichtsmedizinischen anwendung des diatomeen nachweises im grossen kreislauf. Dtsch Z Gesamte Gerichtl Med, 45, 364-369 . Nanikawa, R., Kotoku , S . (1974). Medicolegal observations on a dead body drawn up from the sea bed with special reference to ethanol and diatoms. Forensic Sciences, 3, 225-232. Neidhart, D. A., Greendyke, R.M. (1967). The significance of the diatom demon­ stration in the diagnosi s of death by drowning. American Journal of Clinical Pathology, 18(4),377-382. Neidhart, D. A. (1967 ). The significance of diatom demonstration in the diagno­ sis of death by drowning. Amer J Clin Path, 48, 377-82. Pachar, J. v., Cameron, I .M. (1992). Scanning electron microscopy: Application to th identification of diatoms in cases of drowning. Journal of Forensic Science, 37, 860-866. Pachar, 1. v., Cameron, J.M. (1993) . The Diagnosis of Drowning by the Quantitative and Qualitative Analys is of Diatoms . Medicine, Science, and the Law, 33(4), 291-299 . Patrick, R., Reimer, C.W., Yong, S.I. (1975). The diatoms of North America. Lititz, Pennsylvania: The Academy of Natural Sciences of Philadelphia. Peabody, A. J. (1977) . Diatoms in Forensic Science . Journal of the Forensic Science Society, 17, 81-87. Peabody, A. 1. (1980). Diatoms and Drowning - A Review. Medicine, Science, and the Law, 20(4),254-261. Petersohn, F. (1964). Diatom findings in cadavers of drowned. Deutsche Z Ges Gerichtl Med, 54, 376-378.

152

Forensic Diatomology and Drowning

.5:, :-.cD \·o!:

.:. ~~ II"c

:: L :: : _:".::. L:d.:-i-.

r: . ~ ndu ng de

~ J

dead bod ) _nd diatoms.

.i rom de moo­ _:-. I of Clinical i. :n

the diagno­

: Application of Forensic :- \\ nln g by the ndthe

:: the Foren ic : ;::_~ in e .

Scien e.

Pollanen, M . S. (1997a). The Diagnostic Value of the Diatom Test for Drowning II. Validity. : Analysis of diatoms in the bone marrow and drowning medium. Journal of Forensic Science, 42(2), 286-290. Pollanen, M. S., Cheung, L., Chaisson, D.A. (l997b). The Diagnostic Value of the Diatom Test for Drowning. 1. Utility. : A Retrospective Analysis of 771 cases of Drowning in Ontario, Canada. Journal of Forensic Science, 42(2), 281-285. Porawski, R. (1966) . Investigation on the occurrence of diatoms in organs in deaths from various causes. Journal of Forensic Medicine, 13(4), 134-137 . Ranner, G., Juan, R. , Udermann, R. (1982). [On the evidential value of diatoms in cases of death by drowning]. Z Rechtsmed, 88( 1-2), 57-65. Reh, M . (1968). The diatom question. Dtech Gesmate Gerichtl Med., 63(2), 131­ 133. Round, F. E., Crawford, R.M., Mann , D.G. (1990). The Diatoms: Biology, and morphology of the genera. Cambridge University Press . Schneider, V. (J 967). Studies on the evidence value of diatom demonstration in drowning. Dtech Gesamte Gerichtz Med, 59(2), 58-95. Schneider, V. (1969). Evaluation of the diatom test. Beltr Gerichtz Med, 26, 92-99. Schneider, V. (1980). Remarks on the paper by B. Schell mann and W.SperJ, "Detection of diatoms in the bone marrow (femur) of non-drowning victims" . Z Rechtsmed, 65(4), 315-317. Schneider, V. e. a. ( 1969). [On the determination of radioactively labeled diatoms in the organs]. Beitr Gerichtl Med, 25, 158-164. Sciaudone, G., Palmieri, L. ( 1962). [The study of plankton in the bone marrow as a test of drowning] . Folta med (Napoli), 45, 1361-1382. Spitz, W. U., Schneider, V. (1964) . The significance of diatoms in the diagnosis of death by drowning. Journal of Forensic Science, 9( I), 11-18. Spitz, W. U. (1965). [Studies on air filtration strips from different regions of the Federal Republic for their diatom content. A contribution to the value of diatoms in the diagnosis of death by drowning. Deutsche Z. Ges. Gerichtl. Med, 56, 116-124. Spitz, W. u., Schmidet, H. (1966). Weitere untersuchungen wr diagnostik des estrinkungstodes durch diatomeennachweis. Deutsch Z Ges Gerichtl Med, 58, 195-204. Staak, M. (1968). Critical observations on the specificity of diatom detection. Dtech Gesmate Gerichtl Med., 63(2), 122-126. Tabbara, W., Derobert, L. (1962a) . Le diagnostic medico-legal de 1a submersion par 1a recherche des diatomees dans la moelle osseuse. Ann Med Leg, 42, 374­ 381.

153

SeleCled Bibliography

Tabbara, W., Derobert, L. (l962b). [Technical note on the diatoms]. Ann Me ... Leg . (Paris), 42, 613-615. Tamaska, L. (1949). [Diatom content of bone marrow in corpses in water]. O r Hetil, 16, 509-5 11. Tamaska, L. (1961). [On diatom demonstration in the bone marrow of dro\\ n:: ~ dead bodies]. Deutsc he Z. Ges. Gerichtl. Med ., 51, 398-403. Taylor, J. J. (1994). Diatoms and drowning -- a cautionary case note . Med SL Law, 34(1),78-79. Terazawa, K., Takatori. T ( 1980). Isolation of intact plankton from drownin; lung tissue by centrifugation in a colloidal silica gradient. Forensic Scien ~:: International, 16, 63-66. Thomas, F., Van Hecke, w., Timperman, J. (1961). The detection of diatoms ir. the bone marrow as evidence of death by drowning. Journal of Foren I Medicine, 8(3), 142-144. Thomas, F., Heeke, W., Timperman, J . (1962). D iagnostic medico-legal de ._ mort par submersion par la mi se en evidence de diatomees dans la moelle de_ os longs. Ann. Med. Leg ., 42,369-373. Timperman, J. (1962). The detection of diatoms in the marrow of the sternu m Journal of Forensic Medicine, 9(4), 134-136. Timperman, J. (1968). Observations on the diatom question. Dtech Gesamtc Gerichtl Med. , 63(2),127-128. Timperman, J . (1969). Medico-legal problems in death by drowning: Its diag!1 ~ ­ sis by the diatom method. Journal of Forensic Medicine, 16(2), 45-75. Tomonaga, T (1940). Demostration of plankton in organs of drowned person . Jap. J. Med.Sci., VII, Social Med .and Hyg., 3, 197-198. Tomonaga, T (1963). [On the body of a newborn infant proved by diatom method to have been drowned]. Nagasaki Med J, 38, 671-672. Tomonaga, T, Furuno, Funji, Furukawa, Hiroshi (1964). The diatom findin g_ ~ three infants thrown in water after death. Jap J Leg Med, 18,143-147. Waltz, H. (1965). Zurbeweiskraft von diatomeenbefunden. In Akrnelle frage n ' ~, gerichtlichen medizin Wiss. Zeitschr. der Martin luther-Universitat (pp. 116­ 117). Halle-Wittenberg. Weinig, E., Pfanz, H. (1951). Zur diagnostik des ertrinkungstodes durch d t ~ nachweis von diatomeen im 'Optisch leeren Gewebsschnit'. Deutsch Z Geri . . Med, 40, 664-668. Yoshimura, B., Yoshida, M ., Okii , y , Tokiyasu, T, Watabiki, T, Akanc- . _-. (1995). Detection of green algae (Chlorophyceae) for the diagnosis of dro' ­ ing. Int J Legal Med, I 08( I), 39-42.

154

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n . 1=_

. ' . .'. red

AUTHOR INDEX

Auer, 58

Ludes, 59, 72

Lunetta, 58

Bell, 36

.- (he ternum.

Cameron, 59

Camps, 24, 51

Cox, 126

DiMaio, 50

Dix, 13

J iat m method

Gee, 51

Gettler, 48

Gorgs, 52

Hendey, 56

Montagu , 28

Mueller, 52

Pachar, 59

Peabody, 52,112

Polson, 51

Ranner, 57

Revenstorf, 52

Simpson, 50

Spilsbury, 29

Spitz, 49, 53

Sydney Smith, 9

Incze, 52

Knight, 51

Tamaska, 52

Timperman, 53, 56, 107

155

SUBJECT INDEX

Achnanthes, 146 Achnanthidium, 146 Adipocere, 42, 106 Alveolar macrophages, 49 Amphora, 130 Anoxia, 17, 18 Anoxic encephalopathy, 19 Aquatic decomposition, adipocere, 40 diatom test and, 107 sequence, 40 taphonomy and, 39 Asbestosis, 54 Asphyxia, 21 Asterionella, 136 Atomic force microscope, 101 Alveolar macrophages, 33 Berylliosis, 54 Brides in the Bath case, 9 Cadaveric spasm, 29 Chain of custody, 83, 87 Cocconeis, 77, 138 Criterion of concordance, 56,59, 72 Cutis anserina, 29 Cyciotella, 126 Cymbelia, 77, 128, 130

Daphnia, 49 Diatom blooms, 66

Diatom load, 58

Diatom test,

axiom, 3 bone marrow and, 6,59,85,88 collection of samples, 83-84 contamination, 54, 76 contra-indications for, 106 criterion of concordance, 6, 72 diatom load and, 58 dismembered body parts and, 108 drowning-associated diatoms, 76 experimental models, 57 extraction methods, 58 history of, 52 homicidal drowning and, 113, 115 indications for, 105 interpretation of, 91, 98 percolation and, 6 procedure, 88 sensitivity, 77-78 skeletonized bodies and, 107 specificity, 54, 77-78 suspicious deaths and, 115 157

Forensic Diatomology and Drowning

utility, 65-66

validity, 65, 68

water and, 70

Diatoma, 140

Diatoms,

centric, 96, 126

cuneate, 142

description of, 2

dorsiventral, 130

ecology, 3

features of, 96-97

fossilized, 2-3, 108

heteropolar, 140

naviculoid, 128

pen nate, 96, 146

trace evidence and, 112

Diploenis, 140

Diploneis, 118

Dismembered body parts, 112

Dismemberment, 112

Drowning,

accidental, 8

chemical tests of, 47

epilepsy and, 12

homicidal, 9, 26, 29

hypoxemia, 27

in the bath, 9, 108

manner of death , 1

medicolegal investigation, 2

micro-organism tests, 49

newborns and, 12

pathology of,

air passages in, 30

general, 28

lung weights in, 32

lungs in, 32

perimortem injuries, 29

pleural effusion, 33

temporal bone in, 36

water-related

changes, 30

pathophysiology, 26

su icidal, 8

volu me status, 27

"Dumped" body, 13

Emphysema aquaosum, 33

Epithemia, 132

Federal Ru les of Evidence, 100

Flagilaria, 140, 144

"Floaters", 76

Fluidity of the blood, 26

Frustulin, 103

Frye Test, 100

Gomphonema, 140

Hantzschia, 118- 11 9, 134

Hepatoportal circulation, 56

Hudson ri ver, 118-119

Intracartilaginous laryngeal

hemorrhages, 24

"Major Martin", 28

Manual strangulation, 21, 24

Melosira, 126

Navicula, 58, 77, 128, 130

Near-drowni ng, 33

Nitzschia, 118-119, 134

Oedema aquaosum, 33

Photomicrography, 99

Pink teeth,

Christie case, 38

history of, 36

significance of, 39

Pinnularia, 77 , 128

Polymerase chain reaction, 101

158

Subject Index

Prolonged immersion, 106

Scanning electron microscope, 101

Sphenoid sinuses, 32

Spurious diatoms, 54

Staurone is, 128

Stephanodiscus, 126

Stronti um, 48

Synedra, 77, 140, 144

Tabellaria, 136

Tetrahymena, 49

The Man Who Never Was, 28

Trace evidence, 112

"War-on-diatoms", 49-52

"Washerwoman skin", 30

159