New Developments in Blood Transfusion Research

NEW DEVELOPMENTS IN BLOOD TRANSFUSION RESEARCH

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NEW DEVELOPMENTS IN BLOOD TRANSFUSION RESEARCH

BRIAN R. PETERSON EDITOR

Nova Science Publishers, Inc. New York

Copyright © 2006 by Nova Science Publishers, Inc.

All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. For permission to use material from this book please contact us: Telephone 631-231-7269; Fax 631-231-8175 Web Site: http://www.novapublishers.com NOTICE TO THE READER The Publisher has taken reasonable care in the preparation of this book, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained in this book. The Publisher shall not be liable for any special, consequential, or exemplary damages resulting, in whole or in part, from the readers’ use of, or reliance upon, this material. This publication is designed to provide accurate and authoritative information with regard to the subject matter covered herein. It is sold with the clear understanding that the Publisher is not engaged in rendering legal or any other professional services. If legal or any other expert assistance is required, the services of a competent person should be sought. FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS. LIBRARY OF CONGRESS CATALOGING-IN-PUBLICATION DATA New developments in blood transfusion research / Brian R. Peterson (editor). p. ; cm. Includes bibliographical references and index. ISBN: 978-1-60876-240-8 (E-Book) 1. Blood--Transfusion--Complications. 2. Blood—Transfusion --Research. I. Peterson, Brian R. [DNLM: 1. Blood Transfusion--adverse effects. WB 356 N5325 2006] RM171.N49 2006 615'.39--dc22 2005037436

Published by Nova Science Publishers, Inc. New York

CONTENTS Preface

vii

Chapter I

Blood Transfusion in the Neonate – Where are We Today? Michael S. Schimmel, Michael Kaplan and Roger F. Soll

Chapter II

Health Economics Research on Blood Transfusion Safety Measures -An Introductory Primer Ulf Staginnus

Chapter III

Unique Patient Identification Barcode System for Prevention of Blood Transfusion Errors: A 6-year Experience in a Regional Hospital in Hong Kong Joyce Chan Chee Wun and Raymond Chu

Chapter IV

Perceived Naturalness and Risk of Blood and Blood Substitutes Eamonn Ferguson, Piers Fleming, Ellen Townsend and Kenneth C. Lowe

Chapter V

Prion Biology and the Reduction of the Risk of Transfusion-Transmitted Variant Creutzfeldt-Jakob Disease (vCJD) by Blood Filtration Joseph S. Cervia, Samuel O. Sowemimo-Coker, Girolamo A. Ortolano, Jeffrey Schaffer, Karen Wilkins and Samuel T. Wortham

Chapter VI

Chapter VII

1

15

35 65

77

Allosensitization in Multiply Transfused Sickle Cell Disease Patients Vishwas S. Sakhalkar

103

Acute Normovolemic Hemodilution; Its Role as a Blood Conserving Technique C. F. Weiniger and I. Matot

121

vi Chapter VIII

Chapter IX

Index

Brian R. Peterson A New Technology in Blood Collection: Multicomponent Apheresis Rainer Moog

141

The Use of Recombinant Activated Factor II in Transfusion Medicine Massimo Franchini

157 171

PREFACE Although blood transfusion saves lives and reduces morbidities in many clinical diseases and conditions, it is associated with certain risks. A transfusion-related adverse event, also called transfusion reaction, is any unfavourable event occurring in a patient during or after blood transfusion. About 0.5% to 3% of all transfusions result in some adverse events, but the majority of them are minor reactions with no significant consequences. In general, transfusion-related adverse events are categorized as infectious and noninfectious. However, there are other classifications in the literature based on time of occurrence (i.e. acute versus delayed) or physiological mechanism (i.e. immune mediated versus nonimmune mediated). A significant proportion of adverse events may occur as a result of errors in preparation, ordering or administration of blood and blood products. This new book contains the latest research in this essential field which has been revolutionized in recent decades. Blood transfusion represents a potentially life-saving intervention with well known benefits and risks. Today in neonates, transfusion therapy is indicating mainly to maintain adequate circulating blood volume and its components and to remove harmful substances. Chapter I will emphasize the benefit and the risk of blood transfusion. The risk of transfusion-transmitted infectious disease has been decreased significantly over the past decades through the frequent launches of new blood safety technologies. Previously introduced technologies were designed to screen donated blood for potential contamination thereby exposing blood recipients to a “window period”, determined by the sensitivity of the tests, with the consequence that a residual risk for contamination could not be entirely eliminated. Currently known viral pathogens, bacteria as well as migrating (e.g. SARS) and newly emerging viruses (e.g. Asian flu) therefore continue to threaten the safety of the worldwide blood supply. Latest blood safety technologies such as pathogen inactivation are being designed and clinically tested to overcome the weaknesses of screening systems and to preventively avoid the residual and potential future risks of blood transfusion transmitted disease. With ever tighter healthcare budgets and economic constrains in all health care systems, economic- combined with risk-benefit considerations of new technologies gain broader importance in adoption and reimbursement decision-making. Their economic value must be carefully evaluated before a widespread adoption should be recommended. In chapter II, the health economic aspects of transfusion safety measures and their risk/benefit relations will be discussed. Latest research on the cost-effectiveness of a

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newly developed pathogen inactivation technology and the impact on modern blood banking and transfusion medicine will be reviewed to provide recommendations for future research and to support health policy decision-making. Acute haemolytic reaction arising from human error remains a leading transfusionassociated hazard. Measures to reduce such error continued to be a major issue in the 21st century. As reported in chapter III, in 1998, the Hospital Transfusion Committee of a regional hospital in Hong Kong resorted to develop an electronic barcode system for verification of patient identity at critical points of transfusion process – blood sampling for pre-ttransfusion testing and blood administration. Known as the unique patient identification (UPI) system, a process specific system, it had to be used in conjunction with hospital’s standard transfusion procedures. Among the various choices of patient identity, the eight-digit Hospital Number, unique for each patient and each admission, imprinted on thr patient’s wristband and remained attached to the patient throughout hospital stay, was chosen. The UPI device was hand-held, stand –alone machine with built-in scanner and printer, as well as an in-housed computer programme with two pathways of barcode verification. For both procedures, our patient identification policy mandated patient’s wristband as the starting point. Staff first scan patient’s wristband Hospital Number (prefix WB), then, for blood sampling, the Hospital Number (prefix HN) on patient’s Blood Request Form, or blood administration, the Hospital Number (prefix TN) on the blood unit assssigned to the patient. The second barcode scanning automatically channeled to the pathway of the desired procedure. Verification of correct patient identity was documented by automatic printing out of either a self-adhesive label bearing patient’s Hospital Number (prefix WN) by the device to be affixed onto the sample tube prior to blood sampling; or a special a self-adhesive label to be affixed onto the Blood Transfusion Record prior to blood administration. This UPI system was implemented hospital-wide including wards operating theatres (except the Casuality Department and the outpatient clinics) in May 1999. The first generation device was a ready made one. Its use was terminated in 2003 due to mechanical and battery problems. The second generation device was tailor made with improvements in terms of ergonomics, battery, checking algorithm and data storage. Its implementation in July 2004 included one other regional hospital. From May 1999 to April 2005, for 75,000 blood sampling procedures and 51,000 units of blood administration, no transfusion errors were deteced, compared to 13 errors in blood sampling procedures recoreded from May 1995 to April 1999 when the second checker system was implemented. Monitoring process is in place for further system improvement and enhancement of transfusion safety. While objectively safe the public perceive blood transfusion from donor blood as risky. Indeed, there are a number if minor risks (e.g., transfusion transmitted infections). One solution to this has been to explore the development of blood substitutes. However, would people be willing to use a bio-engineered blood product? There is evidence that people do not look favorably on such options. Chapter IV examines this issue with respect to perceived riskiness and naturalness of donor blood and 3 forms of substitute (bovine Hb, human Hb and GM) and how these are judged relative to a wide range of other natural and engineered medical and food related products. Naturalness is a key construct with respect to the acceptance of bio-technology. Data were collected from a group of 148 undergraduate students on the perceived naturalness and riskiness of 12 products (natural and their bio-

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engineered counter part) – including blood and 3 substitutes. The results show that the more natural the natural product is perceived to be the less natural its GM variants is perceived. Transfusion with these different blood products is rated as risky along with other invasive medical procedures (heart transplant). Ratings of naturalness were unrelated to ratings of risk for invasive procedures, but were related for low risk non-invasive procedures. Furthermore, it was shown that donor blood for transfusion is itself not perceived as very natural. Implications for the health education and promotion with respect to the acceptance of blood substitutes are discussed. Pathogenic prions are infectious proteins that are believed to be responsible for a variety of progressive neurodegenerative diseases. These disorders are referred to as transmissible spongiform encephalopathies (TSE) since the brains of infected individuals have been observed to display a sponge-like morphology upon post-mortem examination. TSEs in humans and other species are invariably fatal. During the 1980’s it became apparent that some cattle in the United Kingdom had become infected by a form of TSE, which became known as bovine spongiform encephalopathy (BSE) or “mad cow” disease. By 1996, it was recognized that ingestion of beef from infected cattle could result in a TSE in humans known as variant Creutzfeldt-Jakob disease (vCJD). Recent incidents of probable transfusiontransmitted vCJD have raised concerns about the safety of the blood supply. The relatively long latency from the time of infection to the onset of symptomatic vCJD as well as the lack of sensitive and specific ante-mortem tests for vCJD, increase the risk that asymptomatic, infected individuals may become blood donors. Until now, donor deferral has been the strategy employed to reduce this risk. Nevertheless, this strategy may be unreliable, and threatens blood availability. Leukoreduction has also been helpful by reducing cell-associated infectious prion, which has been reported to account for up to 42% of infectivity in blood. Unique chemical characteristics of prion surfaces have been determined and proprietary affinity filtration properties developed. These have been successfully adapted to existing high efficiency blood filter matrices for specific reduction of prions present in blood components for transfusion. Validation studies described in chapter V demonstrate that such filtration reduces prions by a factor of 2.9 log or 99.9% when quantified by Western blot analysis while maintaining existing standards for leukoreduction, post-storage hemolysis, and other indices of bio- and hemo-compatability. Definitive bioassay and infectivity studies are ongoing, and are expected to be available by the end of 2005. This prion reduction technology is currently being introduced for use with red cells for transfusion in Europe, and will be available worldwide pending each nation’s regulatory review process and manufacturing capacity expansion. Blood transfusions, sometimes multiple, are commonly used in patients with sickle cell disease for prevention and treatment of complications. Allosensitization leading to antibody formation in blood transfusion recipients can result from exposure to donor’s minor blood types that are not present in the recipient and are not crossmatched for transfusion. The reasons for allosensitization are multifactorial: interracial differences in frequencies of minor blood types in the local population (a major factor), and host factors including genetic predisposition to produce alloantibodies and autoantibodies or react to foreign antigens; recipient’s age, sex, race, etc. The incidence of this problem varies locally and is reportedly between 5-60%. Exposure to donor’s foreign RBC antigens leads to formation of specific

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alloantibodies, sometimes accompanied by autoantibody production possibly due to dysregulation in host immune response. There are no immediate consequences due to the mild and slow rise in titer. The titers drop over a period of time, due to non-exposure to the antigen. Hence, on retesting after few months, the repeat crossmatch may be compatible. However, on re-exposure, there is a brisk anamnestic antibody production and mild to severe delayed transfusion reaction may follow manifesting as an acute or subacute intravascular or extravascular hemolysis leading to indirect hyperbilirubinemia, anemia, pain ‘crisis’, renal failure, and sometimes death. The technical, monetary and health implications for the recipient and institution can be profound. Numerous approaches to this problem have been devised – complete RBC phenotyping of all ‘at risk’ recipients, various levels of pretransfusion extended crossmatch depending on frequency of minor blood groups in the local population (a technically demanding, time consuming, cumbersome, and expensive procedure resulting in an inadvertent overuse of Rh negative blood), autologous blood transfusions, and lifelong avoidance of the particular antigen once the corresponding antibody is demonstrated. While transfusions should be avoided in patients with multiple alloantibodies/autoantibodies, it may be impossible to obtain compatible blood when transfusion is critical for survival. Novel approaches such as hemoglobin polymers, bovine hemoglobins or artificial oxygen carrying complexes can be used. Corticosteroids, immunoglobulins, erythropoietin, hydration, and symptomatic management are also helpful. Chapter VI details all the different aspects of allosensitization and discusses the consequences and approach to the major problem in multicultural communities across the world. Concerns about the risks associated with allogeneic blood transfusion (infectious disease, transfusion reactions, immunomodulation, transfusion-associated lung injury) have led to the development of a variety of blood conserving techniques intended to minimize the need for allogeneic transfusion during surgery. Among these, acute normovolemic hemodilution (ANH) has become accepted. The procedure entails the removal of blood from the patient immediately before operation, and simultaneous replacement with appropriate volume of crystalloid and/or colloid fluids. ANH reduces hematocrit (Hct) so that blood shed during the operative procedure results in less red blood cell mass loss. The removed blood is then reinfused as autologous whole blood after the procedure is completed. The procedure is simple and inexpensive and has the advantage that fresh autologous blood is readily available. The resultant reduction in allogeneic blood transfusion may conserve resources and protect the patient from exposure to the risks of allogeneic blood transfusion. Numerous studies of its efficacy, however, have produced conflicting results, perhaps due to the heterogeneity of the surgeries in which it was employed, differences in study protocol and differences in the definition of outcome variables. Previous mathematical analyses of ANH have shown that ANH may be effective in diminishing the need for allogeneic blood transfusion, and that its efficacy depends on surgical blood loss, initial patient Hct and on the “transfusion trigger” (the Hct at which blood is to be transfused). One meta-analysis of ANH used in 24 quality randomized controlled trials found that ANH reduced the likelihood of exposure to at least 1 unit of allogeneic blood, and had a small positive effect on the perioperative blood loss when compared with patients who did not undergo ANH. Use of ANH is controversial and it is not universally used. The objective of chapter VII is to get the

Preface

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reader acquainted with this blood conserving technique, understand the parameters that determine its efficacy, define which group of patients might benefit from its use, and learn about its potential risks. Multicomponent apheresis procedures offer the possibility of collecting blood components that are standardized, as compared to those available with manual whole blood donations. Recent technologic advances in hemapheresis have made possible the development of systems that can collect different combinations of blood components from the same donor during one collection session as described in chapter VIII. Red blood cells (RBCs) can be concurrently collected with platelets (PLTs) or plasma. Two units of RBCs can also be collected during one apheresis session provided that the donor fulfils the inclusion criteria for that procedure. The hemoglobin content of apheresed RBCs after addition of additive solution is higher than the minimal requirement of whole blood derived RBCs of 40 g per unit due to standardization. A desired PLT yield can be targeted by the algorithm of the blood cell separators after entering donor specific parameters. Automated systems permit predictable collection of blood components with consistent yields and volumes. Previous definitions of blood component yield and volume as well as the flexibility to collect those multicomponents allows the blood centre to collect those components that maximize donors’ contribution and to meet the demands of its area hospitals for blood components. Blood centres are progressively increasing their reliance on apheresis technology to increase the number of blood components collected per donor visit and to reduce the number of donors a patient is exposed to. The use of these systems has shown at the same time that the procedures have been well tolerated by the donors and are cost effective. Recombinant activated factor VII (rFVIIa, NovoSeven® ) is a novel hemostatic agent originally developed to treat bleeding episodes in hemophiliacs with inhibitors against coagulation factors VIII and IX. Successively, rFVIIa has also been employed with benefit for the management of hemorrhages in other congenital and acquired hemostatic abnormalities and nowadays the drug is registered in Europe for the treatment of congenital hemophilia with inhibitors, acquired hemophilia, congenital FVII deficiency and Glanzmann thromboasthenia. More recently, rFVIIa has been utilized to control excessive bleeding, thus reducing the exposure to allogeneic blood, in a wide variety of non-hemophilic bleeding situations unresponsive to conventional therapy including emergency (intracerebral hemorrhage, upper gastro-intestinal bleeds, trauma, oral anticoagulant-induced hemorrhage) or surgery-related (liver resection, orthotopic liver transplantation, neurosurgery, cardiac surgery) bleeds. These latter newer and less well-characterized clinical applications of rFVIIa, basing on a literature search including PubMed, references from reviews and abstracts from the most important meetings on this topic, will be discussed in chapter IX.

In: New Developments in Blood Transfusion Research ISBN 1-59454-962-1 Editor: Brian R. Peterson, p. 1-13 © 2006 Nova Science Publishers, Inc.

Chapter I

BLOOD TRANSFUSION IN THE NEONATE – WHERE ARE WE TODAY? Michael S. Schimmel1,2,*, Michael Kaplan1,3 and Roger F. Soll4 1

Department of Neonatology, Shaare Zedek Medical Center, Jerusalem, Israel Faculty of Health Sciences, Ben Gurion University of the Negev, Beer Sheva, Israel 3 Faculty of Medicine of The Hebrew University, Jerusalem, Israel 4 Department of Pediatrics, University of Vermont College of Medicine, Burlington, Vermont, USA

2

ABSTRACT Blood transfusion represents a potentially life-saving intervention with well known benefits and risks. Today in neonates, transfusion therapy is indicating mainly to maintain adequate circulating blood volume and its components and to remove harmful substances. The following discussion will emphasize the benefit and the risk of blood transfusion.

Keywords: blood transfusion, exchange transfusion, partial exchange transfusion, polycythemia, hyperviscosity, neonatal hyperbilirubinemia

INTRODUCTION Blood transfusion represents a potentially life-saving intervention with well known benefits and risks. The history of blood transfusion in newborn infants goes back to the beginning of the 20th century when Helmholz transfused blood through the superior sagital

*

Corresponding author: Michael S. Schimmel, MD, Department of Neonatology, Shaare Zedek Medical Center, POB 3235, Jerusalem 91031, Israel, Fax 972-2-652 0689, E-mail address: [email protected]

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sinus [1]. Using a similar technique, Hart did the first exchange transfusion in a neonate for hyperbilirubinemia [2]. In neonates today, transfusion therapy is indicated, in neonates, mainly in three clinical situations: 1. To maintain adequate circulating blood volume. 2. To replace specific blood components such as erythrocytes, leukocytes, platelets, plasma etc. 3. To remove harmful substances such as bilirubin or excess red cells (using various techniques of exchange transfusion). The following discussion will emphasize the use of blood transfusion to replace red blood cells (RBC) and exchange transfusion (ET) as a technique to remove excess bilirubin or partial exchange transfusion (PET) to remove excess amounts of RBC.

RED BLOOD CELLS TRANSFUSION Red blood cell transfusion (RBCT) is performed in order to improve oxygen delivery by maintaining a desirable hematocrit (Hct) level appropriate for the clinical status of the newborn. Approximately 75% of all preterm infants born in the USA receive RBCT. Often, the RBCT are multiple small volume transfusions of blood obtained from different donors [3]. In premature infants, two main reasons for the need of RBCT can be identified: 1. "early" (during the first two weeks of life) in order to replace phlebotomy losses for laboratory tests. 2. "late", given to correct "anemia of prematurity". Replacement of blood from phlebotomy losses is the most common indication of RBCT in neonates [4]. The best way to address phlebotomy losses is mainly by minimizing blood drawing. Several technological solutions were developed to reduce phlebotomy losses, including transcutaneous electrodes; the use of minimal blood volume for various blood tests, and indwelling catheters with specific transducers. In many NICU, RBCT is considered if blood drawn reaches 10% of the blood volume or if the Hct is considered suboptimal in an infant who is critically ill. "Anemia of prematurity" is characterized by a low reticulocyte count and delay in renal production of erythropoietin in spite of a low hematocrit. This condition is most probably due to a decreased sensitivity to low hematocrit. Since the early 1990s, exogenous erythropoietin (rhEPO) has been used in an attempt to prevent anemia of prematurity. The results of many of the clinical trails had been promising with very rare adverse effects [5]. However with more restrictive transfusion guidelines, the need for rhEPO may be avoided. Franz et al [6] compared a group of very low birth weight (VLBW) infants (less or equal to 1500gm) born in hospitals with very restrictive guidelines for RBCT to babies who participated in the second European Multicenter Erythropoietin Trail. Franz concluded that under restrictive transfusion

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guidelines, adequate protein and iron supplementation, RBCT can be reduced to the rate reported by the rhEPO trails. The long term adverse effects of using rhEPO have yet to be determined including the effect on iron stores [7]. The appropriate Hct in sick infants is unknown. Little evidence is available to define the best adequate Hct levels in various clinical situations. Thus, many departments have local guidelines with a range of Hct values depending on clinical status that are based on clinical experience and very little evidence. Surrogate markers of anemia include respiratory distress, apnea, tachycardia, poor weight gain and increase blood lactate levels. These parameters may be improved by RBCT, but there is no clear evidence of an associated improved outcome such as reduced mortality or hospital stay. Furthermore, similar benefits may be seen by using volume expansion [8]. Common practice is that in infants with respiratory disease, Hct should be kept above 30% [3,4,9]. In addition, many units maintain infants with cardiopulmonary problems or with growth failure with a Hct above 30%. Other infants, with normal weight gain and head growth and without cardiopulmonary symptoms are transferred to Hct greater than 20% [3,8]. As RBCT is a frequently used procedure, all members of the neonatal team should be familiar with the adverse effects of transfusion. The main unfavorable effects are transfusion associated infections, graft-vs-host disease, electrolyte and acid base imbalance, alloimmunization and fluid overload. [4,10]. To minimize the adverse effects of RBCT various techniques are used. Donor exposure has been reduced by dedicating specific small units to neonates, while taking advantage of longer shelf life and improved viability of RBCs stored in additive solutions [9].

PARTIAL EXCHANGE TRANSFUSION (PET) Neonatal hyperviscosity has been implicated as a cause of long term neurological delay and damage in the growing child [11,12]. Given this, concern neonatologists have developed management protocols to minimize this CNS morbidity. Neonatal polycythemia may influence blood flow. Poiseuille’s equation provides a useful model to explain the physical properties that influence blood flow and can be used to extrapolate the impact of polycythemia on tissue blood flow. Poiseuille’s equation relates flow (F), difference in pressure (∆P), the radius of the tube (r), viscosity (η), and the length of the tube (L) (F α ∆P.r4 / η.L). However, one must remember that the equation was formulated for long, straight tubes, a Newtonian fluid (e.g., water), and a steady laminar flow. Many factors may contribute to increased blood viscosity. Whole blood viscosity is primarily influenced by three blood factors: red cell number, plasma proteins, and erythrocyte deformity [13]. The number of red cells has the greatest influence on blood viscosity. In addition, blood flow is not only influenced by the radius of the vessels, but also by endothelial characteristics, blood pressure and end capillary pressure. In infants with hyperviscosity, there is an increase in internal friction followed by an increase in resistance to blood flow. Decreased blood flow rates, particularly in the microcirculation of vital organs, leads to decreased oxygen delivery and a symptom complex that includes abnormalities of central nervous system function, hypoglycemia, decreased

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renal function, cardiorespiratory distress, and coagulation disorders. Most importantly, hyperviscosity has been reported to be associated with long term motor and cognitive neurodevelopmental disorders [11,12]. The “gold standard” for the measurement of viscosity is performed by using a whole blood viscometer that can accurately measure the viscosity of blood at the low shear rates that occur naturally in the capillary circulation. Unfortunately, whole blood viscometers are not universally available in the clinical setting. As erythrocyte number is the single most important factor affecting viscosity, the measurement of the neonatal Hct is the best clinical screening test for identifying infants with presumed hyperviscosity [14]. Traditionally, polycythemia has been defined as a venous Hct over 65%. This cut off was chosen based on the observation that blood viscosity exponentially increased above an Hct of 65% [15]. However, it is unclear, if this is an appropriate clinical threshold to consider instituting treatment. Gross measured whole blood viscosity in 102 normal and 18 symptomatic infants [14]. All symptomatic infants had hyperviscosity and the Hct of these infants ranged from 63% to 77%. On the other hand, Drew reported that only 47% of infants with Hct over 65% had hyperviscosity and only 23 % of the hyperviscous infants were polycythemic [12]. These findings emphasize the fact that factors other than erythrocyte number determine the degree of blood viscosity and the problematic nature of deciding on therapy based on red cell count alone. The reported incidence of neonatal polycythemia in term newborns varies from 0.4% to 12% [16,17,18]. This wide variation may be due to different screening techniques, sampling sites (capillary versus peripheral or central venous), patient populations, methodology of measuring (Coulter counter or centrifuged capillary blood), and sampling time. Sampling time is the most important source of variation. Shohat demonstrated that the Hct normally rises after birth, reaching a peak at 2 hours postpartum, and then slowly decreases over the next 12 hours [18]. At 2 hours of life, the upper limits (2 S.D.) of a normal capillary Hct was 71%! Thus, simply defining polycythemia as a hematocrit over 65% may not be statistically justified. The causes of hyperviscosity/polycythemia in the neonate are varied. The entity “polycythemia” can be subdivided based on underlying etiology: 1. Increased red cell mass and plasma volume, secondary to “blood transfusion” (delay cord clamping, twin to twin or maternal-fetal transfusion) or maternal diabetes. 2. Increased red cell mass and normal plasma volume associated with a congenital syndrome [Trisomy 13, 18, 21]. 3. Increased red cell mass with normal or decreased plasma volume secondary to intrauterine growth retardation, placental insufficiency, maternal hypertension or smoking [19]. In clinical practice, neonatal polycythemia has been used as a marker for neonatal hyperviscosity and thus clinicians have focused on the newborn infant’s Hct level as the criteria for the therapeutic intervention. Partial exchange transfusion (PET) is traditionally used as the method to lower the Hct and treat hyperviscosity.

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Treatment of Polycythemia Currently, hemodilution by PET is the recommended treatment for the polycythemic infant. PET has been shown to reduce pulmonary vascular resistance [20], and increase cerebral blood flow velocity [21,22]. Bada has documented that PET normalizes cerebral hemodynamics and improves the clinical status of infants with polycythemia [23]. PET is a relatively simple procedure, but has numerous potential complications. Unfortunately, there are no data regarding the incidence of complications of PET; one can only extrapolate from the data on full exchange transfusions performed for neonatal hyperbilirubinemia. Reported complications for whole blood exchange include infections, cardiac arrhythmia, thrombosis, emboli, vessels perforation, necrotizing enterocolitis, accidental hemorrhage, air embolus, hypothermia, reduction in blood pressure and cerebral blood flow fluctuation and even death [24] (more comprehensive discussion follows below). Full exchange transfusion is expected to have a higher incidence of complications than PET, since the amount of blood to be exchanged is almost 9 times higher and the product utilized for the exchange is donor blood. Most of these complications can be avoided by performing the procedure carefully, while monitoring vital signs and adjusting to a standard protocol. However, the relevance of this data in calculating the magnitude of the risk of performing PET is unclear. The statement [25] of the Committee of the Fetus and Newborn of the American Academy of Pediatrics regarding the treatment of neonatal polycythemia with PET reflects both the concern and uncertainty regarding this mode of treatment. “The accepted treatment of polycythemia is partial exchange transfusion (PET). However there is no evidence that exchange transfusion affects the long term outcome. Universal screening for polycythemia fails to meet the methodology and treatment criteria and also, possibly the natural history criterion”. Despite this ambivalent statement, PET in symptomatic babies with a Hct greater than 65% and in asymptomatic babies with a Hct greater than 70% [26,27] is standard practice in many neonatal units. A systematic search of the literature for reports of neonatal hyperviscosity and polycythemia treated with PET revealed very few randomized controlled studies. Some studies could not document any consistent effect of PET on long term neurodevelopmental outcome, especially in asymptomatic infants. An explanation for these findings is offered by Black [28] who hypothesized that in symptomatic infants, the insult to the central nervous system occurs primarily in-utero and thus PET in the neonatal period is too late to affect outcome. Conversely, in asymptomatic infants there is no CNS damage to treat or even to prevent by any postnatal "prophylactic" therapy. No less important is the difficulty for justifying an invasive procedure such as PET when there is no general agreement on the basic diagnostic criteria for defining the condition that needs to be treated. Drew noted that only 23.9% of the patients who had cord hyperviscosity had polycythemia and only 47.8% of the polycythemic babies had hyperviscosity [12]. Thus, utilizing the Hct as a criterion for presumed hyperviscosity and the need for PET is a dubious assumption at best. Thus, Schimmel [29] and others [11,23,30] are skeptical if the minor improvement, if any, reported in some studies justifies the risk of PET, particularly in an otherwise asymptomatic infants.

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Based on these results, our recommendation is that the polycythemic (Hct >70%) asymptomatic infant should be treated according to its specific etiology and not by routine “prophylactic” PET: 1. In an asymptomatic polycythemic infant with presumed normal or increased blood volume status, cardiorespiratory and glucose monitoring would be sufficient. 2. In symptomatic infants with a Hct above 65%, a more aggressive therapeutic approach, i.e. PET with normal saline, should be utilized, in order to enhance the blood flow and reduce possible ongoing tissue injury. 3. If the presumption is that the polycythemia is due to a reduced blood and plasma volume status, such as occurs in an IUGR infant, the infant should be treated with early feeding or plasma expansion with intravenous saline and dextrose as necessary. There is a need for studies comparing the short and long term outcome after PET compared to expectant management in both polycythemic asymptomatic and symptomatic newborns stratified by postnatal age, red blood cell mass and blood volume. Ideally, these studies should include measurement of blood viscosity so that objective criteria can be developed for the use of PET in the newborn period. Until these studies are performed, PET should be reserved only for the symptomatic polycythemic infant or when venous hematocrit is over 75%.

EXCHANGE TRANSFUSION Exchange transfusion was the first definitive therapy for neonatal hyperbilirubinemia. The technique has been instrumental in preventing kernicterus in many neonates since it became available for routine clinical use by Diamond et al in the late 1940s [31,32]. Although in the last few decades its use has diminished drastically due to prevention of Rh isoimmune disease by the use of Rhogam, the universal introduction of phototherapy and more recently, intravenous administration of intravenous immune globulin in cases of isoimmunization, there are still instances in which exchange transfusion may the only method of diminishing the serum total bilirubin concentration in severely hyperbilirubinemic neonates. Knowledge of the procedure is therefore important for all involved in the care of the neonate.

History Although there were some early reports of successful exchange transfusions performed via the saggital sinus and the anterior fontanelle, [33] and later the radial artery/saphenous vein [34], it was the pioneering work of Diamond and colleagues who described their technique utilizing umbilical vein catheterization, that brought the procedure to within reach of the practicing pediatrician [31,32]. By physically removing blood containing high concentrations of bilirubin, in addition to antibody in cases of isoimmunization, severe

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7

hyperbilirubinemia could now be controlled and kernicterus prevented. For several decades, until the introduction of phototherapy, exchange transfusion was the only means of acutely decreasing the serum total bilirubin concentration.

Objectives of Exchange Transfusion The primary objective of exchange transfusion is to control hyperbilirubinemia by physically removing blood containing bilirubin from the body. Presuming a baby's blood volume to be approximately 85 ml/kg body weight, and an exchange transfusion performed using blood aliquots of 10-20 ml, because of dilution with the transfused blood, a single volume exchange can be expected to remove about 70% of the total blood volume, and a double volume exchange about 85% [35]. As further exchange will not significantly increase the volume of blood removed, most exchange transfusions in neonates are limited to double volume. Double volume exchange transfusion can be expected to decrease the serum total bilirubin (STB) by about one half. However, because much of the body bilirubin will be in the extravascular space, a double volume exchange will remove only about 25% of the total body bilirubin. Following completion of the procedure, there is re-equilibration between extravascular and vascular bilirubin, with rebound in the STB to about 75% of the preexchange concentration. Prior to the introduction of phototherapy following completion of the exchange, the STB concentration frequently continued to rise and further exchange transfusions were often required. However, except in conditions of severe isoimmune hemolysis, post-exchange phototherapy is nowadays usually successful in preventing further procedures. In addition to the removal of bilirubin from the body, exchange transfusion may act as an adjunct in the overall therapy of the condition by removing maternal antibody, antibody coated red cells, and increasing the hemoglobin concentration when hemolysis has resulted in anemia.

Indications for Exchange Transfusion For many years exchange transfusion was performed in any neonate in whom the STB concentration exceeded 20.0 mg/dL. This indication was derived from the Rh isoimmunization era in which it was noted that kernicterus rarely occurred in term infants in whom the STB remained below that concentration [36]. This absolute indication is very rarely employed today. In many cases of non-hemolytic jaundice, the STB may be allowed to exceed that point. The Subcommittee on Hyperbilirubinemia of the American Academy of Pediatrics recommends that exchange transfusion should be performed in any neonate in whom the STB exceeds 25 mg/dL despite a trial of intensive phototherapy, or lower according to the infant's age or presence of risk factors. [37]. Furthermore, The National Quality Forum at the Agency for Healthcare Research for Quality has declared kernicterus and STB concentrations in excess of 30.0 mg/dL as "never events" [38]. Unfortunately, there is very little in the way of randomized controlled trials to guide us in our indications for

8

Michael S. Schimmel, Michael Kaplan and Roger F. Soll

performing an exchange transfusion. Current recommendations are as a result not evidence based, but rather based on clinical experience. As no one bilirubin value can be used as a cut off point for the prevention of kernicterus, the indications for exchange transfusion are based on STB concentrations in combination with the chronological age of the infant, gestational age, and risk factors including isoimmune hemolytic disease, glucose-6-phosphate dehydrogenase deficiency, asphyxia, lethargy, temperature instability, sepsis, acidosis and bilirubin/albumin ratio. Comprehensive guidelines for the indications for exchange transfusion for term and near term neonates have recently been published by the Subcommittee on Hyperbilirubinemia [37]. While these guidelines are somewhat arbitrary, neonatal STB concentrations should be closely monitored with the guidelines in mind, and the indications for exchange transfusion enumerated by the Subcommittee should in all probability not be exceeded. Indications for exchange transfusion in low birth weight neonates or those with lower gestational ages than included in the AAP guidelines have also recently been published [39].

Technique of Exchange Transfusion The Subcommittee on Hyperbilirubinemia of the AAP recommends that exchange transfusions be performed only by trained personnel in a neonatal intensive care unit with full monitoring and resuscitation capabilities. Exchange transfusion is usually performed via the umbilical vein, using a size 8 Fr catheter in a term infant, or size 5 in a premature neonate. The procedure of umbilical vein catheterization is well known to neonatologists and pediatricians. Prior to commencing the procedure, the infant should be placed on a radiant warmer and the heart and respiratory rates monitored. Blood for infusion should pass through a blood warmer. Commercially available kits for exchange transfusion are available. These contain infusion sets with a specially designed 4-way stopcock. The stopcock handle points to the port that is open; the operator rotates it in a clockwise direction after withdrawing blood from the umbilical veinous catheter, to a waste bag, to the blood to be infused, and back to the infant. The procedure should be performed slowly, over one to two hours, to prevent sudden hemodynamic changes which may cause wide fluctuations in blood pressure with undesirable effects on cerebral or gastrointestinal blood flow. Isovolumetric exchange, utilizing the umbilical vein and the umbilical artery for blood for simultaneous blood infusion and withdrawal respectively, may facilitate more rapid exchange time, although Patra et al [40] found that the odds for adverse events were greater when this method was used compared to other methods of exchange. The exchange is usually performed by a physician, with the assistance of a nurse whose function it is to carefully record the amounts of blood being withdrawn and infused, and to monitor the infant's cardiorespiratory status, oxygenation, blood pressure and general wellbeing. Following the exchange procedure, blood should be sent for bilirubin determination, complete blood count including platelets, ionized calcium and glucose. In cases in which a second exchange might be necessary, blood should be sent for type and crossmatch.

Blood Transfusion in the Neonate – Where are We Today?

9

Many different combinations of blood components can be used safely and effectively; no one combination or component is unequivocally superior [41]. Red cells reconstituted with 5% albumin or fresh frozen plasma are most frequently used. Citrate anticoagulant may cause hypocalcemia because of its calcium-binding effects. Despite low levels of serum ionized calcium, tetany is rarely encountered, and the practice of administering calcium during the exchange is seldom practiced. Red blood cells for the exchange procedure should be of group O or ABO compatible with maternal and neonatal plasma, Rh negative or Rh identical with the neonate. The blood to be transfused should not contain red cell antigens to which the mother has antibodies. The blood should be collected into CPD anticoagulant, and have a hematocrit of 50-60%. CMV infection of the newborn should be prevented either by using CMV seronegative blood, or by using appropriate blood filters. Irradiation of the blood is essential if the infant had had an intrauterine transfusion. Irradiation is recommended for all exchange transfusions, but in the absence of a previous intrauterine transfusion may be omitted should the irradiation process result in significant clinical delay [8]. As both stored whole blood and reconstituted blood are deficient in platelets, in infants who are severely thrombocytopenic or who have a bleeding tendency, platelet transfusion should be considered following the exchange transfusion.

Complications of Exchange Transfusion Exchange transfusion is an invasive procedure and complications may be related to the blood transfusion itself, catheter-related complications and those related to the procedure [42]. With regard to the blood transfusion per se, severe hemolysis due to infusion of incompatible red blood cells may be life-threatening. Although infections should be screened for, there is a small chance of viral or bacterial infection being transmitted via the transfused blood. Hyperkalemia may result from infusing blood that has been stored for a long period of time. The glucose load during exchange transfusion may be high, resulting in insulin secretion and subsequent rebound hypoglycemia. Graft-versus-host disease is unusual, but may occur in premature infants or those who have had in-utero transfusions. Insertion of an umbilical venous catheter may be associated with air embolism, hemorrhage or introduction of infection. During the procedure itself, there may be fluctuations in blood volume resulting in hypovolemia or hypervolemia, intracranial blood pressure fluctuations or diminished bowel perfusion with resultant necrotizing enterocolitis. Serious complications of exchange transfusion were commonly encountered in the preintensive care era. Nowadays, mortality should be very low in healthy, term neonates, but may still be encountered in sick or extremely premature infants. Of 106 neonates who had undergone 140 exchange transfusions between 1980 and 1995, the overall mortality was 2%, but increased to 8% in the subset of infants who were ill [43]. In a much smaller series, Chima et al reported no serious adverse effects of death among 22 term neonates who underwent 26 exchange transfusions between 1990 and 1998 [44]. Patra et al [40] recently reviewed charts of 55 neonates who had undergone 66 exchange transfusions between 1992 and 2002. Seventy-four percent of exchange transfusions were associated with some form of adverse event, the most common of which included thrombocytopenia, hypocalcemia and

10

Michael S. Schimmel, Michael Kaplan and Roger F. Soll

metabolic acidosis. Seizures occurred in one infant, and there was one death in an extremely low birth weight, 25 week gestation infant. Most complications were treatable. Sepsis, necrotizing enterocolitis and cardiac arrest were not encountered in this series. In conclusion, the technique of exchange transfusion has facilitated the treatment of hyperbilirubinemia and prevention of kernicterus and for many years was the only therapeutic modality available for this purpose. Although it is an invasive procedure with the potential of serious and even lifethreatening complications, exchange transfusion has an important place in our therapeutic regimen. While the decision to perform an exchange should not be made lightly, when indicated, if performed under intensive care facilities and with appropriate expertise, its advantages should clearly outweigh the small risk of serious complications.

CONCLUSION Newborn are the most heavily transfused patient group with the greatest potential for longevity; as such, it should be kept in mind that blood transfusion, including it's various forms, is a double edge sword. It is an essential therapy but, its adverse effects should always be remembered.

REFERENCES [1] [2] [3]

[4] [5] [6]

[7] [8]

Zuelzer WW. Pediatric Hematology in historical Perspective. In Hematology of Infancy and Childhood. Ed. Nathan and Oski. W.B. Sanders Company 1974 p.1. Hart AP. Familial icterus gravis of the newborn and its treatment. Can Med Assoc J 1925;15:1008. Simon TL, Alverson DC, AuBuchon J, Cooper ES, DeChristopher PJ, Glenn GC, Gould SA, Harrison CR, Milam JD, Moise KJ Jr, Rodwig FR Jr, Sherman LA, Shulman IA, Stehling L. Practice parameter for the use of red blood cell transfusions: developed by the Red Blood Cell Administration Practice Guideline Development Task Force of the College of American Pathologists. Arch Pathol Lab Med. 1998;122:130-138. Strauss RG. Transfusion therapy in neonates. AJDC 1991;145:904-11. Ohls RK. Human recombinant erythropoietin in the prevention and treatment of anemia of prematurity. Pediatr Drugs 2002;4:111-121. Franaz AR, Pohlandt F. Red blood cell transfusions in very and extremely low birth weight infants under restrictive transfusion guidelines – is exogenous erythropoietin necessary? Arch Dis Child Fetal neonatal Ed 2001;84:F96-F100. Kling PJ and Winzerling JJ. Iron status and the treatment of anemia of prematurity. Clin Perinatology 2002;29:283-294. Gibson BE, Todd A, Roberts I, Pamphilon D, Rodeck C, Bolton-Maggs P, Burbin G, Duguid J, Boulton F, Cohen H, Smith N, McClelland DB, Rowley M, Turner G; British Commitee for Standards in Haematology Transfusion Task Force: Writing group. Transfusion guidelines for neonates and older children. Br J Haematol. 2004;124:43353.

Blood Transfusion in the Neonate – Where are We Today? [9] [10]

[11]

[12]

[13]

[14] [15]

[16] [17] [18] [19] [20]

[21]

[22]

[23]

[24] [25]

11

Roseff SD, Luban NLC and Manno CS. Guidelines for assessing appropriateness of pediatric transfusion. Transfusion 2002;42:1398-1413. Sloan SR, Benjamin RJ, Friedman DF, Webb IJ and Silberstein L. Transfusion Medicine. In Hematology of Infancy and Childhood. Ed. Nathan DG, Orkin SH, Ginsburg D and Look AT. 2003, 6th edition, Saunders Company p.1709. Delaney-Black V, Camp BW, Lubchenco LO, Swanson C, Roberts L, Gaherty P, et al. Neonatal hyperviscosity association with lower achievement and IQ scores at school age. Pediatr 1989;83:662-667. Drew JH, Guaran RL and Hobbs JB. Neonatal whole blood hyperviscosity, the import factor influencing later neurological function is viscosity and not polycythemia. Clin Hemorheol Microcirc 1997;17:67-72. Muller R. Haemorheology and peripheral vascular disease, a new therapeutic approach. J Med 1981;12: 209-236) (Gross GP, Hathaway WE and McGaughey HR. Hyperviscosity in the neonate. J Pediatr 1973;82:1004-1012. Gross GP, Hathaway WE and McGaughey HR. Hyperviscosity in the neonate. J Pediatr 1973;82:1004-1012. Nelson NM. Respiration and circulation before birth. In: Smith CA and Nelson NM editors. Physiology of the newborn infant 4th edition. Springfield Charles C Thomas; 1976. p 17. Reisner SH, Mor N, Levy Y, Merlob P. Incidence of neonatal polycythemia. Isr J Med Sci 198319:848-9. Wiswell TE, Cornish JD, Northam RS. Neonatal polycythemia: frequency of clinical manifestations and other associated findings. Pediatrics 1986;78:26-30. Shohat M, Merlob P and Reisner SSH. Neonatal polycythemia: I. Early diagnosis and incidence relating to time of sampling. Pediatric 1984;73:7-10. Al- Alawi E and Jenkins D. Does maternal smoking increase the risk of neonatal polycythemia? Ir Med J 2000;93:175-176. Murphy DJ Jr, Reller MD, Meyer RA, Kaplan S. Effects of neonatal polycythemia and partial exchange transfusion on cardiac function: an echocardiographic study. Pediatrics. 1985;76:909-13. Rosenkrantz TS, Oh W Cerebral blood flow velocity in infants with polycythemia and hyperviscosity: effects of partial exchange transfusion with Plasmanate J Pediatr. 1982;101:94-8. Maertzdorf WJ, Tangelder GJ, Slaaf DW, Blanco CE. Effects of partial plasma exchange transfusion on cerebral blood flow velocity in polycythaemic preterm, term and small for date newborn infants. Eur J Pediatr. 1989;148:774-778. Bada HS, Korones SB, Pourcyrous M, Wong SP, Wilson III WM, Koloni HW et al. Asymptomatic syndrome of polycythemic hyperviscosity – effect of partial plasma exchange transfusion. J Pediatr 1992;120:579-585. Merchant RH and Gupta SC. Neonatal exchange transfusion. Indian Pediatr 1986;23:459-465. American Academy of Pediatrics Committee on Fetus and Newborn: Routine evaluation of blood pressure, Hct, and glucose in newborns. Pediatrics. 1993; 92:474476.

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[26] Roithmaier A, Arlettaz R, Bucher HU, Krieger M, Duc G and Versmold HT. Randomized controlled trail of Ringer solution versus serum for partial exchange transfusion in neonatal polycythemia. Eur J Pediatr 1995;154:53-56. [27] Acunas B, Celtik C, Vatansever U, Karasalihoglu S. Thrombocytopenia: an important indicator for the application of partial exchange transfusion in polycythemic newborn infants? Pediatr Int. 2000;42:343-347. [28] Black VD, Lubchenco LO, Koops BL, Poland RL, Powell DP. Neonatal hyperviscosity: randomized study of effect of partial plasma exchange transfusion on long-term outcome. Pediatrics 1985;75:1048-1053. [29] Schimmel MS, Bromiker R and Soll RF. Neonatal polycythemia: Is partial exchange transfusion justified? Clinics in Perinatology. Clin Perinatol. 2004;31:545-553. [30] Rothenberg T. Partial plasma exchange transfusion in polycythemic neonates. Arch Dis Child 2002; 86:60-62. [31] Diamond LK, Allen FH, Thomal WO. Erythroblastosis fetalis. VII. Treatment with exchange transfusion. N Eng. J Med 1951:244:39-49. [32] Diamond LK. Erythroblastosis foetalis or haemolytic disease of the newborn. Proc. Royal Soc. Med. 1977;40:546-550. [33] Hart AP. Familial icterus gravis of the newborn and its treatment. Can Med Ass J 1925;15:1008-1011. [34] Wiener AS, Wexler IB, Grundfast GH. Therapy of erythroblastosis with exchange transfusion. Bull NY Acad Med 1947;23:207-220 [35] Veall N, Mollison PL. The rate of red cell exchange in replacement transfusion. Lancet 1950;2:792-797. [36] Hsia DY, Allen FH, Gellis SS, Diamond LK. Erythroblastosis fetalis VII. Studies of serum bilirubin in relation to kernicterus. N Engl J Med 1952;247:668-671. [37] AAP Subcommittee on Hyperbilirubinemia, American Academy of Pediatrics. Clinical Practice Guideline: Management of hyperbilirubinemia in the newborn infant 35 or more weeks of gestation. Pediatrics 2004;114:297-316. [38] A National Framework for Healthcare Quality Measurement and Reporting: A Consensus Report. National Quality Forum. Available at http://www.qualityforum.org [39] Maisels MJ, Watchko JF. Treatment of jaundice in low birthweight infants.Arch Dis Child Fetal Neonatal Ed. 2003;88:F459-463. Watchko JF, Maisels MJ. Jaundice in low birthweight infants: pathobiology and outcome. Arch Dis Child Fetal Neonatal Ed. 2003;88:F455-458. [40] Patra K, Storfer-Isser A, Siner B, Moore J, Hack M. Adverse events associated with neonatal exchange transfusion in the 1990s. J Pediatr. 2004;144:626-631. [41] Technical Manual Committee, Vengelen-Tyler V, Chair and Editor, American Association of Blood Banks. Neonatal and pediatric transfusion practice, Technical Manual of the American Association of Blood Banks, Bethesda, MD, 1999; 513-530. [42] Watchko JF. Exchange transfusion, in Maisels MJ, Watchko JF, eds. Neonatal Jaundice, Harwood Academic Publishers, Amsterdam, 2000;169-176. [43] Jackson JC. Adverse events associated with exchange transfusion in healthy and ill newborns. Pediatrics. 1997;99:E7.

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[44] Chima RS, Johnson LH, Bhutani VK. Evaluation of adverse events due to exchange transfusions in term and near-term newborns. Pediatr Res 2001;49:324 [Abstract].

In: New Developments in Blood Transfusion Research ISBN 1-59454-962-1 Editor: Brian R. Peterson, pp. 15-34 © 2006 Nova Science Publishers, Inc.

Chapter II

HEALTH ECONOMICS RESEARCH ON BLOOD TRANSFUSION SAFETY MEASURES -AN INTRODUCTORY PRIMER Ulf Staginnus∗ Managing Director, European Health Economics

ABSTRACT The risk of transfusion-transmitted infectious disease has been decreased significantly over the past decades through the frequent launches of new blood safety technologies. Previously introduced technologies were designed to screen donated blood for potential contamination thereby exposing blood recipients to a “window period”, determined by the sensitivity of the tests, with the consequence that a residual risk for contamination could not be entirely eliminated. Currently known viral pathogens, bacteria as well as migrating (e.g. SARS) and newly emerging viruses (e.g. Asian flu) therefore continue to threaten the safety of the worldwide blood supply. Latest blood safety technologies such as pathogen inactivation are being designed and clinically tested to overcome the weaknesses of screening systems and to preventively avoid the residual and potential future risks of blood transfusion transmitted disease. With ever tighter healthcare budgets and economic constrains in all health care systems, economic- combined with riskbenefit considerations of new technologies gain broader importance in adoption and reimbursement decision-making. Their economic value must be carefully evaluated before a widespread adoption should be recommended. In this chapter, the health economic aspects of transfusion safety measures and their risk/benefit relations will be discussed. Latest research on the cost-effectiveness of a newly developed pathogen inactivation technology and the impact on modern blood banking and transfusion medicine will be reviewed to provide recommendations for future research and to support ∗

Correspondence concerning this article should be addressed to Ulf Staginnus, Managing Director, European Health Economics - Spain. Paseo de los Parques 27, 1B; 28109 Alcobendas, Madrid; Tel/Fax: +34 91 650 9125; Mobile: +34 638 029 982 Email: [email protected] www.healtheconomics.es

Ulf Staginnus

16 health policy decision-making.

Keywords: blood safety, economics, cost-effectiveness, risk-benefit

INTRODUCTION Blood transfusion has revolutionized modern medicine. By maintaining blood volume, replacing deficient blood components and improving oxygen transportation, transfusion has expanded the boundaries of modern medicine, allowing many crucial surgical procedures, organ transplants, and cancer therapies to be performed. As a result, blood transfusions save many lives every year. Blood transfusions, however, are not risk-free. Despite significant improvements in safety measures, blood transfusions are still associated with a residual risk of infection by various pathogens many of which are serious and life threatening. New safety measures are required to address these existing needs. The economic value of healthcare products, such as that improving blood safety, is of increasing concern as healthcare systems struggle to deliver the highest quality services within their budgetary constraints.[1] As a result, governments and other decision-making bodies increasingly require formal health technology assessments that evaluate the therapeutic as well as economic value of new interventions, such as improvements in transfusion safety, as a condition of market access and reimbursement. Health economics is a discipline that is commonly concerned with the calculation of the added clinical and quality-of-life value of making a specific incremental financial investment. That is, if a certain sum of money is spent in a particular area, what will be the return in terms of improved health outcomes, thereby balancing cost, benefit, and risk considerations? This cost-to-benefit relationship is typically measured using a cost-effectiveness ratio, such as life year (LY) or quality-adjusted life year (QALY) saved per cost incurred. The lower the costeffectiveness ratio (e.g. the lower the cost per ‘unit’ of health gained), the more ‘costeffective’ the intervention is thought to be. Preventive measures such as improved blood screening and testing typically have higher and, therefore, less favorable cost-effectiveness ratios than other health interventions. This high cost-effectiveness ratio occurs because relatively few recipients gain a perceived benefit from the intervention, driving the average benefit down. Higher cost-effectiveness thresholds may apply when evaluating their economic benefit, however, as preventive measures focus on preventing injuries and deaths before they occur. Cost-effectiveness alone may not be a sufficient concept in the medical device area where technologies are often implemented on a center by center basis offering individual benefits and financial incentives depending on the organizational structure and processes used in the respective institution. More traditional cost impact studies on an account level might be more appropriate to accurately reflect the value of introducing novel blood safety devices and procedures. Apart from economic constrains, risk/benefit relations is an important dimension linked to the economic value of safety measures in the area of transfusion medicine. The risk of becoming infected while receiving blood components is nowadays low; therefore any new

Health Economics Research on Blood Transfusion Safety Measures

17

technology should be carefully evaluated for its risk/benefit profile in order to ensure that technologies with the highest net benefit to patients are implemented. In this chapter, I introduce the concepts of health economics research commonly used in the area of blood transfusion safety, review the cost-effectiveness of various blood safety interventions and put those into context using an example of a newly developed and - in several markets - already approved and utilized pathogen inactivation technology.

TRANSFUSION SAFETY – UNMET NEEDS The risk of disease transmitted by blood transfusion has generally been controlled, still breakthrough infections occur since blood is not tested for many potentially hazardous pathogens.[2] New and old infectious diseases emerge frequently and every so often spread rapidly over the globe, in numerous occasions the medium of transmission being the transfusion of blood components.[3]

Viral Infections Although the risk of viral infection has been enormously reduced, it is still a hazard confronting transfusion recipients (table 1). Nucleic acid testing has enhanced the ability to detect HCV and HIV in recently infected donors. The pre-antibody seroconversion window periods for HIV and HCV have been reduced from 22 to 12 days and from 70 days to 10–14 days, respectively.[4] While these advances have reduced the risks of viral infection, the risk has not been eliminated. Additional interventions are needed to eliminate the detection step of screening, and focus instead on directly eliminating the pathogen. Furthermore, these measures can also address increasing concerns about new viral strains.[5] Table 1. Residual risk of viral infections due to blood transfusion. Country

China

Rates of viral contamination 1/x (post NAT)

Reference

HIV

HCV

HBV

HTLV

50.000 – 1.000.000

5.000 -

5.000 –

n.a.

[6,7,8]

50.000

50.000

Germany

10.753.000

1.157.000

1.582.000

n.a.

[9]

Japan

2.669.000

348.000

52.000

n.a.

[10,11]

Spain

1.420.000

237.000

177.000

250.000

[12]

USA

2.135.000

1.935.000

205.000

2.993.000

[13]

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Ulf Staginnus

Bacterial Infections Bacterial infections, though usually attracting less attention than viral infections, are the most common microbiological cause of transfusion-associated morbidity and mortality.[14] They are both under-recognized and under-reported.[18] Bacterial contamination, either directly from a donor or from an external source, is most common in platelets. This occurs because platelets are stored for a maximum of 5 days at 22 °C, a temperature that facilitates bacterial growth. As a result, bacterial infections are more common among patients receiving platelets than among those receiving red blood cells, which are stored at 4 °C. To decrease the risk of bacterial contamination, blood can either be stored at lower temperatures or for shorter periods of time. However, platelet storage at lower temperatures is not possible because of the irreversible damage that occurs to microtubules. This damage essentially prevents the in vivo ‘survival’ of platelets. Moreover, storage times have already been reduced from 7 to 5 days based on study results documenting that bacteria can rapidly grow under these conditions.[16] While further decreases in storage time may decrease bacterial infections, they will also adversely limit and impact the blood supply. New interventions such as the inactivation of bacterial pathogens in blood could address this issue and reduce the current number of bacterial infections, allowing the extension of platelet storage to more than 5 days, and also helping to alleviate shortages currently experienced by some blood banks. Recent studies have estimated the risk of sepsis infection in apheresis platelets, ranging from 1:19,500 (USA) [17] to 1:31,500 (France) [18] units transfused. According to the UK SHOT report, a blood transfusion surveilence, bacterial contaminations constituted 58% of documented transfusion transmitted disease betwen 1995 and 2003.[19] Overall, as many as 1 in 1.000 to 3.000 platelet units are contaminated with bacteria in the US.[20] Recent European data on systematic bacterial cultures for more than 130.000 platelet components indicated the infectivity rates is about 7 in cases per 1000 components.[21,22] These rates of contamination are much greater than many transfusion-associated viral infections. Furthermore, mortality associated with sepsis infection is high, estimated at 14–26%.[23] Red blood cell units (allogenic and autologous) and plasma products have also been associated with bacterial infections, though at lower levels than that of platelets.

Protozoan Infections Blood screening and testing procedures focus mainly on prevention of the transmission of Plasmodium sp., the aetiological agent causing Malaria. Each year, 100 cases of malaria are reported in the USA, with approximately 3 cases related to infected blood product exposure.[24] Few safety measures are directed towards preventing infections with the pathogens responsible for diseases such as Chagas disease or Babesiosis. Estimates from the US indicate that 1 in 25.000 blood donors is infected with T. cruzi, the parasite responsible for Chagas disease, an increasing trend to migrations of the Hispanic population from Latin America to the US and Europe. Seroprevalence studies, although limited suggest that rates are in the range of 0,3% to 4,3% of US donors in epidemic areas.[25] New safety measures to protect the blood supply from these and other serious protozoan infections are needed. A task

Health Economics Research on Blood Transfusion Safety Measures

19

group, assembled by the US Assistant Secretary for Health and Surgeon General, found that restrictions on donations from some potential donors may contribute to blood shortages. Recently, stricter donor restrictions were put in place in the USA to protect against the possible transmission of new variant Creutzfeld-Jakob disease (nvCJD) infections. Specifically, people who resided in or travelled to the UK for a total of 3 months or more between 1980 and 1996 were prohibited from donating blood and plasma. These additional donor restrictions may exacerbate already existing blood supply shortages. New blood safety measures that pre-emptively inactivate pathogens would be a vital strategy [26] to battle the mutational capabilities of infectious agents and perhaps allow relaxation in donor restrictions to ensure sufficient blood supply.

Emerging and Migrating Pathogens Current safety measures do not guard against infections from newly emerging bloodtransmitted agents that have long incubation periods or which remain dormant for long periods of time.[27] Already, novel hepatitis agents have been identified; patients with acute post transfusion hepatitis have tested negative for all known hepatitis agents.[28] Thus, at least one as yet unknown hepatitis virus exists and can be transmitted by blood transfusion. Scientists have identified transfusion-transmitted virus (TTV) and the SEN-V virus as candidates possibly responsible for these infections. While both have been shown to be transmittable by transfusion, neither has yet been demonstrated as a causative agent of nonA–E hepatitis. Furthermore increased international air travel increases the risk of spreading viruses such as SARS via the blood transfusions.[29] The recent West-Nile virus infections due to blood transfusions (risk of a donation contaminated with West Nile in the US ranges from 1:1.000 and 1:6.000 during summer) have document the risk of emerging pathogens [30] and the difficulty to develop additional safety layers in form of testing to safeguard the bloodsupply. New safety measures that prevent these risks are required.[34,2]

HEALTH ECONOMICS OF BLOOD TRANSFUSION SAFETY MEASURES The risk of complications and the negative publicity after blood transfusion is a major concern to the general public and health policy makers. The perception of these risks and potential judicial consequences cause decision makers to favor implementation of procedures to further improve blood transfusion safety. Therefore, the goal in blood donation and transfusion medicine has primarily been to maximize safety. Major progress has been made during the last decade in the safety of allogeneic blood products, such as plasma products, platelets and red cells. A number of approaches have been utilized, including extended testing for viruses and bacteria, treatment of blood products (e.g. leucoreduction) and strategies to limit the number of donors to which a recipient is exposed. This latter category includes reduction in the use of allogeneic blood transfusion. These conservation strategies comprise autologous transfusion, use of blood growth factors

20

Ulf Staginnus

(erythropoietin), perioperative cell salvage, artificial blood and the development of guidelines for minimal use of blood transfusion.[31] From a health economics perspective it is now relevant to consider which technologies that would offer further increase of transfusion safety thereby addressing the unmet needs outlined above is worthwhile to be implemented (i.e. whether financial benefits of averted transfusion complications are worth the costs).

Elements of an Economic Evaluation All economic studies investigate the balance between inputs (the consumption of resources) and outcomes (improvements in the state of health of individuals and/or society). The Input Although the unit price of a blood safety procedure is often a prime factor in decisionmaking, economic outcomes research provides a more comprehensive interpretation of cost. This is accomplished by determining the overall cost of a given diagnostic or therapeutic process from the initiation of diagnosis until a final outcome is achieved. The various types of costs can be grouped under the following categories: direct medical costs; direct nonmedical costs; and indirect costs. Direct Medical Costs Direct medical costs are defined as those resources used by the provider in the delivery of medical care. As an example, direct medical costs for a blood bank include: medical stuff time for personnel; screening test and procedures; supplies; use of other equipment etc. These costs can be directly related to the care of patients. Other costs of operating a blood bank include plant maintenance and repairs, utilities, telephone, accounting, legal fees, insurance, taxes, real estate costs, and interest expense. In general, most economic studies do not factor general operating costs into the dollar value assigned to the cost of resources expended for a given medical intervention. Direct Non-Medical Costs The economic literature generally defines direct nonmedical costs as out-of-pocket expenses paid by the patient for items outside the healthcare sector. This category includes such costs as: (1) travel to and from the hospital, clinic, or doctor’s office; (2) travel and lodging for family members who live elsewhere; (3) domestic help or home nursing services; (4) insurance co-payments and premiums; and (5) the treatment not covered by third-party payers. Although these costs are generally classified as “nonmedical” to the patient they are real and often substantial costs of medical care. What makes them “non-medical” is that they are not costs incurred by the healthcare provider, and are somewhat difficult to measure. These costs are however by and large of lesser relevance for evaluations of blood safety technologies.

Health Economics Research on Blood Transfusion Safety Measures

21

Indirect Costs One definition of indirect costs is the overall economic impact of illness on the patient’s life. These include: (1) loss of earnings due to temporary, partial, or permanent disability; (2) unpaid assistance by family members in providing home healthcare; and (3) loss of income to family members who forfeit paid employment in order to remain at home and care for the patient. Like direct non-medical costs, indirect costs are real to the patient, but abstract to the provider—but may impact the provider’s direct medical costs. For example, patients who cannot earn income may not be able to pay their bills—including medical bills. Economic hardship may result in poor compliance with drug therapies as patients reduce doses or fail to refill prescriptions in order to save money. The medical provider may have to bear the additional costs of managing complications. Economic destitution may also result in missed follow-up appointments leading to the same types of problems for providers as described previously with direct non-medical costs. The Output: Consequences and Outcomes Final states or outcomes can be negative: • • • • •

death; disability (patient is permanently disabled and unable to return to work or school, perform household chores, etc.); discomfort (patient is in constant state of moderate to high level of hurt); dissatisfaction (patient is not satisfied with the course of treatment or services provided); and disease (patient’s condition is not being controlled resulting in frequent relapses, rehospitalization, and expenditure of additional resources).

There are also positive outcomes: • • • •

patient is cured; patient is able to resume normal functions; improved or satisfactory quality of life; or patient’s medical condition is successfully managed or stabilized by continued drug therapy.

The use of outcomes research represents an important advance in medical economic analysis because of the relationship between the final state and overall cost-effectiveness. If one can demonstrate that a product will achieve cost-effective positive outcomes, one will increase the chances of making the technology available and reimbursed.

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Economic Evaluation Methodology The most common methods employed by health economists are classical research designs such as cost of illness, cost–benefit, cost-effectiveness, cost–utility, costminimization and cost-of-illness analyses.[32] Cost-of-Illness Studies In the economic literature one will also find references to “burden of illness”. Definitions vary, but generally “cost-of illness” refers to all the costs, as they are borne by society. The cost-of-illness to society is reflected by such factors as loss of productivity in the work force and loss of income by the patient, which results in the loss of tax revenues and inability to purchase the goods and services that drive the economy. The important point is that everyone in society bears the cost healthcare providers, patients, third-party payers, and business and industry. An example for the area of transfusion medicine would be to evaluate the cost of illness due to HIV transmission via blood transfusion in sub-Saharan Africa. Cost-Minimization Analyses (CMA) Cost-minimization analysis is concerned with comparing the costs of different treatment modes, which produce the same result. For example, this form of analysis could be used to compare the cost of two viral screening programs. Both have the same outcome in terms of the sensitivity and specificity of the test, but the first program might require more logistical changes or cosumes additional resources. Given these two alternatives, the search would be for the least costly treatment. When the cost of two interventions is being compared, costminimization analysis often assumes they lead to the identical health outcome. Studies of this nature should report evidence to support the contention that outcome differences are nonexistent or trivial in nature. In most cases, however, the issues are more than one of solely cost. It is rarely the case where two therapies having the same indication produce identical health outcomes in every respect. Cost–Benefit Analyses (CBA) As applied to healthcare, cost–benefit analysis (CBA) measures all costs and benefits of competing therapies in terms of monetary units. Generally, a ratio of the discounted value of benefits to costs (the present value of both) is calculated for each competing therapy. The ratios for each of the competing therapies and for competing programs (e.g., intensive care unit versus new diagnostic equipment) can be readily compared. CBA has the shortcoming of requiring the assignment of a dollar value to life and to health improvements including quality of life variables. This presents equal benefit issues as well as substantial measurement problems. CBA, for these reasons, has not been widely used in recent years for evaluating of blood safety measures although can be found in older literature.[33] Cost-Effectiveness Analyses (CEA) a Cost–Utility Analyses (CUA) The relationship of cost to benefit is typically measured with a cost-effectiveness ratio, such as LY saved per cost incurred. The lower the cost-effectiveness ratio (e.g., the lower the cost per unit of health gained), the better is the value of the intervention. Cost-effectiveness

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analysis is used to allow comprehensive comparisons between alternative medical technologies (e.g., NAT testing versus pathogen inactivation), combining clinical effectiveness and cost. Cost–utility analysis as a form of cost-effectiveness analysis compares the added costs of therapy with the number of quality-adjusted life years gained. The quality adjustment weight is a utility value, which can be measured as part of clinical trials or independently. The advantage of cost–utility analysis is that therapies, which produce different or multiple results can be compared. Cost–utility analysis is an improvement over cost effectiveness analysis because it can measure the effects of multiple outcomes (such as the impact of blood safety measures on both morbidity and mortality). In cost–utility analysis reductions in mortality and morbidity are combined in a single index. The most used index is the quality adjusted life years. QALYs combine changes in quantity and quality of life (QoL) into one composite measure that is independent of program or disease. The quality adjustment factors (or utilities) are weights ranging from 0 to 1 (1 = optimal health, 0 = health state judged equivalent to death). They should reflect aggregated preferences of individuals for the outcomes. The factors have been measured directly on patients or the general public. Cost-Consequence and Cost-Impact Analysis The economic impact of introducing a new technology can be expressed in a so-called cost-consequence analysis. This analysis includes the initial cost of implementing the technology as well as potential downstream savings associated with use if adopted. By presenting the relevant parameters and their cost, decision makers and payers can estimate the net impact of an intervention, thereby facilitating assessment of a health technology intervention without necessarily combining all cost and outcome categories into a single ratio of cost-effectiveness, such as cost per life-year (LY) gained or cost per quality-adjusted lifeyear (QALY) gained as used in traditional cost-effectiveness analysis. In a cost-consequence analysis, the relevant parameters must be identified and cost values assigned. This type of analysis is especially useful for the evaluation of the cost-impact of a new blood safety technology from operators (e.g. individual blood bank) perspective.[34] The Importance of the Perspective The choice of the perspective is the single most important point in the analysis, as it determines which costs should be included and how they should be valued. The answer to the question whether or not a new blood safety technology is cost-effective may depend on who is asking it. Patient, society in general or third party payer may reach different judgments about specific costs. In the comprehensive societal perspective, all costs and benefits should be identified, regardless of who incurs the costs and who receives the benefits. Hospitalization costs, effectiveness parameters, and prices as well as incidence of transfusion-transmitted disease are main determinants in the cost-effectiveness equation. Preferably the societal perspective should be used since it captures the cost and consequences that are relevant to different groups however separate reporting of results with and without indirect cost to allow for comparisons should be the gold standard.[35]

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COST-EFFECTIVENESS OF BLOOD SAFETY INTERVENTIONS Currently, a variety of interventions are available to improve the safety of blood transfusions. Taken together, their use and cost-effectiveness reflect society’s willingness to allocate healthcare resources to achieve greater blood safety. It is interesting to observe that blood safety seems to be valued more highly than other types of health interventions. This may reflect the history and potential threat of HIV infection and the ability of these interventions to prevent infections by these agents. In a review of cost-utility studies on clinical preventive services in the USA, blood safety measures were found to have high costeffectiveness ratios relative to other healthcare interventions. For example, interventions that screen blood donors had a median cost/QALY of $355,000.– and ranged from ‘cost-saving’ to $8,700,000.–/QALY. Interventions that involved autologous blood donation had a median cost/QALY of $730,000.– and ranged from $46,000.– to $27,000,000.–/QALY.[36] Together, these results demonstrated that the distribution of blood safety interventions currently available is heavily dominated by interventions with net cost of up to several million $US per. QALY gained. These include both testing procedures for transfusion donations (such as viral antibody screening procedures for HIV, HBV and HCV) and medical procedures (such as autologous blood transfusion in elective surgery, solvent detergent treatment of plasma and epoietin therapy).[37,42] Before we can analyze the implications of potential future blood safety technologies, we need to understand what cost-effectiveness actually means and how the data that are being presented to the public can be interpreted. Cost-effectiveness ratios can be viewed in two ways. First, if national health policy makers have established a certain threshold of costeffectiveness, new technologies could be evaluated against that cutoff point. Several countries have determined “official” criteria for cost-effectiveness. For example, in the United States interventions that yield a cost-effectiveness ratio of more than US$50,000 per QALY gained are considered unfavorable and should ideally not be implemented.[38] The United Kingdom National Institute of Clinical Excellence uses a similar threshold of approximately £30,000 per QALY gained.[39] According to these “decision rules,” none of the recently used blood safety measures should have been implemented because their cost-effectiveness ratios are much higher than the designated thresholds. Cost-effectiveness ratios for new safety procedures such as DNA nucleic acid testing are high for a variety of reasons. Every unit of donated blood is tested, and as a result, bears the additional cost of the test. In addition, because recent safety measures have already decreased infection risks, few units actually contain pathogens. As a result, the cost-effectiveness of novel safety measures is driven down. This is particular true in recent improvements in blood safety, some even contend that little additional value exists in adopting additional safety measures such as HBV DNA NAT in today’s cost cautious healthcare environment.[40] However, while the risks have been greatly reduced, transfusion recipients continue to face the risks of both known and unknown bacterial and viral infections, signifying the need for additional safety measures. It is evident that the general public values improvements in blood safety, even when they are costly. Society’s motivation to invest significant amounts of resources to meet this goal is obvious by looking at the cost-effectiveness ratios of commonly

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accepted blood safety precautions. While regulatory and societal decisions may take costeffectiveness into account when evaluating new testing procedures, they are also likely to be influenced strongly by a concern with patients’ overall safety. This is especially true in cases where safety improvements are marginal at best (i.e. p24 HIV-1 antigen test). Moreover, though risks of transfusion-transmitted infections are diminutive, safety is still dominant in the policy decision-making process in many countries.[41] This suggests that higher costeffectiveness thresholds apply when evaluating new technologies that improve blood safety. In fact, a recent study similarly concluded that while the cost-effectiveness ratio of minipool nucleic acid testing was high ($ 2,900,000.–/LY gained), it was not necessarily unreasonable given the cost-effectiveness of other blood safety interventions and society’s preference to further reduce transfusion-related risks.[42] Thus, given these factors, it seems that a more elevated threshold value than the subjective benchmark of $50,000.–/QALY may better represent society’s preference for blood safety improvements and should be used to evaluate innovative interventions. Costeffectiveness is a relative concept and should be applied to in-group comparisons. In other words, new blood safety technologies should be evaluated against technologies in the same area (e.g., NAT screening, pathogen inactivation treatment). Given the zero risk policy for blood safety in most countries, transfusion medicine is in a situation in which additional benefits gained for protection against selected pathogens are marginal and come at a cost leading to higher cost-effectiveness ratios than other medical interventions.

NEW BLOOD SAFETY TECHNOLOGIES Bacterial Screening Systems Among the screening systems, apart from improved NAT tests for viral contamination (e.g. single or minipool NAT) bacterial detection systems (e.g. BacT/ALERT,[43] Pall eBDS,[44] Scansystem [45]) have been widely introduced into many blood banking operations world wide. Bacterial detection systems are being implemented to greatly contribute towards the reduction of bacterial sepsis, a serious consequence often arising when bacteria are transmited to recipients of platelet transfusions. Bacterial screening has been shown to be an effective measure against bacterial infections, however these systems are less successful at preventing transfusion of many infected units due to increased time required for detection, and demonstrated a significant rate of false-positive and false-negative results.[30] While the former increases product loss thereby reducing the economic efficiency of the detection system, the later can lead to serious consequences when tainted units are already transfused, or at the least require an intensive and very costly recall route in case a positive culture has finally been confirmed in the lab but the platelet unit has already left the blood bank. In that case life threatening bacterial sepsis may occur despite negative screening test results.[46] False-positive results may be caused when slow-growing bacterial species do not reach the systems sensitivity threshold within the incubation period.[47]

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Cost-Effectiveness of Bacterial Screening Systems Due to their comparatively moderate additional cost ($20-25 per single donor platelet in the US) [48] and relatively high sensitivity and specifity, bacterial testing systems are expected to be cost-effective screening methods. Until today, only one published study of the Bact/ALERT has been presented. In a model for Coronary Artery Bypass Graft patients in the Netherlands, the screening of platelets for bacterial contamination resulted in a costeffectiveness ratio of $14.000 to $62.000 per life-year gained.[49]. According to our own unpublished analysis [50], taking into account important additional variables that reflect the various issues of bacterial detection systems such as recent data [29] on rates of falsepositive, false-negative test results,[30] the cost of timely and untimely recall, the cost of false positive (e.g. blood waste, platelet product cost, retest cost) as well as platelet loss due to sampling, the cost-effectiveness of bacterial testing systems in random donor platelets appears to be more in the region of $500.000 per QALY (10 year old pediatric leukemia patient) to $1.450.000 per QALY (Non-Hodgkin’s lymphoma patient, 50 years of age) gained. Less favorable results can be expected in single donor platelets. Bacterial testing systems seem therefore not to demonstrate superior cost-effectiveness than currently employed safety measures discussed before.

Pathogen Inactivation Systems Various pathogen inactivation technologies are currently under development. The most advanced and already implemented into routine praxis in various European countries [51] through CE (Conformité Européne) Mark registration is the Intercept Blood System (IBS) for platelets, using photochemical decontamination with psoralen (S59) that has been shown to inactivate a broad spectrum of pathogens [52]. Similar systems for plasma and red blood cells are currently in various development phases. The potential economic impact of pathogen inactivation systems can be evaluated in two ways. First, traditional cost-effectiveness analysis can be conducted to compare its costeffectiveness to other safety measures and secondly, cost-consequence and cost impact analysis on an individual blood banking level maybe more appropriate since implementation and budgeting can be decided in many countries directly by the blood bank or hospital itself. Such analysis reflects more the actual situation in each center and allows for a detailed evaluation of the cost offset potential in the respective institution. We will therefore discuss both approaches with specific examples.

Cost-Effectiveness of Pathogen Inactivation Systems As with any new technology pathogen inactivation bears an initial investment and cost to the healthcare system. Many authors have listed the further increase of the cost of blood products due to the introduction of pathogen inactivation as a major concern and hurdle for its adoption among considerations about its risk-benefit profile.[53] An assessment of cost-

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effectiveness and risk-benefit is therefore essential to implement blood safety technologies that offer the greatest health benefit to recipients. Pathogen inactivation is intended to proactively improve blood safety hence certain procedures or tests (e.g. gamma irradiation, bacterial screening) that potentially can be omitted would greatly contribute to improve the cost-effectiveness of such a technology without compromising patients safety.[54] However, this seems to still be a thorny concept since it affects blood banking logistics but more importantly requires a shift in re-thinking the screening and layering of safety technologies pattern. Various cost-effectiveness analyses have been conducted on the Intercept blood system for platelets (IBS) in several countries, taking the above considerations into account and examining cost-effectiveness under different scenarios and with extensive sensitivity analysis, which have been published elsewhere.[55,56,57,58,59] The main benefit of pathogen inactivation and its relation can be derived from the prevention of bacterial infection and associated mortality, the prevention of migrating and/or emerging virus as well as from benefits derived from replacing current procedures and test, or even potential future tests (such as West Nile screening). Cost-effectiveness ratios of pathogen inactivation are high as expected and compared to general accepted threshold for cost-effectiveness, however the cost-benefit relation of IBS is by far more favorable as compared to most other blood safety measures currently in place (plasma inactivation, NAT testing) due to the combination of preventive effects (i.e. inactivation of various types of pathogens) in one technology. Cost-effectiveness results from various geographical regions are depicted in table 2. Table 2. Cost-effectiveness of Intercept blood system for platlets. Cost-effectiveness of IBS in various countries and subpopulations in Cost (USD 2005) per QALY (Baseline analysis) Populations receiving platelet transfusions ALL CABG 60 years NHL 50 years pediatric 10 years Platelets produced as: Reference Country RDP SDP RDP SDP RDP SDP China 4.697 N.A. 18.990 N.A. 24.500 N.A. [59] Spain 474.228 1.327.714 856.873 2.061.537 1.445.518 3.441.819 [57] Japan N.A. 900.409 N.A. 2.391.996 N.A. 3.940.882 [56] USA N.A. 1.308.833 N.A. 2.664.880 N.A. 4.451.650 [55]

ALL = Acute Lymphatic Leukemia CABG = Coronary Artery Bypass Graft NHL = Non Hodgkin’s Lymphoma RDP = Random Donor Platelet SDP = Single Donor Platelet QALY = Quality Adjusted Life Year

Cost-effectiveness is generally more favorable in random donor platelets as compared to single donor (aphaeresis) platelets due to the fact that pooling of platelet units from various donors bears an increased risk of transfusion transmitted disease which in turn when

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prevented yield a better economic profile. These data are baseline data without incorporating any additional benefits such as avoided procedures, extended platelet shelf-life and/or the potential prevention of an emerging virus. Inclusion of the full potential of benefits significantly improved the cost-effectiveness of IBS and in some patient population (e.g. pediatrics) would actually lead to overall net savings.[64] When comparing pathogen inactivation technology with IBS to viral screening methods as well as to the cost-effectiveness of lately developed bacterial screening tests, healthcare budgets would be more economically efficient allocated by implementation of pathogen inactivation due to the technology’s more or at least equal overall economic profile. Moreover, looking at an example of China, where in many rural areas the risk of transfusion transmitted disease is many fold higher than in Western Europe, Japan, or the US, hence introduction of a comprehensive pathogen inactivation method would be highly cost-effective and cost saving solution under the circumstances.[8,67] Considering the increasing air travel and population migrations, pathogen inactivation maybe an essential opportunity to prevent spread of deadly diseases from areas where standards of blood safety are still lacking the levels of sophistication from those in the developed world.

Cost-Consequence and Cost Impact Analysis of Pathogen Inactivation Systems The economic value of pathogen inactivation not only depends on the products ability to inactivate pathogens and its additional procedure cost but as well as to how the technology is applied. The problem in blood banking is that any procedure that has been adopted to further enhance safety has always been an add-on to already existing technologies leading to a layering of safety measures that in the end only will diminish the safety returns while increasing cost enormously as it was the case with advancing the NAT technologies.[60] Inasmuch as prevalence of viral transmission decreases due to advanced testing technologies, cost-effectiveness of such tests becomes more unfavorable because the increment of prevented infections becomes smaller with each test being adapted however still all blood units need to be tested thereby increasing the marginal cost and driving the marginal benefit down. Furthermore these tests usually focus on a single pathogen, which explains the high and unfavorable cost-effectiveness ratios of screening methods in transfusion medicine. This will become partially true for pathogen inactivation as well, especially in the developed world. In light of this, analyses of the direct cost impact for a blood center to adopt pathogen inactivation technology may become more relevant than a traditional cost-effectiveness analysis that might be less relevant for an individual implementation decision. If, for example, by introduction of pathogen inactivation other procedures and test can be eliminated, direct cost of the adaptation of the technology would be mitigated. Immediate cost offset potentials that could be realized with pathogen inactivation technology for platelets would be the cost otherwise associated with bacterial testing, CMV testing, and gamma irradiation of conventional platelet products. While each of these procedures addresses only on single recognized risk associated with platelet transfusion, pathogen inactivation has been shown to be effective against all three [61,62,63] and therefore each of

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the three procedures maybe replaced by one single step thereby resulting in cost-savings for the blood bank.[56] Additional benefits such as prolonged shelf-life and thus less complex inventory management will add to the savings potential.[64] In our cost consequence and subsequent budget impact analysis for IBS implementation in Japan we calculated that in the most conservative scenario about 60% of the initial cost for pathogen inactivation would be offset leading resulting in a net budget increase for blood products of only 0.89%.[64] This conservative calculation did not yet include additional benefits such as shelf life extension and reduced transfusion reactions which would further reduce the net cost impact for the blood banking operation and hospital. More cost-impact studies should be conducted with data from real life experience with the technology in order to overcome hastily created ramifications about the overall economic impact of an innovative system in an environment of increasing budget constraints.

RISK-BENEFIT CONSIDERATIONS Every economic analysis is a function of competing risks: one hand of the balance the benefits in terms of risk avoidance (residual risk of pathogen transmission) due to the technology, on the other hand potential risks due to the usage or implementation of that technology. The overall economic value is than a combination of three dimensions: cost, benefits, and risks whereby the one should to adopt technologies with low cost, high benefits and the lowest risk. In other words, the net benefit of the various technologies should be compared. Since risk of currently known and tested viruses are minimal (often in the magnitude of a risk of being hit by lightening), at least in the Western world, questions arose whether such a novel pathogen inactivation technology would potentially add a new risk dimension itself simply by its application due to the chemical compound (amatosolan) used. Theoretical toxicity concerns for transfusion recipients have caused anxiety and intense discussions in the blood transfusion community. Any economic evaluation that has been conducted in the area of pathogen inactivation was performed under the assumption that there are no negative external effects with the usage of pathogen inactivation as demonstrated in animal toxicity models and later also in routine use, although long term follow-up data is not yet available.[65,66] Indeed, these are important and sound considerations since it cannot be meaningful to perhaps replace one risk while at the same time introducing another, although only theoretical at this point. As far as blood platelets are concerned, the greatest risks are infections due to bacterial contamination. Future data from implementation trials and routine use can be used for supplementary technology assessments. In the light of increased sensitivity towards innovative medical technologies, yet one must not forget that safety considerations should not be limited to the pathogen inactivation methods, but also to other systems. Screening procedures such as recently put into place against bacterial contamination pose their own risk to patients, e.g. by falsely reassuring the transfusion physician and patient about the safety of a platelet unit when actually perhaps false-negative units have been already transfused due to the technical limitations of the screening device. The Center for Disease Control and

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prevention in the US recently discussed this limitation of bacterial testing systems and recommended caution about the possibility of false-negative results, since two people have died after negative bacterial test results.[67] Thus, the risk of acquiring severe sepsis combined with the anxiety of the patient may be a factor of greater magnitude than the theoretical and so far non-proven risk of toxicity from pathogen inactivation with photochemical compounds. In the end of the day, only long term experience and data would tell as it is the case with any new innovative drug, procedure or device in medicine. Post marketing hemovigilance studies will be able to address those questions and should be conducted with caution and under heightened scrutiny to ensure patients’ safety at all times.

CONCLUSIONS The risk for transfusion-transmitted infections has been greatly reduced by the introduction of new blood safety measures. However, new safety interventions that can effectively reduce new and existing pathogen transfusion risks are still needed. Our society places a high value on preventing accidental deaths and injuries, as evidenced by the relatively high cost-effectiveness ratios of existing technologies. New transfusion safety measures should be evaluated using cost-effectiveness thresholds that are higher than those typically used by healthcare decision makers, but which reflect accurately the high value society places on reducing unintentional deaths and infections, related to blood transfusions. In addition, cost-effective analyses must be done accurately and comprehensively to fully appreciate the value of new blood safety measures. Economic studies need to be conducted to investigate the true economic impact of bacterial testing systems from a cost-effectiveness viewpoint as well as from the individual blood banking perspective. Specifically, given their preventive and comprehensive potential to deal with the challenge of multiple pathogens, future health economics research in optimizing blood transfusion safety should focus on cost-impact studies of pathogen inactivation methods in local blood centers with related potential of replacing current testing and in evaluating the technologies value of eliminating potentially redundant blood safety measures. Concurrently, decision makers should consider those other benefits of blood safety measures such as pathogen inactivation that are not so easily incorporated into cost-effectiveness analyses. These include benefits such as increased shelf life or the increase in productivity typically lost due to lethal virus infections such as HIV. These aspects, and the peace of mind that increased safety offers to all transfusion recipients, should also be considered when evaluating new blood safety interventions. According to our analyses in the various regions, realizing the full potential of pathogen inactivation technology reduces the additional cost to a rather small amount considering the extensive array of benefits that allow cost offsets elsewhere in the blood banking operating scheme. If the early European experience, that use of the IBS did not require collection of more platelet doses, is confirmed with broader practice, then the net cost impact of pathogen inactivation on health care resources is very reasonable. The application of pathogen inactivation may initiate a necessary future paradigm shift from testing to prevention, thereby harnessing additional long-term cost savings far beyond the immediate benefits of disease

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prevention. Development and utilization of pathogen inactivation systems may avoid implementation of multiple new screening tests, extend the shelf life of blood components, and broaden the donor base by avoiding travel-related donor exclusions. The main benefit however from such a technology comes from its potential to prevent emerging and migrating pathogens and from its ability to address multiple risks in one process.

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[35] Drummond M, McGuire A. Economic Evaluation in Health Care, Merging Theory with Practice. Oxford University Press 2001. [36] Yeh JM, Botteman M, Pashos CL, et al. Economics of Transfusion. Infus Ther Transfus Med. 2002;29:219-225. [37] Van Hulst M, Wolf JTM, Staginnus U, et al. Pharmaco-economics of blood transfusion safety: review of the available evidence. Vox Sanguinis 2002;83:146-155. [38] Winkelmayer WC, Weinstein MC, Mittleman MA, et al. Health economics evaluations: The special case of end-stage renal disease treatment. Med Dec Making 2002;22:417430. [39] www.nice.org.uk. [40] Busch MP. Should HBV DNA NAT replace HbsAG and/or anti-HBc screening of blood donors? Transfus Clin Biol. 2004;11:26-32. [41] Cyranoski D. Tainted transfusion leaves Japan scrambling for safer blood tests. Nat Med. 2004;10:217. [42] Marshall DA, Kleinman SH, Wong JB, et al. Cost-efectiveness of nucleic acid testing of volunteer blood donations for hepatitis B, hepatitis C and human immunodeficiency virus in the Unites States. Vox Sanguinis 2004;86(1):28-40. [43] www.industry.biomeriueux-usa.com/industry/food/bacy_alert3d/ [44] www.pall.com/datasheet_medical_28112.asp. [45] www.hemosystem.com. [46] Te Boekhorst PAW, Beckers EAM, Vermeij VH et al. Clinical significance of bacteriologic screening in platelet concentrates. Transfusion 2005;45:514-519. [47] Jacobs MR, Bajaksouzian S, Windau A, et al. Evaluation of the Scansystem method for detection of bacterially contaminated platelets. Transfusion 2005;45:256-259. [48] Benjamin RJ. Bacterial detection in platelet components and the rationale for pathogen inactivation: A blood center perspective. Journal of Clinical Apheresis 2005;20(2):117122. [49] Postma MJ, De Natris T, Smit Sibinga CTH et al. Pharmacoeconomics of bacterial screening of platelets. VII. European Congress of the International Society of Blood Transfusion 2001 (abstract). [50] Premor Associates. Data on file. New York 2005. [51] Janetzko K, Lin L, Eichler H, Mayaudon V, Flament J, Kluter H. Implementation of the Intercept Blood System for platelets into routine blood bank manufacturing procedures: evaluation of apheresis platelets. Vox Sang. 2004;86:239-245. [52] Wollowitz S. Targeting DNA and RNA in pathogens: mode of action of amotosalen HCl. Transfus Med Hemother. 2004;31(suppl 1):11-6. [53] AuBuchon JP. Pathogen reduction technologies: what are the concerns? Vox Sanguinis 2004;87(Suppl. 2):S84-S89. [54] Osselar JC. Doyen C. Sonet A et al, Routine use of platelet components with photochemical treatment (Intercept platelets): Impact on clinical outcomes and cost. Presented at American Society of Hematology (ASH), 46th Annual Meeting, December 4, 2004 - December 7, 2004.

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[55] Bell CE, Botteman MF, Gao X, et al. Cost-effectiveness of transfusion of platelet components prepared with pathogen inactivation treatment in the United States. Clin Ther. 2003;25:2464-2486. [56] Staginnus U, Corash L. Economics of pathogen inactivation technology for platelet concentrates in Japan. International Journal of Hematology 2004;80(4):317-324. [57] Villaescusa RG, Barallobre J, Staginnus U. Coste efectividad de las transfusiones de componentes plaquetarios preparados con tratamiento de inactivación de patógenos en Espana. Rev Esp Econ Salud 2003;2(2):46-54. [58] Moeremans K, Warie H, Annemans L. Assessment of the Economic Value of the INTERCEPT Blood System for Platelets in Belgium. Presented at American Society of Hematology (ASH), 46th Annual Meeting, December 4, 2004 - December 7, 2004. [59] Staginnus U. Russell S. Cost-effectiveness of transfusing blood platelets prepared with pathogen inactivation treatment in China. Value in Health 2005;8(3):282. [60] Schlenke P. Kirchner H. Corash L. Concerning Caspari et al: Pathogen inactivation of cellular blood products – More security for the patient or less? Transfus Med Hemother 2005;32:1-5. [61] Lin L. Inactivation of cytomegalovirus in platelet concentrates using Helinx technology. Semin Hematol 2001;38(suppl 11):27-33. [62] Grass JA, Hei DJ, Metchette K. Inactivation of leucocytes in platelet concentrates by psoralen plus UVA. Blood 1998;91:2180-2188. [63] Lin L, Dikeman R, Molini B, et al. Photechemical treatment of platlet concentrates with amotosalen and UVA inactivates a broad spectrum of bathogenic bacteria. Transfusion 2004;44:1496-1504. [64] Picker SM, Speer R, Gathof BS. Functional characteristics of buffy-coat PLTs photo chemically treated with amotosalen-HCI for pathogen inactivation. Transfusion 2004;44:320-329. [65] Dayan AD. The Science of Safety: Toxicological Review of Amotosalen HCl. Transfus Med Hemother. 2004;31(suppl 1):17-23. [66] Benoit Y, Van Haute I, Vandecruys E, et al. Safety and efficacy of pathogeninactivated platelets trasnfused in routine use to pediatric patients: An interim report. Presented at American Society of Hematology (ASH), 46th Annual Meeting, December 4, 2004 - December 7, 2004 [67] CDC. Fatal bacterial infections associated with platelet transfusions – Unites States 2004. MMWR weekly 2005;54(07):168-170.

In: New Developments in Blood Transfusion Research ISBN 1-59454-962-1 Editor: Brian R. Peterson, pp. 35-64 © 2006 Nova Science Publishers, Inc.

Chapter III

UNIQUE PATIENT IDENTIFICATION BARCODE SYSTEM FOR PREVENTION OF BLOOD TRANSFUSION ERRORS: A 6-YEAR EXPERIENCE IN A REGIONAL HOSPITAL IN HONG KONG Joyce Chan Chee Wun1 and Raymond Chu2 1

Department of Medicine Department of Clinical Pathology Pamela Youde Netersole Eastern Hospital, Hong Kong 2

ABSTRACT Background Acute haemolytic reaction arising from human error remains a leading transfusionassociated hazard. Measures to reduce such error continued to be a major issue in the 21st century.

Methods In 1998, the Hospital Transfusion Committee of a regional hospital in Hong Kong resorted to develop an electronic barcode system for verification of patient identity at critical points of transfusion process – blood sampling for pre-ttransfusion testing and blood administration. Known as the unique patient identification (UPI) system, a process specific system, it had to be used in conjunction with hospital’s standard transfusion procedures. Among the various choices of patient identity, the eight-digit Hospital Number, unique for each patient and each admission, imprinted on thr patient’s wristband and remained attached to the patient throughout hospital stay, was chosen. The UPI

Joyce Chan Chee Wun and Raymond Chu

36

device was hand-held, stand –alone machine with built-in scanner and printer, as well as an in-housed computer programme with two pathways of barcode verification. For both procedures, our patient identification policy mandated patient’s wristband as the starting point. Staff first scan patient’s wristband Hospital Number (prefix WB), then, for blood sampling, the Hospital Number (prefix HN) on patient’s Blood Request Form, or blood administration, the Hospital Number (prefix TN) on the blood unit assssigned to the patient. The second barcode scanning automatically channeled to the pathway of the desired procedure. Verification of correct patient identity was documented by automatic printing out of either a self-adhesive label bearing patient’s Hospital Number (prefix WN) by the device to be affixed onto the sample tube prior to blood sampling; or a special a self-adhesive label to be affixed onto the Blood Transfusion Record prior to blood administration.

Results This UPI system was implemented hospital-wide including wards operating theatres (except the Casuality Department and the outpatient clinics) in May 1999. The first generation device was a ready made one. Its use was terminated in 2003 due to mechanical and battery problems. The second generation device was tailor made with improvements in terms of ergonomics, battery, checking algorithm and data storage. Its implementation in July 2004 included one other regional hospital. From May 1999 to April 2005, for 75,000 blood sampling procedures and 51,000 units of blood administration, no transfusion errors were deteced, compared to 13 errors in blood sampling procedures recoreded from May 1995 to April 1999 when the second checker system was implemented. Monitoring process is in place for further system improvement and enhancement of transfusion safety.

BACKGROUND Manual Second Checker System Establishment of a Hospital Transfusion Committee (HTC) has been a requirement for all public hospitals with transfusion service in Hong Kong since the establishment of the Hong Kong Hospital Authority in 1990. As a regional hospital situated on the eastern part of the Hong Kong Island and equipped with an emergency service and 1600 beds, our hospital was officially opened in the fall of 1993 and a HTC was formed in early 1994. Among its many charged duties, a key function of the HTC is to minimize the risk of mistransfusion, i.e. giving the patient the incorrect unit of blood. In order to reduce human errors in patient identification and to ensure compliance with standard operating procedures, the HTC introduced a ‘second checker’ system, a dual witness verification of identification system for blood transfusion in May 1995. Two qualified persons were required to verify patient identity for blood sample collection and for blood administration. Implementation of such a labourintensive system was not without difficulty in an already manpower-tight busy hospital. Our hospital is the first, and up to now, one of the three hospitals in Hong Kong to have implemented such a system hospital-wide. Credits undoubtedly go to the high level of

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37

commitment of our Hospital Chief Executive to risk management, and to the beloved dedication of our nursing staff to high quality patient care. This in turn owes deeply to the heritage of the Christian spirit and tradition of the century-old Nethersole Hospital whose entire medical and nursing staff moved to our hospital after demolition of the Nethersole Hospital building.

Turning Point to Electronic Barcode System Monitoring the performance of the ‘second checker’ system by the HTC revealed reduction in transfusion incidents related to non-compliance to standard operating transfusion procedures. Circumvention of protocols however remained inevitable. Numerous factors, psychological and environmental, contributed to inattention and distraction and resulted in failure to follow procedure instructions. In 1997, the HTC proposed to resort to technologybased solutions to remove human factors from the patient identification process, believing that technologies would be of assistance in the repetitive tasks of blood sample collection and blood administration as they are not subject to most of the psychological or environmental factors that provoke human errors. This project was supported by the hospital management and the hospital Information Technology (IT) team. A task group with membership consisting of Chairman of the HTC, lead consultant for the transfusion medicine, representatives from the central nursing division, blood bank manager, front line medical and nursing staff, hospital management and IT representatives, was formed. Introducing a new system requires a comprehensive review of the available technologies as well as all the processes involved in blood transfusion. The barcode system is the oldest and the commonest system among all machine-readable automatic identification systems. Its use in healthcare was first described over 30 years ago in clinical laboratories and blood banks. In Hong Kong, barcode systems are widely used by the Hong Kong Hospital Authority. Examples include the Integrated Patient Administration System (IPAS) and the Laboratory Information System (LIS). This technology thus has demonstrated its ease for healthcare operators to use. A careful review of the hospital’s complex transfusion process showed two critical points where human errors are most likely to occur – the bedside blood sample collection for pre-transfusion compatibility testing, and the blood administration. Thus the same two points addressed by the manual ‘second checker’ system would be tackled by the barcode system. It is to be used in conjunction with, and not replacing the hospital’s standard transfusion procedures, and is to be a process specific application for blood transfusion, named as the Unique Patient Identification (UPI) system.

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Joyce Chan Chee Wun and Raymond Chu

METHODS I. Development of UPI System: 1st Generation UPI Device System Components

Unique Patient Identifier The foremost important task was for the task group to decide what should be used as a patient’s unique identifier. The final decision was to use a patient’s Hospital Number. Each patient on admission to a hospital under Hong Kong Hospital Authority is allocated a Hospital Number which is unique for each patient and each admission. This is an 8-digit number with a prefix HN. It is produced by the computerized IPAS of the hospital at the Admission Office and is printed out on a wristband label as well as on ‘3 x 6 cm’- sized selfadhesive labels known as the ‘gum label’. This unique Hospital Number on the wristband label appears in numeric form as well as in a linear D1 barcode format. Other relevant patient information on the wristband label such as the name, gender, date of birth, Hong Kong Identity number, date and time of admission, admitting department and ward are not accompanied by barcodes. The wristband remains attached to the patient throughout his/her hospital stay and is only removed upon his/her discharge from hospital. The ‘gum label’ contains two strips of linear barcodes, one for the patient’s unique Hospital Number and the other for the Hong Kong Identity number. All other information is without barcodes. This ‘gum label’ is used as a patient’s identity on the Blood Request Form, the Medication Administration Record, the Medical Progress Sheet, and other documents during a patient’s hospital stay.

Modification of Prefix of Hospital Number on Wristband In order to make the Hospital Number on patient’s wristband unique, i.e. such number would only appear on patient’s wristband and nowhere else, a small modification was made to the prefix of the Hospital Number on the wristband label. A computer was installed and linked to the IPAS to produce and print out a wristband label bearing a Hospital Number which is numerically the same as that on the ‘gum label’, but is prefixed with WB instead of HN.

Electronic Matching Device The electronic matching device chosen was a commercially available portable hand-held scan-and-print electronic device (PathFinder Ultra; Monarch UPN Alliance, Dayton, Ohio, United States). It weighed 1kg and was a stand-alone, battery-operated device with three major components – a laser barcode scanner for scanning and verifying the barcodes to be matched; a thermal label printer for printing out labels as documentation of matched results; and a display screen for conveying battery condition, error messages, time and date, etc.

Unique Patient Identification Barcode System for Prevention of Blood …

39

Bedside: Blood Sample Collection for Pre-transfusion Compatibility Testing Transferring Patient Identity from Wristband / Blood Request Form to Blood Sample Tube Verification and Documentation of Patient Identity with UPI device

System Use Medical staff first scans the Hospital Number barcode (prefix WB) on patient’s wristband, then the Hospital Number barcode (prefix HN) on the ‘gum label’ of the Blood Request Form (BRF). If the two barcodes do not match, an error message will appear on the display screen and the process of patient identification has to be repeated. If the two barcodes match, the device will print out a self-adhesive ‘UPI label’ bearing a barcoded Hospital Number (prefix HN) and date and time of blood sampling. This ‘UPI label’ is affixed onto the sample tube and blood sampling then starts. The sample tube bearing the ‘UPI label’ together with the completed Blood Request Form are sent to the blood bank. Wristband Barcode

Gum label Barcode (on BRF)

Tai-man Chan

Tai-man Chan

= WB12345678

HN12345678

Hand-held UPI device ↓ UPI barcode label

Sample Tube

⇒ HN12345678 date/time

Blood Bank: Issue of Blood Unit, Transferring Patient Identity from Blood Sample Tube and Blood Request Form to Blood Unit, Verification and Documentation of Patient Identity with LIS

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Blood bank staff first visually checks the information on the ‘UPI label’ of the blood sample tube against that on the ‘gum label’ of the Blood Request Form, and then uses the LIS to scan the two labels. Only if the two barcodes match, will further blood tests be processed. If a blood unit is to be issued, an LIS barcoded label is generated and affixed onto the blood unit allocated to the patient. This ‘LIS label’ contains the patient’s Hospital Number (prefix HN), in barcode and in numeric format, as well as patient’s other identity information.

LIS label

Blood unit

⇒

Bedside: Blood Administration, Matching Patient Identity on Blood Unit to Wristband, Verification and Documentation of Patient Identity with UPI device Wristband Barcode LIS Barcode on blood unit Tai-man Chan

Tai-man Chan

= WB12345678

HN12345678

Hand-held UPI device Blood Transfusion Record Verified barcode Transfusion label

⇒ HN12345678 date/time

Medical staff scans the Hospital Number barcode (prefix WB) on patient’s wristband, then the Hospital Number barcode (prefix HN) on the LIS label of the blood unit. If the two barcodes do not match, an error message is generated and the process of patient identification

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41

has to be repeated. If the two barcodes match, a self-adhesive ‘verified transfusion label’ is generated by the UPI device. This ‘verified transfusion label’ is then affixed onto the patient’s Blood Transfusion Record and blood transfusion then begins. System Implementation

Staff Training Training of nurses and doctors took place at ward level. They were given written instruction and were supervised by ward managers until they were competent with the UPI procedure. Operation of the UPI system was included in the orientation programme for newly recruited hospital staff.

User Protocol A handbook containing the standard blood transfusion procedures using the UPI system as well as the conventional ‘second checker’ system was prepared by the task group. Electronic version was put on hospital web page and a hard copy was kept in each ward, operating theatre and other patient care areas.

Backup System The conventional ‘second checker’ system remained a contingency back-up system, which could be resorted to whenever the UPI system failed or the UPI device was not available for use.

Scope of Use After a trial period of nine months, the UPI system was rolled out for use hospital-wide in May 1999. Areas of use included all wards of departments of Medicine, Paediatrics, Surgery, Orthopaedics, Gynaecology and Obstetrics, and Clinical Oncology; the Intensive Care Unit; operating theatres; and day ward service. One UPI device was placed in each ward or patient care area, and in total, 51 UPI devices including three spare ones were installed. Areas excluded from use were psycharic wards where blood transfusion was infrequent and whenever indicated, patients were transferred to medical wards; the Accident and Emergency Department where a different patient identification system (AE number instead of Hospital Number) was used and that number would be changed to Hospital Number when the patient is admitted to hospital; and the Specialist Outpatient Clinics where no wristband was allocated to outpatients. System Cost

Capital Cost Initial capital cost for purchase of 51 UPI devices (HK$ 20,000/device) and installation of computers and barcode label printers (two in the Admission Office) amounted to HK$ 1,000,000. This was supported by the hospital.

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Joyce Chan Chee Wun and Raymond Chu

Recurrent Cost This included cost for battery maintenance and paper supply for label printing. The average annual cost was HK$ 50,000. System Monitoring

Feedbacks: Staff Acceptance Feedbacks from end users were collected by members of the HTC and the task group and were discussed in each quarterly HTC meeting. Both the nursing and the medical staff found no difficulty in using the UPI device. New staff required supervision only during their initial two to three episodes of use.

Surveys: System Performance Towards the end of its 3-year implementation, a survey was conducted from February to April 2002 to evaluate the performance of the UPI device. All staff using the UPI device for either blood sample collection or blood administration had to record problems they encountered during the procedure in a pre-prepared problem sheet. Evaluation revealed the overall compliance rate of using the UPI system for transfusion process approached 90%. In approximately 10% of occasions, the conventional ‘second checker’ system had to be resorted to because of UPI problems. The survey also showed that on average, an intern took six minutes to complete a blood sampling procedure with either the UPI system or the conventional ‘second checker’ system. Scanning and barcode label printing normally took 30 seconds. Analysis of the survey results showed problems occurred in 12% of episodes of use of the device and most problems were related to battery failure leading to scanning and printing defects. End users’ chief comments were that the system still required staff signature and manual recording of the blood unit’s serial number. The system could be fooled by scanning barcode labels bearing the same prefix (HN), for example, scanning of the wristband label (prefix WB) plus either the ‘LIS label’ (prefix HN) or the ‘gum label’ (prefix HN) could generate a ‘verified transfusion label’ for blood administration. This fault, however, did not happen in practice as there was no incentive for staff to depart from the scanning protocol. Another survey in May and July 2002 assessed interns’ performance of blood sampling procedures – five weeks (in May) towards the end and four weeks (in July) at the beginning of their internship. It showed that interns at the beginning of their career (July of each year) needed substantial guidance for familiarity with transfusion procedures.

Analysis of Transfusion Incidents: Effectiveness From May 1999 to May 2002, no transfusion incidents of either blood being transfused to wrong patients, or wrong labelling of sample tubes and Blood Request Form, occurred with 41000 blood sampling procedures and 27000 units of blood administration. From May 1995 to April 1999 when the ‘second checker’ system was in place, a total of 13 transfusion incidents – wrong labels on sample tubes and on Blood Request Forms were recorded.

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43

Table 1 Overall Comparison of the UPI System and the Conventional ‘Second Checker’ System.

Cost Development Recurrent (labels, batteries) Effectiveness (number of cases) Blood transfused to wrong patient (mistransfusion) Wrong labeling of blood sample tubes/blood request forms Acceptance Time taken for one procedure (minutes) Compliance rate

Second Checker May1995 -April 1999

UPI system May1999 -April 2002

0 0

HK$ 1,000,000 HK$ 50,000/year

0 13

0 0

6 90%

6 90%

System Modification

Revision of UPI Device Model On the basis of the survey results, the HTC was convinced that the electronic UPI barcode system was effective in reducing human errors related to transfusion procedures and decided to tailor-make a second-generation UPI device in order to overcome some of the problems of the first generation UPI devices. Design of the new device was based on the original model with emphasis on handiness, efficient scanning/printing mechanics, and the use of re-chargeable, cheaper batteries. A few new functions were to be incorporated.

II. Improvement of UPI System: 2nd Generation UPI Device Tailor-made Model of UPI Device

Design and Production This was a joint project between the UPI task group, the Hong Kong East Cluster Blood Transfusion Committee (the HTC of individual hospitals within the cluster combined to form the cluster-based hospital transfusion committee in 2003) and the Hong Kong Polytechnic University (PolyU). The project was commenced in the 4th quarter of 2002 but was interrupted for almost one year because of the outbreak of SARS in Hong Kong in 2003. Mechanical design, identification algorithm, programmes, circuit design, plastic tooling and PCB tooling were jointly worked out.

Features The new UPI device weighs 500 gm and is used again as a process specific application for patient identification for the purpose of blood transfusion. It is a compact design with all components being assembled in one piece - a handheld barcode scanner, a built-in printer, a computer, and a LCD screen. It is a standalone device and requires four AA-sized rechargeable batteries.

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Joyce Chan Chee Wun and Raymond Chu

New Functions (i) Product Identity: The ‘verified transfusion label’ for blood administration accommodates additional information about the blood unit, the serial number and the ABO blood group, so as to spare staff from recording these data manually on the patient’s Blood Transfusion Record thereby eliminating documentation error and saving time. (ii) Staff Identity: Both the ‘verified transfusion label’ for blood administration and the ‘UPI label’ for blood sample tube include staff ID barcode number thereby rendering staff signatures, often illegible, unnecessary. (iii) Patient Identity: In addition to patient’s Hospital Number on wristband, patient’s Hong Kong Identity Number is also scanned and recorded on the ‘UPI label’, which acts as the second patient identifier (for visual but not for electronic verification) in the blood sampling procedure. (iv) Automatic Serial Change of the Prefix of Hospital Number In order to prevent staff from taking short cuts, the prefix of the unique Hospital Number in different stages of the transfusion process is automatically and serially changed by the electronic device, such that the prefixes appear as WB on wristband, HN on Blood Request Form, WN on ‘UPI label’ for blood sample tube, TN on LIS label of blood unit, and TN on ‘verified transfusion label’. (v) Memory for Uploading The device has a memory of 1000 transaction records for capturing and storing each blood sample collection and blood administration events, and data can be uploaded via USB port to a central computer so that data can be used for further processing /analysis. Each device is uniquely numbered so that events can be traced.

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45

(vi) Triple Matching for Blood Administration For more accurate verification of patient identity, a matching among patient’s Hospital Number on wristband, on blood unit, and on Blood Transfusion Record prior to blood administration is incorporated. With the first generation device, matching between patient’s Hospital Number on wristband and on blood unit only was required. System Use Flow Chart UPI System – Flow Chart Verification and Documentation of Unique Patient Identity for Transfusion

Blood Sampling from a patient with unique Hospital Number HN04041735(8) on wristband Blood sampling

Patient’s Wrist band Wrist- Band HN04041735(8) Barcode:WB04041735(8)

=

Gum Label on Blood Blood Request Request Form Form /BTR HN04041735(8) Barcode: HN04041735(8)

↓ Verification: UPI Scanner by blood taker Documentation of verification: UPI printer

↓ Blood bank

UPI Label on Sample Tube HN04041735(8) Barcode: WN04041735(8)

Gum Label on Blood Blood Request Request Form Form /BTR HN04041735(8) Barcode: HN04041735(8)

= ↓

Verification by Blood Bank Staff Documentation of verification

↓ Blood administration

Patient’s WristWristBandBand HN040041735(8) Barcode:WB04041735(8)

=

=

LIS Label on Blood Blood Unit Bag HN04041735(8) Barcode: TN04041735(8)

=

Gum Label on Blood Request Form HN04041735(8) Barcode: HN04041735(8)

↓ Verification: UPI scanner by blood giver Documentation of verification: UPI printer

↓ UPI Verified Transfusion Transfusion Label WT04041735(8) Label TN04041735(8)

Blood Transfusion to a patient with unique Hospital Number HN04041735(8) on wristband

Joyce Chan Chee Wun and Raymond Chu

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System Implementation

Installation Additional hard ware is required and includes a standalone computer for capture of uploaded data from individual UPI devices in hospital blood bank, battery chargers and rechargeable batteries. Consumables include paper rolls for printing out ‘UPI labels’ and ‘verified transfusion labels’.

Scope of Use The programme was rolled out in our hospital and another acute regional hospital in the cluster, Ruttonjee Hospital, in July 2004. A total of 75 UPI devices were manufactured – 55 for our hospital and 20 for Ruttonjee Hospital.

Staff Training and Training Tools A total of six identical training sessions were organized by the UPI task group (two in our hospital, four in Ruttonjee Hospital) and training included live demonstration and handon practice for nurses, interns, doctors and blood bank staff. Training tools included video and pocket-sized handbooks. The latter is prepared by the UPI task group and the central nursing division and includes clear description of steps of transfusion process, maintenance of the UPI device, and management of shoot-up problems of UPI device. Electronic version of both the video and the handbook guidelines are also available from the cluster web page. Logistics of Maintenance and Feedback End-users (nurse, doctor) 1 UPI device and 1 battery charger in each ward/patient care area

Material Management Department Keep supply of consumables (labels and batteries)

Blood Bank Collect, check, repair faulty device Liaison with PolyU for repair Keep spare devices Monitor UPI data upload

Central Nursing Division UPI Task Group/HKE Cluster Transfusion C i Ad hoc problems Overall monitoring and review

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System Cost Table 2 Set up cost amounted to HK$ 970,800 and recurrent cost for the two hospitals is estimated to be HK$ 11,088 per year. Set up Development Production Accessories - batteries - chargers Two barcode label Printers Upgrade (Mid- improvement) Total

HK$ 640,500 259,500 (3460/device) 3,000 6,000 (80/charger) 27,600 34,200 970,800

Recurrent Unit price of paper roll (110labels/roll) Total cost of paper roll (70 rolls/month)

HK$ 13.2 11,088

System Evaluation, Mid-point Improvement Strategy, Re-evaluation

Initial Survey: Problems, Improvement strategy An evaluation survey in the form of problem log was carried out from 26 July to 29 August 2004 and identified problems, mainly printing and scanning problems, in 20% of episode-use. The mid-point strategy was to improve the device mechanics.

Second Survey: Satisfaction

System

Performance,

Technical

Acceptance,

Staff

A second evaluation survey was carried out from 16 December 2004 to 9 January 2005 which also covered a period of switching of internship. The second evaluation consisted of three parts. (i) Technical evaluation by the Medical Physics Department of the hospital confirmed software validation, equipment safety, and fulfillment of the requirements stipulated in the tender specification. The result of the technical acceptance test of the device for use in blood transfusion was satisfactory. (ii) End users’ problem log showed 93% of performance was without problems. (iii) End users’ satisfaction survey showed most scored point 3 to 4 out of a 1-5 point-scale.

RESULTS As a result of the introduction of the UPI system for blood transfusion, the current procedures for bedside blood sample collection for pre-transfusion compatibility test and for blood administration are as follows. For the sake of completeness of the transfusion chain, steps in the blood bank are included.

Joyce Chan Chee Wun and Raymond Chu

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I. Steps for Blood Sample Collection for Type and Screen Step 1 - Ensure ‘Type and Screen’ is ordered •

Check patient’s Medical Record for the ‘Type and Screen’ order

Step 2 - Assemble materials for ‘Type and Screen’ • • • • • • • • •

Blood Sample tube Syringe Alcohol swab Touniquet Kidney dish Specimen carrier bag Blood Request Form Medical record and gum label UPI device

Step 3 - Identify the Correct Patient •

Identify the patient by his/her unique HOSPITAL NUMBER and his/her unique HONG KONG IDENTITY (HKID) NUMBER pre-printed on his/her wrist-band label Wristband Unique Patient Identity HKID number

Hospital Number barcode reads as WB04041735(8)

Hospital Number

Step 4 – Obtain patient’s gum label • • •

Check the ‘gum label’ belongs to the patient who is going to be sampled for Type and Screen Affix the ‘gum label’ onto the Blood Request Form (BRF) Complete the Blood Request Form (BRF)

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49

Gum Label Unique Patient Identity HKID number barcode

Hospital Number barcode reads as HN04041735(8)

Step 5 –Verify patient identity with UPI device •

Use the UPI Device to scan (i) Wristband Hospital Number barcode (ii) Gum label Hospital Number barcode on Blood Request Form (BRF) (iii) Gum label HKID number barcode on Blood Request Form (BRF) (iv) Blood taker’s staff ID number barcode Wristband

Gum label on BRF (iii)

=

(i) (ii)

•

If the patient’s (i) wristband Hospital Number barcode and (ii) the ‘gum label’ Hospital Number barcode on the Blood Request Form (BRF) do not match, an error message will be issued, and steps 3, 4 and 5 have to be repeated.

Joyce Chan Chee Wun and Raymond Chu

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Step 6 – Document the verification with UPI device

•

•

•

If the patient’s (i) wristband Hospital Number barcode matches (ii) the ‘gum label’ Hospital Number barcode on Blood Request Form (BRF), the UPI Device will print out a ‘UPI label’ The ‘UPI label’ contains (i) UPI Hospital Number barcode WN04041735(8) (ii) Hospital Number HN04041735(8) (iii) HKID Number (iv) Date and time of Type and Screen (v) Blood taker’s staff ID number (vi) Device identity number Stick the self-adhesive ‘UPI label’ generated onto the sample tube UPI label (i) (vi)

(iv)

(ii) (v) (iii)

Sample Tube Step 7 - Take blood from patient Step 8 - Send the following to the hospital blood bank • •

Blood sample tube affixed with the ‘UPI label’ Completed Blood Request Form (BRF) with patient’s ‘gum label’ affixed

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Special Precautions • •

Complete steps 1 to 8 for Type and Screen in one go, steps 3 to 7at patient’s bed side Perform one patient’s blood sampling at one time

Conventional Type and Screen Procedures without UPI Device - Second Checker system (when the UPI system is not available) (for details, please refer to HKEC Transfusion Manual at the HKE Cluster web page) • • •

• • •

Steps 1,2, 3, 4 as above Step 5 – Affix a patient’s ‘gum label’ onto the sample tube Step 6 – Verify visually with the help of a second checker that the ‘gum labels’ on the sample tube and on the Blood Request Form (BRF) bear the same Hospital Number and the same HKID number as on patient’s wristband. Blood taker please sign the ‘gum label’ on the sample tube Step 7 as above Step 8 - Send blood sample tube affixed with a ‘gum label’, and Blood Request Form (BRF) affixed with an identical ‘gum label’ to the hospital blood bank

II. Steps in Blood Bank Step 1 •

Check, visually as well as by using barcode scanning, the ‘UPI label’ on the sample tube and the ‘gum label’ on the Blood Request Form (BRF) belong to the same patient

Step 2 •

When blood is to be issued, generate a ‘LIS label’ which contains: (i) Hospital Number HN04041735(8) (ii) Hospital Number barcode TN04041735(8) (iii) ABO blood group of blood unit (iv) Serial number of blood unit (v) Hong Kong Identity number

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LIS label (iii)

(ii)

(iv)

(i)

(v)

Step 3

•

Stick the ‘LIS label’ onto the blood bag of the blood unit assigned to the patient LIS Label

Serial number & barcode Expiry date & barcode

Blood group & barcode

Blood unit

III. Steps for Blood Administration Step 1 - Ensure that blood transfusion is ordered •

Check patient’s Medical Record and Medication Administration Record (MAR) for blood transfusion order

Step 2 – Obtain informed consent from patient for blood transfusion •

Give ‘Transfusion Information Leaflet for Patient’ to the patient

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53

Explain to patient the indication, hazards, and alternatives of blood transfusion, with reference to the ‘Transfusion Information Leaflet for Staff’ Hard copy available in ward. Electronic version at HKE Cluster web page.

• •

Step 3 - Identify the correct patient who is going to receive blood •

Check visually the patient’s unique Hospital Number and other identification information on his/her wristband against that on the ‘LIS label’ of the blood unit Check visually patient’s blood group and blood unit’s serial number on the Blood Transfusion Record against that on the blood unit Check expiry date on blood unit Always ask a patient to state his/her name first if possible

• • •

Step 4 – Verify patient identity with UPI device •

Use the UPI Device to scan (i) Wristband Hospital Number barcode (ii) LIS label Hospital Number barcode on blood unit (iii) ‘Gum label’ Hospital Number barcode on Blood Transfusion Record (BTR) (iv) Serial number barcode on blood unit (v) Blood group barcode on blood unit (vi) Blood giver’s and checker’s staff ID number barcode LIS Label Wristband (i)

(ii)

(iv)

=

Blood Transfusion Record

(v)

(iii)

•

If the patient’s (i) Hospital Number barcode on the wristband, (ii) the ‘LIS label’ Hospital Number barcode on the blood unit and (iii) patient’s ‘gum label’ Hospital Number barcode on Blood Transfusion Record (BTR) do not match, an error message will be generated, and steps 3 and 4 have to be repeated.

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Step 5 – Document the verification with UPI device •

Upon correct completion of the scanning process, the UPI device will generate a self-adhesive ‘verified transfusion label’, which contains: (i) Date and time of transfusion (ii) ABO and Rh(D) blood group of blood unit (iii) Serial number of blood unit (iv) Hospital Number with prefix TN04041735(8) (v) Blood giver’s and checker’s staff ID number (vi) Device Identity number

Verified Transfusion Label

(i) (vi)

(iv) (ii)

(iii) (v)

• • •

Check the ‘Verified transfusion label’ contains all the items required Affix the ‘Verified transfusion label’ onto the Blood Transfusion Record (BTR) Sign on the BTR by blood giver and checker

Step 6 - Start blood transfusion Step 7 – Monitor patient’s condition • •

Record patient’s transfusion reactions during the course of transfusion Record the volume of blood transfused at the end of transfusion

Special Precaution •

Complete steps 1 to 6 in one go

Conventional Blood Administration Procedures without UPI device – Second Checker system (when the UPI system is not available) (for details, please refer to HKEC Transfusion Manual at HKE Cluster web page) •

Steps 1, 2, 3 as above

Unique Patient Identification Barcode System for Prevention of Blood … •

•

•

55

Step 4 – Verify visually, with the help of a second checker, that the Hospital Number on the ‘LIS label’ on the Blood Transfusion Record is identical to the Hospital Number on patient’s wrist-band Step 5 – Manually enter (i) date and time of transfusion (ii) ABO and Rh(D) blood group (iii) serial number of blood unit (iv) initials of blood giver and checker, on to the Blood Transfusion Record (BTR) Steps 6, 7 as above

DISCUSSION Do We Have Exact Figures of Mistransfusion Incidents? Mistransfusion, i.e., transfusion of blood to the wrong patient, is the most important serious hazard of transfusion [1-6] globally. The risk of mistransfusion is many times greater than that due to the transmission of HIV or HCV infection by blood [1]. There are many reports on the estimation of mistransfusion rates in different localities. Invariably there is a wide variation in such estimation results attributing not only to differences in transfusion systems adopted by individual country or institution, but also to methodological differences in analysis. These figures are therefore not strictly comparable. Mistransfusion rate as high as 1 in 400 has been reported in Belgium [7]. Linden [8] reported a 10-year experience on transfusion errors in New York State and came up with a figure of erroneous administration of blood of 1 in 19,000. This study is representative and reliable as the study duration was 10 years and there were 9,000,000 transfusions given during the study period. Inojie and Urbaniak [9] highlighted the fact that the true incidence of transfusion errors is much higher than that of the actual mistransfusion events. In this paper, the rate of mistransfusion equated to 1/8,610 compatability procedures, and 1/27,007 units of blood issued, whereas the number of true transfusion errors equates to 1/2,153 compatability procedures and 1/6,752 units of blood issued. It is thus alarming to note that the true incidence of errors is at least four times the actual mistransfusion events detected. In Hong Kong, two major mistransfusion incidents were recorded in the past decade. One resulted in transfusion of a wrong blood unit to a patient due to failure of checking blood group prior to blood administration. The other mistransfusion resulted from wrong labeling of blood sample and Blood Request Form. Considering an annual consumption of nearly 200,000 blood units in Hong Kong, this figure is likely an underestimation. In most series, mistransfusion incidents arise as a result of checking errors at bedside [13]. According to the Serious Hazards of Transfusion (SHOT) 2003 annual report [4], about 70% of errors occurred in clinical areas (30% prescription, sampling, request; 40% collection, administration) and 30% in blood bank. The commonest error (26.5%) was failure of pretransfusion bedside check. The Linden study [8] showed a similar picture with about half of the errors occurring outside blood bank (administration to the wrong patient 38%; phlebotomy errors 13%), while the blood bank errors accounted for 29% of the events. Multiple errors occurred in 52% of the SHOT study and 15% of the Linden study.

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Do We Have Sound Solutions to Prevent Mistransfusion? It is apparent from these studies that the current patient safeguards to prevent mistransfusion are clearly inadequate. Adequate staff training on proper procedures for patient identification together with measures to ensure staff’s strict adhesion to the standard operating procedures cannot be emphasized enough. However, they are inadequate by themselves, as human errors cannot be completely avoided without major system changes. This fact was highlighted by Sherwood [21], who stated in the Transfusion editorial in 1990 that “Since it is not reasonable to expect human error to occur with a frequency of less than one in 10,000, the only way to improve procedural error rate, i.e., the final product error rate, is by making major system changes.” Various approaches have been adopted to tackle this problem but none is entirely satisfactory at present. Some hospitals require patients who have no prior blood transfusion history to have another blood specimen taken at a later time to confirm the ABO grouping. This may be of help to a certain extent but would certainly double the workload for blood bank staff and the person performing the venepuncture with added patient inconvenience. As venepuncture has to be done twice, there would be a delay in getting blood for the patient. Furthermore, the staff who performs the venepuncture can come up with ways to bypass the tedious task of double venepuncture by doing it once and putting blood into two sample tubes and sending them out separately at different time. It is standard practice to have two nursing staff to double check before blood administration but only a single staff for the blood sampling procedure for pre-transfusion testing. Taking blood from the right patient but wrongly labeled with another patient’s demographic data or taking blood from the wrong patient but labeled with the intended patient’s demographic data is one of the commonest causes of mistransfusion. Some hospitals require two staffs to perform the pre-transfusion blood sampling procedure with one staff acting as a witness of the whole procedure and doing the counterchecking. This so-called ‘buddy’ or ‘second checker’ system is not widely practised because it puts further strain on the already tense staffing situation of the hospital, and it also raises the concern that some staff may coerce others into countersigning even though the other staff might not have witnessed the whole procedure. As mentioned previously, our hospital is the first and one of the three hospitals in Hong Kong to have implemented such a ‘second checker’ system hospital-wide so far. In some European countries, e.g. France, it is mandatory to have the pretransfusion bedside compatibility test before red cell transfusion. The test allows verification of the ABO compatibility between donor blood and recipient blood immediately before transfusion. When performed correctly, the test can prevent errors that may have occurred at any point during the transfusion chain. However, errors in interpretation of the test result may prop up, and the residual risks are still high, especially when it is performed by inexperienced staff [10-12]. In the last two decades, system change with the use of information technology has been promising in tackling the mistranfusion problem. Wenz [13] in 1991 and AuBachon [14] in 1996 described the use of mechanical barrier system in preventing transfusion errors. An orthopedic institute tried this system and found it to be useful in preventing potential

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transfusion-associated fatalities [13]. The system however was perceived by some staff to be inconvenient as locking and unlocking of the system were required. As a result, this system was not widely adopted. Jensen [14] in 1996 described the use of a portable barcode scanner for bedside verification between patient identification and blood unit identification, coupled with the use of a host computer system that was capable of accepting transfusion data from the scanner. However, the device was not tried for ensuring correct blood sampling for pretransfusion testing, one of the common procedures with a high risk of human error. More recently, two papers [17,18] described the use of barcode scanners in both blood sampling for pretransfusion testing and blood administration. However, they had been used in limited areas (haematology outpatient clinic) [17] or only for study purpose [18]. Lau [19] described the use of a specially designed transfusion wristband with unique transfusion barcode printed on each transfusion label and the corresponding wristband, which had the potential for further development by incorporating a barcode reader to facilitate counterchecking of the transfusion code and recording of the transfusion process. Again, this study was done as a trial in a limited area of the hospital. We published our three-year experience in using an electronic barcode patient identification system for bedside blood transfusion procedures in 2004 [20]. This is the only paper that described sustained, large-scale, hospitalwide use of an electronic barcode system for ensuring patient identity from blood sample collection to blood administration.

What are the Merits of our UPI System? Safeguards to Bedside Transfusion Procedures The design of our UPI system pivots on the unique patient Hospital Number barcode, which is imprinted on every patient’s wristband label at the time of admission to hospital. This Hospital Number is unique to each patient as it is different for each patient and each admission. The wristband remains attached to the patient throughout his/her hospital stay and is removed only upon his/her discharge. This fact is of paramount importance as the ‘sessile’ and unique location of the wristband dictates the checking procedures for blood sample collection and blood administration to be done at the designated patient’s bedside. For both procedures, the system mandates patient’s wristband as the starting point of checking. This means that the first scanning is always and must be a patient’s Hospital Number barcode on the wristband. Scanning of barcodes other than the wristband Hospital Number barcode, whose unique prefix WB the device has been programmed to recognize, will not initiate further process, but an error message will appear on the LCD screen of the UPI device to alert the operator. The second scanning, either the Hospital Number barcode on the Blood Request Form, or the Hospital Number barcode on the blood unit, will automatically channel the process to the desired pathway, i.e. either the blood sample collection or the blood administration, two built-in process pathways in the computer of the UPI device. The system also ensures absolute correctness of the specimen label on the blood sample tube for pretransfusion testing, as the ‘UPI label’ for blood sample tube can only be obtained after scanning confirms matching of the Hospital Number barcodes on the wristband and the Blood Request Form. This step of matching automatically leads to the printing out of a ‘UPI

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label’ by the UPI device. Though the UPI device is not used in the blood bank, the LIS of the blood bank will double check the patient identity (Hospital Number) on the UPI label of the sample tube with that on the Blood Request Form, and transcribe electronically the patient identity information to the blood unit for the intended patient. Blood administration at bedside will commence only after matching is confirmed by scanning of the Hospital Number barcodes on the wristband and the blood unit, and verification is documented by the printing out of a ‘verified transfusion label’ by the UPI device. The fundamental merits of our UPI system thus lie in its obligate requirement for the performance of blood sampling procedure to be done at bedside; absolute guarantee of a correct label on the blood sample tube; and matching of the blood unit with the intended patient. We achieve our system objectives through accurate transfer of a patient’s identity, in this case, the unique Hospital Number, from a patient’s wristband, to the Blood Request Form, to the blood sample tube, and to the blood unit, coupled with documentation of the verification procedures at two critical points known to be associated with high incidence of human error - pre-transfusion blood sampling for compatibility test and blood administration. Manpower Saving compared with the Second Checker System Long before the implementation of the UPI system, we required two staff in both the blood administration procedure and the blood sampling procedure. After the implementation of the UPI system, only one staff is required for the blood sampling procedure. This saves a lot of manpower. We are in the process of reviewing the possibility of requiring one staff instead of two in the blood administration procedure. The conventional ‘second-checker’ system is not to be totally abandoned as it remains a contingency back-up system, which could be resorted to whenever the UPI system fails.

Simple, Inexpensive, Large Scale implementation compared with Other Electronic Systems There are a number of commercial bar coding systems for patient identification available in the market, e.g. Olympus, Becton Dickinson. They usually require network connection to the hospital’s Patient Administration System, and the initial capital cost is usually enormous. For example, installation of the Becton Dickinson system for an average 400-bed US hospital is estimated to cost between US $250,000 and $350,000. Our UPI system does not require an infrastructure or network connection with the hospital computer system. This is what we intentionally tried to avoid from the very beginning as it would invariably involve time, cost and inconvenience. Thus at times when the hospital computer system is down or in maintenance, the operation of our UPI system remains unaffected. Compared to most commercial systems, our UPI system is much less expensive but is capable of achieving the same objectives. The design and development cost of our UPI system amounted to US $82,000. Depending on the number of UPI devices made, the cost per device can further go down to US $400, which is much lower than those commercially available products. Because of the low cost, we can afford to implement the UPI system in most of our patient care areas, including the operating theatres. Even for our hospital, which is a major regional hospital

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with over 1,000 hospital beds, the number of UPI devices required is about 50 as only one device is required per clinical ward or operating theatre with spare devices kept centrally. Other capital setup cost includes cost of two barcode printers (US $3,500) for printing patient wristband in the Admission Office and two computers (US$15,00) – one in the Admission Office for printing barcoded patient wristbands and the other in blood bank for downloading data from all the UPI devices within the hospital. Because of ease of operation of the UPI device, large scale use of the device does not encounter resistance from staff. Overcoming staff or professional barrier is an important determining factor in successful implementation of a new system. Effectiveness Effectiveness of the UPI system is born out by a reduction in the transfusion errors, in terms of wrong labeling of blood sample tubes and Blood Request Forms, and transfusion of blood to the wrong patient. Other errors, such as out of forgetfulness, sending blood sample tubes without UPI labels to blood bank, still occur from time to time. These however are easily picked up at the blood bank level. Although the scale of reduction of transfusion errors is small when compared with the ‘second checker’ system, it is important to remember that the human error allowed in the field of transfusion is zero.

Is there Continuous Quality Improvement for our UPI System? Simple as our system is, its ultimate success pivots on the proper functioning of the UPI device. Our first generation UPI device, a commercially ready-made product, was put to use hospital-wide in May 1999. As every new system may generate problems on its own, our first generation UPI device was no exception. It was heavy. Batteries ran out of power easily. As a measure of continuous quality improvement, we collaborated with a local university to tailormake our second generation UPI device based on the problems encountered on extensive use of the first generation device. Similar to our first generation device, the second generation UPI device is also an integrated device with built-in laser barcode scanner and printer. The advantages over the first generation device are that it is more compact in size, ergonomically designed, and utilizes easily available AA sized alkaline or rechargeable batteries. The checking logistics behind both systems are very similar with improved checking algorithm and more detailed documentation of the processes with the second generation device. Three additional items are included in the scanning/recording process. First, we include product documentation – documentation of the blood group and serial number on the ‘verified transfusion label’ which is to be affixed onto the Blood Transfusion Record. Staff no longer have to do the recording manually and transcription errors can be avoided. Second, we include documentation of staff ID number on the ‘UPI label’ (blood sample tube) and the ‘verified transfusion label’(Blood Transfusion Record). Staff signature is no longer required. Third, we add scanning of patient’s Hong Kong Identity number barcode with printing out of the number on the ‘UPI label’ for the blood sample tube. This together with the patient’s Hospital Number serves to satisfy the requirement of major peer organizations such as AABB and JCAHO that two patient identifiers for positive identification of their blood samples are

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needed. Other major improvements concern the design of the computer programme. The foremost important one is for the second generation UPI device to have a built-in programme for manipulating the prefix of the Hospital Number. The different prefixes for different barcodes (WB for patient wristband, HN for Blood Request Form, WN for label on blood sample, TN for label on blood units) serve the purpose of identifying the processes uniquely such that it would be difficult and inconvenient for staff to bypass the usual checking steps. Another improvement worth mentioning is that the UPI devices are so programmed that it has the intelligence of sensing unexpected types of barcode when being scanned and would alert staff if the wrong type of barcode is being scanned. For example, if an operator accidentally scans the blood unit expiry date barcode instead of the intended ABO blood group barcode, the device would produce an audio and visual alarm such that one cannot proceed further until the right barcode is being scanned. To facilitate staff’s performance, the LCD screen of the UPI device displays the correct sequence of scanning items so that staff could follow the proper steps for both the blood sample collection and blood administration procedures. The last but not the least improvement of the second generation UPI device is its non-volatile memory that can hold more than 1000 transaction records. These data can be uploaded to a computer for further analysis. This is very useful for look back purpose in the event of occurrence of transfusion incidents. The data can also be used for auditing blood transfusion processes. Merging the uploaded data on blood administration procedure with patient’s transfusion record in the blood bank computer system can update the latest status of the blood units issued. This is helpful as with the current practice, the blood bank only assumes blood units issued to patients are transfused but this may not be the case all the time. The second generation UPI device was put to use in July 2004, and we are still closely monitoring its performance. Counting from May 1999 when the first generation UPI device was put to use, to the time of writing (August 2005), we have been using our electronic UPI system in blood transfusion practice for over six years. We have extended the use of the second generation UPI device to two regional hospitals and we are in the process of rolling out the system to a third hospital within the cluster.

What are the Shortcomings of our UPI System? Clearly there are steps in the transfusion loop which are beyond the safeguard of our UPI system, and areas where the UPI system is not applicable. As stated previously, the starting point of the UPI system is patient’s wristband. Here comes the importance of the correctness and the integrity of the wristband. It requires the same wristband to stay with the patient throughout blood sampling to blood administration. Therefore one of the essential elements for the system to function is to ensure the right wristband is in place all the time during a patient’s hospital stay. The wristband we use is water-resistant and is readable by barcode scanner after many weeks of repeated use. If the wristband for any reason becomes unreadable, a new wristband label has to be generated from the Admission Office with proper documentation from clinical wards so as to ensure the right wristband is being used for the right patient. If for one reason or another, a wrong patient is approached for blood sampling, the wrong patient’s identity will be used throughout the transfusion process, and this

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constitutes an area where the UPI system is unable to detect the error. Accurate identification of the intended patient remains a crucial step in the transfusion process and relies entirely on medical staff’s prudence and awareness of the importance of this very first step. Again as the system relies on the presence of Hospital Number barcode on the wristband which is issued only to patients admitted to hospital, patients in the Specialist Outpatient Clinics and patients in the Accident and Emergency area without wristbands are not benefited by the system. The most affected group are those requiring blood transfusion on planned or regular basis, such as surgical patients pending elective operation, and transfusion dependent thalassaemic patients. As the issue of blood units and subsequent blood administration require the presence of a Hospital Number, blood sampling for pre-transfusion compatibility test can only be done after these patients are admitted. Fortunately some of these patients can be admitted to the day ward where patients are issued wristbands with Hospital Number and the UPI system is applicable. Although the effectiveness, acceptance and the improved functionality of our second generation UPI device has proved our UPI system to be a success, it has cost and manpower implication. From a transfusion service perspective, the true benefit of introducing such a system calls for a proper cost-benefit analysis, probably also taking into consideration of the legal costs associated with major transfusion incidents. Our system lacks such an evidence based analysis so far.

What are our Future Directions? The UPI devices, with wear and tear, and repeated use by a multitude of experienced as well as inexperienced staff, are expected to have a life expectancy of three to five years. We anticipate further improvement with the system, such as extension of its use to Specialist Outpatient Clinics which is strongly desired by some sector of the professionals. We also strive for further upgrading the mechanical function of our UPI device. One of the current problems of our second generation UPI device concerns the printing speed, which is considered at least by some staff to be too slow. Label jamming is another problem, which still occurs from time to time in spite of our mid-point improvement strategy. With a compact integrated device, these problems seem inevitable. One solution is to separate the printer from the scanner so that a faster, better quality printer could be connected to the scanner either by wire or by various wireless links. In that case, one has to carry two devices to patient’s bedside. We have considered this option both at the beginning of the project in 1997 and in 2002 when the system was being upgraded. Frontline staff on both occasions preferred to have one integrated device. Another option is to do without the printer and to document the verification processes in the scanner memory and to upload such data onto computers for archival and look-back purposes. With the dropping cost of radio frequency identification (RFID) devices and tags, there is a potential for substituting RFID technology for barcode technology in the near future for patient identification purposes.

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CONCLUSION In summary, our UPI system is a cost-effective bar coding system in tackling two recognized weaknesses in the transfusion process - patient identification for collection and labeling of blood samples, and administration of blood. Our over 6-year experience of implementing this innovative system hospital-wide in major regional hospitals proves the underlying principle or working logistics of the system is sound, or at least applicable in the acute hospital setting in Hong Kong. It enhances blood transfusion safety by decreasing human errors. Through software modification, the functionality of the device could be further improved and even widened to other applications. The initiation and the successful implementation of the project depend very much on the commitment and vision of the senior management of the hospital and the HTC, the resources provided by the hospital management, and the concerted effort of all professionals and staff of the hospital. We shall continue to strive for a full quality system in transfusion practice embracing the loop from patient identification to transfusion and its subsequent monitoring. Implementation of the long overdue patient-safety mechanisms is never too late.

REFERENCES [1]

Dzik WH: Emily Cooley lecture 2002: Transfusion safety in the hospital. Transfusion 2003; 43; 1190-1198. [2] McClelland DBL, Phillips P: Errors in blood transfusion in Britain: Survey of hospital haematology departments. BMJ 1994: 308: 1205-1206. [3] Sazama K: Reports of 355 transfusion-associated deaths: 1976 through 1985. Transfusion 1990: 30: 583-590. [4] Serious Hazards of Transfusion. Annual report 2003: Serious hazards of transfusion scheme, Manchester, UK, 2004 (www.shotuk.org). [5] Robilliard P, Itaj NK, Corriveau P: ABO incompatible transfusions, acute and delayed hemolytic transfusion reactions in the Quebec hemovigilance system- year 2000 [abstract]. Transfusion 2002; 42(suppl): 25S. [6] Andreu G, Morel P, Forestier F, et al.: Hemovigilance network in France: organization and analysis of immediate transfusion incident reports from 1994 to 1998. Transfusion 2002; 42: 1356-1364. [7] Baele PL, De Bruyere M, Deneys V, et al.: Bedside transfusion errors. A prospective study by Belgium SAnGUIS Group. Vox Sang 1994: 66: 117-121. [8] Linden JV, Wagner K, Voytovich AE, Sheehan J: Transfusion errors in New York State: an analysis of 10 years’ experience. Transfusion 2000; 40: 1207-1213. [9] Ibojie J, Urbabiak SJ: Comparing near misses with actual mistransfusion events: a more accurate reflection of transfusion errors. Br J Haematol 2000; 108: 458-460. [10] Dujardin P-P, Salmi LR, Ingrand P: Errors in interpreting the pretransfusion bedside compatability test. An experimental study. Vox Sang 2000; 78: 37-43.

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[11] Ingrand P, Surer-Pierres N, Houssay D, Salmi LR: Reliability of the pretransfusion bedside compatability test: Association with transfusion practice and training. Transfusion 1998; 38: 1030-1036. [12] Migeot V, Ingrand I, Salmi LR, Ingrand P: Reliability of bedside ABO testing before transfusion. Transfusion 2002; 42: 1348-1355. [13] Wenz B, Burns ER: Improvement in transfusion safety using a new blood unit and patient identification system as part of safe transfusion practice. Transfusion 1991; 31: 401-403. [14] Aubuchon JP, Littenberg B: A cost-effectiveness analysis of the use of a mechanical barrier system to reduce the risk of mistransfusion. Transfusion 1996; 36: 222-226. [15] Mercurilali F, Inghilleri G, Colotti MT, Podico M, Biffi E, Fare M, Vinci A, Scalamogna R: One-year use of the Bloodloc system in an orthopedic institute. Transfus Clin Biol 1994; 1(3): 227-230. [16] Jensen NJ, Crosson JT: An automated system for bedside verification of the match between patient identification and blood unit identification. Transfusion 1996; 36: 216221 [17] Turner CL, Casbard AC, Murphy MF: Barcode technology: its role in increasing the safety of blood transfusion. Transfusion 2003; 43: 1200-1209. [18] Marconi M, Langeberg AF, Sirchia G, Sandler SG: Improving transfusion safety by electronic identification of patients, blood samples and blood units. Immunohematol 2000; 16(2): 82-85. [19] Lau FY, Wong R, Chui CH, Ng E, Cheng G: Improvement in transfusion safety using a specially designed transfusion wristband. Transfus Med 2000;10: 121-124. [20] Chan JCW, Chu Rw, Young BWY, Chan FL, Chow CC, Pang WC, Chan C, Yeung SH, Chow PK, Lau J, Leung PMK: Use of an electronic barcode system for patient identification during blood transfusion: 3-year experience in a regional hospital. Hong Kong Med J 2004;10: 166-171. [21] Sherwood WC: To err is human… Transfusion 1990; 30: 579-580.

In: New Developments in Blood Transfusion Research ISBN 1-59454-962-1 Editor: Brian R. Peterson, pp. 65-76 © 2006 Nova Science Publishers, Inc.

Chapter IV

PERCEIVED NATURALNESS AND RISK OF BLOOD AND BLOOD SUBSTITUTES Eamonn Ferguson1,∗, Piers Fleming1, Ellen Townsend1 and Kenneth C. Lowe2,∗ 1

Risk Analysis, Social Processes and Health Group, School of Psychology, University of Nottingham, Nottingham, UK, 2School of Biology, University of Nottingham, Nottingham, UK.

ABSTRACT While objectively safe the public perceive blood transfusion from donor blood as risky. Indeed, there are a number if minor risks (e.g., transfusion transmitted infections). One solution to this has been to explore the development of blood substitutes. However, would people be willing to use a bio-engineered blood product? There is evidence that people do not look favorably on such options. This chapter examines this issue with respect to perceived riskiness and naturalness of donor blood and 3 forms of substitute (bovine Hb, human Hb and GM) and how these are judged relative to a wide range of other natural and engineered medical and food related products. Naturalness is a key construct with respect to the acceptance of bio-technology. Data were collected from a group of 148 undergraduate students on the perceived naturalness and riskiness of 12 products (natural and their bio-engineered counter part) – including blood and 3 substitutes. The results show that the more natural the natural product is perceived to be the less natural its GM variants is perceived. Transfusion with these different blood products is rated as risky along with other invasive medical procedures (heart transplant). Ratings of naturalness were unrelated to ratings of risk for invasive procedures, but were related for low risk non-invasive procedures. Furthermore, it was shown that donor blood for transfusion is itself not perceived as very natural. Implications for the health ∗

Correspondence concerning this article should be addressed to Eamonn Ferguson, email: [email protected], tel. - +44-(0)-115-951-5327, fax - +44-(0)-115-951-5324, or Kenneth C. Lowe e-mail : [email protected], tel. - +44-(0)-115-951-3311, fax - +44-(0)-115-951-3251.

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Eamonn Ferguson, Piers Fleming, Ellen Townsend et al. education and promotion with respect to the acceptance of blood substitutes are discussed.

Keywords: Perceived risk, Naturalness, Blood substitutes, Blood transfusion

BACKGROUND AND CONTEXT Blood for transfusion is a major commodity used by all health services. However, a number of practical concerns relating to psychological/behavioural phenomena face the transfusion services, most important amongst these are (1) recruitment and retention of donors to maintain an adequate blood supply (Ferguson, 1996; Ferguson and Bibby, 2002), (2) the extent to which patients perceive transfusion with donor blood as a risk (Finucane et al., 2000; Ferguson et al., 2001; Lowe and Ferguson, 2003; Ferguson et al, 2005), and (3) the potential for medical problems arsing from transfusion, such as cross-match reactions (Lowe et al., 2001; Lowe and Ferguson, 2003). As well as behavioural and education procedures to aid the recruitment of donors (Ferguson, 1996, Ferguson and Bibby, 2002, Ferguson and Chandler, 2005) and to educate the public about the objectively low risks posed by transfusion (Farrell et al., 2001; Lee and Mehta, 2003; Lee et al., 2003), biotechnological approaches have also been explored. One such biotechnological option is to devise and develop so called ‘blood substitutes’. Such substitutes are injectable artificial oxygen carrying fluids that can be used as a temporary transfusion intervention, often referred to as an ‘oxygen bridge’ or ‘transfusion bridge’. This is because some such materials have been used in patients to oxygenate their tissues during surgery, thereby conserving the patients own, pre-donated (autologous) blood for subsequent transfusion (Lowe, 2003, 2004). Blood substitutes have a number of potential direct and indirect advantages, which include (1) they can be developed to reduce the risk of transfusion related infections, (2) the chances of cross-reactivity are reduced, and (3) donor blood, needed to treat a much larger spectrum of clinical issues, is conserved. Thus, blood substitutes would appear to be an important and viable option for use in transfusion medicine. There are, however, several important psychological issues that need to be considered if blood substitutes are to be more widely accepted in clinical practice. While objectively such substitutes may meet the needs of transfusion specialists, patient groups and the general public may not find them so acceptable. Indeed, there is evidence that people perceive biotechnology to be risky and that people have a more negative attitude towards it (Gaskell et al., 1999; Siegrist, 2000). Thus, the public may not be so willing to accept blood substitutes as transfusion specialists and biotechnologists might expect. One key reason why people may be less accepting of blood substitutes is the fact that they may be perceived as unnatural (Fife-Schaw and Rowe, 1996; Midgley, 2003; Verhoog, 2003; Tenbult et al., 2005). This Chapter, therefore, explores the constructs of risk and naturalness as they pertain to transfusion medicine and presents preliminary data on risk and naturalness with respect to a variety of hypothetical transfusion options.

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Blood Substitutes – Options Currently, blood substitutes include materials based on (1) the naturally-occurring oxygen carrier, haemoglobin (Hb) processed from human or animal blood, (2) synthetic, oxygen-carrying fluorinated compounds [perfluorochemical (PFC) liquids] or (3) encapsulated Hbs (Alayash, 2004; Buehler and Alayash, 2004; Niiler, 2002; Chang, 2003, 2004, 2005; Lowe, 2003, 2004; Vandegriff et al., 2003; Winslow, 2003). Advanced clinical trials have been performed with the first two types of substitutes and one bovine Hb-based product, known commercially as Hemopure (Biopure Corporation, USA), has received regulatory approval in South Africa for clinical use as an alternative to blood during surgery in adults. Hemopure consists of polymerised bovine Hb (13 g dL-1) dispersed in a modified Lactated Ringer’s (electrolyte) solution. It is perhaps not surprising that Hemopure has been approved for use in a country where a large proportion of the adult population are infected with the human immunodeficiency virus (HIV) and where there are serious risks of spreading the disease through the transfusion of blood (Lowe, 2004). Oxygent™ (Alliance Pharmaceutical Corporation, USA) is the most advanced product based on the synthetic PFCs compounds. Multi-centre European Phase III clinical studies with Oxygent™ in surgical patients have been reported (Spahn et al., 2002). Infusion of Oxygent™ not only maintained adequate tissue oxygenation but, importantly, reduced both the frequency and volume of intra-operative blood use over an initial 24 h study period. The study encountered some scepticism from the clinical community (e.g. Tremper, 2002) who highlighted the limited ability of PFCs to contribute to oxygen transport coupled with the crucial requirement for patients to breathe additional oxygen. Specific reference was made to the increased frequency of serious adverse events, notably those involving the digestive system (post-operative ileus) in patients receiving Oxygent™. Paradoxically, a related paper reported that infusion of Oxygent™ during surgery involving cardiopulmonary bypass (CPB) significantly improved post-operative gastrointestinal tract function (Frumento et al., 2002). In 2001, Alliance voluntarily suspended enrolment in a Phase III cardiac surgery study with Oxygent™ to evaluate an unexpected increased incidence of stroke in recipient patients (Niiler, 2002). In March 2002, Alliance announced a re-structuring of European Phase III studies with Oxygent™ to focus any future use of the drug in surgical patients (with no ANH) as an alternative to donor blood. In April 2004, the Alliance web site announced a licensing, development and marketing agreement for Oxygent™ with the IL Yang Pharmaceutical Company (Korea) for the further clinical and regulatory development of the drug in Asia.

Transfusion Medicine, Perceived Risk and Naturalness When people think about biotechnology they may make a number of judgements about it, usually along a number of dimensions that help them to understand its nature and the extent to which it is hazardous. One such dimension that has been discussed in the literature, but received very little in the way of empirical investigation, is that of naturalness (Verhoog, 2003). In one recent psychometric study examining the perceived risks from food

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biotechnology, Townsend et al (2004) found that GM food was rated as unnatural relative to other current risk issues. Naturalness This is seen as related to risk perception, especially as it pertains to genetically modified products and has previously been explored with respect to GM food products (Fife-Schaw et al., 1996; Breakwell, 2000; Tenbult et al., 2005; Verhoog, 2003). Verhoog (2003) sees naturalness as one of the main intrinsic ethical concerns in the debate about GM. These may be related to a sense of disgust or the so called ‘yuk-factor’ (Midgley, 2003; Verhoog, 2003). In a recent study, however, neither GM plants nor GM animals were rated as particularly disgusting, either by people who reported that they would purchase GM food if it became available in future, or by those who would not purchase GM (Townsend and Campbell, 2004). However, non-purchasers disgust ratings for GM were significantly higher than those of purchasers. One way in which people may make judgements about naturalness is by the extent to which they perceive some degree of relatedness between the ‘natural product’ and a engineered counterpart (cf. Verhoog, 2003). As such, naturalness can be studied in a bioengineering context by comparing the extent to which the natural component and its synthetic counterpart are perceived as natural. Indeed, there is some evidence that there may be a proportional relationship between the degree of perceived naturalness between natural and bioengineered variants (Tenbult et al., 2005), such that the more the natural component is viewed as natural the less the bioengineered counterpart is perceived as natural. For example, the prediction would be that the difference in naturalness rating for items that are more natural (e.g. an apple) and their GM version should be greater than for products perceived as less natural and their bio-engineered counterparts (e.g. donor blood and modified blood). Thus, a positive correlation should be observed between the rating of naturalness for the natural product and the mean difference in the naturalness rating for its bioengineered counterpart. Unlike many food products (which have been the sole focus of study to-date), bioengineered blood comes in the different forms outlined above, that in themselves may vary with respect to degrees of relatedness and hence perceived naturalness. Thus, while all such substitutes are unnatural to some extent (i.e. they are modified), the products based on synthetic chemicals (e.g. PFCs) may be perceived as less natural than products based on modified biological compounds (e.g. Hb). This issue has not been explored before and, consequently, is addressed in this chapter. Perceptions of naturalness for donor blood, blood substitutes and some other medical treatments derived from biotechnology, that are in common usage, are studied here. This makes it possible to identify whether the perceptions of blood substitutes are influenced by the fact that they are relatively new and less familiar to the public than other types of modified product used more frequently in medical interventions. To explore this further, therapeutic treatments, such as GM insulin, as well as non-natural products based upon nonbioengineered procedures (e.g. mechanical heart valves), are used as points of comparison

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Risk While objectively safe, people still view donated blood as risky (Finucane et al., 2000; Ferguson et al., 2001; Lowe and Ferguson, 2003). However, a recent study has shown that blood substitutes are perceived as more risky than natural donor blood (Ferguson et al, 2005). One question is: are such perceptions of risk associated with perceptions of naturalness? Furthermore: are blood products, as suggested by Tenbult et al., (2005) that are viewed as less natural also perceived to be more risky? To our knowledge, there have been no studies to date that have explored the relationship between perceived risk and naturalness for bioengineered medical interventions. Importantly, the results generated from the investigation reported here will inform the development of novel educational materials that aim to reduce the perceptions of risk about blood transfusion and the use of potential substitutes.

METHODS Participants An opportunity sample of 148 adult undergraduate students (49 men, 92 women, 7 undeclared; mean ± s.d. age = 19.7 ± 1.2) took part in the study that was conducted at the University of Nottingham, UK, in January 2005.

Measures Information was collected on ratings of 12 treatments, including 4 options for blood transfusion (i.e. donor blood, bovine Hb, human Hb and GM-derived blood substitutes). These were rated across two dimensions: naturalness and perceived risk. These were assessed on 7-point Likert-type scales using the following questions (1) How risky do you think this is (1 – not at all, 7 very risky) and (2) How natural do you think this is (1 = not at all, 7 = completely natural). The 12 treatments were categorized as natural (N) and non-natural (N-N) and whether or not they were medically invasive (I) or not (NI), and these are listed in Table 1.

RESULTS Natural Versus Modified Treatments Table 2 shows the results from the pair-wise T-test comparisons for the natural product versus its modified form. Previous research has shown that the more natural the natural component is perceived to be, the less natural its modified version is perceived to be (Tenbult et al., 2005). The original work was conducted with food and its GM variants. The pattern of findings reported in Table 2 shows that this replicates, to an extent, in the domain of blood substitutes. The apple was perceived overall as the most natural with a mean rating of 6.7 out

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of a maximum of 7 and its GM variant as the least natural (mean score = 2.8). This was the greatest mean difference and supports the idea that the extent of unnaturalness of the GM variant is proportional to the extent to which the natural variant is perceived to be natural. For example, the next largest mean difference was for Chamomile versus Steroid Cream. The same pattern was seen also for non-GM modifications in the domain of blood and blood substitutes and with respect to synthetic variation, such as the mechanical heart valve. The correlation (Spearman’s ρ) between the mean for the natural counterpart and the mean difference is ρ (6) = .87, p = 0.025. Thus, the difference in naturalness rating for GM variants and their natural counterparts is proportional to the naturalness rating of the natural product. Table 1 Treatments studied.

Natural

Invasive Donor blood for transfusion

Non-Invasive Eating an traditionally grown apple Applying chamomile ointment Eating Yogurt Eating a GM apple

Donor heart valve for transplant NonNatural

Chemical blood substitute for transfusion GM blood substitute for transfusion Bovine blood substitute for transfusion Mechanical heart valve for transplantation Using GM insulin

Applying steroid cream

Table 2 Differences in ratings of naturalness. Treatment Apple GM Apple Chamomile Steroid Cream Donor Heart Mechanical Heart Donor Blood Chemical Blood Donor Blood Bovine Substitute Donor Blood GM Substitute

Mean 6.7 2.8 5.5 3.7 3.8 2.6 4.0 2.3 4.0 2.8 4.1 2.9

SD 1.0 1.6 1.6 1.7 1.7 1.6 1.8 1.4 1.7 1.4 1.7 1.5

Mean Difference 3.9

T-test 20.8***

1.8

11.8***

1.2

7.9***

1.7

12.4***

1.2

8.7***

1.3

7.4***

Note: N = 127 – 135, * p < 0.05, ** p < .01, *** p < 0.001

For blood substitutes, it is noteworthy that, in all cases, the use of donor blood for transfusion was not itself perceived as extremely natural. Indeed, the mean score of 4 for this

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option on a 7-point scale places it approximately in the middle of the rating scale. Thus, while the product (i.e. the blood) may be natural, the intervention (i.e. transfusion) is not. Furthermore, this procedure is perceived to be more unnatural when the material for transfusion is modified, as in the case of the different blood substitutes offered. There were also minor differences in mean ratings between donor blood and the blood substitutes. That is, the mean difference between donor blood and the two biological manipulations (bovine and GM haemoglobin) is smaller than that for the blood substitute derived from chemicals. It appears that just the biological aspect was related to perceptions of naturalness.

Associations Between Naturalness and Risk Table 3 contains the results from Principal Components Analysis followed by a Varimax Rotation of the ratings of naturalness and riskiness of the 12 treatments (KMO = 0.77, Bartlett’s test of sphericity = 1120.8, p < 0.0001). Three factors were extracted on the basis of the Scree test and Parallel Analysis that accounted for 47% of the variance. The first point to emphasise is that for the more riskier and invasive types of treatments (i.e. transplants, transfusions) loaded on separate factors for risk and naturalness. Interestingly, ratings of risk and naturalness for the GM apple load with the more risky medical procedures. However, for the less risky and more topical (non-invasive) procedures, risk and naturalness were correlated. That is, higher risk ratings were related to lower ratings of naturalness. Thus, the extent to which naturalness and risk are correlated depends on the nature of the treatment or hazard being studied: associations were only seen for low risk, non-invasive procedures. This finding indicates that perceived naturalness for invasive procedures, in general, is unrelated to perceived risk. Both donor blood and the blood substitutes load with the medical invasive procedures. To examine if risk and naturalness were separate for blood products specifically, correlations were calculated between the rating of risk and naturalness for each of the blood products (donor blood and the three substitutes). There were no significant associations between the ratings of risk and naturalness (rs range from -0.13 to 0.11). This confirmed that, for the blood substitutes, risk and naturalness ratings were unrelated.

CONCLUSIONS AND IMPLICATIONS Several conclusions may be drawn from this research with respect to donor blood and blood substitutes for transfusion. First, the degree of naturalness may not be related to the biological character of the modification, with all substitutes perceived as significantly less natural than donor blood. Secondly, the degree of naturalness was not related to the degree of risk associated with a number of transfusion options, and this may reflect their invasive nature. Finally, transfusion with donor blood is itself not perceived as being natural. The theoretical and practical implications of these findings merit further discussion.

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Table 3 Varimax Rotated Factor Matrix. Treatment Naturalness GM Blood Naturalness GM insulin Naturalness Mechanical heart Naturalness Donor Heart Naturalness Bovine Substitute Naturalness Steroid cream Naturalness donor blood Naturalness Chemical Blood Naturalness GM apple Risk GM blood Risk GM insulin Risk Bovine Blood Risk Chemical Blood Risk Donated Blood Risk Mechanical Heart Risk Donor Heart Risk GM Apple Naturalness apple Risk Chamomile Naturalness Chamomile Risk Apple Naturalness Yoghurt Risk Yoghurt Risk Steroid Cream

NaturalnessMedical/Invasive 0.78 0.77 0.76 0.76 0.75 0.75 0.74 0.72 0.58 0.04 0.11 0.02 0.13 0.08 0.07 -0.14 -0.07 -0.12 0.14 0.36 0.03 0.48 0.10 0.00

RiskMedical/Invasive 0.04 -0.16 0.14 0.08 -0.06 0.05 0.06 -0.09 -0.15 0.79 0.69 0.67 0.58 0.54 0.53 0.52 0.35 0.12 0.30 0.18 0.03 00.10 0.23 0.28

Naturalness/ Risk–Topical -0.01 0.00 0.13 -0.01 0.04 -0.18 -0.08 0.06 0.20 0.08 0.09 0.19 0.19 0.07 -0.18 -0.15 0.05 -0.70 0.66 -0.65 0.60 -0.55 0.51 0.47

Note: Transfusion option in italics, loadings on target factors in bold and cross loadings underlined.

Dimensions of Risk and Naturalness Previous related work has shown that, for food related hazards, perceived naturalness may be considered a dimension of risk (Fife-Schaw et al., 1996; Breakwell, 2000). However, these earlier investigations focused primarily on food products that are non-invasive and the results from the present study show that whether or not a hazard is invasive or non-invasive impacts on the extent to which risk and naturalness are correlated. This is important because it means that it is not possible to generalise from one type or category of hazard to another. This has implications for the development of risk communication, as discussed later. It is pertinent to ask why risk and naturalness may be unrelated for invasive procedures but related for non-invasive ones. It may be that the nature of the invasive procedure is very psychologically salient. That is, by their nature these procedures are life saving and rare.

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Therefore, when a person is faced with a decision about having such a treatment, the extent of naturalness or other decision making dimensions are likely to be less salient. However, for more mundane everyday experiences, that are not either life threatening or saving, a person may wish to use as much information as possible to reach a decision. Therefore, for noninvasive procedures these dimensions are linked. Invasive procedures also probably have a very strong visual component. People are used to seeing images of transplantation and transfusion in the media and such images are often very powerful. Consequently, a person’s thinking may be so strongly focused that they just think about the procedure and not about other issues pertaining to it, such as, for example, where the products used came from (Loewenstein et al., 2001).

Risk Communication The present results provide information to inform how physicians’ dialogue and public information might be presented to patients and the public about blood substitutes. Such information could, for example, be used to design new decision aids to assist patients in making informed choices between transfusion options that might be available to them. Related materials have been produced by others to help patients decide whether to pre-donate their own blood for autologous transfusion during heart surgery (Grant et al., 2001). It was reported that such a decision aid improved knowledge and risk perceptions of blood donation and transfusion and, importantly, increased the percentage of patients willing to accept autologous transfusion. A further advantage of using decision aids is that they offer a multiformat approach for communicating information on healthcare issues that appeal to people with different educational backgrounds. Indeed, Lee and Mehta (2003) evaluated the impact of a visual risk communication tool on knowledge and perception of risk associated with blood transfusion among the Canadian public. Risk communication with both written and visual presentational formats increased knowledge of transfusion risk and decreased the perceived dread and severity of the transfusion risk. However, neither format changed the perceived knowledge and control of transfusion risk, nor the perceived benefit of transfusion. The authors recognized that risk communication materials using a multi-format approach may provide added value to patients. One important finding from the present research was that transfusion with donated blood (that is, the natural product) is itself not perceived as natural, in the same way that, for example, heart valves transplants are not. This information could be used to encourage people to think about transfusion as a life saving procedure (i.e. a transplant) and, as with the mechanical heart valve, focus on how some of the basic mechanical functions of blood can be mimicked. While these results show that, for transfusion options, naturalness and risk are not related, the data on naturalness does highlight ways in which that information can be used to communicate with people on the uses and characteristics of blood substitutes. One issue about communicating information on blood substitutes, is to consider renaming this general category of products. Blood for people has often powerful social and emotional connotations, pertaining as it does to the source of life (in literature and religion), death (infections), family/social relations (‘blood feud’, ‘bad blood’, ‘blood is thicker than

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water’) and mystical properties (vampires, rejuvenation). Blood substitutes have none of these links, since they are technically simply temporary oxygen carriers. Because the word blood carries so many cultural and social connotations, some of which may lead people to be worry about receiving possible substitutes, re-naming should be considered. There is, in fact, lengthy and on-going debate within the ‘blood substitutes’ community about the terminology that should be used to accurately describe these materials, with alternatives such as ‘red cell substitutes’, ‘artificial oxygen carriers’, ‘anti-hypoxic drugs’ and ‘oxygen therapeutics’ having been proposed as alternatives (Lowe, 2003). The latter currently attracts the most support but it could be argued that this phrase is too technical for the general public to understand what the products actually do. Nevertheless, all of the suggested alternative descriptions imply that the treatment is one that is designed to enhance specific blood functions, but not to be a substitute for it. It is suggested that, in order to enhance public understanding that these products, they could be referred to as ‘blood oxygen boosters’ or ‘blood oxygen enhancers’. This would emphasise that they are boosting a natural process, like antibiotics they are helping the body’s natural processes. It is predicted, from the results reported here and elsewhere, that such a change in terminology would lead people to perceive the substitutes as less risky and more of a supplement to the natural blood system. This is, of course, an empirical question, but one that must be answered if blood substitutes and to become more widely accepted in transfusion medicine. The present results also show that perceptions of risk are not related to naturalness for transfusion options. This highlights the importance of studying risk perception, specifically for transfusion medicine rather than relying on generalising from other areas of risk perception (e.g. GM food), when developing communication strategies. Again the risk data showed that blood transfusion options were seen as similar to other invasive procedures and, as such, the points of comparison suggested above with respect to naturalness are also pertinent here. Finally, it will be important to consider other possible emotional underpinnings of perceived risk in relation to blood products. For example, the potential disgust reactions to certain types of blood substitutes now require investigation as such feelings are potentially potent drivers of risk-judgements and acceptance of these technologies.

ACKNOWLEDGEMENT This work is part of the Euroblood Substitutes project funded by the European Commission.

REFERENCES Alayash, A.I. (2004) Oxygen therapeutics: can we tame hemoglobin? Nature Reviews, 3, 152-159. Breakwell, GM. Risk communication: factors affecting impact. British Medical Bulletin, 56, 110-120.

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Buehler, P.W. and Alayash, A.I. (2004) Toxicities of hemoglobin solutions: in search of invitro and in-vivo model systems. Transfusion, 44, 1516-1530. Chang, T.M.S. (2003) Future generations of red blood cell substitutes. Journal of Internal Medicine, 253, 527-535. Chang, T.M.S. (2004) Hemoglobin-based red blood cell substitutes. Artificial Organs, 28, 789-794. Chang, T.M.S. (2005) Therapeutic applications of polymeric artificial cells. Nature Reviews Drug Discovery, 4, 221-235. Farrell, K., Ferguson, E., James, V. and Lowe, K.C. (2001) Confidence in the safety of blood for transfusion: the effect of message framing. Transfusion, 41, 1335-134. Ferguson, E. and Bibby, P.A. (2002) Predicting future blood donor returns: past behavior, intentions and observer effects. Health Psychology, 21, 513-518. Ferguson, E. (1996) Predictors of future behaviour: A review of the psychological literature on blood donation. British Journal of Health Psychology, 1, 287-308 Ferguson, E., Farrell, K., Lowe, K.C. and James, V. (2001) Perception of risk of blood transfusion: knowledge, group membership and perceived control. Transfusion Medicine, 11, 129-135. Ferguson, E., Leaviss, J., Townsend, E., Fleming, P. and Lowe, K.C. (2005) Perceived safety of donor blood and blood substitutes for transfusion: the role of informational frame, patient groups and stress appraisals. Transfusion Medicine, in press. Fife-Schaw, C. and Rowe, G. (1996) Public perceptions of food related hazards: a psychometric study. Risk Analysis, 16, 487-500. Finucane, M.L., Slovic, P. and Mertz, C.K. (2000) Public perception of the risk of blood transfusion. Transfusion, 40, 1017-1022. Frumento, R.J., Mongero, L., Naka, Y. and Bennett-Guerrero, E. (2002) Preserved gastric tonometric variables in cardiac surgical patients administered intravenous perflubron emulsion. Anesthesia and Analgesia, 94, 809-814. Gaskell, G., Bauer, M.W., Durant, J. and Allum, N.C. (1999) Worlds apart? The reception of genetically modified foods in Europe and the US. Science, 285, 384-387. Grant, F.C., Laupacis, A., O’Connor, A.M., Rubens, F. and Robblee, J. (2001) Evaluation of a decision aid for patients considering autologous blood donation before open-heart surgery. Canadian Medical Association Journal, 164, 1139-1144. Lee, D.H., Mehta, M.D. and James, P.D. (2003) Differences in the perception of blood transfusion risk between laypeople and physicians. Transfusion, 43, 772-778. Lee, D.H., Mehta, M.D. (2003) Evaluation of a visual risk communication tool: effects on knowledge and perception of blood transfusion risk. Transfusion, 43, 779-787. Loewenstein, G. F., Weber, E. U., Hsee, C. K., and Welch, N. (2001) Risk as feelings. Psychological Bulletin, 127, 267-286. Lowe, K.C. and Ferguson, E. (2003) Benefit and risk perceptions in transfusion medicine: blood and blood substitutes. Journal of Internal Medicine, 253, 498-507. Lowe, K.C. (2003) Engineering blood: synthetic substitutes from fluorinated compounds. Tissue Engineering, 9, 389-399. Lowe, K.C. (2004) Blood substitutes. Education in Chemistry, 41, 95-97.

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Lowe, K.C., Farrell, K., Ferguson, E.M.P. and James, V. (2001) Current perceived risks of transfusion in the UK and relevance to the future acceptance of blood substitutes. Artificial Cells, Blood Substitutes, and Immobilization Biotechnology, 29, 179-189. Matheson, B., Raznaka, A., Kwansa, H. and Bucci, E. (2002) Vascular response to infusions of a nonextravasating hemoglobin polymer. Journal of Applied Physiology, 93, 14791486. Niiler, E. (2002) Setbacks for blood substitute companies. Nature Biotechnology, 20, 962963. Siegrist, M. (2000) The influence of trust and perceptions of risk and benefits on the acceptance of gene technology. Risk Analysis, 20, 195-203. Spahn, D.R., Waschke, K.F., Standl, T., Motsch, J., Van Huynegem, L., Welte, M., Gombotz, H., Coriat, P., Verkh, L., Faithfull, S. and Keipert, P. (2002) Use of perflubron emulsion to decrease allogeneic blood transfusion in high-blood-loss non-cardiac surgery. Anesthesiology, 97, 1338-1349. Tenbult, P., de Vries NK., Dreezens, E., and Martijn, C. (2005) Perceived naturalness and acceptance of genetically modified food. Appetite, 45, 47-50. Townsend, E. and Campbell, S. (2004) Psychological determinants of willingness to taste and purchase genetically modified food. Risk Analysis., 24, 1385-1393. Townsend, E. Clarke, D.D. and Travis, B. (2004) Effects of context and feelings on perceptions of genetically modified food. Risk Analysis, 24, 1369-1384. Tremper, K.K. (2002) Perfluorochemical “red cell substitutes”: the continued search for an indication. Anesthesiology, 97, 1333-1334. Vandegriff, K.D., Malavalli, A., Woodridge, J., Lohman, J. and Winslow, R. (2003) MP4, a new nonvasoactive PEG-Hb conjugate. Transfusion, 43, 509-516. Verhoog, H. (2003). Naturalness and the genetic modification of animals. Trends in Biotechnology, 21, 294-297. Winslow, R.M. (2003) Current status of blood substitute research: towards a new paradigm. Journal of Internal Medicine, 253, 508-517.

In: New Developments in Blood Transfusion Research ISBN 1-59454-962-1 Editor: Brian R. Peterson, pp. 77-102 © 2006 Nova Science Publishers, Inc.

Chapter V

PRION BIOLOGY AND THE REDUCTION OF THE RISK OF TRANSFUSION-TRANSMITTED VARIANT CREUTZFELDT-JAKOB DISEASE (VCJD) BY BLOOD FILTRATION Joseph S. Cervia,∗,1,2 Samuel O. Sowemimo-Coker,1 Girolamo A. Ortolano1, Jeffrey Schaffer,1 Karen Wilkins1 and Samuel T. Wortham1 1

Pall Corporation, 2Albert Einstein College of Medicine, New York

ABSTRACT Pathogenic prions are infectious proteins that are believed to be responsible for a variety of progressive neurodegenerative diseases. These disorders are referred to as transmissible spongiform encephalopathies (TSE) since the brains of infected individuals have been observed to display a sponge-like morphology upon post-mortem examination. TSEs in humans and other species are invariably fatal. During the 1980’s it became apparent that some cattle in the United Kingdom had become infected by a form of TSE, which became known as bovine spongiform encephalopathy (BSE) or “mad cow” disease. By 1996, it was recognized that ingestion of beef from infected cattle could result in a TSE in humans known as variant Creutzfeldt-Jakob disease (vCJD). Recent incidents of probable transfusion-transmitted vCJD have raised concerns about the safety of the blood supply. The relatively long latency from the time of infection to the onset of symptomatic vCJD as well as the lack of sensitive and specific ante-mortem tests for vCJD, increase the risk that asymptomatic, infected individuals may become blood donors. Until now, donor deferral has been the strategy employed to ∗

Correspondence concerning this article should be addressed to: Joseph S. Cervia, M.D., Medical Director, Pall Corporation, 2200 Northern Boulevard, East Hills, NY 11548

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Joseph S. Cervia, Samuel O. Sowemimo-Coker, Girolamo A. Ortolano et al. reduce this risk. Nevertheless, this strategy may be unreliable, and threatens blood availability. Leukoreduction has also been helpful by reducing cell-associated infectious prion, which has been reported to account for up to 42% of infectivity in blood. Unique chemical characteristics of prion surfaces have been determined and proprietary affinity filtration properties developed. These have been successfully adapted to existing high efficiency blood filter matrices for specific reduction of prions present in blood components for transfusion. Validation studies described herein demonstrate that such filtration reduces prions by a factor of 2.9 log or 99.9% when quantified by Western blot analysis while maintaining existing standards for leukoreduction, post-storage hemolysis, and other indices of bio- and hemo-compatability. Definitive bioassay and infectivity studies are ongoing, and are expected to be available by the end of 2005. This prion reduction technology is currently being introduced for use with red cells for transfusion in Europe, and will be available worldwide pending each nation’s regulatory review process and manufacturing capacity expansion.

Keywords: prion, blood, filtration,transfusion, vCJD

INTRODUCTION Prion diseases are a growing concern.[1] Prions are conformational alterations of normally occurring proteins.[2] The normal proteins are concentrated in the brain but can be found in peripheral tissues at lower levels.[3] Blood cells are associated with prions also.[4] Although B-lymphocytes were thought to contain the highest levels among the cellular elements of blood prompting the universal adoption of leukocyte reduction of transfused blood products in the UK,[5] recent evidence for the presence of normal cellular prion (PrPC) on platelets has also emerged.[6] When these proteins undergo abnormal folding they cause transmissible spongiform encephalopathies (TSE) in humans[7,8] and animals.[9,10] TSEs are characterized by progressive neurologic damage. In these syndromes, brain tissue develops the appearance of sponge upon histopathologic examination. These disorders have been universally fatal. The names of the prion diseases change with the species afflicted; and, examples include cervids (deer and elk where the disease is referred to as chronic wasting disease, CWD), mink (mink spongiform encephalopathy; MSE), sheep (scrapie), cattle (bovine or BSE) and humans. The disease in humans can take several forms including kuru (known to affect the Indians of Papua, New Guinea), Creutzfeldt-Jacob disease (CJD) which can derive sporadically (sCJD), after exposure with contaminated instruments or tissue such as corneal or dura matter transplants or human growth hormone where the disease is referred to as iatrogenic (iCJD) or following the ingestion of meat from afflicted cattle where the symptoms vary enough from sCJD that the name variant or vCJD has been ascribed. The latter, vCJD, has recently been shown to very likely come about from the transfusion of blood from a asymptomatic infected donors.[11,12] The history of the prion as a pathogenic agent warranted a Nobel prize awarded to Dr. Stanley Prusiner for his insight into, what must have been at the time, a heretic view that a disease could be caused by a replicating protein.[13,14] Until then, infectious diseases were only known to occur by organisms or molecules involving nucleic acid reproduction. In fact,

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the nature of the protein in its normal state, referred to as the PrPC isomer (because it was initially discovered as a cell-associated protein), remains poorly understood. The pathogenic protein is identical in amino acid composition to PrPC. It is believed that PrPC is transformed by the infectious agent and initiates a cascade forming aggregates of the pathogenic protein.[15] Although many investigators accept the idea of an infectious protein may exist, Prusiner and co-workers continue to challenge their own findings, and have thus far, failed to reveal a role for nucleic acids in prion disease processes.[16]

PRION STRUCTURE AND FUNCTION The normal function of PrPC is not well understood. Genetically altered mice having the gene sequence for PrPC removed (so-called knockout mice) can reproduce and appear to grow normally. There are some animal studies suggesting PrPC may be required for sleep regulation and normal neural function. Interestingly, some forms of prion disease have a component of insomnia. The syndrome fatal familial insomnia is an example.[17] There is some evidence that PrPC may play a role as an anti-oxidant.[18,19] There are data to support an increase in oxidants like hydrogen peroxide in those afflicted with prion disease and whether this is related to the cause of symptoms or simply an epiphenomenon awaits resolution.[20] Behaviorally, evidence suggests that mice deficient in the prion gene handle stress differently than do wild-type mice.[21] The primary structure of PrPC is known in humans and many animals. In general the protein is about 254 amino acids in length varying slightly in number of amino acids across species. There are two sulfide-containing amino acids at positions 178 and 213 which form a disulfide bond that restricts movement of the protein in this region. PrPC undergoes posttranslational modification whereby the first 23 and last 22 amino acids are removed and a chain approximating 210 amino acids is modified further in two important ways. A glycophosphatidylinositol (GPI) moiety on the C-terminus occurs; and, this is used to anchor the molecule to cells. This GPI moiety may play a role in the manifestation of prion disease.[22] Within the molecule, however, two asparagine sites at positions 181 and 197 in the human (and not far away from these sites in animals) undergo fairly extensive glycosylation adding about 3 kilodaltons to the molecular mass for each site. These glycosylations appear to have considerable implications for the manifestations of disease[23] and some believe they contribute to the species barrier. A recent report offers the view that pathogenic forms of the protein are often presented more in the unglycosylated form.[24] Others suggest the proportional distribution of glycoforms is species-specific.[25] However, the determinant of clinical illness appears to lie in the composition of amino acids that comprise PrPC. Polymorphism within the human PrPC gene (PRNP) predispose to vCJD and the most notable is codon 129 homozygosity for methionine (Met).[26,27] Transfusion-transmitted vCJD in a Met homozygote has been reported,[28] but also a suspected case occurred in a Met/valine (Val) heterozygote.[29] Inasmuch as prions affect nervous tissue, it should be of little surprise that nervous tissue function appears related to polymorphisms within the PRNP gene too. For example, recent evidence shows long term

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memory is more robust in Met/Met and Met/Val alleles at codon 129 than seen among those homozygous for Val.[30] Amino acid sequences are considered determinants of the species barrier. The species barrier relates to the observation that pathogenic prion is not comparably transmissible from one species to another. For example, it is thought that scrapie-infected sheep elaborating PrPSc are the source of prions leading to the elaboration of PrPBSE in cattle that can transmit disease to humans manifested as vCJD through the ingestion of beef. Sheep to human TSE has not been reported.[31] Laboratory studies show the CWD cannot be easily transmitted to humans; because, humanized transgenic mice do not develop disease, yet cervidized mice do.[32] And in a recent study of CWD prion injected intracerebrally into cattle, there was evidence of prion elaboration in brain tissue, though recipient cattle were not symptomatic.[33] Although there is considerable support for the amino acid sequence serving as a determinant to the species barrier, it should be noted that the origin of the infectious agent causing vCJD is not entirely resolved. In fact, it has been suggested that vCJD may have come from human corpses from India that contaminated feed for livestock and in turn infected humans.[34] The very thought of such a mechanism emphasizes the enigma surrounding the pathogenesis of vCJD. Recent work shows a shift in the focus from the polymorphisms in the PRNP gene to polymorphisms that exist in the PRNP gene promoter.[35] Disease transmission is thought to occur when pathogenic prion aligns with normal prion protein and induces a conformational change resulting in the pathogenic isoform. This serves to seed the continued conversion of PrPC to the pathogenic form. Structurally, the PrPC is rich in alpha helices with a low percentage of beta-sheet formation. After infections, the pathogenic form contains a much greater percentage of beta-sheet formation and lower amount of alpha helices.[36] The pathogenic form is relatively insoluble in aqueous media and is relatively resistant to proteinase-K. The hydrophobic palindrome within the prion molecule PrP (112-AGAAAAGA-119), appears critical to the progression of proteinase-K resistant prion.[37] Once the process is initiated, aggregates of the pathogenic prion form. In a recent study, these aggregates were partially disrupted, segregated based upon size and each size range tested for infectivity and the ability of fragments to convert the nonpathogenic protein to the pathogenic isomer. There is an optimal size range of aggregate to cause disease with protein oligomers ranging from 14-28 molecule aggregates or 300-600 kDa conferring the greatest infectivity.[38]

BRIEF HISTORY OF TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHIES (TSE) In 1759, a bizarre affliction of sheep was described. Affected sheep would appear to scrape their sides along the fences of their pens. Thus the condition was commonly referred to as ‘scrapie’. Although experimental transmissibility of the scrapie agent was not reported until 1936, post mortem examination revealed that the brains of infected animals were sponge-like. Later, the term ‘transmissible spongiform encephalopathy’ was applied to

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describe a common and recurring characteristic of this type of disease in sheep and other animals. Early in the 1920s, two separate reports appeared describing a progressive neurodegenerative disease in humans presenting with central nervous system dysfunctions reminiscent of those seen in scrapie. The first was reported by Hans Creutzfeldt (1920) [39]; and, the second by Anton Jakob (1921) [40]. Thus, the constellation of symptoms, which they described, became known as Creutzfeldt-Jakob disease (CJD). In 1966, Alder and co-workers provided the first clue to demonstrate the unique pathogenesis associated with TSE. They showed that chemical manipulations resulting in the destruction of nucleic acids, known to be the infective component of viruses, did not alter prion infectivity. It was Pruisner, however, who is credited with providing overwhelming evidence suggestive of the radical idea that a protein was the infective agent – an effort he published in 1982 and for which he was subsequently awarded a Nobel Prize.[41] Another form of human TSE referred to as kuru reached epidemic proportions with the practice of ritual cannibalism among the Fore people of Papua New Guinea uncovered in the 1950’s. As this practice subsided, the incidence of kuru among these people has decreased. The cultural practice reflected a devotion to deceased ancestors by the ingestion of their bodies. Since brain matter was predominantly consumed by the women and children, while the men consumed muscle tissue, women and young people were disproportionately afflicted with kuru. This finding further implicated neural tissue as a major vehicle for the transmission of TSE.[42] In the mid 1980s, the United Kingdom experienced an outbreak of TSE among cattle. Symptoms included awkward movements and abnormal gait with exaggerated behaviors characterized by some as madness – hence the term ‘mad cow disease’ was coined for bovine spongiform encephalopathy (BSE). At first, it was thought that scrapie was passed to cattle because they were fed sheepderived meat and bone meal. In 1989, selected parts of slaughtered cattle (brain, spinal cord, intestine, thymus, tonsils and spleen collectively referred to as Specifed Bovine Offal or SBO that had been used in feed) were banned from becoming byproducts to be fed to cattle thus preventing prions from entering the human food chain. As would later be discovered however, human infection with the agent of BSE (termed variant Creutzfeldt-Jakob Disease, vCJD) had already occurred.[43] More recently, there has been some concern over the rising incidence of prion disease in other animal species, such as deer and elk (called ‘chronic wasting disease’ or CWD).[44] It is also appreciated that many animals are capable of acquiring TSEs including cats (feline or FSE), mink (transmissible mink encephalopathy or TME), and a number of exotic zoo animals as well.

CLINICAL FEATURES OF HUMAN TSE Symptoms characterizing different clinical TSE syndromes vary; however, the brains of afflicted individuals show remarkable similarity upon gross inspection. Ordinarily, the brain degenerates so visibly as to appear, upon gross inspection and histopathologic examination,

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like a sponge. Damage is confined largely to the gray matter. This is often accompanied, as in vCJD with abundant amyloid plaque formation.[45] Amyloid plaque is a proteinaceous translucent substance with a waxy consistency. It is made of protein in combination with polysaccharides and may be associated with Alzheimer’s disease and other disorders. It has been noted that polymorphisms in codon 129 of the human prion coding region (i.e. methionine or met homozygosity, versus valine or val homozygosity, versus met/val heterozygosity) along with the types of bands present on Western blot (i.e. type 1, 21 kDa, versus type 2, 19 kDa) may be used in a system to characterize the clinical and histopathological manifestations of various subtypes of spontaneously occurring or sporadic CJD (sCJD). Occurring in roughly one in one million persons, sCJD is generally characterized by varying degrees of awareness or cognitive impairment and psychosis, along with a lack of coordination or ataxia, visual field defects, and other neurological manifestations, with onset most commonly in the seventh decade of life. Subtypes of sCJD include one characterized by homozygosity for methionine and Type 1 (19 kDa) migration on Western blot analysis, referred to as MM1. Another subtype is characterized by homozygosity for valine and Type 2 (21 kDa) migration is denoted as VV2. Finally, heterozygosity at codon 129 with Type 1 migration, referred to as MV1, completes the 3 most frequently encountered subtypes. These subtypes run their courses to a fatal outcome in a matter of 4-6 months, while other subtypes may proceed more gradually over 15-17 months on average.[46] Some prion diseases appear to occur on a familial basis. Familial CJD (fCJD) is very similar to sCJD in terms of its clinical and histopathological manifestations. Familial fatal insomnia (fFI) is another such disorder characterized by intractable insomnia, muscle twitches or myoclonus, and autonomic dysfunction with a mean age at onset of 49 years and a duration of 11-23 months. Finally, a familial prion disease due to a PRNP open reading frame mutation is known as Gerstmann-Straussler-Scheinker Syndrome (GSS). It is characterized by a gradual progression of cerebellar ataxia, dementia, spastic paraparesis or weakness of the legs, and extrapyramidal signs over 5-6 years beginning in the 5th decade. Extrapyramidal signs include involuntary movements of the mouth, lips and tongue as well as tremors, body restlessness or rigidity among other things. Most importantly, a number of prion diseases have a recognized mode of transmission. Kuru was one of the first to be uncovered. The Fore people, indigenous to Papua New Guinea, gave this disorder its name, which means, “to shiver.” Transmitted by ritual cannibalism, and characterized by progressive and ultimately fatal cerebellar ataxia over 6-9 months, onset was usually in the 2nd to 4th decades. It is possible to contract prion disease from contaminated surgical instruments as well as biological preparations and these are referred to as iatrogenic CJD (iCJD). Depending upon the source of infection, the onset varies among those contracting iCJD such as for neurosurgical instrumentation (with onset in 12-28 months), human growth hormone (50-450 months), corneal transplantation (16-320 months), dural patches (18-216 months), and human gonadotropin (144-192 months).[46] Of greatest concern, however, is vCJD, which occurs years after the ingestion of meat products containing traces of neural tissue from cows infected with BSE. Despite this long latency period, there has been a mean age at onset of 28 years. vCJD is manifested by

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psychiatric and sensory disturbances, and dementia that progresses to its inexorably fatal outcome in an average of just over one year. It is believed that neuroinvasion of pathogenic prion is facilitated by white blood cells, particularly B-lymphocytes, which are present in intestinal Peyer’s patches and in areas of inflammation.[47] Recently, two cases of vCJD transmitted by blood transfusion have been reported.[48] This has raised concerns about protecting the blood supply from prion diseases, which are, by nature and as the name of the family of diseases implies, transmissible. In addition, though a number of BSE diagnostic tests are in various stages of development, screening methods for prion detection in blood have thus far been elusive. Adding to the level of concern is the fact that the incidence of progressive neurological diseases, such as Alzheimer’s Disease, have increased dramatically in recent years, even when adjusted for our increasing population of elderly individuals. These progressive disorders are diagnosed almost exclusively based upon the clinical observation of signs and symptoms that overlap with those of CJD. This may have important implications in the potential for underreporting of prion diseases. Recently, histopathological examinations of the brains of patients thought to have died from Alzheimer’s disease have revealed that some of those patients actually died of prion disease.[48,49,50,51,52,53,54,55] Therefore, TSE may be pose a more prevalent risk to the blood donor pool than has previously been recognized.

CONCERNS ABOUT TRANSFUSIONTRANSMITTED VCJD (TT-VCJD) Factors Indicating a Risk Providing safe blood for transfusion remains a challenge. Despite advances in preventing transmission of hepatitis B, hepatitis C, HIV, and transfusion-transmitted bacterial infection, other risks such as variant Creutzfeldt Jakob Disease (vCJD) remain worrisome. Considering the lengthy, asymptomatic incubation period of vCJD in people, no one can accurately determine the magnitude of the next possible outbreak. Individuals who will develop CJD can remain without symptoms for decades and then progress rapidly to dementia, severe loss of coordination and death. Due to lack of sensitive and specific ante-mortem tests for vCJD, we also do not know how many people may be harboring the infectious prions and also donating blood. The risk of transmission of CJD through blood transfusion has not been definitively established, but it cannot be completely ruled out. Concerns about the potential transfusion transmissibility of vCJD emerged soon after the discovery of the disease in 1996.[56] These concerns prompted three large UK recalls of blood products that originated from individuals who later developed vCJD.[57] The disease has a long incubation period, and studies and surveillance programs have not lasted long enough or included sufficient numbers of cases to conclude that CJD is transmitted through blood transfusion.[58] Further, the transmissibility of CJD cannot be supported by look-back investigations and studies on CJD mortality trends.[59,60] It has been speculated that the transmissibility of vCJD through

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blood may be greater than that of classic CJD.[61] The UK has reported two presumptive transfusion transmitted cases of vCJD. (Table 1) More recently, an alarming situation occurred in August 2005. About 100 people who donated blood in the UK were told they may be carriers of vCJD. Letters were sent to blood donors who may have transmitted the disease to three people who later developed vCJD. However, it is not known whether the source of vCJD in these three people who later developed the disease is related to the blood they received. David Salter, MD, acting chief medical officer for Wales, said, “Until a reliable blood screening test becomes available, it is sensible to proceed with highly precautionary measures such as this to rule out any possibility of onward transmission of the disease.” (Source: Western Mail, August 22, 2005) Table 1 Scientific Evidence for Possible Transfusion of vCJD. Level of evidence Cohort studies Case-control studies Ecological evidence Case series Case reports

Animal evidence

Biological models

Evidence None None None None Case report of vCJD infection identified at autopsy of patient who received transfusion from donor with preclinical vCJD Human case report of vCJD in recipient of transfusion from donor with preclinical vCJD In primates, efficiency of BSE transmission via transfusion shown to be at least as effective as by oral route Sheep-to-sheep transfusion transmission shown in animal models of preclinical and clinical disease. Animal-model evidence of transfuion transmission of TSE Prion identified in lymphoid tissue Theoretical transfusion-transmission risk suspected on basis of demontration of oral of prion

Year 2004

2004[70] 2004[71]

2000[72] 2002[73] 1998[74] 1997[65] 1996[75]

Levels of infectious prions in blood are generally too minute to be detected with biochemical methods. However if prions could be identified in the blood, it might be possible to treat the condition at an early stage, before permanent brain damage occurs. The problem with current tests is they have low sensitivity. They can detect animals that are symptomatic or about to manifest signs of the disease; however, the clinically asymptomatic latency period from the time of infection to the time of the development of clinical symptoms can last several years.

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Case Reports of TT-vCJD Variant CJD has killed about 150 people worldwide (Tables 2,3, and 4). Symptoms can take years to develop. In addition to improving blood safety, a practical test could help find infected people and animals before they show symptoms. This is critical because one or two decades from now (when symptoms would be expected to manifest themselves) it could be too late to adequately respond to the ensuing health crisis. If we know today there are many people infected, biotech companies will step up efforts to research effective therapies. In a paper published in the August 28th 2005 issue of Nature Medicine, a research team described a new technique called protein misfolding cyclic amplification (PMCA) technology, that can be automated and optimized for high-efficiency amplification of PrPSc (infectious prions). These findings represent the first time that prions have been detected biochemically in blood, offering hope that a noninvasive method for early diagnosis of prion diseases can be developed. Table 2 Reported Mortality Due to vCJD Worldwide. Location United Kingdom France Republic of Ireland Italy Canada United States Japan Total

Deaths 150 6* 2* 1 1* 1* 1* 162

*Three vCJD cases in France and one case of the disease in Canada, the United States, Japan and Ireland are considered to be a result of exposures in the United Kingdom.

In the US, there is a constant monitoring and trending of the incidence of CreutzfeldtJakob disease by the Centers for Disease Control and Prevention (CDC) by analyzing death certificate information from U.S. multiple cause-of-death data compiled by the National Center for Health Statistics, CDC. The average annual CJD death rate in the U.S. has remained relatively stable at about one case per million population per year.

EARLY EFFORTS AT RISK REDUCTION AND THEIR LIMITATIONS Until now, donor deferral has been the strategy employed to reduce risk. Blood centers worldwide have instituted criteria to reject donors who may have been exposed to vCJD. The UK has been the most aggressive in managing the risk, instituting such policies as importing plasma to manufacture fractionated products and deferring donations from some individuals who have received a transfusion.[63,64] At the time that these policies were introduced, the

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risk of vCJD was considered to be theoretical on the basis of the existence of infectious prion in reticuloendothelial tissue, and the demonstration of peripheral transmission via the oral route.[65] Table 3. Creutzfeldt-Jakob Disease in the UK. (By Calendar Year) Referrals of Suspected CJD Year Referrals

Year

Sporadic

Iatrogenic

Familial

GSS

vCJD

1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005* Total Referrals

1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 Total Deaths

28 32 45 37 53 35 40 60 63 62 50 58 72 77 52 35 799

5 1 2 4 1 4 4 6 3 6 1 4 0 5 2 1 49

0 3 5 3 4 2 2 4 3 2 2 3 4 4 3 2 46

0 0 1 2 3 3 4 1 2 0 1 2 1 2 1 1 24

3 10 10 18 15 28 20 17 18 9 2 150

53 75 96 78 118 87 133 162 154 170 178 179 163 162 114 84 2006

Deaths of Definite and Probable CJD Patients Total Deaths 33 36 53 46 61 47 60 81 89 85 82 87 94 106 67 41 1068

*As of September 2005[62] Referrals-a simple count of all the cases that have been referred to the Surveillance Unit for further investigation in the year in question. Sporadic- classic CJD cases with typical EEG and brain pathology. Iatrogenic- where infection with classic CJD has occurred accidentally as the result of a medical procedure. Familial- cases occurring in families associated with mutations in the PrP gene. GSS- Gerstmann-Straussler-Scheinker syndrome-an exceedingly rare autosomal dominant disease, typified by chronic progressive ataxia and terminal dementia.

Table 4 Summary of vCJD cases. Deaths from definite vCJD (confirmed): Deaths from probable vCJD (without neuropathological confirmation): Deaths from probable vCJD (neuropathological confirmation pending): Number of deaths from definite or probable vCJD (as above): Number of definite/probable vCJD cases still alive: Total number of definite or probable vCJD (dead and alive):

108 42 0 150 7 157

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Previous measures taken in the UK to improve the safety of blood in relation to vCJD include the following: • • • •

• •

• •

Since 1998, plasma derivatives, such as clotting factors, have been prepared from plasma imported from the USA; Since October 1999, white blood cells (which may carry a significant risk of transmitting vCJD) have been removed from all blood used for transfusion; In August 2002, it was announced that fresh frozen plasma for treating babies and young children born after 1 January 1996 would be obtained form the USA; On December 16, 2002, the Department of Health completed its purchase of the largest remaining independent US plasma collector, Life Resources Incorporated. This secured long-term supplies of non-UK blood plasma for the benefit of UK patients; In April 2004, individuals who had themselves received a transfusion of whole blood components since January 1980 were excluded from donating blood; The exclusion criteria for blood donation was extended on August 2,2004 to include two new groups, who have received transfusions of whole blood components since 1980: Previously transfused apheresis donors; and Donors who were unsure if they had previously had a blood transfusion.

Canadian Blood Services (CBS) is changing its donor deferral criteria to reflect the latest research on risks to the blood supply. Since September 30, 1999 safeguards have been in place to protect the blood system from the risk of transmission of variant Creutzfeldt-Jakob disease (vCJD). The following changes reflect safeguards the UK, France, and Western Europe have put in place to protect cow and human populations: •

•

•

Donors who have received a blood transfusion or received medical treatment with a product made from blood in the UK, France or Western Europe since January 1, 1980 will now be deferred indefinitely. Previously this deferral was limited to the UK. Donors who have spent a cumulative total of three months or more in France or in the UK between January 1, 1980 and December 31, 1996 will be deferred indefinitely. In the past, donors who have spent a cumulative total of three months or more in France or the UK since January 1980 were deferred. Donors whose cumulative three-month travel period to the UK or France did not occur between January 1, 1980 and December 31, 1996, will again be eligible to donate. The 1996 cut-off date reflects the period between January 1980 and December 1996 when the bovine spongiform encephalopathy epidemic (BSE) was at its peak in the UK and France. Since that time, BSE cases have continued to decline, and BSE monitoring and control mechanisms have been implemented to stop the spread of the disease in the bovine population and thereby decreasing the risk of transmission of vCJD to humans. (Source: Canadian Blood Services press release, August 15, 2005)

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Currently, there is no screening test for the disease, and while blood transfusions have never been definitively shown to transmit any form of the disease, as a precaution the Food and Drug Administration (FDA) prohibits blood donation by individuals who may be at risk. Since the UK has reported two presumptive transfusion transmitted cases of vCJD, FDA blood donor deferral policies seek an optimal balance between vCJD risk reduction and blood supply preservation. These policies are under constant review by FDA as we learn more about vCJD and BSE. The FDA recommends that the following donors be deferred indefinitely due to vCJD risk: • • •

• •

Donors who spent a total of three months or more in the United Kingdom (UK) from the beginning of 1980 through the end of 1996; Donors who have spent a total of five years or more in Europe from 1980 to the present; Current or former US military personnel, civilian military employees and their dependents who resided at US military bases in Northern Europe (Germany, UK, Belgium, and the Netherlands) for a total of six months or more from 1980 through 1990, or elsewhere in Europe (Greece, Turkey, Spain, Portugal, and Italy) from 1980 through 1996. Donors who have received any blood or blood component transfusions in the UK between 1980 and the present; Donors who have injected bovine insulin since 1980, unless it is possible to obtain confirmation that the product was not manufactured after 1980 from cattle in the UK.

The Department of Defense (DoD) has a slightly different policy summarized here. The DoD implemented its set of donor deferral rules in October 2001. All active-duty military personnel, civil service employees, and these two groups’ family members will be deferred indefinitely due to vCJD risk if they are: • • • •

Donors who traveled or resided in the UK for a cumulative total of three months or more at any time from 1980 through the end of 1996; Donors who have received a blood transfusion in the UK at any time from 1980 to the present; Donors who have traveled to or resided anywhere in Europe for a cumulative total of six months or more at any time from 1980 through the end of 1996; Donors who traveled to or resided anywhere in Europe for a cumulative total of five years or more at any time from Jan. 1, 1997, to the present.

Donor deferral has been the strategy to reduce risk; but, it is not very reliable, and at the same time reduces blood availability. In many European countries, public health control measures including elimination of sick animals, bans on specified risk materials and increased BSE surveillance are aimed at preventing contamination of the human food chains. vCJD transmission risk via blood transfusion is being minimized by a number of measures including a ban on blood donation from people that lived for 3 months or more in the UK

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between 1980 and 1996. Universal leukocyte reduction was introduced in the UK as a preventative measure to reduce the risk of transmission of vCJD through blood transfusion.[66,67] However, recent reports from animal studies suggest that leukocyte reduction is only about 42% effective in reducing the risk of transmission of infectious prion, since not all prions found in the blood are cell-associated.[68] New technology that removes both leukocytes and prions are in development and have been shown to prevent the transmission of prion disease in an animal model.[69] In Canada for example, precautionary measures against the theoretical risk of vCJD transmission through transfusion have been implemented. In addition, a universal program of pre-storage leukoreduction implemented in 1999 may play an important role in further reducing the theoretical risk.

DEVELOPMENT OF A FILTER FOR REMOVING INFECTIOUS PRIONS FROM RED CELL CONCENTRATES There are neither diagnostic tests for the detection of infectious prion in blood of potential blood donors who are asymptomatic for the disease, nor can infectious prions be inactivated or destroyed by currently available technologies without destroying the essential therapeutic product. To deal with these challenges, several precautionary measures have been introduced in many countries to reduce the risk of transmission of vCJD through blood transfusion including donor deferral and, in the UK, the implementation of universal leukoreduction for all transfused blood products[66,67]. Since leukocytes have been identified as the main cells that are associated with prion infectivity[76,77], removal of leukocytes is a prudent step to minimize the risk of transmission of vCJD. However, there is evidence to indicate that some infectivity present in blood may not be removed with the current generation of leukocyte reduction filters[68]. To address the limitation of the current leukocyte reduction filters, a new filter has been developed specifically to remove infectious prions from red cell concentrate (RCC), which remains the most widely transfused blood component. The scientific basis for the design of this new filter relies on the distinct differences in the biochemical and biophysical properties between the non-pathogenic cellular prion (PrPC) and the pathogenic isoform (PrPSc). Although the two conformers share the same primary amino acid sequence, PrPSc can be distinguished biochemically from PrPC by its resistance to digestion by proteinase K (PK) enzyme, and insolubility in nondenaturing detergents.[78] Fourier-transformed infrared spectroscopy (FTIR) and circular dichroism (CD) has also shown that PrPC had a high (about 42%) alpha-helical content, and was almost devoid (3%) of beta-sheet secondary structure. In contrast, PrPSc exhibited a higher amount of beta-sheet (43%), and decreased levels of alpha helix (32%)[79]. Therefore, these differences in the secondary structures of PrPC and PrPSC may alter: (1) the distribution of the negatively and positively charged residues leading to significant modification in electrostatic interaction; (2) the intermolecular recognition by changing surface hydrogen bonding patterns; and, (3) the increased hydrophobic interaction (i.e.prion proteins contain hydrophobic domains which play important roles in the interaction of these proteins with different surfaces). In addition, these differences in secondary structure may be responsible for the existence of the majority of PrPSc mainly as aggregates, while PrPC are monomers.[80]

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Therefore, the following two main mechanisms were used to remove infectious prions from RCC[81]: 1. Ion Exchange, Ionic Interaction, and Hydrogen Bonding: Both normal and abnormal prions are removed by cation/anion exchange, non-specific ionic interaction, as well as hydrogen bonding through the amine and hydroxyl groups with the polyester fibers. The Leukocyte Affinity Prion Reduction Filter (LAPRF, Pall Medical) contains polyester fibers that are surface modified with a proprietary chemistry to allow the above reactions to occur. 2. Removal of Cell Associated Prions-Leukocyte Reduction : Since prion infectivity is associated with leukocytes, non-woven polyester fibers are specially constructed with proprietary technology for optimum binding of leukocytes (Figure 1). The main mechanisms utilized in removing prions here involve mechanical trapping and sieving of PrPSc aggregates, PrPSc associated with leukocytes, and other prion containing heterotypic aggregates by the polyester fibers. Surfaces of these fibers are also chemically modified to enhance the binding of the various populations of leukocytes to the fibers through a specific activation-dependent mechanism.

A

B

Figure 1. Electron micrographs of polyester fibers that are configured to enhance the binding and removal of various populations of leukocytes and aggregates.

VALIDATION OF PRION REMOVAL WITH LAPRF The methodologic approach to validate reduction of prion from blood with filters has been described.[82] The following two main methods were used to obtain the PrPSc that was used in validating the performance of LAPRF in terms of removal from RCC: 1. Exogenous Study – PrPSc obtained from homogenized brain tissue; 2. Endogenous Study – PrPSc obtained from peripheral blood.

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Exogenous (Spiking Study) This study is designed to establish the capacity of the filter to remove (scrapie) PrPSc. Scrapie is a progressive neurodegenerative disease of sheep caused by infectious prions that replicate in the lymphoreticular tissue and central nervous system. The agent used in our validation study is the hamster-adapted strain 263K. Although it would be better to use human derived material for our validation study, it is very difficult, indeed almost impossible, to obtain enough brain materials from vCJD patients to conduct the validation study. Therefore, 263K strain of scrapie from infected hamster brain homogenate was used as a source of PrPSc. Prion protein is well conserved with more than 90% homology between human and hamster protein[83,84]. In addition, PrP from different species share similar physicochemical properties, antigenicity and different degrees of resistance to PK digestion[85]. Therefore, these properties of hamster PrPSc support its use as a suitable model for prion clearance studies. In the hamster model, intracerebral injection of high titer of 263K scrapie into Syrian hamsters results in the generation of scrapie in the animals within 60-80 days. During the clinical phase of the prion disease, the hamsters are killed and their brains are removed and homogenized in phosphate buffered saline to prepare 10% (w/v) scrapie infected hamster brain homogenate (SIHBH). Brain-derived material is one of the most widely used preparation for validating processes or methods for specific removal of PrPSc because of the high concentrations of the infectious agent. In the exogenous PrPSc removal study, 10mL of SIHBH were added into a unit (270mL) of RCC in additive solution (SAGM) or citrate phosphate-dextrose with or without adenine (CPDA-1, CPD) anticoagulant. The RCC were filtered at room temperature at a filtration head height of 30 inches. The concentrations of PrPSc in the RCC before and after filtration were measured either with a bioassay or a Western blot assay, using 3F4 as the primary monoclonal antibody, and goat anti mouse IgG conjugated to horse radish peroxidase with a chemiluminiscent substrate (Figure 2). Results of the Western blot assay show that LAPRF removed 2.75 ±0.72 logs of proteinase K resistant infectious prions (PrPres). In the bioassay, the pre-filtration control red cell concentrate samples were diluted with phosphate buffered saline (PBS) 10 fold to produce samples with serial dilutions between 105 and 10-9. The post-filtration red cell concentrates were also serially diluted 10 fold to obtain samples between 10-2 and 10-8. Six hamsters were injected intracerebrally with 40 µL of either pre- or post-filtration serial dilution of red cell concentrate. The animals are currently being maintained and examined at weekly intervals; and, the titration will be terminated when the hamsters show clinical signs of scrapie disease (head bobbing, wobbling gait, weight loss, etc). The concentration of infectious prions in the sample will be calculated from the score at the highest dilution at which there is a positive result using the Reed-Muench method.[86] The results will be calculated and expressed as log reduction.

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220kD 120kD 100kD 80kD

Filtration 1

Filtration 2

Filtration 3

Filtration 4

Filtration 5

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50kD 40kD

Pre

st Po

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st Po

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st Po

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Figure 2. Western blot of PrPres in RCC before (Pre=Prefiltration) and after (Post = Postfiltration) with LAPRF. The extracted PrPres in the pre- and post-filtration samples were treated with PK enzyme resulting in complete digestion of cellular prion and non-prion protein and conversion PrPSc 33-35 into PrP27-30 PK resistant fraction.

Endogenous Infectivity Study: RCC from Scrapie Infected Hamsters Each of 300 weanling normal Syrian hamsters was inoculated intracranially using 40 µL of a 10% (w/v) brain homogenate in phosphate buffered saline with a titer of 9.2 log10LD50/mL. At the onset of the clinical symptoms (wobbling gait and head bobbing) about 65-80 days after inoculation, blood samples were collected from the animals into CPD or CPDA-1 anticoagulant. A total of 500 mL whole blood was collected from the 100 hamsters (about 4-5 mL per hamster) into 70 mL CPD anticoagulant and processed into a single unit (300 mL) RCC by centrifugation at 5000g for 20±5 minutes. The RCC was then either adjusted to 55-60% hematocrit with the supernatant plasma, or resuspended in 100 mL SAGM additive solution in the same manner as single units of human RCC are prepared from anticoagulated whole blood according to Council of Europe guidelines. The blood bag containing a full unit of RCC was attached to Pall Leukotrap Affinity prion reduction filter, and then, filtered at room temperature at a filtration height of 30 inches. Aliquots of 40 µL of the pre- and post filtration RCC samples were injected intracranially into both sides of the brain of normal Syrian hamsters for each treatment condition (200 hamsters for control unfiltered red cells and 400 hamsters for filtered red cells). The animals are currently being maintained, and will be monitored for 400 days; those that develop clinical symptoms of scrapie will be sacrificed, and the brains of all animals (including survivors) will be tested for the presence of PrPSc by Western blot using 3F4 monoclonal antibody. The Pall Leukotrap Affinity Prion Reduction Filter was developed specifically in response to the specificity of the current leukocyte reduction filters. This new filter concurrently removes leukocytes and all types of prions, cell-associated and non-cell associated, in a single step. It is based on a proprietary surface modification technology that

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removes all types of prions. The use of this type of filter will improve the safety of the blood supply by reducing the risk of human vCJD through transfusion.

BIO- AND HEMO-COMPATIBILITY OF PRION REDUCTION BY FILTRATION Prion reduction filtration is a new technology and there are currently no guidelines to specify the quality of resultant blood components either immediately after filtration or during component storage. The most relevant guidelines are therefore those for leukocyte depleted blood components. These vary, however, from country to country. The American FDA standards[87] specify that leukocyte depleted red cells should contain less than 5 x 106 leucocytes/unit, red cell recovery should be >85% and that hemolysis at the end of storage should be less than 1.0% red cell mass. Council of Europe Guidelines[88] specify a leukocyte residual of less than 5 x 105 leucocytes/unit (for 90% of units tested), that each unit should contain a minimum of 40g/unit hemoglobin, and that hemolysis should be less than 0.8% at end of storage. Yet another example are the UK guidelines[89], which specify that 99% of red cells should contain less than 5 x 105 leucocytes/unit (at 95% confidence levels), and that 75% of units should contain more than 40 g/unit hemoglobin. To further compound matters, each donor whole blood unit contains a different amount of hemoglobin, and differences as a result of local processing variations are inevitable. The above-mentioned guidelines/standards do not completely address the issue of the ‘red cell storage lesion’. It is well documented that red cells undergo marked changes during storage. Red cell adenosine triphosphate (ATP) levels drop by up to 50%[90,91,92] and it is considered that ATP levels of 2-3 µmol/g Hgb are normal at the end of red cell shelf life.[93] There is also a marked decrease in 2,3-diphosphoglycerate (2,3-DPG) with levels falling at a linear rate to zero after approximately 2 weeks of storage.[94] When the red cells are transfused, however, the stored cells regenerate ATP and 2,3-DPG resuming normal energy metabolism and hemoglobin function as they circulate in the recipient. It takes about 24 hours for severely depleted red cells to restore 2,3-DPG and normal hemoglobin function.[95,96] Supernatant potassium levels are known to increase during red cell storage due to the paralysis of the Na/K pump at the low storage temperature.[97] Levels of up to 75 mmol/L have been reported for CPDA-1 red cells.[8] Studies with red cells stored in additive solutions have given slightly lower K+ levels.[90,91,98] Red cells also undergo marked morphological changes during storage. The cells develop finger-like projections which are initially reversible but then become permanent as the projections bud off to form microvesicles; approximately 25% of membrane phospholipid is lost during 42 days of storage.[99] The result of this is that the cells become less deformable. Formation of the microvesicles is a complex process that depends on changes in the interactions occurring between the cytoskeleton, peripheral membrane proteins and lipids. The quantity and structure of the microvesicles depends upon the storage medium. It has been suggested that lower loss of critical membrane proteins is an indicator of improved red cell preservation.[100] It has also been shown that standard leukocyte depletion filters do not give rise to significant cellular microvesiculation.[101] Traditionally blood set/filter manufacturers

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have tested their products with regard to above parameters; and, it would seem logical that any new prion reduction filter should be tested in the same way. The Pall Leukotrap Affinity Prion Reduction Filter has been tested and shown to meet all relevant guidelines in this respect.[102] This filter has also been investigated for any effects on band 3 protein, CD47 expression and red cell antigen expression. Band 3 forms the central element of a macrocomplex of integral and peripheral proteins in the red cell membrane. It is speculated that this complex may have a central role in red cell CO2/O2 gas exchange.[103] CD47 is an integrin-associated protein and has a crucial inhibitory role as a marker for self. Red cells that lack CD47 are recognized and eliminated by spleen red pulp macrophages. CD47 levels have been shown to decrease during red cell storage.[104] The Pall Leukotrap Affinity Prion Reduction Filter has been shown to have no significant effect on band 3 or CD47 levels compared to controls and to have no effect on red cell antigen expression (manufacturer’s data). In vivo red cell recovery studies are a final indicator of red cell quality. 24 hour in vivo red cell recoveries for the Pall Leukotrap Affinity Prion Reduction Filter have been shown to be well above FDA and Council of Europe requirements.[105]

PRACTICAL IMPLEMENTATION OF FILTRATION IN REDUCING THE RISK OF TT-VCJD Overview of Regulatory Issues The age of meaningful preventive medicine is only dawning. As a result, our regulatory product approval systems are challenged in attempting to validate claims for preventive medical technologies. Our medical system is much more applauded and experienced concerning interventional technologies. This makes the regulatory pathway, and eventual use of prion removal in transfused blood products, difficult or at least uncertain in its final product approval considerations. Certainly it is now possible to quantify prion removal from blood. The effectiveness in human disease prevention, however, is quantitatively indeterminable for the foreseeable future. Somewhere in the process of implementation, judgment, leadership and logic-based decision-making will determine the speed of utilization. There will also be a technology value judgment made first by the companies which develop the technology, and will promote expanded use though patient benefits; as well as, the regulatory agencies, which have unwritten yet very specific public and political duties concerning the generally acknowledged benefit of spent healthcare monies. This is clearly the case in the present comparison between some countries within the European Union which have an informed public concerning TSE disease, and are expediting use of prion filtration to achieve a safer blood supply; while in the United States, the public is less informed and even comforted by government assurances of isolation from TSE exposure. As a result, decisions to use preventive technology for TSE in the United States lack impetus awaiting definitive scientific data, and/or, public outcry for safety measures. Both of these will be reactionary, as the addressed disease has been medically identified some fifteen years

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ago and has a period of between five and thirty years of dormancy prior to exhibiting obvious physical distress. Without definitive diagnostic tests, which might not be widely used even if available in order to spare the patient angst over having contracted a fatal disease without hope of intervention, it is likely that the public in some nations will remain generally uninformed regarding technology advances, and, comprehensive risk-benefit to cost evaluations. Product submissions for regulatory approval will be able to provide not only quantitative prion removal data, but also disease transmission prevention data from extensive animal studies. This will allow not only efficacy judgments, but also, a beginning of value assessments. When the disease threat is yet un-quantified, safety and efficacy are first priorities for developers and regulatory approval bodies alike. After these are judged to be established, it is then the obligation of technology developers and regulatory gatekeepers together to guide, and provide, advances in transfusion safety technology for clinical use within reasonable product approval processes and timelines.

Cost Effectiveness With claims for absolute prevention of transfusion-transmitted prion disease lacking, too much cost will cause the clinician to avoid use of the product even if readily available. And, while not specifically addressed in most regulatory approval processes, product cost does factor into the regulatory process of approval and decision-making. The preferred product iteration then, will be an addition of prion removal to existing leukocyte-reduction blood filter construction. This serves to isolate product development cost, and eventual additional selling price to the attributes necessary for prion removal, and avoids much of the manufacturing engineering cost associated with a total new design, or separate single-function device. Under extreme regulatory labeling restrictions, the most innocuous claim and indication for use could be simply the recognition of prion removal in efficiency terms followed by an acknowledgement that prions have been shown to be associated with TSE diseases. This, of course, might be perceived by bedside practitioners as less effective technology than the product really merits. Historically, leading practitioners and clinical researchers have undertaken definitive patient studies to clarify what product development and approval systems were unable to address. With prion diseases however, having no diagnostic tests and extremely few autopsies, and after years of multiple avenues of exposure during the course of a patient's life, companies, government agencies and the public at large will need to come to the decision on utilization of prion removal in transfused blood through new channels. Some nations are pursuing decisions for TT-TSE prevention based on the doctrine that a known threat which can be reasonably avoided dictates a decision to do so wherever and whenever feasible. At the highest organizational level, this is a commitment of a government to its citizens; and, a policy for utilization follows. Other nations have little history of decision making in advance of strong public and political outcries, or, reaction to the actual realization of the previously identified threat. In

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both cases, there is an economic price to be paid. If the prevention technology were free, after proof of safety and efficacy, everyone would use it with immediacy. If the technology added 1% of additional cost to each blood unit, then universal use would be a short time coming. However, at an additional 10% cost to each blood unit, the blood providers and governments will find it difficult to assimilate the additional cost, and thus, will likely extend the universal use decision, while subjecting interim transfused patients to possible TT-TSE exposure which may result in much greater cost later. Under these circumstances some policy makers will be forced to triage patients favoring the very young for protection, for example, while awaiting funding and new policies for the average transfused patient. The public will no doubt respond as they become aware; and, public pressure may speed availability to larger populations of transfused patients. The strong scientifically supported reasoning that removal of 99.9% of prions carried in blood transfusions will avoid transmission of TSE to at least some, if not all, transfusion recipients is compelling for many populations around the world. The use of prion reduction technology in these populations immediately should diminish the TT-TSE exposure risk worldwide. With broad use of this technology, and the resulting documentation of realized benefits, the standard of care would be elevated for all.

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[79] Alonso DOV, DeArmond SJ, Chen, FE, Daggett V. Mapping the early steps in the Phinduced conformational conversion of the prion protein. Proc Natl Acad Sci 2001; 98:2985-2989. [80] Prusiner SB. Prions. Proc Natl Acad Sci 1998; 95:13363-13383. [81] Sowemimo-Coker SO. Making blood Safe-A filtration technology for removing infectiousprions from red cell concentrates. Biochemist 2005;27(4):29-32. [82] Ortolano GA, Wilkins K, Cervia JS. Characterization of prion removal devices for blood products. Trans Med Hematotherapy 2005; (in press) [83] Lee DC, Stenland CJ, Miller JLC, Cai K, Ford EK, Gilligan KJ, Hartwell RC, Terry JC, Rubenstein R, Fournel M, Petteway Jr. ST. A direct relationship between the portioning of the pathogenic prion protein and transmissible spongiform encephalopathy infectivity during the purification of plasma proteins. Transfusion 2001; 41:449-455. [84] Liao YC, Lebo RV, Clawson GA, Smuckler EA. Human prion protein cDNA:molecular cloning, chromosomal mapping, and biological implications. Science 1986;233:364-367. [85] Bendheim PE, Bockman JM, McKinley MP, Kingsbury DT, Prusiner SB. Scrapie and Creutzfeldt-Jakob disease prion proteins share physical properties and antigenic determinants. Proc Natl Acad Sci USA 1985;82(4):997-1001. [86] Reed, Muench. A simple method for estimating fifty percent endpoint. American Journal of Hygiene. 1938. 27(3):493-7. [87] Food and Drug Administration Department of Health and Human Services Recommendations and licensure requirements for leukocyte reduced blood products. Memorandum to all registered blood establishments, May 29, 1996. [88] Guide to the preparation, use and quality assurance of blood components (11th ed.). Council of Europe Publishing 2004. [89] Guidelines for the Blood Transfusion Services in the United Kingdom (6th ed.). The Stationary Office, Norwich. 2002. [90] Heaton WAL, Holme S, Smith K, Brecher ME, Pineda A, AuBuchon JP, Nelson E. Effects of 3-5 log10 pre-storage leukocyte depletion on red cell storage and metabolism. Br J Haematol 1994: 87, 363-368. [91] Pietersz RNI, de Korte D, Reesink HW, Dekker WJA, van den Ende A, Loos JA: Storage of whole blood for up to 24 hours at ambient temperature prior to component preparation. Vox Sang 1989; 56, 145-150. [92] Hogman CF. Liquid-stored red blood cells for transfusion. Vox Sang 1999; 76, 67-77. [93] Hess JR, Kagen LR, van der Meer PF, Simon T, Cardigan R, Greenwalt TJ, AuBuchon JP, Brand A, Lockwood W, Zanell A, Adamson J, Snyder E, Taylor HL, Moroff G, Hogman C. Interlaboratory comparison of Red-cell ATP, 2,3-diphosphoglycerate and hemolysis measurements. Vox Sang 2005; 89, 44-48. [94] Collection, preparation, storage and distribution of components from whole blood donation. – Chapter 8. AABB Technical Manual 15th edition; Brecher ME (ed) 2005; pp. 175-202. [95] Hogman CF, Merryman HT. Storage parameters affecting red cell survival and functions after transfusion. Transfus Med Rev 1999; 13, 275-276.

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[96] Heaton A, Keegan T, Holme S. In vivo regeneration of red cell 2,3-diphosphoglycerate following transfusion of DPG-depleted AS-1, AS-3 and CPDA-1 red cells. Br J Haematol 1989; 71, 181-186. [97] Hogman CF. Preparation and preservation of red cells. Vox Sang 1998; 74 (Suppl. 2), 177-187. [98] Greenwalt TJ, Zehner Sostok C, Dumaswala UJ. Studies in red blood cell preservation. 1. Effect of other formed elements. Vox Sang 1990; 58, 85-89. [99] McLellan SA, Walsh TS, McClellan DBL. Should we demand fresh red blood cells for perioperative and critically ill patients? Br J Anaesth 2002; 89, 537-540. [100] Dumaswala UJ, Dumaswala RU, Levin DS, Greenwalt TJ. Improved red blood cell preservation correlates with decreased loss of bands 3, 4.1, acetylcholinesterase and lipids in microvesicles. Blood 1996; 87, 1612-1616 [101] Krailadsiri P, Perry R, Drummond O, Smith K, Hockly D, Seghatchian J, Sprin F, Macgregor I, Williamson L, Prowse C, Anatole Lubenko, Anstee D, Barrowcliffe T, Turner M. The effects of leukocyte depletion on the generation and removal of microvesicles and prion related protein in blood components. Transfus Apheres Sci 2001; 25, 177-178. [102] Saunders C, Herbert P, Rowe G, Hayward M, Wilkins K, Milligan J, Stenning M, Seacombe M, Prowse C. In-vitro evaluation of PALL Leukotrap Affinity Prion Reduction Filter as a secondary device following primary leucoreduction. Vox Sang 2005; in press. [103] Bruce LJ, Beckmann R, Ribeiro ML, Peters LL, Chasis JA, Delaunay J, Mohandas N, Anstee DJ, Tanner MJ. A band 3-based macrocomplex of integral and peripheral proteins in the RBC membrane. Blood 2003; 101, 4180-4188. [104] Stewart A, Urbaniak S, Turner M, Bessos H. The application of a new quantitative assay for the monitoring of integrin-associated protein CD47 on red blood cells during storage and comparison with the expression of CD47 and phospatidylserine with flow cytometry. Transfusion 2005; 45, 1496-1503. [105] Nelson E, Taylor H, Whitley P, Lieu T. Evaluation of in vivo red blood cell recovery after processing with a new filter designed to remove prions. Vox Sang 2005;89(suppl. 2), 20.

In: New Developments in Blood Transfusion Research ISBN 1-59454-962-1 Editor: Brian R. Peterson, pp. 103-120 © 2006 Nova Science Publishers, Inc.

Chapter VI

ALLOSENSITIZATION IN MULTIPLY TRANSFUSED SICKLE CELL DISEASE PATIENTS Vishwas S. Sakhalkar∗ Department of Pediatrics, Division of Hematology/Oncology, Children’s Hospital of Shreveport, Louisiana State University Health Sciences Center, Shreveport, LA, USA.

ABSTRACT Blood transfusions, sometimes multiple, are commonly used in patients with sickle cell disease for prevention and treatment of complications. Allosensitization leading to antibody formation in blood transfusion recipients can result from exposure to donor’s minor blood types that are not present in the recipient and are not crossmatched for transfusion. The reasons for allosensitization are multifactorial: interracial differences in frequencies of minor blood types in the local population (a major factor), and host factors including genetic predisposition to produce alloantibodies and autoantibodies or react to foreign antigens; recipient’s age, sex, race, etc. The incidence of this problem varies locally and is reportedly between 5-60%. Exposure to donor’s foreign RBC antigens leads to formation of specific alloantibodies, sometimes accompanied by autoantibody production possibly due to dysregulation in host immune response. There are no immediate consequences due to the mild and slow rise in titer. The titers drop over a period of time, due to non-exposure to the antigen. Hence, on retesting after few months, the repeat crossmatch may be compatible. However, on re-exposure, there is a brisk anamnestic antibody production and mild to severe delayed transfusion reaction may follow manifesting as an acute or subacute intravascular or extravascular hemolysis leading to indirect hyperbilirubinemia, anemia, pain ‘crisis’, renal failure, and sometimes death. The technical, monetary and health implications for the recipient and institution can be profound. Numerous approaches to this problem have been devised – complete RBC phenotyping of all ‘at risk’ recipients, various levels of pretransfusion extended ∗

Correspondence concerning this article should be addressed to Dr. Vishwas S. Sakhalkar, M.D. Assistant Professor of Pediatrics, Division of Hematology/Oncology LSUHSC, P. O. Box 33932, 1501 Kings Highway, Shreveport, LA 71130-3932, USA. Tel: 1(318) 813-1080; Fax: 1(318) 813-1088; E-mail: [email protected].

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Keywords: Alloantibody, autoantibody, transfusion reactions, extended crossmatch, multiple blood transfusions, allosensitization, febrile, allergic, sickle cell disease, thalassemia.

INTRODUCTION The phenomenon of allosensitization due to exposure to repeated blood transfusions in patients with sickle cell disease (SCD) and other conditions (e.g. thalassemia) requiring multiple blood transfusions, has been well documented in literature [1-10]. Over their lifetime, about ninety percent of patients with sickle cell disease (SCD) receive blood transfusions to prevent complications of their disease [2,3,11-16]. For many acute conditions encountered in SCD patients that are emergent (such as acute chest syndrome and priapism), or elective (e.g. surgery), blood transfusions are considered essential for a favorable outcome [3,5,11-20]. Chronic transfusions, a unique form of therapy for SCD, are frequently used for secondary prevention of life-threatening events, such as neurologic injury, acute chest syndrome, chronic pain, leg ulcers, and end-stage organ failure [21-24]. More recently, red cell transfusions have also proven to be effective in the primary prevention of stroke in asymptomatic patients with abnormal transcranial doppler studies [25]. Hence blood transfusions are now increasingly used for primary prevention of other causes of morbidity in SCD [15,18,19]. However, transfusions are complicated by a high incidence of RBC alloimmunization and transfusion-related complications which has been studied and published since the 1980s [5,13,20,26-49]. The goal of this article is a comprehensive review of literature on the topic of allosensitization in SCD. It details the different aspects of allosensitization and discusses the consequences and approach to the major problem in multicultural communities across the United States and the rest of the world.

Incidence and Causal Factors for Allosensitization – Recipient/Donor Racial Heterogeneity Allosensitization leads to alloantibody formation in the blood transfusion recipients. It occurs due to recipient’s immune response to donor’s minor blood type antigens that are not

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present in the recipient and are not crossmatched for the transfusion. The reasons for allosensitization are multifactorial: Interracial differences in frequencies of minor blood types in the local population resulting in significant difference in RBC antigen profile of the donor and recipient is probably the most important reason for allosensitization [30,47-49]. In a comparative study of SCD patients in Manchester, United Kingdom (UK) and Jamaica [49], the author notes that immune antibodies occurred in three Jamaicans (2.6% of those transfused) and 16 UK subjects (76% of those transfused). Multiple antibodies (a particularly menacing condition causing a nightmare to blood bank staff and physicians when obtaining compatible blood in an emergency) occurred in 10 (63%) UK patients, but not in any patients from Jamaica. In another study in Brazil [47], fifteen alloantibodies were detected in 11 patients, most (80%) of these involved antigens in the Rhesus and Kell systems. This observed RBC alloimmunization rate (of 12.9%) in SCD patients in Brazil was lower than that reported by studies from North America, suggesting that the requirement for extended antigen-matched RBC transfusion for SCD patients in the setting of a RBC phenotype concordant donor-recipient populations may not be cost effective. As there was no significant racial and RBC antigen profile differences occurring in patients with chronic anemia and blood donors, but significant difference between patients with SCD and the donor population, studies comparing allosensitization in patients with other conditions requiring multiple transfusions to patients with SCD showed much higher rate of alloimmunization in SCD patients over their counterparts [30,50].

Allosensitization – Host Genetic Factors Genetic predisposition of host to produce antibodies to react to foreign antigens [51], variable immunogenicity of the various ‘minor’ blood group antigens, immune competence of the recipient at time of transfusion (cancer patients, irrespective of race, being immunosuppressed have much lesser incidence of allosensitization than SCD patients), age and sex of recipient, type of blood product transfused (e.g. leukoreduced or not, amount of plasma contamination?) etc. Incidence of alloimmunization is particularly low in thalassemia patients probably because they are usually transfused from a young age and there is some evidence that alloimmunization is more probable in patients receiving a first transfusion at a later age [50,52,53]. Alloimmunization to human platelet antigen 1a is strongly associated with HLADRw52a (HLADR3*0101),[54] which is carried by a third of people in the United Kingdom. Whether such association exists that predisposes to alloimmunization to RBC antigens needs to be studied.

Allosensitization – Other Factors in the Host Incidence of alloimmunization increases with number of transfusions, as many as 60% of SCD patients may be allosensitized as adults [13]. Women have higher incidence of allosensitization than men by virtue of greater antigen exposure through pregnancy, miscarriages and parturition [13,55]. While young children are more tolerant, older children

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have more likelihood of alloantibody formation when compared to adult males [50]. In our study [1], women and children has 1½ times more likelihood of forming alloantibodies per transfusion as compared to adult males (p = NS). Erythrocytopheresis performed instead of regular manual exchange results in a greater annual unit exposure than simple transfusions, but interestingly did not increase alloimmunization according to a study [56]. Due to the above variables, the incidence of allosensitization in different studies varies markedly, and is reportedly between 5 - 60% [38].

Platelet Allosensitization Allosensitization to RBCs is not the only form of allosensitization that occurs by transfusions. Allosensitization occurs to other components of blood as well. Allosensitization to platelets can be a major problem if these multiply transfused patients later require platelet transfusions. In one interesting study comparing RBC and platelet allosensitization and is durability,[44] sera collected from 47 transfused and 14 untransfused SCD patients were screened for HLA and platelet-specific antibodies. Transfusion and RBC antibody histories were reviewed. A subset of the patients was rescreened one year later. Eighty-five percent of patients with at least 50 RBC transfusions (22 of 26), 48% of patients with less than 50 transfusions (10 of 21), and none of 14 untransfused patients demonstrated platelet alloimmunization (P < .05). Platelet alloimmunization was more prevalent than RBC alloimmunization (20% to 30%). Eighteen of 22 patients (82%) on chronic RBC transfusion remained platelet-alloimmunized 11 to 22 months after initial testing. In summary, 85% of heavily transfused SCD patients are alloimmunized to HLA and/or platelet-specific antigens. These patients may be refractory to platelet transfusion, a condition that would increase their risk during BMT. Leukodepletion in the transfusion support of SCD patients helps prevent platelet alloimmunization.

Autoantibody Formation and its Etiology An interesting and commonly documented complication of allosensitization is simultaneous or near simultaneous autoantibody formation around the time an alloantibody is detected [31,57]. Also on many occasions not one but multiple alloantibodies are detected simultaneously, often associated with autoantibody detection [1]. In blood transfusion recipients with sickle cell disease (SCD) autoantibody formation probably occurs due to dysregulation in immune response that also leads to formation of multiple alloantibodies [1]. That autosensitization results in autoantibody formation after blood transfusions was first noted in humans sixty years ago [24,] and rabbits as much as 87 years ago [58,59]. Though numerous observations of autoantibody formation against red blood cells (RBCs) induced by transfused RBCs from same and different species of animals were made in 1950s and later [60-62], and in humans in early 1980s, the significance of this phenomenon was poorly understood until the late 1990s [31,58-62]. There are various reasons for autoantibody formation: interracial differences in frequencies of minor blood types in the local population,

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genetic predisposition of host to produce autoantibodies and alloantibodies or react to foreign antigens (minor blood groups), immunocompetence of the individual at time of transfusion, age and sex of recipient, viral and other infections, etc [58]. Patients with HLA phenotypic expression of DR3 are associated with increased incidence of antibody formation [54]. Fourfold increase in antinuclear antibody (ANA) titers from baseline was observed in those situations where the recipient already had antinuclear antibodies and developed autoantibodies after blood transfusion suggesting that there are alterations in self-reactive antibody repertoires of plasma IgM and IgG that are independent of the presence of a specific immune response to RBC antigens [31,59].

Incidence of Autoantibody Formation Autoantibody formation occurs at different frequencies in different studies, depending on the above outlined factors and is reported between 20-50% in SCD patients with alloantibodies.[35,48] These are mainly warm autoantibodies. In patients that do not form alloantibodies despite multiple blood transfusions, the frequency of autoantibody formation is much lower (about 5%) and they are mostly cold agglutinins,[1] suggesting that there may be different immunological processes involved, probably related to host immune factors. In a large single hospital study, autoantibody formation has been noted to be associated with alloantibody formation in about a third of cases of autoantibody detection giving an idea of the significance of the role of allosensitization in autoantibody formation on the whole [64]. Autoantibody formation is thus significantly higher when alloantibody formation is documented.[1,64] We have also noticed increased association of allergic reactions with alloantibody formation. These observations suggest that once the blood product recipient’s immune system is stimulated due to allosensitization or other methods such as donor leukocyte contamination, it leads to a dysregulation of immune response leading to multiple abnormalities in different aspects of immune system (alloantibody and autoantibody formation to various antigens in the body, allergic reactions, etc).

Etiology and Clinical Manifestations of Autoantibodies Alloantibody formation probably upregulates/dysregulates the immune system, and the antigen alloantibody complex and host immune response exposes autoantigens. A combination of these factors in a patient that is genetically and immunologically vulnerable may lead to formation of autoantibodies. Interestingly, in spite of such a high incidence of autoantibody formation, the patients are usually asymptomatic – a possible reason behind the delay in scientific community trying to decipher the problem [31,59]. A symptomatic patient can present with anemia because of the additive combination of four hemolytic mechanisms; the baseline hemolysis due to sickle hemoglobinopathy, alloantibody induced donor cell hemolysis, ‘bystander’ hemolysis of autologous red cells sometimes seen with allosensitization and autoantibody induced autologous red cell hemolysis. These processes are compounded by reticulocytopenia (see ‘DHTR’ below). “Bystander’ hemolysis is

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probably due also to an autoantibody that cannot be identified by conventional means and not merely due to alloantibodies and complement activation as speculated. The autoantibody titers also drop over period of time, in many cases with drop of alloantibody titers and activity. The clinical manifestations vary from mild, with only a surprise autoantibody detection during crossmatch before blood transfusion, to severe involving acute or sub-acute hemolysis leading to a Coomb’s positive (and occasionally Coomb’s negative) hemolytic anemia, indirect hyperbilirubinemia, renal failure, and can be, at times, fatal.

Types of Autoantibodies The type of autoantibody falls into three main categories; IgG in warm autoantibody induced and spleen mediated extravascular hemolysis, IgG with complement induced intravascular hemolysis that is sometimes associated with intrahepatic extravascular hemolysis in paroxysmal cold hemoglobinuria and IgM cold antibody induced intravascular hemolysis. The specificities are numerous depending on the antibody category and other factors varying from IgG type panagglutinins to IgM type anti-I or anti-i antibodies. In a study involving 184 pediatric patients who received multiple erythrocyte transfusions,[35] 14 children (7.6%) developed warm (IgG) erythrocyte autoantibodies. Median transfusion exposure at the time of autoantibody formation was 24 erythrocyte units, range 3-341 units. The autoantibody reacted as a panagglutinin in 11 cases but had anti-e specificity in three patients. Surface complement also was detected in five patients. Clinically significant hemolysis was documented in four patients, each of whom had both surface IgG and C3 detected. The development of erythrocyte autoantibodies was associated with the presence of erythrocyte alloantibodies. Presence of surface C3 was associated with significant hemolysis. Hence the characteristics of the autoantibody influence the severity of its manifestations in the patient.

Prevention and Management of Patient with Autoantibody Formation Numerous therapeutic approaches include methods towards primary avoidance of alloantibody formation (see management of allosensitization). High dose steroids, other immunosuppressive agents and intravenous immunoglobulins are particularly helpful in IgG mediated hemolysis.[65] Avoiding cold (e.g. transfusing blood at body temperature) is helpful in IgM induced hemolysis. Plamsapheresis is more effective in severe cases of IgM mediated hemolysis as the antibody is mainly confined to the intravascular compartment and is not bound to the red cell membrane at core body temperatures. Though best avoided, at times with severe anemia, blood transfusions may be required. There are unique issues in providing blood for patients who have RBC autoantibodies. Algorithms for excluding the presence of "hidden" alloantibodies are available when all units appear to be incompatible due to the autoantibody [46]. Clinicians should be aware of these approaches and not necessarily use "the least incompatible unit." Novel approaches such as

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hemoglobin polymers and bovine hemoglobins can be used in life-threatening situations [65]. Artificial oxygen carrying complexes are still in an experimental stage of development.

Donor and Blood Unit Characteristics and Allosensitization Whether characteristics of the donor other than the RBC phenotype affect the alloantibody and autoantibody formation (i.e. sex, age, HLA type and make up) is not well known and is open to speculation. The possibility that blood transfusion might mediate donor-specific immunologic tolerance was first suggested by Billingham et al in 1953 on the basis of some experimental evidences in the mouse system [66]. Thereafter, in the 1970s, several lines of evidence indicated that donor-specific transfusion, as well as allogeneic blood transfusion, performed before renal allograft or during surgery improved graft survival [67]. Leukocytes in the donor blood have been found to be essential for this effect, [68,69] and that the immunomodulatory effect of allogeneic blood transfusion is linked to the presence of donor leukocytes.[68-72] Soluble human leukocyte antigen-1 (sHLA-1) and soluble Fas Ligand (sFasL) are functional soluble molecules that are found in some blood components [70-77]. Elevations in concentrations of sHLA-1 and sFasL molecules is proportionate to the amount of leukocytes in each blood component and to the length of storage; and sHLA-I and sFasL molecules detected in blood components are functional and play immunomodulatory effects in vitro [78]. According to Ghio et al, [78] HLA class I and FasL antigens are released from residual donor blood leukocytes membrane during storage and that the immunomodulatory effect of allogeneic blood transfusion is linked to the presence of these functional molecules. The following potential immunomodulatory effects of allogeneic blood transfusion should be taken into account in clinical practice according to a study by Ghio et al [78] (1) transfusion of fresh RBC containing viable donor leukocytes and a low amount of soluble molecules can cause alloimmunization but may also induce anergy or tolerance; (2) transfusion of nonleukodepleted or poststorage leukodepleted RBC containing dead donor leukocytes and a high amount of functional soluble molecules is more likely to induce strong immunosuppression; (3) transfusion of prestorage leukodepleted or W-RBC should prevent the immunosuppressive effect; and (4) transfusion of random-donor platelets, which contain viable donor leukocytes and elevated concentrations of soluble molecules, can induce alloimmunization and immunosuppression as well. Researchers have also noted decreased allosensitization in patients after using leukodepleted red cells [79]. Whether other factors, such as donor cytokines or other factors present in donor products are also involved in this phenomenon, remains to be studied. Multiple (liberal) blood transfusions have also been implicated in poor survival in patients in intensive care and chronically transfused cancer patients pointing to influence of donor factors other than simply transfused red cells on the host.

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Hemolysis Following Alloantibody Formation The consequences of alloantibody formation have been described variously ranging from mild, with only a surprise detection of an alloantibody during the crossmatch before blood transfusion, to severe involving acute or sub-acute intravascular or extravascular hemolysis (see DHTR below). The mechanism involves exposure of the recipient to donor’s foreign RBC antigens leading to formation of antibodies (that were absent before) without any immediate consequences due to the mild and slow rise in titer. However, on re-exposure to the particular antigen by transfusion, there is a brisk anamnestic antibody production and mild to severe hemolysis can occur that usually takes 7-10 days to manifest clinically. This “delayed hemolytic transfusion reaction (DHTR)” can be life-threatening [26,29,34,36-38]. Severe anemia can be compounded by hemolysis of recipient’s RBCs, also known as the ‘bystander phenomenon’ which can sometimes be fatal. The monetary, technical and health implications for the recipient and the patient’s institution can be profound. In our study we found that once an alloantibody was formed, the probability of second and subsequent antibody formation is incrementally increased [1]. Subsequent alloantibodies were formed after fewer transfusions, and sometimes many antibodies were formed simultaneously.

Prevention and Management of Allosensitization – Pretransfusion Extended Crossmatch Numerous approaches to prevent allosensitization or manage its effects have been devised. A common approach consists of various levels of extended crossmatch of donor’s and recipient’s blood depending on frequency of minor blood groups in the local donor population before each transfusion. This approach is helpful in markedly decreasing the rate of allosensitization. In addition to the universal ABO and Rh crossmatch, the Rh antigens ‘C’ and ‘E’ and another Kell antigen ‘K’ are associated with a very high incidence of alloantibody formation. Most extended crossmatch protocols include C, E, and K antigen crossmatch in their protocols [1,80]. A recent study [44] showed that if all transfusions had been selected by limited phenotype matching (C, c, E, e, and K, as well as for ABO and D), all alloantibodies would have been prevented for more than half (51%) of their alloimmunized patients. If all transfusions had been matched for C, c, E, e, K, S, Fya, and Jkb, all antibodies would have been prevented for 70.8 percent of the 137 alloimmunized patients. However, only 13.6 percent of random white blood donors would be expected to match a limited phenotype-matching protocol, whereas only 0.6 percent would match an extended phenotype-matching protocol. Thus limited phenotype matching would have prevented all alloantibodies in 53.3 percent of the patients who formed alloantibodies and requires RBCs that are readily available. Extended phenotype matching (additionally S, Fya, and Jkb) would have prevented alloimmunization in 70.8 percent of patients who formed alloantibodies. However, this would require phenotypes that are 22.7 times less prevalent among random blood donors and is therefore impractical for a long-term strategy. A questionnaire was mailed recently to 50 academic medical centers in the US and Canada regarding their routine practice concerning the use of prophylactic phenotypically

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matched RBCs for transfusion [80]. In spite of the various complications related to regular cross-matching for chronic transfusions, almost 3/4 institutions (27 of 37 responders) routinely provided extended crossmatch for their SCD patients. Of the 3/4 that did perform an extended crossmatch, four hospitals performed extended crossmatch after the development of the first alloantibody and two hospitals performed extended crossmatch after the development of the second alloantibody. The centers practiced varying degree of extended crossmatch involving different antigens (presumably unique to their set-up). Of the 27, 24 (89%) match for C, E, and K; 15 of these 24 match for additional antigens (most commonly c and e). The reasons for the varied approach are probably related to the unique situation at each of these institutions mostly depending on the antigen variation perceived among donors and recipients. Prophylactic phenotypic matching for blood group antigens for RBC transfusion in sickle cell disease to prevent alloimmunization still remains controversial [13,30,44,8083]. These data suggest that there is currently no single standard of care in academic medical centers in the US and Canada. The reason for the tremendous differences in this practice is not surprising – the cost, technical difficulties, the availability of resources and personnel and requisite blood units can be intimidating (see below). Hence the most common approach is pretransfusion phenotypic matching for C, E, and K.

Issues Related to Pretransfusion C, E, and K Antigen Crossmatch In our study,[1] patients receiving CEK crossmatched red cells were 3 times less likely to form non-CEK alloantibodies (p = 0.03) and 10 times less likely to form autoantibodies (p = 0.005) than the corresponding group of patients that received regular (ABO and Rh) crossmatched blood. However, finding a C, E, K antigen (CEK) matched blood for every SCD transfusion for a large sickle cell program can be daunting. Each antigen testing requires about $14 worth of reagents. On an average, our study noted that additional CEK matching results in 30 minute extra time of a skilled technologist and $153 extra CEK reagent cost per unit (for screening and crossmatching multiple units before finding the appropriate unit) to find CEK matched pRBCs for every transfusion for these multiply transfused patients. As Rh D negative units are more likely to be C, E and K negative, this method also results in a marked overuse of Rh negative pRBCs. In this era of cost efficiency, further study to determine the impact of using CEK matched pRBCs for all patients after development of their first alloantibody may be helpful as this may decrease use of the scarce CEK negative (Rh negative) units in two-third of our patients. One third of our patients never formed an antibody in spite of multiple exposures, some of these patients had received over 100 transfusions, giving credence to the idea of performing extended crossmatch after the detection of the first antibody. The downside of this approach is that some patients develop multiple antibodies at the time of the first antibody detection itself, some patients develop as many as 4-5 antibodies, with simultaneous autoantibody formation (in about one sixth of patients in our study [1]), making it extremely difficult to find a compatible donor unit for subsequent transfusions.

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Primary and Secondary Prevention of Allosensitization There are many other approaches to prevent allosensitization or manage its effects. A pre-transfusion ‘complete’ red blood cell phenotyping of all ‘at risk’ recipients (with SCD, thalassemia, cancer, etc) helps in easier identification of an alloantibody when it occurs. Red cell phenotyping of the regular or directed blood donors helps in quick and easy identification of compatible blood units. Autologous blood transfusions can be performed after elective blood collection from these patients and freezing the blood units. Erythropoietin can also be administered and blood collection performed at an appropriate time before elective surgical procedures. Blood can be frozen and stored for over 10 years. Lifelong avoidance of the particular antigen once the corresponding antibody is demonstrated is warranted in case of clinically significant antigens.

Novel Therapeutic Approaches Erythropoietin coupled with corticosteroids has been used for stimulating erythropoiesis and suppression of the immune response, as well as to avoid additional transfusions [82,84]. Hemoglobin polymers and bovine hemoglobins have been used in heavily alloimmunized patients when compatible blood is unavailable but transfusion is critical for survival [65]. Artificial oxygen carrying complexes are still in an experimental stage of development. Two processes, in development, produce red cells that can be described as "universal" donor or "stealth" red blood cells (RBCs). The first process involves changing group A, B, or AB RBCs into group O RBCs by removing the immunospecific sugars responsible for A and B specificity by using specific enzymes. The second process involves covering all blood group antigens on the RBC surface using polyethylene glycol [46]. In one case report, peripheral blood stem cell transplantation by use of a new nonradiation-based conditioning regimen enabled successful engraftment of allogeneic donor peripheral blood stem cells and the elimination of alloantibody in a SCD patient with multiple RBC antibodies [85]. Patients having stroke, ulcers, etc. and multiple alloantibodies creating extreme difficulty finding compatible blood have been treated with Hydroxyurea which led to improvement in their underlying condition and baseline hemoglobin level [86,87]. Some studies tried to directly tackle the issue that in practice blood from white donors (with a higher incidence of certain Rh, Duffy, Kell, and Kidd blood group antigens) is being transfused to black patients often lacking these antigens. Hence they proposed a model to reduce alloimmunization in patients with SCD by providing them with blood from black random donors only. Rationale is shown by examining calculations based on the phenotype E-, C-, Fy(a-), K-, and Jk(b-). There is a 7% probability that this phenotype belongs to a white donor, while there is a 93% probability that this phenotype belongs to a black donor. The probability of selecting blood from a black donor identical with the above phenotype for black recipients from an all black population and from a typical urban blood inventory population (90% white, 10% black) is 1/4 and 1/33, respectively. An 8-fold greater chance of selecting antigen non-identical blood occurs if blood is obtained from a typical urban donor population as compared to a black population. Based on these calculations, alloimmunization

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can be reduced prospectively in patients with SCD by meeting their transfusion requirements with blood selected from random black blood donors [30,88]. Luban et al [48] noted that children of SCD with non-American ethnic origins had a 42.9% incidence of alloimmunization compared to 17.6% in American counterparts with SCD. Antigen frequencies were similar in the two groups. Fewer transfusions resulted in more antibodies in the non-American children.

Transfusion Reactions Many sickle cell disease (SCD) patients require numerous blood transfusions in their lifetime. This leads to exposure of various blood products with resultant transfusion related complications. Among the most serious are delayed hemolytic transfusion reactions (DHTRs). Cytokines released from donor white blood cells (WBCs) commonly cause febrile reactions that can be minimized with acetaminophen administration before transfusion. Plasma proteins in donor blood can cause allergic reactions, which can sometimes be minimized by antihistaminic administration before transfusion. Febrile reactions can be minimized using third generation WBC filters that are most efficient when used near the time of blood collection from donor. Allergic reactions can be avoided by washing the red cells with saline before transfusion. All transfusion reactions have to be investigated according to local blood bank protocols.

Delayed Hemolytic Transfusion Reactions (DHTRs) The incidence of DHTR is between 1-5% [26,29,34,36-38,89-93]. DHTR involves exposure of the recipient to donor’s RBC antigens leading to formation of antibodies. This leads to antibody formation to the foreign antigen found on the donor’s RBCs, usually without any immediate consequences due to the mild and slow rise in titer. The titers drop over period of time, due to non-exposure to the donor antigen. Hence, on retesting after few months, the crossmatch may be compatible. However, on reexposure to the particular antigen, there is a brisk anamnestic antibody production and mild to severe delayed transfusion reaction can follow after few days to 3 weeks, usually after 7-10 days of exposure. The consequences have been described variously ranging from mild, with only a surprise alloantibody detection during crossmatch before blood transfusion, to severe involving acute or sub-acute intravascular hemolysis of the donor red blood cells (RBCs), labeled as a DHTR, leading to indirect hyperbilirubinemia, anemia, increased lactic dehydrogenase, renal failure, and sometimes death. The etiology of this syndrome is multifactorial. The factors may include a poorly understood genetic predisposition, [51,54] bystander hemolysis caused by activation of the complement system, [29,92] suppression of erythropoiesis, [38] and hyperactivity of reticuloendothelial cells [93]. DHTR often presents as a painful crisis in a patient with SCD [26]. DHTR syndrome in pediatric SCD patients typically presents 1 week after transfusion with back, leg, or abdominal pain; fever; and hemoglobinuria that may mimic pain crisis. Hemoglobin is often lower than it was at the time of original transfusion,

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suggesting the hemolysis of the patient's own RBCs (bystander effect) [38] in addition to hemolysis of the transfused RBCs; a negative DAT and reticulocytopenia are often present. Severe complications including acute chest syndrome, congestive heart failure, pancreatitis, and acute renal failure can be associated with DHTR syndrome. DHTR is a serious and potentially life-threatening complication of RBC transfusion. It is important to avoid additional transfusions in these patients, if possible, because these may exacerbate the hemolysis and worsen the anemia. DHTR syndrome must be included in the differential of a patient who has SCD and vasoocclusive crisis who has recently had a transfusion. In one 10 year single hospital study of 78 SCD children that received 1860 transfusions over 10 years [38], seven patients developed DHTR and/or serologic transfusion reactions, two with hyperhemolysis, two with clinical evidence of hemolysis, and three with serologic evidence only. The two patients with hyperhemolysis had received extended antigen-matched RBC transfusions to provide blood compatible with their existing antibodies. Hence the authors noted that hyperhemolysis, which may be triggered by a transfusion, was not prevented by matching for RBC antigens. In most instances, the cause of hyperhemolysis was multifactorial. Studies on DHTR are limited and because of the retrospective nature of the studies, they may have missed additional cases of DHTR. Because the diagnosis must be made with a high index of suspicion, there may be underrepresentation of the incidence of DHTR. Conversely, in the absence of a positive DAT, it is possible that there is overestimation of the frequency of DHTR as there may be some other unknown cause of accelerated hemolysis such as glucose-6-phosphate dehydrogenase deficiency. Hence in all of the patients with DHTR, glucose-6-phosphate dehydrogenase deficiency and other causes of hemolysis have to be investigated. It is a consistent finding in most patients with DHTR that they have lower hemoglobin than the starting pretransfusion level. Bystander hemolysis may occur by the development or augmentation of RBC autoantibodies (epitope spreading) as a result of alloimmunization from the transfusion [89]. Garratty [92] suggested another possible mechanism that may worsen the hemolysis by the reaction of alloantibodies with transfused RBCs, which leads to the attachment of activated complement components to autologous RBCs resulting in their lysis. Conversely, he proposed, in the absence of RBC alloantibodies, that other antibody reactions with transfused foreign antigens (e.g., HLA and plasma proteins) may cause complement activation. These antibodies may be present in patients who have received multiple transfusions, which may lead to immune complex formation. Inappropriately low reticulocytes counts are seen in the majority of patients with DHTR. The mechanism of the reticulocytopenia is unclear. Erythropoiesis can be suppressed by the transfusion or concurrent illness (viral infection), which can exacerbate the anemia. Other possible mechanisms of reticulocytopenia include accelerated destruction of reticulocytes as a result of selective antibody targeting of reticulocytes or decreased levels of erythropoietin secondary to kidney damage [65]. Numerous approaches have been devised to minimize the incidence of DHTRs – complete red blood cell phenotyping of all ‘at risk’ recipients before the first transfusion to document and record the RBC antigenic profile, various levels of extended crossmatch of donor’s and recipient’s blood depending on frequency of minor blood groups in the local

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donor population before transfusion, autologous blood transfusions, and lifelong avoidance of the particular antigen once the corresponding antibody is demonstrated.

CONCLUSION Though a lot of work has been done, much still remains to be learnt about causes, pathophysiology, manifestations, and management of RBC allosensitization in SCD. Breakthroughs in understanding normal and abnormal human immune response to foreign and self antigens would make its understanding and treatment easier. Because of multiple major exogenous factors such as donor/recipient composition, availability of blood products, etc. there is no consensus for the approach to the issue of preventing allosensitization.

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[29] King KE, Shirey RS, Lankiewicz MW, et al. Delayed hemolytic transfusion reactions in sickle cell disease: simultaneous destruction of recipients' red cells. Transfusion 1997;37:376-81. [30] Vichinsky EP, Earles A, Johnson RA, et al. Alloimmunization in sickle cell anemia and transfusion of racially unmatched blood. N Engl J Med 1990;322:1617-21. [31] Garratty, G. Autoantibodies induced by blood transfusion Editorial. Transfusion 2004;44:5-9. [32] Davies SC, McWilliam AC, Hewitt PE, et al. Red cell alloimmunization in sickle cell disease. Br J Haematol 1986;63:241-5. [33] Ambruso DR, Githens JH, Alcorn R, et al. Experience with donors matched for minor blood group antigens in patients with sickle cell anemia who are receiving chronic transfusion therapy. Transfusion 1987;27:94-8. [34] Lankiewicz MW, Shirey RS, Ness PM, Charache S. Delayed hemolytic transfusion reactions (DHTR) in sickle cell disease (SCD): evidence for autologous RBC destruction (abstract). Transfusion 1993;33S:22S. [35] Castellino SM, Combs MR, Zimmerman SA, et al. Erythrocyte autoantibodies in paediatric patients with sickle cell disease receiving transfusion therapy: frequency, characteristics and significance. Br J Haematol 1999;104:189-94. [36] Anderson RR, Sosler SD, Kovach J, DeChristopher PJ. Delayed hemolytic transfusion reaction due to anti-Js(a) in an alloimmunized patient with a sickle cell syndrome. Am J Clin Pathol 1997;108:658-61. [37] Sosler SD, Perkins JT, Saporito C, et al. Severe autoimmune hemolytic anemia induced by transfusion in two alloimmunized patients with sickle cell disease (abstract). Transfusion 1989;29S:49S. [38] Petz LD, Calhoun L, Shulman IA, et al. The sickle cell hemolytic transfusion reaction syndrome. Transfusion 1997;37:382-92. [39] Issitt PD. Race-related red cell alloantibody problems. British J Biomedical Science 1994;51:158-67. [40] Morel PA, Garratty G, Perkins HA. Clinically significant and insignificant antibodies in blood transfusion. American J Medical Technology 1978;44:122-9. [41] Noizat-Pirenne F, Lee K, Le Pennec P, et al. Rare RHCE phenotypes in black individuals of Afro-Caribbean origin: identification and transfusion safety. Blood 2002;100:4223-4231. [42] Cox JV, Steane E, Cunningham G, Frenkel EP. Risk of alloimmunization and delayed hemolytic transfusion reactions in patients with sickle cell disease. Arch Intern Med 1988;148:2485-9. [43] Diamond WJ, Brown FL Jr, Bitterman P, et al. Delayed hemolytic transfusion reaction presenting as sickle-cell crisis. Ann Intern Med 1980;93:231-4. [44] Castro O, Sandler SG, Houston-Yu P, Rana S. Predicting the effect of transfusing only phenotype-matched RBCs to patients with sickle cell disease: theoretical and practical implications. Transfusion 2002;42:684-90. [45] Young PP, Uzieblo A, Trulock E, Lublin DM, Goodnough LT. Autoantibody formation after alloimmunization: are blood tranfusions a risk factor for autoimmune hemolytic anemia? Transfusion 2004;44:67-72.

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[46] Garratty G, Telen MJ, Petz LD. Red cell antigens as functional molecules and obstacles to transfusion. Haematology 2002;122:445-62. [47] Moreira G, Bordin JO, Kuroda A, Kerbauy J. Red blood cell alloimmunization in sickle cell disease: the influence of racial and antigenic pattern differences between donors and recipients in Brazil. American J Hematol 1996;52:197-200. [48] Luban NL. Variability in rates of alloimmunization in different groups of children with sickle cell disease: effect of ethnic background. American Journal of Pediatric Hematology-Oncology 1989;11:314-9. [49] Olujohungbe A, Hambleton I, Stephens L, Serjeant B, Serjeant G. Red cell antibodies in patients with homozygous sickle cell disease: a comparison of patients in Jamaica and the United Kingdom. British J Haematol 2001;113:661-5. [50] Hmida S, Mojaat N, Maamar M, Bejaoui M, Mediouni M, Boukef K. Red cell alloantibodies in patients with haemoglobinopathies. Nouvelle Revue Francaise d Hematologie 1994;36:363-6. [51] Alarif L, Castro O, Ofosu M, et al. HLA-B35 is associated with red cell alloimmunization in sickle cell disease. Clin Immunol Immunopathol 1986;38:178-83. [52] Michail-Merianou V, Pamphili-Panousopoulou L, Piperi-Lowes L, Pelegrinis E, Karaklis A. Alloimmunization to red cell antigens in thalassemia: comparative study of usual versus better-match transfusion programmes. Vox Sang 1987;52:95-8. [53] Spanos T, Karageorga M, Ladis V, Peristeri J, Hatziliami A, Kattamis C. Red cell alloantibodies in patients with thalassemia. Vox Sang. 1990;58:50-5. [54] Valentin N, Vergracht A, Bignon JD, et al. HLA-DRw52a is involved in alloimmunization against PL-A1 antigen. Human Immunol 1990;27:73-9. [55] Rayment R, Birchall J, H Yarranton, et al. Neonatal alloimmune thrombocytopenia BMJ 2003;327:331–2. [56] Wallhermfechtel MA, Pohl BA, Chaplin H. Alloimmunization in patients with warm autoantibodies. A retrospective study employing three donor alloabsorptions to aid in antibody detection. Transfusion 1984;24:482-5. [57] Adams DM, Schultz WH, Ware RE, Kinney TR. Erythrocytapheresis can reduce iron overload and prevent the need for chelation therapy in chronically transfused pediatric patients. J Pediatr Hematol Oncol 1996;18:46-50. [58] Rous P, Robertson OH. Free antigen and antibody circulating together in large amounts (hemagglutinin and agglutinogen in the blood of transfused rabbits). J Exp Med 1918;27:509-17. [59] Stahl D, Desmazes-Lacroix S, Sibrowski W, et al. Red blood cell transfusions are associated with alterations in self reactive antibody repertoires of plasma IgM and IgG, independent of the presence of a specific immune response toward RBC antigens. Clin Immunol 2002;105:25-35. [60] Ovary Z, Speigelman J. The production of "cold auto-agglutinins" in the rabbit as a consequence of immuni-zation with isologous erythrocytes. Ann N Y Acad Sci 1965; 124:147-53. [61] Zmijewski CM. The production of erythrocyte autoanti-bodies in chimpanzees. J Exp Med 1965;121:657.

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[62] Liu CK, Evans RS. Production of positive antiglobulin serum test in rabbits by intraperitoneal injection of homologous blood. Proc Soc Exp Biol Med 1952;79:194. [63] Naysmith JD, Ortega-Pierres MG, Elson CJ. Rat erythrocyte-induced anti-erythrocyte autoantibody production and control in normal mice. Immunol Rev 1981;55:55-87. [64] Branch DR, Petz LD. Detecting alloantibodies in patients with autoantibodies. Transfusion 1999;39:6-10. [65] Janssen van Doorn K, Diltoer M, Servotte S, et al. Transfusion of polymerized bovine haemoglobin in a patient with sickle cell anaemia and severe allo-immunization: a case report. Acta Clinica Belgica 2001;56:191-4. [66] Billingham RE, Brent L, Medawar PB. ‘Actively acquired tolerance’ of foreign cells. Nature 1953;172:603. [67] Opelz G, Mickey MR, Terasaki P: Effect of blood transfusions on subsequent kidney transplants. Transplant Proc 1973;5:253. [68] Persijn GG, Cohen B, Lansbergen Q, Van Rood JJ: Retrospective and prospective studies on the effect of blood transfusions in renal transplantation in The Netherlands. Transplantation 1979;28:396. [69] Jonker M, Persijn GG, Parlevliet J, Frederiks E, Van Rood JJ: The influence of previous immunization on skin graft survival. Transplantation 1979;27:250. [70] van de Watering LMG, Hermans J, Houbiers JG, van den Broek PJ, Bouter H, Boer F, Harvey MS, Huysmans HA, Brand A: Beneficial effects of leukocyte depletion of transfused blood on postoperative complications in patients undergoing cardiac surgery. A randomized clinical trial. Circulation 1998;97:562. [71] Shanwell A, Kristiansson M, Remberger M, Ringde´n O: Generation of cytokines in red cell concentrates during storage is prevented by prestorage white cell reduction. Transfusion 1998;37:678. [72] Mincheff M: Changes in donor leukocytes during blood storage. Implications on posttransfusion immunomodulation and transfusion associated GVHD. Vox Sang 1998;(suppl 2)74:189. [73] Westhoff U, Grosse Wilde H: Soluble HLA class I and class II concentrations in factor VIII and PCC preparations.Vox Sang 1995;68:73. [74] Grosse-Wilde H, Blasczyk R, Westhoff U: Soluble HLA class I and class II concentrations in commercial immunoglobulin preparations. Tissue Antigens 1992;39:74. [75] Blasczyk R, Westhoff U, Grosse-Wilde H: Soluble CD4, CD8, and HLA molecules in commercial immunoglobulin preparations. Lancet 1993;341:789. [76] Santoso S, Kiefel V, Volz H, Mueller-Eckhardt C: Quantitation of soluble HLA class I antigen in albumin and immunoglobulin preparations for intravenous use by solidphase immunoassay. Vox Sang 1992;62:29. [77] Klu¨ter H, Schlenke P, Mu¨ller-Steinhardt M, Paulsen M, Kirchner H: Impact of buffy coat storage on the generation of inflammatory cytokines and platelet activation. Transfusion 1998;37:362. [78] Ghio M, Contini P, Mazzei C, et al. Soluble HLA Class I, HLA Class II, and Fas ligand in blood components: A possible key to explain the immunomodulatory effects of allogeneic blood transfusions. Blood 1999;93:1770-7.

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[79] Friedman DF, Lukas MB, Jawad A, Larson PJ, Ohene-Frempong K, Manno CS. Alloimmunization to platelets in heavily transfused patients with sickle cell disease. Blood 1996;88:3216-22. [80] Afenyi-Annan A, Brecher, ME. Pre-transfusion phenotype matching for sickle cell disease patients Transfusion 2004;44:619. [81] Tahhan HR, Holbrook CT, Braddy LR, et al. Antigen-matched donor blood in the transfusion management of patients with sickle cell disease. Transfusion 1994;34:5629. [82] Ness PM. To match or not to match: the question for chronically transfused patients with sickle cell anemia. Transfusion 1994;34:558-60. [83] Wayne AS, Kevy SV, Nathan DG. Transfusion management of sickle cell disease. Blood 1993;81:1109-23. [84] Telen MJ, Combs M. Management of massive delayed hemolytic transfusions reactions in patients with sickle cell disease. Transfusion 1999;39S:97. [85] Bartholomew A, Sher D, Sosler S, et al. Stem cell transplantation eliminates alloantibody in a highly sensitized patient. Transplantation 2001;72:1653-5. [86] Monsaingeon-Lion A, Le Pennec PY, Bridey F et al. A sickle cell homozygote with transfusion deadlock. Favorable outcome with hydroxyurea treatment. Revue Francaise de Transfusion et d Hemobiologie. 1993;36:477-84. [87] Sumoza A, de Bisotti R, Sumoza D, Fairbanks V. Hydroxyurea (HU) for prevention of recurrent stroke in sickle cell anemia (SCA). American J Hematol 2002;71:161-5. [88] Sosler SD, Jilly BJ, Saporito C, Koshy M. A simple, practical model for reducing alloimmunization in patients with sickle cell disease. Amer J Hematol 1993;43:103-6. [89] Talano JA, Hillery CA, Gottschall JL, Baylerian DM, Scott JP. Delayed hemolytic transfusion reaction/hyperhemolysis syndrome in children with sickle cell disease. Pediatrics 2003;111:e661-5. [90] Telen MJ, Combs M. Management of massive delayed hemolytic transfusions reactions in patients with sickle cell disease. Transfusion 1999;39S:97. [91] King KE, Shirey RS, Lankiewicz MW, Young-Ramsaran J, Ness PM. Delayed hemolytic transfusion reactions in sickle cell disease: simultaneous destruction of recipients' red cells. Transfusion. 1997;37:376-81. [92] Garratty G. Severe reactions associated with transfusion of patients with sickle cell disease (letter). Transfusion 1997;37:357-61. [93] Win N, Doughty H, Telfer P, et al. Hyperhemolytic transfusion reaction in sickle cell disease. Transfusion 2001;41:323-8.

In: New Developments in Blood Transfusion Research ISBN 1-59454-962-1 Editor: Brian R. Peterson, pp. 121-140 © 2006 Nova Science Publishers, Inc.

Chapter VII

ACUTE NORMOVOLEMIC HEMODILUTION; ITS ROLE AS A BLOOD CONSERVING TECHNIQUE C. F. Weiniger and I. Matot∗ Department of Anesthesiology and Critical Care Medicine, Hadassah Hebrew University Medical School, Jerusalem, Israel.

ABSTRACT Concerns about the risks associated with allogeneic blood transfusion (infectious disease, transfusion reactions, immunomodulation, transfusion-associated lung injury) have led to the development of a variety of blood conserving techniques intended to minimize the need for allogeneic transfusion during surgery. Among these, acute normovolemic hemodilution (ANH) has become accepted. The procedure entails the removal of blood from the patient immediately before operation, and simultaneous replacement with appropriate volume of crystalloid and/or colloid fluids. ANH reduces hematocrit (Hct) so that blood shed during the operative procedure results in less red blood cell mass loss. The removed blood is then reinfused as autologous whole blood after the procedure is completed. The procedure is simple and inexpensive and has the advantage that fresh autologous blood is readily available. The resultant reduction in allogeneic blood transfusion may conserve resources and protect the patient from exposure to the risks of allogeneic blood transfusion. Numerous studies of its efficacy, however, have produced conflicting results, perhaps due to the heterogeneity of the surgeries in which it was employed, differences in study protocol and differences in the definition of outcome variables. Previous mathematical analyses of ANH have shown that ANH may be effective in diminishing the need for allogeneic blood transfusion, and that its efficacy depends on surgical blood loss, initial patient Hct and on the “transfusion trigger” (the Hct at which blood is to be transfused). One meta-analysis of ANH used in 24 quality randomized controlled trials ∗

Correspondence concerning this article should be addressed to Idit Matot, MD Department of Anesthesiology and Critical Care Medicine, Hadassah Hebrew University Medical School, Ein Kerem POB 12000, Jerusalem 91120 Israel. Email: [email protected]; Tel: 972-050-7874307; Fax: 972-2-5337418

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C. F. Weiniger and I. Matot found that ANH reduced the likelihood of exposure to at least 1 unit of allogeneic blood, and had a small positive effect on the perioperative blood loss when compared with patients who did not undergo ANH. Use of ANH is controversial and it is not universally used. The objective of this review is to get the reader acquainted with this blood conserving technique, understand the parameters that determine its efficacy, define which group of patients might benefit from its use, and learn about its potential risks.

INTRODUCTION Concerns about the risks associated with allogeneic blood transfusion (infectious disease, transfusion reactions, immunomodulation, transfusion-associated lung injury), and more specifically the AIDS epidemic, triggered the development of a variety of blood conserving techniques intended to minimize the need for allogeneic transfusion during surgery. Several methods have been described during the perioperative period in order to avoid or reduce the reliance on allogeneic blood products. These include preoperative autologous blood donation, intra- and post-operative blood salvage, ANH and the following pharmaceuticals: aprotinin, epsilon-aminocaproic acid, tranexamic acid, desmopressin and recombinant human erythropoietin. There is no one single universal blood conservation strategy that is applicable to all patients and populations. Clearly, factors such as preexisting disease will alter the approach, as well as financial, administrative and religious considerations. Little is known about current utilization in the United States of technologies or techniques to reduce allogeneic blood transfusion. The results of a survey sent to 1,000 hospitals in United States reported that preoperative blood donation and cell salvage were used most frequently. Moreover, hospitals reported use of techniques significantly more than pharmaceuticals [1]. ANH was described to be useful as a blood conservation strategy more than 30 years ago, yet seldom is practiced today. Emerging clinical studies now show that ANH is equivalent to pre-donated autologous blood in reducing allogeneic blood exposure in patients undergoing elective surgery, and moreover it is less costly and there is no possibility of administrative error [2,3]. The objective of this chapter is to review ANH as a blood conserving technique, to understand the parameters that determine its efficacy, define which group of patients might benefit from its use, and learn about its potential risks.

TECHNIQUE ANH involves the removal and simultaneous infusion of clear fluids to maintain isovolemia. The replacement fluid may be either crystalloid [4] (3 ml crystalloid for every 1ml of blood withdrawn) or colloid solution (1 ml colloid for every 1ml of blood withdrawn). No significant difference has been found among dilutents [5]. Blood is withdrawn after induction of anesthesia, prior to blood loss, through a large venous catheter, into citratephosphate-dextrose storage bags, which are numbered and maintained at room temperature up to 6 hours [4,6]. Atallah suggested that ANH might be safely initiated prior to induction of

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anesthesia, in order to increase the amount of time available in order to perform ANH prior to the start of surgery [7]. During collection, the bags should be periodically mixed. One unit of blood should take up to 10 minutes to withdraw. Monitoring should include direct arterial blood pressure monitor, which also facilitates repeated measures of the Hct. Up to 2000 ml may be removed for ANH, which will result in up to 5 autologous blood bags [4,6]. Each bag should contain from 300-450 ml of blood. The time of blood withdrawal, the volume withdrawn and the fluid replacement volume should be recorded on the anesthesia chart [4]. The removed blood is re-infused as autologous whole blood after the procedure is completed in reversed order. In summary, ANH entails removal of blood of a higher Hct concentration at the start of surgery and that is saved for later transfusion. The theoretical advantage of ANH as a blood conservation method is that each unit of blood lost during surgery contains a lower Hct and therefore is of a lower hemoglobin concentration [4]. At the end of the operation the patient is transfused with autologous blood with high Hct levels.

MATHEMATICAL MODEL In order to predict the efficacy of ANH, it can be described using mathematical analysis. Mathematical computation may determine the actual volume of blood which may be safely removed prior to surgery by measuring the initial Hct. The target Hct is determined as the Hct at which autologous blood will be transfused back to the patient. A high initial Hct level, a low target Hct, and a high surgical blood loss are required in order to make the procedure most efficacious [8]. The original mathematical model was described by Bourke and Smith [9]:

V=

EBV x (Ho − Ht) Hav

where V = blood volume removed, EBV = estimated blood volume, Ho = initial Hct, Ht = target Hct, and Hav = mean of Ho and Ht. This equation implies that a small change in initial Hct level should equal a small change in blood volume from the initial blood volume. This formula should be used in clinical practice by defining the target Hct, and then calculating V, the volume of blood to be removed by ANH. The main fixed parameter of this formula, EBV, varies by body weight, however actual preoperative blood volume is unknown, and may vary greatly among patients [8]. Using the basic formula for a 70 kg adult, with an initial Hct of 0.45, and using a target Hct of 0.35 will allow a removal of 1250 ml of blood, whereas if the initial Hct is lower at 0.35, and the target Hct more extreme at 0.25, the blood volume removed will be only 1000 ml. The usefulness of mathematical modeling is the ability to measure the savings in allogeneic blood. This will depend greatly on the target Hct, which may be 0.25, or more

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extreme at 0.20 [10] or lower 0.15 [11]. It is important to note that moderate normovolemic hemodilution (i.e. a post-hemodilution Hct value above 24%) is of minimal value [11]. When using the mathematical model in order to determine the maximum saving on allogeneic blood, or the minimal Hct for transfusion [11,12] the results are conflicting with regard to the saving of allogeneic blood. Currently, removal of only 2 or 3 units of blood is common [4,13]. With the removal of only 2 units, and a target Hct of 0.26, successful ANH requires an initial Hct of at least 0.46 [14]. The removal of 1 extra unit allows a 4% increase in target Hct, or a 4% decrease in initial Hct. These authors suggest that a 70-kg patient with an initial Hct of 0.4, and a target Hct of 0.26 must have 5 units of blood removed to achieve maximum benefit. One examination of the maximum potential reductions in allogeneic transfusions used an exponential equation to describe the dilution of hemoglobin [11]. It was found that greater the initial Hct, and the greater the blood volume, the greater blood loss necessary to reach target Hct from the initial Hct. By extending the dilution target from a Hct of 0.3 down to 0.25, the first unit of autologous blood replaced will cover 857 ml of surgical blood loss, while at a target Hct of 0.15 it will replace 1428 ml. Thus hemodilution from 45 to 15 percent potentially saves 3.9 units of allogeneic blood being transfused [11]. The value in this model is the removal of the last of unit of blood, with a Hct itself close to the target Hct. By further decreasing the circulating Hct, the relative Hct value of the other removed bags is increased. Although 15 percent may be considered an extreme target Hct, current recommendations from the consensus conference of the National Institutes of Health recommend that hemoglobin concentrations higher than 7 g/dl do not require blood transfusion [15]. Using a different model, with a calculation for the patient’s red blood cell volume on a minute-minute basis, the transfusion of autologous blood was begun when the target Hct was reached [12]. Units of ANH blood were transfused such that the lower Hct unit was transfused first. In this study, maximum savings of allogeneic blood were only 1.2 units. In order to be efficacious the initial Hct must be sufficiently high, the target Hct sufficiently low along with a sufficient expected blood loss for the surgery. Previously it was suggested that ANH is appropriate when the expected blood loss is 20% of the estimated blood volume [10]. This blood removal target, however, has been challenged as mathematically unproven [16]. Weiskopf developed a mathematical analysis [11] which examined the efficacy of ANH as a function of the fraction of blood volume lost during surgery. The author suggests that 50% of the EBL must be lost in order to comply with National Consensus guidelines by administering blood only when the hemoglobin falls to 7g/dl [15]. This would imply that the target estimated blood loss is not 20% but closer too 70% of the total estimated blood volume. Weiskopf concluded that the expected surgical blood loss should be 0.71–1.20 or more of the patient’s blood volume for ANH to be efficacious (Figure 1). A lesser blood loss does not require blood transfusion. As the author noted, a loss of 20% of blood volume would reduce an initial Hct of 0.45 to 0.37, a value not ordinarily requiring transfusion or an erythrocyte-saving strategy [16]. This finding is supported by Kick and Daniel [14], who suggested that many patients, with a high initial Hct can tolerate up to 50% loss of blood volume due to surgical bleeding without ANH. Thus patients for whom the estimated blood loss exceeds 1500-2000 ml, with no possibility of PABD will most likely benefit from ANH [14].

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Thus using the mathematical model will guide the clinician with regard to the blood volume to be removed prior to surgery, but it may also be used to prove, or not, the efficacy of ANH.

Table 1 Parameters to be considered for ANH. Patients' factors * Absence of restrictive or obstructive lung disease Absence of renal failure Absence of coagulopathies Absence of ischemic heart disease in patients undergoing non cardiac surgery Absence of cirrhosis Absence of infection Factors that determine whether ANH will benefit the patient High initial Hct Low target Hct Withdrawal of sufficient volume of blood Surgical blood loss is within the range of potential efficacy * There are no absolute contraindications for ANH. Factors that may be considered of concern are mentioned in the table.

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ELIGIBILITY From the mathematical analysis it can be seen that the main eligibility criteria are a high initial Hct, low target Hct and a sufficiently high expected blood loss (Table 1). Anemia (hemoglobin < 11 g/dl) is the main contraindication to ANH [4,10], since the initial Hct is too low. Cardiac disease would preclude ANH due to the deleterious effects of increasing cardiac output as a response to ANH. The risk of silent ischemia is frequent in cardiac patients, even without hemodilution. This means that ANH is a risky procedure for patients with existing cardiac disease. Other contraindications include restrictive or obstructive lung disease, renal disease, cirrhosis, coagulopathies and infection [17]. Appropriate vascular access and monitoring capabilities are necessary for performing ANH. Age is not a limiting factor when deciding to use ANH [10].

PHYSIOLOGY Cardiovascular Physiological compensatory mechanisms for ANH may actually confer an advantage to the use of this blood conservation technique. Following removal of blood from the patient prior to surgery, the oxygen carrying capacity of the blood decreases. The main components of oxygen carrying capacity are cardiac output and hemoglobin. The other mechanism for maintaining tissue oxygenation is the tissue oxygen extraction ratio. Nunn described the surplus hemoglobin in the body, which implies a significant oxygen reserve in the healthy patient [18]. This implies both that the patient can well tolerate not only ANH, but also surgical blood loss. As the Hct is reduced to target Hct (0.2-0.25), while isovolemia is maintained, the cardiac output increases by 15-20% during anesthesia [19]. Even without this increase in cardiac output, the oxygen extraction ratio increases. Elderly patients demonstrated increased oxygen extraction without an increase in cardiac output, with a target Hct of 0.28 during hip arthroplasty, which suggests the safe use of ANH in this group of patients [6]. The prime source of the increased cardiac output is the decreased viscosity resulting from ANH. The total peripheral resistance falls as the vascular tone falls due to the decreased red cell mass. This in turn decreases afterload, which results in an increased cardiac output. The heart rate does not increase with ANH as long as normovolemia is maintained. However there is a cardiac compensatory mechanism for the decreased Hct. The venous return increases, afterload falls, and resulting stroke volume raises the cardiac output. If hypovolemia occurs in addition, the resulting fall in cardiac output will decrease overall oxygen delivery [20]. Thus patients with cardiac disease will not tolerate ANH, and may respond with myocardial ischemia. Under anesthesia with enflurane, moderate ANH (to Hct of 0.3) was not associated with an increase in cardiac output among a group of patients undergoing hip arthroplasty [21]. In this group a fall in oxygen delivery occurred, however, an increase in oxygen extraction was observed. The proposed mechanism for the failure to raise cardiac output included a limit in increased venous return due to the depressant effect of enflurane on

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venous motor tone, and a poor adrenergic response to ANH due to negative inotropic effect of enflurane. Thus volatile anesthesia may interfere with the normal cardiac response to ANH, which may result in tissue hypoxia. Tissue oxygen extraction ratio is not measurable at the individual tissue level. It is a calculated value, whereby CaO2 - CvO2 (arterial and venous oxygen content, respectively) is the measure of adequate oxygenation. If CaO2 -CvO2 increases, more oxygen is utilized in the tissues, reflecting a decrease in oxygen reserve. Hct as low as 0.2 has been shown to either maintain, or even decrease oxygen utilization. Thus it may be proposed that ANH increases oxygen delivery to the tissues, secondary to capillary recruitment [22]. In normal volunteers, who undergo hemorrhage without volume replacement, the oxygen extraction increases more than the cardiac output [23]. Another proposed mechanism for increased oxygen utilization is a shift in the oxygen dissociation curve to the right [24,25,26]. However this has only been demonstrated as actually occurring in animals. Awake patients with moderate ANH increase both the stroke volume and heart rate in order to maintain tissue oxygenation, and in addition a significant rise in oxygen extraction ratio has been demonstrated. Anesthetized patients are less able to increase the cardiac index as a response to ANH [27]. The small rise in cardiac index in anesthetized patients is due to a rise in stroke index alone. Tissue oxygenation is maintained by an increase in oxygen extraction. This difference is probably related to anesthesia medication. Patients with cardiac disease may also tolerate moderate ANH (Hct 0.28) by increasing the stroke volume and preload indices [28]. Under anesthesia patients with coronary disease undergoing moderate ANH maintain left ventricular systolic and diastolic function. The reduced viscosity improves cardiac output, as is seen in normal patients [28]. In addition, although overall oxygen delivery falls due to the reduced hemoglobin and result in fall in oxygen carrying capacity, this does not cause myocardial ischemia. Similarly, patients with obstructed left ventricular outflow due to critical aortic stenosis demonstrated an ability to tolerate moderate ANH. Using transesophageal echocardiography in anesthetized patients these patients demonstrated increased stroke volume, while mean arterial pressure decreased together with heart rate [29]. Interestingly, among these patients with critical aortic stenosis the increased preload indices contributed to the increased cardiac output. However, normally the reduced viscosity with ANH would accelerate blood flow from the left ventricle, but there was an impaired response to the reduced viscosity due to the outflow obstruction from the stenosed valve.

Nervous System Human response time slows as a result of severe ANH (target hemoglobin of 5g/dl) [30]. ANH also degrades memory, and decreases energy levels. These effects may be reversed by the addition of oxygen supplement to raise arterial oxygen content.

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COAGULATION ANH may provide an advantage in that the blood removed and stored at room temperature contains normal concentrations of platelets and other clotting factors, which are lacking in stored blood [31]. However a lower Hct has also been associated with a prolonged prothrombin time, and partial thromboplastin time, but without clinical effects [32].

Gastrointestinal Splanchnic oxygen uptake represents 25% of normal total body oxygen consumption. Gastric mucosal oxygenation has not been shown to be affected by ANH, or reducing Hct even to severely low values of 0.2 [33]. Hepatic arterial blood flow rises 86% during ANH, as does small intestinal perfusion, but to a lesser degree [34].

Pulmonary Pulmonary function has not been shown to be affected by ANH.

SAFETY The removal of substantial quantities of blood and the reduction of the hemoglobin concentration to values sufficient to produce efficacy has the potential to threaten patient safety. Previous studies of ANH did not evaluate a sufficient number of patients to document its safety. To document an increase in the incidence of an adverse event, such as myocardial ischemia, inclusion of hundreds of patients will be required. A recent meta-analysis of 42 randomized controlled trials reported death, myocardial infarction, cardiac ischemia, venous thromboembolism, cerebral infarction and hypotension in patients with ANH [35]. Transfusion reaction to autologous blood has not been demonstrated. The incidence of adverse events, however, was not significantly different from that observed in the control group, suggesting the relative safety of this technique.

Technical Safety The blood should be kept in the same room as the patient in order to avoid erroneous administration. Refrigeration may be used if the bags are standing more than 6 hours, but ideally the bags should be kept at room temperature to preserve platelet function. Refrigeration should be provided with wet ice, with attention paid to uniform chilling. Dry ice may result in non-uniform cooling. Units not used within 24 hours should be discarded [4].

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Threshold Hemoglobin Concentration The lowest safe hemoglobin concentration in humans is not known. To preserve oxygen delivery to and oxygenation of critical organs, normovolemia must be maintained. Hemodilution, however, by decreasing the oxygen carrying capacity and oxygen content increases the potential for critical organ ischemia. However, it should be noted that healthy, resting humans tolerate hemoglobin concentrations of 5 g/dl and oxygen delivery as low as 7.3 ml O2 · kg-1 · min-1 without evidence of inadequate systemic oxygenation [36]. The indications for autologous transfusion are not definitive. Unlike the awake patient, symptoms of oxygen transport deficit are unclear in the anesthetized patient [11]. Blood gas measurements including base excess, lactate, and even invasive measurements such as cardiac index and oxygen extraction ratio may give a clearer indication of the need for blood transfusion. Hemoglobin levels may not reflect an oxygen carrying deficiency [11]. Practice guidelines suggest transfusing below a hematocrit of 0.21. The safe limits of hemodilution for patients who cannot increase blood flow sufficiently to critical organs (e.g., because of arterial stenosis, vasculitis, or impaired cardiac function) are not known. Hemodilution has not been associated with systemic or cardiac markers of inadequate oxygenation in patients undergoing coronary artery surgery [37,38]. The coronary vasculature has a reserve dilatory capacity, which, in response to acute anemia, can increase blood flow by several-fold. However, data from laboratory studies clearly demonstrate that at very low hemoglobin concentrations (below 3–5 g/dl), the myocardium becomes hypoxic, with decreased contractility, and that the hemoglobin value at which this occurs is higher when coronary artery blood flow is limited by a stenosis [39-41]. Postoperatively, the hemoglobin concentration should be high enough to facilitate increased oxygen consumption due to shivering, increased metabolism, and basic physical activities. A low target Hct (0.15-0.2) should be increased by autologous transfusion, prior to extubation [42].

SURGICAL MODALITIES FOR USE OF ANH ANH has been described among many types of surgery. Most commonly it is used in cardio-pulmonary bypass surgery, however, it has been described and studied in major vascular surgery, primary and revision of hip surgery, major spine surgery, hepatic resection, and radical pelvic surgery [4]. The universal factor among these surgical procedures is the large expected peri-operative blood loss. Both children [43-46], and elderly patients were studied [6,47]. ANH was evaluated as a single blood conservation technique or combined with preoperative autologous blood donation (PABD).

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Table 2 Study characteristics when ANH performed prior to cardio-pulmonary bypass for cardiac surgery (coronary artery-bypass graft or valve surgery). N in ANH group (total N) Hallowell 1972 [54] Boldt 1988 [55] Dietrich 1989 [56]

25 (50)

Patient age (mean or range, years)

Initial Hct

Target Hct

Mean Blood volume withdrawn

Efficacy vs control for reduction in allogeneic blood transfusion

38

1252 ml

3 unit saving in blood bank products, p = 0.01, No significant difference† Significant reduction with ANH†, and advantage enhanced when cell saver used, p < 0.01 No significant difference† No significant difference No significant difference, p=0.58 Significant reduction† with ANH alone. When ANH combined with cell saver p = 0.001 Significant reduction with ANH, p=0.03 Significant reduction with ANH, p < 0.05 with both degrees of ANH No significant difference, p=0.9 No significant difference† Significant when cell saver used, p = 0.015, versus control, regardless of use of ANH

15(30)

42

42

25

10 ml/kg

25 (100)

55

42

30

731 ml

Boldt 1991 [57] Vedrinne 1992 [58] Triulzi 1995 [59] Tempe 1996 [60]

15(45)

N/A

41

27

10 ml/kg

30(90)

58

42

24

400 ml

18(70)

60

50(150)

27

36

25

303 mL

Herregods 1997 [51] Kahraman 1997 [61]

39 (71)

62

40

34

750 ml/kg

14 (42) patients per group, 3 groups 103 (181)

41-70

42

30

5-8 ml/kg, 12-15 ml/kg

63

42

20

500 ml/kg

40 (80)

63

43

17

1099 ml

86 (256)

63

14 g/dL

20

10 ml/kg

Casati 2002 [62] Hohn 2002 [63] McGill 2002 [53]

924 ml

N = Number of patients; N/A = not available; Using Medline (1966-2005), studies using ANH versus no ANH, published in English were included. Only studies comparing ANH with no treatment (or in combination therapy) were included. Studies that recorded a saving in allogeneic blood transfusion were included; † = no p value given.

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Cardiac Surgery (Table 2) Cardio-pulmonary bypass uses a prime solution, which when connected to the patients’ circulation increases the overall blood volume. Thus a large degree of hemodilution is to be expected in all patients undergoing cardio-pulmonary bypass. This hemodilution demonstrates specific physiological advantages similar to those seen in ANH, in that the lower blood viscosity facilitates improved organ perfusion. Currently in adults and large children a clear prime solution is used. Children under 35 kg still receive some allogeneic blood in the prime solution due to the small blood volume relative to the prime volume, and the overall aim is to maintain the Hct above 0.18 with the initiation of bypass. During the hypothermia performed on bypass, the blood viscosity increases and this may reduce organ perfusion. Excessive hemodilution (greater than 50% fall in hemoglobin concentration) may cause critical ischemia, as the blood flow cannot increase further to compensate for reduced oxygen carrying capacity. Although oxygen requirements decrease during the hypothermia, during re-warming sufficient hemoglobin concentration must allow adequate oxygen delivery. Patients with critical stenosis may be less able to increase blood flow to compensate for reduced hemoglobin, although this effect may be offset by the decreased viscosity. Patients who are hypothermic, alkalotic or who have reduced 2,3-diphosphoglycerate levels will have a left-shift in the oxygen dissociation curve, with subsequent reduction in the amount of oxygen released by the hemoglobin to the tissues. Patients undergoing cardiopulmonary bypass procedures are susceptible to a variety of hemostatic derangements that lead to the need for frequent transfusions of allogeneic blood products. ANH may be performed in addition to hemodilution from the use of pump prime. Cardiac patients, with critical stenosis or severe coronary artery disease may not endure removal of autologous blood during anesthesia induction prior to the surgery. Thus it is usually performed once the patient is cannulated and connected to the bypass machine. The blood withdrawn for ANH is removed as it makes the first pass through the roller pump (in order to remove blood of a sufficiently high Hct). It should be noted that the blood removed following cannulation is heparinized, and this may affect the quality of the blood removed by reducing platelet function [48,49]. ANH performed during cardiac surgery does not appear to increase postoperative complications when compared with patients who do not undergo ANH [50]. The volumes of blood withdrawn during cardiac surgery in randomized control trials are presented in Table 2. As can be seen, a wide range of target Hct and removed blood volumes have been described. To date, the published trials derive no uniform conclusions. Conflicting results are apparent, with some trials which describe reduction of the need for allogeneic transfusion [51] while other studies describe partially positive effects only on patients at high risk for excessive postoperative bleeding [52], or a lack of effect of ANH [53]. It is of importance to note that the negative results of several studies clinically confirm the theoretical conclusions of mathematical analysis on ANH, [16] which state that only high volume ANH in the presence of significant intraoperative bleeding significantly reduces the need for allogeneic transfusions. During off-pump surgery, one study demonstrated with moderate ANH a significant reduction in the allogeneic blood transfusion requirements, suggesting that this patient population may benefit from the use of ANH [64]. Another recent

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study advocated a multi-modality approach to blood conservation during cardiac surgery including identification of those patients at greater risk for transfusion [52]. Thus in cardiac surgery, although the theoretical advantages of ANH with regard to savings of allogeneic blood are clear, the actual benefit appears to be still in question.

Radical Pelvic Surgery (Table 3) Several studies have described the use of ANH either during radical prostatectomy or hysterectomy [13,42,65-68]. Goodenough did not demonstrate a saving in allogeneic blood transfusion with ANH in patients undergoing radical prostatectomy when compared to the patients with no autologous blood available [13]. Monk [67], however, demonstrated a 44% reduction in the cost of blood therapies using ANH during radical prostatectomy which translated to a 0.6 unit saving in allogeneic blood. Removal of 30% of the patients’ blood volume saved 145 ml red blood cells, whereas removal of 16% of the patients’ blood volume saved 95 ml red blood cells. As with ANH in cardiac surgery, these studies demonstrate clinically what has been suggested in theoretical mathematical models, that increasing the volume of blood removed increases the efficacy of ANH. Patients undergoing radical prostatectomy are typically older (> 60 years of age), yet no cardiovascular complications were demonstrated. Among 15 patients undergoing radical hysterectomy, ANH (target Hct 0.24) was shown to reduce the surgical loss of erythrocytes [42]. The authors suggested that use of ANH in combination with PABD could promote economical treatment of patients’ preoperatively donated erythrocytes. One potential pitfall of radical hysterectomy may be that these patients are often anemic. This may preclude the use of ANH. One study examined 4 patients undergoing radical hysterectomy, with an initial Hct below 0.33, while ANH was performed to a target Hct of 0.16. Savings of erythrocytes (maximum 148 ml) were demonstrated, and a reduction of 30% surgical red cell loss was seen [66]. In summary, for radical pelvic surgery with an expected blood loss of up to 2000 ml, the savings in allogeneic blood are small (typically less than 1 unit) when up to 30% of the blood volume is removed during ANH. The expected blood loss during radical pelvic surgery is sufficient to justify the use of ANH, yet studies have not yet demonstrated the efficiency of ANH as a blood conservation method, primarily because the blood volume removed has been conservative.

Orthopedic Surgery ANH has been described in several orthopedic procedures; primary and revision hip surgery and spine surgery [46,69]. As emphasized previously, the estimated blood loss for an orthopedic procedure to benefit from ANH should be sufficiently large. In studies of joint surgery, patients who had at least 1000 ml autologous blood withdrawn, and used a transfusion protocol demonstrated the greatest benefit from ANH [69]. Despite a higher patient age among these orthopedic patients [21,70], ANH did not appear to effect the cardiovascular stability.

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Table 3 Study characteristics when ANH performed during radical pelvic surgery versus no ANH. Surgery performed

Mean N Age of patients

Rehm 2000 [66]

Radical hysterectomy

47

Rehm 2000 [42]

Radical hysterectomy

Goodenough 1994 [13]

Radical retropubic prostatectomy Radical retropubic prostatectomy Radical retropubic prostatectomy

Monk 1997 [67] Monk 1995 [68]

Blood volume removed ml 1300 (40%)

EBL during Blood surgery ml saving

4

Hct Transtarget fusion (%) trigger Hct 20 16

44

15

24

1150

727±726

60

11

24

1000

1731

60

250

28

25

1622 (29.6%)

1555

64

30

29

25

1740 (32%)

20

80-149 ml (max 1 unit blood) 117 ml (1 unit of blood) 95 (0.48 unit) 112 (0.6 unit) 143 ml

Medline (1966-2005) was used to identify studies published in English, where ANH was compared to no therapy (control). N = Number of patients; ml = milliliter; Hct = hematocrit; ANH = acute normovolemic hemdilution; PABD = pre-operative autologous blood donation

During hip surgery, Shulman [71] described a reduction in allogeneic transfusion from 2.4 to 0.6 units, and Van der Linden [21] also found a reduction in allogeneic blood products when ANH was compared to a group where the baseline Hct was maintained by allogeneic transfusion. ANH has been compared in hip surgery to preoperative autologous blood donation [70], and found to be equivalent with regard to allogeneic blood exposure, yet more cost effective. In this study only two units of autologous blood were procured during surgery, and the Hct decreased only from 0.4 to 0.31. Thus the saving may have been greater if the ANH was less conservative. Spine surgery may expect particularly high blood loss, and thus in theory ANH should be a suitable procedure for blood conservation. Martin [46] described extreme ANH in spine surgery in children as compared with a more liberal transfusion protocol (from Hct 0.27). In this study, Hct was reduced to 0.2 by ANH, and allowed to fall to 0.15 prior to re-transfusion of autologous blood. The patients had no signs of cardiac distress and extreme ANH was well-tolerated. The advantage of ANH was demonstrated by the fact that patients in the ANH group received 6 times less allogeneic blood than the more liberal transfusion group. Another study looking at 43 adolescents undergoing spine surgery demonstrated that ANH to a target Hct of 0.3 (in conjunction with hypotensive anesthesia and cell saver) was more effective than hypotensive anesthesia and cell saver alone in reducing the red cell loss, and thereby decreasing transfusion requirements [72]. The authors did note that the effectiveness of ANH may be hampered by preoperative autologous blood donation which reduces the initial Hct

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and therefore the amount of red cells which may be harvested from ANH at the start of the anesthesia. In summary, as with cardiac surgery, also for hip surgery, the use of ANH has not been shown to conclusively reduce the risk of allogeneic blood transfusion. However this may be due to the conservative target Hct used in many of the current studies. [73,74].

Hepatic Surgery Patients undergoing major gastrointestinal surgery or liver resection frequently receive blood transfusions. Blood loss may reach 10 liters, yet often only two units of blood are needed [75]. Thus ANH may be a suitable blood conservation technique. A larger study compared ANH versus no ANH among patients who underwent major gastrointestinal surgery [76]. One hundred and sixty-eight patients were included in the study, which concluded that ANH (removing 3 units of blood, maintaining hemoglobin above 8g.dl-1) in this population did not reduce the need for allogeneic blood transfusion. Conversely, another randomized study [75] used ANH as the single blood conservation technique, with a target Hct of 0.24 to collect autologous blood. Large volumes of autologous blood were collected (2000 ml), and this was not associated with cardiovascular instability. Significantly fewer patients in the ANH group received allogeneic blood (4 as compared with 14 patients). These findings were supported by other studies [77]. Children undergoing hepatic resection have also been shown to benefit from ANH, with regard to allogeneic blood [44]. The evidence for the beneficial effect of ANH in these patients is stronger compared to other surgical procedures in part because the studies have used a lower target Hct, with the aim of removing maximum blood volume prior to the surgical blood loss.

SUMMARY OF EVIDENCE In addition to the mathematical model, another measure of the efficacy of ANH has been the meta-analysis. Two recent meta-analyses have reached similar conclusions that ANH only saves 1 or 2 units of allogeneic blood [35,78]. Segal [35] analyzed 42 quality randomized controlled trials in which ANH was used as the single blood conservation method, up until 2002. The evidence suggested that the efficacy of ANH is likely to be small. It appears to modestly reduce bleeding and the volume of allogeneic blood requirements, but its efficacy with regard to avoidance of allogeneic transfusion was unproven. However, the authors suggest that on closer examination many studies were flawed in design, such as not using a protocol to manage ANH, which may have accounted for the results. In addition, the target Hct was not uniform, and was as high as 0.3. Thus the volume of blood withdrawn ranged from 500 to 2000 ml. These authors noted that the allogeneic blood saving were directly related to the blood volume withdrawn. This is in agreement with the mathematical models that indicate that a maximum blood volume removed converts to maximum blood savings. Another meta-analysis [78] of all trials evaluating ANH use regardless of quality suggested that the risk of allogeneic transfusion was similar among patients receiving ANH and those

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receiving usual care. The results from this meta-analysis are in agreement with Segal et al [35] as it shows that ANH reduces the number of allogeneic units by 1 or 2 and blood loss during the peri-operative period.

CONCLUSION Use of ANH is still limited, despite 30 years of practice. While it may not be the most efficacious method of blood conservation in every surgical circumstance, its use is certainly appropriate in situations in which significant intraoperative bleeding is expected, as outlined in this review. The advantages of reducing allogeneic transfusion are clear. Moreover, the cost–benefit ratio has to be considered as well. In the ANH procedure the patient is the donor and recipient of his own blood without additional cost for matching, storing, delivery and labeling. In addition, the indirect benefit is avoidance of eventual complications related to allogeneic transfusion. The lack of current evidence to support widespread use of ANH, either on its own or in combination with other techniques, may simply be due to failure to fulfill one or more of the critical criteria. All of these criteria: a relatively homogeneous population of patients so that blood loss is reasonably uniform; prospective random allocation of patients to groups with or without ANH; sufficiently high initial Hct; sufficiently low Hct (“target”) after ANH; withdrawal of a sufficient volume of blood; prospective transfusion criteria, uniformly and consistently applied; surgical blood loss that is within the range of potential efficacy; and a sample size sufficiently large to have a reasonable expectation of detecting a difference, should one exist [79] are required to provide a valid test of hemodilution. In conclusion, further large carefully controlled prospective randomized clinical trials are needed. Until this is done, ANH as a blood conserving technique may be considered primarily in subgroups of surgical population in which this technique has been shown to efficacious (i.e. patients undergoing liver resection) or in patients in whom there is contraindication to the use of allogeneic blood transfusion.

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[22] Rieger H, Kohler M, Schoop W, Schmid-Schonbein H. Normovolemic hemodilution in peripheral arterial disease. Ann Clin Res 1981; 13: 78-83 [23] Watkins GM, Rabelo A Jr, Bevilacqua RG, Brennan MF, Kagey KS, Anderson WP, Sheldon GF, Moore FD. Controlled anemia in normal man. Cardiac output, oxygen transport and extraction. Surg Forum. 1972; 23: 187-9 [24] Gaspard DJ, Cohen JL, Gaspar MR. Aortoiliofemoral thromboendarterectomy vs bypass graft. Arch Surg 1972; 105: 898-901 [25] Priebe HJ. Hemodilution and oxygenation. Int Anesthesiol Clin 1981; 19: 237-55 [26] Klovekorn WP, Pichlmaier H, Ott E, Bauer H, Sunder-Plassmann L, Jesch F, Messmer K. Acute preoperative hemodilution in surgical patients. Bibl Haematol 1975; 41: 24859 [27] Ickx BE, Rigolet M, Van der Linden PJ. Cardiovascular and metabolic response to acute normovolemic anemia: effects of anesthesia. Anesthesiology 2000; 93: 1011-6 [28] Licker M, Ellenberger C, Sierra J, Christenson J, Diaper J, Morel D. Cardiovascular response to acute normovolemic hemodilution in patients with coronary artery diseases: Assessment with transesophageal echocardiography. Crit Care Med 2005 ; 33 : 591-7 [29] Licker M, Ellenberger C, Murith N, Tassaux D, Sierra J, Diaper J, Morel DR. Cardiovascular response to acute normovolaemic haemodilution in patients with severe aortic stenosis: assessment with transoesophageal echocardiography. Anaesthesia 2004; 59: 1170-7 [30] Weiskopf RB, Aminoff MJ, Hopf HW, Feiner J, Viele MK, Watson JJ, Ho R, Songster C, Toy P.. Acute isovolemic anemia does not impair peripheral or central nerve conduction. Anesthesiology 2003; 99: 546-51 [31] Gillon J, Thomas MJG, Desmond MJ. Acute normovolaemic haemodilution. Transfusion 1996; 36: 640-3 [32] (32 ) Kloevekorn WP, Pichlmaier H, Ott E, Bauer H, Sunder-Plassman, Messmer K. Acute preoperative hemodilution-possibility for autologous blood transfusion. Chirurg 1974; 45: 452-8 [33] Kleen M, Habler O, Hutter J, Podtschaske A, Tiede M, Kemming G, Welte M, Corso C, Messmer K. Effects of hemodilution on gastric regional perfusion and intramucosal pH. Am J Physiol 1996; 271(5 pt 2): H1849-55 [34] Habler O, Kleen M, Hutter J, Podtschaske A, Tiede M, Kemming G, Corso C, Batra S, Keipert P, Faithfull S, Messmer K. Effects of hemodilution on splanchnic perfusion and hepatorenal function. Renal perfusion and hepatorenal function. Eur J Med Res 1997; 2: 419-24. [35] Segal JB, Blasco-Colmenares E, Norris EJ, Guallar E. Preoperative acute normovolemic hemodiltion: a meta-analysis. Transfusion 2004; 44: 632-44. [36] Lieberman JA, Weiskopf RB, Kelley SD, Feiner J, Noorani M, J. L, Toy P, Viele M: Critical oxygen delivery in conscious humans is less than 7.3 ml O2-1 ⋅ kg -1 ⋅ min-1. Anesthesiology 2000; 92: 407–13. [37] Spahn DR, Schmid ER, Seifert B, Pasch T: Hemodilution tolerance in patients with coronary artery disease who are receiving chronic beta-adrenergic blocker therapy. Anesth Analg 1996; 82: 687–94.

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[38] Doak GJ, Hall RI: Does hemoglobin concentration affect perioperative myocardial lactate flux in patients undergoing coronary artery bypass surgery? Anesth Analg 1995; 80: 910–6. [39] Jan KM, Chien S: Effect of hematocrit variations on coronary hemodynamics and oxygen utilization. Am J Physiol 1977; 233: H106–13. [40] Crystal GJ, Salem MR: Myocardial oxygen consumption and segmental shortening during selective coronary hemodilution in dogs. Anesth Analg 1988; 67: 500–8. [41] Levy P, Kim S, Eckel P, Chavez R, Ezz F, Gould S, Salem M, Crystal G: Limit to cardiac compensation during acute isovolemic hemodilution: Influence of coronary stenosis. Am J Physiol 1993; 265: H340–9. [42] Rehm M, Orth V, Kreimeier U, Thiel M, Haller M, Brechtelsbauer H, Finsterer U. Changes in intravascular volume during acute normovolemic hemodilution and intraoperative retransfusion in patients with radical hysterectomy. Anesthesiology 2000; 92: 657-64. [43] Schaller RT, Schaller J, Morgan A, Furman EB. Hemodilution anesthesia: a valuable aid to major cancer surgery in children. Am J Surg 1983: 146: 79-84. [44] Schaller RT, Schaller J, Furman EB. The advantages of hemodilution anesthesia for major liver resection in children. J Pediatr Surg 1984; 19: 705-10. [45] Kraft M, Dedrick D, Goudsouzian N. Haemodilution in an eight-month old infant. Anaesthesia 1981; 36: 402-4 [46] Martin E, Ott E. Extreme hemodilution in the Harringtom procedure. Biblthca Haemat 1981; 47: 322-37. [47] Vara-Thorbeck R, Guerrero-Fernandez Marcote JA.. Hemodynamic response of elderly patients undergoing major surgery under moderate normovolemic hemodilution. Eur Surg Res 1985; 17: 372-6. [48] Heiden D, Mielke CH, Rodvien R. Impairment by heparin of primary haemostasis and platelet [14C] 5-hydroxytryptamine release. Br J Haematol 1977; 36: 427-36. [49] Fernandez F, N'guyen P, Van Ryn J, Ofosu FA, Hirsh J, Buchanan MR. Hemorrhagic doses of heparin and other glycosaminoglycans induce a platelet defect. Thromb Res 1986; 43: 491-5. [50] Niinikoski J, Laato M, Laaksonen V, Meretoja O, Vanttinen E, Arstila M, Inberg MV. Effects of extreme haemodilution on the immediate post-operative course of coronary artery bypass patients. Eur Surg Res. 1983; 15:1-10. [51] Herregods L, Moerman A, Foubert L, Den Blauwen N, Mortier E, Poelaert J, Struys M. Limited intentional normovolemic hemodilution: ST-segment changes and use of homologous blood products with left main coronary artery stenosis. J Cardiothor Vasc Anesth 1997; 11: 18-23. [52] Moskowitz BM, Klein JJ, Shander A, Cousineau KM, Goldweit RS, Bodian C, Perelman SI et al. Predictors of transfusion requirements for cardiac surgical procedures at a blood conservation center. Ann Thorac Surg 2004: 77: 626-34. [53] McGill N, O'Shaughnessy D, Pickering R, Herbertson M, Gill R. Mechanical methods of reducing transfusion in cardiac surgery: a randomized controlled trial. BMJ 2002; 324: 1299.

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[54] Hallowell P, Bland JH, Buckley MJ, Lowenstein E. Transfusion of fresh autologous blood in open-heart surgery. A method for reducing bank blood requirements. J Thorac Cardiovasc Surg 1972; 64: 941-8. [55] Boldt J, Bormann BV, Kling D, Scheld H, Hempelmann G. Influence of acute normovolemic hemodilution on extravascular lung water in cardiac surgery. Crit Care Med 1988;16: 336-9. [56] Dietrich W, Barankay A, Dilthey G, Mitto HP, Richter JA. Reduction of blood utilization during myocardial revascularization. J Thorac Cardiovasc Surg 1989; 97: 213-9. [57] Boldt J, Kling D, Weidler B, Zickmann B, Herold C, Dapper F, Hempelmann G. Acute preoperative hemodilution in cardiac surgery: volume replacement with a hypertonic saline-hydroxyethyl starch solution. J Cardiothorac Vasc Anesth 1991;5: 23-8. [58] Vedrinne C, Girard C, Jegaden O, Blanc P, Bouvier H, Ffrench P, Mikaeloff P, Estanove S. Reduction in blood loss and blood use after cardiopulmonary bypass with high-dose aprotinin versus autologous fresh whole blood transfusion. J Cardiothorac Vasc Anesth 1992; 6: 319-23. [59] Triulzi DJ, Gilmor GD, Ness PM, Baumgartner WA, Schultheis LW. Efficacy of autologous fresh whole blood or platelet-rich plasma in adult cardiac surgery. Transfusion 1995; 35 : 627-34. [60] Tempe D, Bajwa R, Cooper A, Nag B, Tomar AS, Khanna SK, Satsangi DK, Gupta BK, Nigam M, Lall NG. Blood conservation in small adults undergoing valve surgery. J Cardiothorac Vasc Anesth 1996; 10: 502-6. [61] Kahraman S, Altunkaya H, Celebioglu B, Kanbak M, Pasaoglu I, Erdem K. The effect of acute normovolemic hemodilution on homologous blood requirements and total estimated red blood cell volume lost. Acta Anaesthesiol Scand 1997; 41: 614-7. [62] Casati V, Speziali G, D'Alessandro C, Cianchi C, Grasso MA, Spagnolo S, Sandrelli L. Intraoperative low-volume acute normovolemic hemodiltion in adult open-heart surgery. Anesthesiology 2002; 97: 367-73. [63] Hohn L, Schweizer A, Licker M, Morel DR. Absence of beneficial effect of acute normovolemic hemodilution combined with aprotinin on allogeneic blood transfusion requirements in cardiac surgery. Anesthesiology 2002; 96: 276-82. [64] Casati V, Benussi S, Sandrelli L, Grasso MA, Spagnolo S, D'Angelo A. Intraoperative moderate acute normovolemic hemodilution associated with a comprehensive bloodsparing protocol in off-pump coronary surgery. Anesth Analg 2004; 98: 1217-23. [65] Boldt J, Weber A, Mailer K, Papsdorf M, Schuster P. Acute normovolaemic haemodilution vs controlled hypotension for reducing the use of allogeneic blood in patients undergoing radical prostatectomy. Br J Anaesth 1999; 2:170-4. [66] Rehm M, Orth V, Kreimeier U, Thiel M, Haller M, Brechtelsbauer H, Finsterer U. Four cases of radical hysterectomy with acute normovolemic hemodilution despite low preoperative hematocrit values. Anesth Analg 2000 ; 90: 852-5. [67] Monk TG, Goodnough LT, Brecher ME, Pulley DD, Colberg JW, Andriole GL, Catalona WJ. Acute normovolemic hemodilution can replace preoperative autologous blood donation as a standard care for autologous blood procurement in radical prostatectomy. Anesth Analg 1997; 85: 953-8.

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[68] Monk TG, Goodnough LT, Birkmeyer JD, Brecher ME, Catalona WJ. Acute normovolemic hemodilution is a cost-effective alternative to preoperative autologous blood donation by patients undergoing radical retropubic prostatectomy. Transfusion 1995; 35: 559-65. [69] Tenholder M, Cushner FD. Intraoperative blood management in joint replacement surgery. Orthopedics 2004; 27: S663-8. [70] Goodnough LT, Despotis GJ, Merkel K, Monk TG. A randomized trial comparing acute normovolemic hemodilution and preoperative autologous blood donation in total hip arthroplasty. Transfusion 2000; 40: 1054-7. [71] Shulman G, Grecula MJ, Hadjipavlou AG. Intraoperative autotransfusion in hip arthroplasty. Clin Orthop Relat Res 2002; 396: 119-30. [72] Copley LA, Richards BS, Safavi FZ, Newton PO. Hemodilution as a method to reduce transfusion requirements in adolescent spine fusion surgery. Spine 1999; 24: 219-22. [73] Lisander B, Jonsson R, Nordwall A. Combination of blood-saving methods decreases homologous blood requirements in scoliosis surgery. Anaesth Intensive Care 1996; 24: 555-8. [74] Karakaya D, Ustun E, Tur A, Baris S, Sarihasan B, Sahinoglu H, Guldogus F. Acute normovolemic hemodilution and nitroglycerin-induced hypotension: comparative effects on tissue oxygenation and allogeneic blood transfusion requirement in total hip arthroplasty. J Clin Anesth 1999; 11: 368-74. [75] Matot I, Scheinin O, Jurim O, Eid A. Effectiveness of acute normovolemic hemodiltion to minimize allogeneic blood transfusion in major liver resections. Anesthesiology 2002; 97: 794-800. [76] Sanders G, Mellor N, Rickards K, Rushton A, Christie I, Nicholl J, Copplestone A, Hosie K. Prospective randomized controlled trial of acute normovolaemic haemodilution in major gastrointestinal surgery. Br J Anaesth 2004; 93: 775-81. [77] Johnson LB, Plotkin JS, Kuo PC. Reduced transfusion requirements during major hepatic resection with use of intraoperative isovolemic hemodilution. Am J Surg 1998; 76: 608-11. [78] Bryson GL, Laupacis A, Wells GA. Does acute normovolemic hemodilution reduce perioperative allogeneic transfusion? A meta-analysis. Anesth Analg 1998; 86: 9-15. [79] Weiskopf RB. Hemodilution and candles. Anesthesiology 2002; 97: 773-5.

In: New Developments in Blood Transfusion Research ISBN 1-59454-962-1 Editor: Brian R. Peterson, pp. 141-156 © 2006 Nova Science Publishers, Inc.

Chapter VIII

A NEW TECHNOLOGY IN BLOOD COLLECTION: MULTICOMPONENT APHERESIS Rainer Moog∗ Institute for Transfusion Medicine, University Clinics Duisburg-Essen, Germany.

ABSTRACT Multicomponent apheresis procedures offer the possibility of collecting blood components that are standardized, as compared to those available with manual whole blood donations. Recent technologic advances in hemapheresis have made possible the development of systems that can collect different combinations of blood components from the same donor during one collection session. Red blood cells (RBCs) can be concurrently collected with platelets (PLTs) or plasma. Two units of RBCs can also be collected during one apheresis session provided that the donor fulfils the inclusion criteria for that procedure. The hemoglobin content of apheresed RBCs after addition of additive solution is higher than the minimal requirement of whole blood derived RBCs of 40 g per unit due to standardization. A desired PLT yield can be targeted by the algorithm of the blood cell separators after entering donor specific parameters. Automated systems permit predictable collection of blood components with consistent yields and volumes. Previous definitions of blood component yield and volume as well as the flexibility to collect those multicomponents allows the blood centre to collect those components that maximize donors’ contribution and to meet the demands of its area hospitals for blood components. Blood centres are progressively increasing their reliance on apheresis technology to increase the number of blood components collected per donor visit and to reduce the number of donors a patient is exposed to. The use of these systems has shown at the same time that the procedures have been well tolerated by the donors and are cost effective.

∗

Correspondence concerning this article should be addressed to Rainer Moog, Institute for Transfusion Medicine University Clinics Essen, Hufelandstrasse 55, Germany. Phone: +492017231558; Fax: +492017235945; email: [email protected].

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Keywords: multicomponent donation erythro-platelet-plasmapheresis – 2-units RBC collection – side effects

INTRODUCTION In the 1960s, blood component preparation was developed to separate blood products from whole blood donation. Red blood cells (RBCs) and plasma are commonly prepared by separation after a centrifugation step. The separation is usually performed within a few hours after blood donation. It requires blood bank equipment such as manual or automated plasma extractors and large centrifuges for the centrifugation of blood bags. RBCs, plasma and platelet concentrates are provided in a fixed ratio (mainly, one unit each). As interest grew in cellular products, the plasmapheresis technique was adapted to collection of platelets (PLTs) [1-4], granulocytes [5,6] and later on RBCs [7,8]. The scope of multicomponent apheresis has now expanded to include plasma, PLTs and RBCs, and multiple combinations of each [7-14]. Multicomponent donation encompassing RBCs along with PLTs was practiced firstly in autologous perioperative donation systems. In this condition apheresis apparatuses were employed both for preoperative platelet poor plasma or RBC collections or for intentional hemodilution with sequestration of different blood components along with RBC concentrates. The original system used a discontinuous semiautomated apparatus for intraoperative blood salvage immediately prior to operation: the patient’s blood under the action of a peristaltic pump was sent to a 225 ml bowl where separation of plasma from cellular components took place [15]. Plasma came out of the bowl first and was sent to a dedicated bag. PLTs could be collected afterwards and the RBCs, which remained in the bowl, could be reinfused to the patient or collected into a separate bag. The cycle of separation-collection could be repeated 2 – 3 times depending on the patient’s hematological condition and the amount of component collection required. An average of 220 ml of RBC (hematocrit 60 %) and 250 – 300 ml of plasma could be collected per pass and employed for specific component autologous transfusion, during or after operation depending on the patient’s specific transfusion needs. Developed for cardiac surgery the system has been expended to major orthopedic and plastic surgery without any undesired effect or complication. This concept of multicomponent collection has expanded from the field of autologous to the field of homologous donations. In 1988 Valbonesi et al. reported on the concurrent collection of platelet concentrates in 30 minutes along with plasma and RBCs in allogeneic donors [16]. Using the Eccentriplate cell separator (Dideco Spa, Mirandola, Italy), a technique was developed that allowed the collection of 3.68 x 1011 PLTs, 250 ml of plasma, and 225 ml RBCs with a hematocrit (Hct) of 66.5 %. Compared with traditional manual whole blood collection, automated systems of collecting and separating RBCs, plasma and / or PLTs from the donor increase the versatility of apheresis donation without any requirement for additional processing [17]. Less technical staff is required for the production of multicomponents than for the production of components from manual whole blood donations. On the other hand there is an increased demand for well-trained cell separator operators, because the extracorporeal circulation

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during apheresis has to be monitored more closely and the donation time is longer than in manual whole blood donation. The blood collection facility of the future may utilize apheresis technologies to collect various components from donors based on their blood groups. Such a program would collect apheresis RBCs from group 0 donors especially if two units of RBCs could be collected during one donation. Group AB donors would undergo plasmapheresis for regular or jumbo (2-units) plasma products. Plateletpheresis procedures could be performed on donors from all AB0 blood groups but particularly on donors with higher platelet counts. Keller and Brainbridge made a model for improved donor utilization by extending the concept of apheresis [18]. Hundred percent of male donors and 95 percent of female donors were eligible for plateletpheresis or platelet plus plasma collection. Other possible component preparations from 362 donors of 7 apheresis centers are shown in table 1. Estimates from the Indiana Blood Center, Indianapolis, are that a 50 % conversion of whole blood collections to RBC apheresis would result in a 68 % decrease in component preparation, a 21 % decrease in component labelling and a 21 % decrease in the number of donor test profile required [19]. Table 1 Model for improved donor utilization by multiple product collection according to Keller and Bainbridge [18]. SDP: single donor platelets, PLS: plasma; DPP: double platelets, DPLS: double plasma, RBC: red blood cells, DRBC: double red blood cells.

SDP SDP-PLS DPP DPP-PLS SDP-DPLS SDP-RBC DPP-RBC SDP-PLS-RBC DRBC

Men (%) 100 100 70 68 98 100 64 95 94

Women (%) 95 95 51 22 36 86 10 22 22

With respect to the quality of RBC units multicomponent donation is also advantageous. Because anticoagulant is metered at a defined rate in proportion to whole blood withdrawal, apheresis avoids the “lesion of collection” that occurs in manual whole blood collection, in which the first RBCs collected are exposed to a higher concentration of anticoagulant and are therefore prematurely destroyed [20]. This may explain the improved cellular functionality of apheresed RBCs compared with manual collected RBCs [9,21,22]. Apheresis systems permit standardized collection of RBC units with consistent hemoglobin content by targeting automatically a pre-defined product independent of the donors’ baseline values. Higher variations of hemoglobin content are observed in manually collected RBC products due to varying donor’s pre-donation hemoglobin levels. Reduced donor exposure of the transfusion recipient is also an important issue in multicomponent donation. Transfusion services can implement systems to transfuse components obtained from the same donor to the same recipient during the same or

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subsequent transfusion events [17]. Oncology patients often need RBC as well as platelet transfusions during or after chemotherapy and are therefore optimal candidates for such transfusion programs. The consequent transfusion of multicomponents from one donor will result in a reduced patient’s risk of acquiring transfusion transmitted diseases as well as a lower rate of immunization due to less antigenic donor cells.

SPECIAL MULTICOMPONENT APHERESIS DEVICES MCS The MCS (Haemonetics, Braintree, USA) blood cell separator was designed for the collection of various blood components from one donor. The MCS is a portable, table-top machine that can be used with various disposable bowls (e.g. Latham blow, blow molded bowl). All separations are single-needle, intermittent flow procedures. The device allows the collection of one component such as PLTs [23-26], plasma or RBCs or multiple components during one apheresis [7,9,27] (Fig. 1).

MCS protocols Plasma protocols

FFP

PPP

Platelet protocols

LDP

LDP-RBC

RBC protocols

SDR

RBCP+

Figure 1. MCS+ protocols for multicomponent collection. FFP: fresh-frozen plasma, PPP; platelet poor plasma, LDP: leuko-depleted platelets, LDP-RBC: leuko-depleted platelets and red blood cells, SDR: single donor red cells (2 units), RBCP+: red blood cells and plasma.

Zeiler and Kretschmer investigated different protocols for the concurrent collection of RBCs and plasma [7]. In the first cycle of the procedures only plasma was collected. Plasma collection was stopped when the bowl optics indicated (which is triggered by the ascending platelet and white blood cell fraction in the bowl) or when enough plasma has been collected according to the preset value (e.g. 400 ml). The maximum plasma limit for the first cycle was calculated from donor Hct and desired plasma yield, because the second cycle had to be a complete cycle with overflow of the buffy coat into the collection bag. After the centrifuge had stopped, RBCs were returned to the donor and simultaneously mixed with 230 ml of saline solution. During the second cycle, the RBCs and a second unit of plasma were collected. In the first protocol (blow-molded bowl, centrifugation at 7000 rpm), the packed RBCs in the bowl were pumped into the transfer bag at the end of the second cycle and resuspended in 80 ml of additive solution. Then the RBCs were filtered in a closed system by gravitation through an in-line leukodepletion filter into a transfer bag and stored. In the second protocol (Latham bowl, centrifugation at 4800 rpm) one unit of buffy coat-poor RBCs and two units of plasma were collected. At the end of the second collection cycle, the buffy

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coat was discarded into a waste bag. The buffy coat-poor RBCs in the bowl were resuspended in additive solution and pumped into a transfer bag. The RBC volume was 336 ± 9 ml (first protocol), and 337 ± 7 ml (second protocol) with a Hct of 59 ± 2 and 53 ± 3 percent, respectively. On day 49, hemolysis was 0.24 ± 0.1 percent (first protocol), and 0.33 ± 0.32 percent (second protocol). The filtered RBC unit met the international standards for WBC-reduced RBCs [28]. The in vivo RBC values (lactate dehydrogenase, 2hydroxybutyrate dehydrogenase, hemolysis, potassium, adenosine triphosphate (ATP), 2,3diphosphoglcyerate (DPG)) were at least equal to those RBCs collected by manual whole blood donation. Two units of plasma were collected with each collection (first protocol: 420 ± 55 ml, 5.4 ± 7 WBCs/µl, 6500 ± 5000 PLTs/µl; second protocol: 440 ± 33 ml, 5 ± 12 WBC/µl, 3400 ± 3500 PLTs/µl). Moog and co-workers evaluated the concurrent collection of in-line filtered PLTs and RBCs [22]. The LDPRBC protocol (Fig. 2) aimed at collecting a programmed yield of PLTs and one RBC unit with automatic addition of RBC conservation solution (saline-adenineglucose-mannitol, SAGM). Anticoagulated whole blood (ratio 1:9) was drawn from the donors and centrifuged with 4800 rpm in the Latham bowl. After all the sterile air had been displaced from the bowl, the plasma began to exit the bowl through the effluent port. As soon as 30 g of plasma had been collected, the MCS+ activated the transfer pump, which began maintaining the ideal flow in the bowl. With this plasma transfer, a stable layer was achieved in the bowl even if there was an insufficient blood flow from the donor. After approximately 1 minute, a bowl optics reference was taken. The bowl optics reference was a reference voltage proportional to the optical density of the donor plasma. When the plasma/buffy coat interface was detected by the bowl optics, the line sensor reference was taken. This initiated dwelling, during which the plasma was re-circulated at 100 ml/min through the bowl in order to enhance the separation of cell layers. After dwelling, the surge phase began, during which plasma was rapidly pumped from the plasma bag into the Latham bowl. The speed of recirculation increased until the effluent line sensor detected the presence of PLTs. When this occurred, the platelet valve opened and PLTs were collected. This continued until the end of collection point was recognized by the line sensor. At this time, the blood pump gradually reduced speed and the platelet collection ended closing the platelet valve. The platelet products were in-line filtered during the last pass of the procedure. Using the RBC aliquot option, a programmed volume of RBCs was collected each cycle. This volume was calculated by the machine to reach the target RBC volume of 230 ml. When the return mode started, plasma from the plasma bag and RBCs from the bowl were pumped simultaneously to the donor. When the preset volume of RBCs had been emptied from the bowl, the transfer pump stopped and the blood pump continued to empty 5 ml RBC from the bowl to the donor. By this means, the common tube to the donor and to the RBC bag was primed with RBCs avoiding dilution with plasma. Then the whole blood valve closed, the blue valve opened, and the calculated volume of the RBC aliquot was collected into the RBC bag. After collection, SAG-M preservative solution was automatically added to the RBCs. Thereafter, the RBCs were in-line leukodepleted by gravity filtration at room temperature. PLTs and RBCs were subsequently stored at 22 ± 2 °C for 5 days and 4 ± 2 °C for 35 days, respectively. An average platelet dose of 2.47 ± 0.74 x 1011 was collected in a product volume of 232 ± 43 ml. The RBC volume averaged 280 ± 20 ml and the hemoglobin content

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was 56.8 ± 2.4 g per unit. The leukocyte contamination of the platelet product was 0.44 ± 0.56 x 105 and the residual leukocyte content of the RBC product was 0.28 ± 0.02 x 105. Storage data showed no relevant drop in pH. Day 35 results of the RBC products showed that all of the units had less than 0.8 percent hemolysis.

Figure 2. LDPRBC disposable set with two leukoreduction filters for the MCS+ blood cell separator. 1 pump cartridge, 2 bowl, 3 plasma bag, 4 platelets collection bag, 5 system pressure monitor connector, 6 donor pressure monitor connector, 7 blood filter, 8 bacterial barrier filters, 9 platelet bags, 10 injection port, 11 needle, 12 presample pouch, 13 platelets leukocyte reduction filter, 14 RBC collection bag, 15 RBC storage bag, 16 RBC leukocyte reduction filter.

Trima The COBE Trima (COBE BCT, Lakewood, CO, USA) is a new concept single needle apparatus set up with the unique purpose of collecting single donor PLTs alone or in combination with one or more blood components from the same donor during the same session [13]. The collection procedure uses a disposable tubing set with a cassette that facilitates the loading and unloading of the tubing in different pumps and valves (Fig.3). As suggested by the computer at the beginning of the operation, multiple combinations are possible by taking into account specific donor characteristics such as gender, body weight, Hct, and platelet pre-count. These data are processed by the computer and a list of priorities is produced. The operator can modify the priorities according to the needs of specific products. Priorities can be ranked according to the standards of each country or blood center. No priming of the machine is needed except for the one with the anticoagulant. Whole blood is pumped into the system and mixed with the anticoagulant at a controlled ratio near the

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access needle. The blood enters the separation channel in the centrifuge, where blood is separated into RBCs, PLTs, and plasma. These components are collected into separate storage bags. The components not collected are returned to the donor via the return pump. Leukocyte contamination is kept in the same low range of the sister apparatus Spectra since the Trima belt is very similar to the Spectra belt, leukocyte reduction system (LRS) included [29-34]. When PLTs and RBCs are concurrently collected, a single packed RBC unit is collected after the collection of a single-donor platelet unit. The additional time typically needed to collect the packed RBC unit was approximately 12 to 15 minutes. When RBCs are collected, addition of the additive solution SAG-M is done immediately after preparation thus taking advantage of a dedicated sterility filter.

RBC bag

Plasma bag

Platelet bags

Filter

AC line Cassette Access line

Channel

Figure 3. Trima disposable for the collection of PLTs, plasma and RBCs with the single stage separation channel.

Amicus The Amicus single needle (SN) plateletpheresis procedure separates ACD-A anticoagulated whole blood into all of its components and returns all of the RBCs while collecting a high percentage of PLTs and the volume of plasma desired. A new protocol, software version 2.43, was designed for the collection of PLTs, plasma and RBCs with the Amicus separator’s SN procedure (Baxter, Deerfield, IL, USA). With this software, the SN platelet collection procedure remains relatively unchanged, but allows for the concurrent collection of an RBC product. The separator determines when to begin collecting the RBC

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product by using the whole blood processed volume, and stores the RBCs in the SN kit’s return pack. When the RBC collection volume is reached, the Amicus Separator exits the RBC/platelet collection mode and returns any overcollected blood components as well as saline to counteract the blood volume deficit. An average procedure time of 74 ± 9 minutes was necessary to collect in average a packed RBC concentrate of 239 ± 11 ml, with an average platelet product of 3.9 ± 0.9 x 1011 PLTs/product [35]. Whole blood processed with the Amicus separator averaged 3,613 ± 433 ml. Post-collection, the return pack was aseptically removed from the SN kit and was sterile connected to an Amicus RBC Collection Set. The Amicus RBC Collection Set contains SAGM preservation solution, a Sepacell R-3000 leukocyte reduction filter, and a RBC storage container. The SAG-M was added to the ACD-A RBC product and after mixing leukocyte filtration was started. For all the filtered units, the leukocyte log reduction was in average 4.68 ± 0.20. The quality of the RBC product was analysed for 42 days. The supernatant hemoglobin level increased significantly throughout the storage period in all RBC units. None of the RBC units tested showed individual values higher than 0.8% hemolysis at the end of the storage period. The potassium level increased uniformly during the storage period in all RBC units tested. The pH values decreased for all RBC units tested throughout the storage period. The effect of storage was statistically significant. However, all units individually had a pH value higher than 6.5 at day 42. ATP behaved similarly in all RBC preparations throughout storage period showing similar values up to day 42. ATP values were noted to be higher on day 21 of storage than day 0 and decreased during the remainder of the storage period. The ANOVA with repeated measurement over the storage period showed that only the storage effect was statistically significant. When expressed as a percentage of day 0 values and analyzed individually, ATP values showed a significant statistical difference at day 21 of storage. All units showed in average more than 70 % ATP maintenance at the end of the 42 day storage period. 2,3-DPG concentrations decreased consistently and significantly to reach undetectable levels at day 21 of storage. All of the RBC products passed sterility testing after 42 days of storage. The hemapheresis unit of the University Clinics Essen, Germany has successfully implemented erythro-plateletpheresis with an extra gain of ~ 400 RBC units per year [21]. In the year 2002, an RBC by-product was collected in 68.2 percent of SN plateletphereses. A mean of 1.8 ± 0.9 RBC units were collected from each donor per year. Two hundred and twenty one donors donated one RBC unit, 117 two units, 54 three units, and 12 four RBC units per year. Most frequent reasons for the non-collection of an RBC unit were a donation interval of less than 3 months (20.5 percent) and low pre-hemoglobin values (17.4 percent).

2-UNITS RBC COLLECTION The first 2-unit RBC apheresis instrument to be granted clearance by the FDA in the United States was the Haemonetics MCS+ [36]. Based on the Haemonetics submission for licensure, FDA criteria for allogeneic 2 unit RBC apheresis donation became more stringent

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than for whole blood donation. For both sexes, a minimum Hct of 40 % (or hemoglobin of 13.3 g/dl) is required. The donation intervals must be at least 16 weeks. Men must meet a minimum height of requirement of 5 feet 1 inch and weight of 130 pounds and women a minimum height of 5 feet 6 inches and a weight of 150 pounds. The MCS+ is designed to collect RBC units on the basis of absolute RBC volume, with the term “absolute” meaning a Hct of 100 percent [36]. The programmable absolute RBC volume ranges from 90 – 210 ml per unit, with a standard deviation in practice of < 6 percent. This ability to collect a specified absolute RBC volume represents a significant advantage over traditional whole blood collection, in which the absolute RBC volume varies widely, depending on donor Hct and blood volume. After a single-site venipuncture with the 18gauge needle already connected to the disposable kit, the 2 units of RBCs are collected in two cycles, each cycle consisting of draw, transfer and return phases. In the draw phase, whole blood withdrawn at a rate of 20 – 100 ml/min mixes with the anticoagulant/stabilizator solution at a ratio of 1 part of anticoagulant/stabilizator solution to 15 part of whole blood. The anticoagulated blood is drawn into the blow-molded bowl, which is spinning at 7000 rpm to separate the plasma from the RBCs. The plasma is diverted into the plasma bag while RBCs packed at a Hct of 84 percent remain in the bowl. Once the bowl completely fills to a volume of 250 ml with packed RBCs at an 84-percent Hct, the machine stops drawing blood and stops the centrifuge. The transfer phase is then initiated. The programmed absolute RBC volume is routed from the bowl into one of the RBC bags filled with 100 ml additive solution. Once the target absolute RBC volume has been transferred into the RBC bag, the return phase is initiated. Plasma from the plasma bag is returned to the donor through the bowl with any excess of RBCs in the bowl and half of the normal saline return, which has a programmable range of 130 – 750 ml. This entire cycle is simply repeated to collect the second RBC unit, and a final rinse with part of the programmed saline return volume through the disposable kit reduces the absolute number of RBCs wasted in the equipment tubing to about 5 – 10 ml. The typical time required for the 2-unit RBC apheresis is 45 – 60 minutes, and it varies depending on the programmed saline return volume, draw speed, and return speed. In contrast, the typical collection time for whole blood collection is 10 – 15 minutes [37]. Another system designed for the collection of two RBC units (equivalent of 360 ml of absolute RBC with a theoretical Hct of 100 %) is the ALYX device (Baxter, Deerfield, USA) [38,39]. It is a single needle procedure using several collection and return cycles (Fig. 4). The donor extracorporeal volume is low (about 400 ml) due to the short collection cycles with a low volume of blood collected per cycle and saline injection at each return cycle, all based on donor height, weight and hemoglobin. Blood components are separated by a continuous-flow centrifuge. All fluid flow during the procedure is controlled by a pneumatic pump system. Internal sensors monitor the weight of blood and collected components as well as consumption of each fluid to ensure proper function during the collection procedure. The disposable set consists of a rigid-wall separation chamber, a cassette that interfaces with pneumatic pumps to control the fluid pathways, a 2-unit RBC reduction filter, and collection bags including the two final storage containers. All necessary fluids such as ACD-A, additive solution, and normal saline are integrally attached to the collection set by the manufacturer.

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Good quality of the collected RBC units was found up to 42 days of storage [38,39] and mean 24-hour recovery after infusion for leukoreduced units at the end of storage was 80.9 ± 6.9 percent [38]. No serious adverse events were observed in the donors during the procedure and during the following five days after collection. Schooneman et al. reported that all donors confirmed they would agree to donate two RBC units again with this system [39].

Figure 4. Draw and return phase of the ALYX system. Anticoagulated whole blood (WB) is pumped to the centrifuge and separated into its components in the draw phase. Plasma and RBCs are transferred to the containers. In the return phase, saline is substituted to the donor. Leukodepletion by filtration is performed after the end of the procedure.

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One benefit of 2-unit RBC collection for the donor is to give an equivalent number of RBC units as in manual whole blood donation, but with less frequent visits to the blood center. For the recipient, one benefit is reduced donor exposure, if the 2 units are transfused to the same patient. Another benefit is that the expected rise in Hct after transfusion should be more predictable, because RBC apheresis unit contain a defined amount of RBC per unit, rather than the variable amount in whole blood units.

FUNCTIONALITY OF RBCS COLLECTED BY APHERESIS Studies show that, even after 42 days of storage, RBCs collected by apheresis are as functionally intact as those collected by manual whole blood donation, as measured by rates of hemolysis, ATP levels [9,14], and 24-hours percentage of in-vivo recoveries. In the study by Smith et al., even though the plasma potassium rose from 2.6 ± 0.3 on day 0 to 48.7 ± 6.1 mEq per L on day 42, the percentage of hemolysis was stable at 50 percent on both days 0 and 42 [40]. In vivo survival of autologous RBCs obtained by the MCS cell separator was compared with RBCs collected manually in the study of Regan et al. [41]. In this cross-over controlled study, five male volunteers donated one unit of RBCs by MCS and one unit of whole blood by the manual method, 3 months apart. After storing donations in SAG-M for 35 days under standard conditions, radioactive (51Cr)-labelled autologous RBCs were injected into each donor. The post-transfusion recovery of RBCs at 24 and 48 hours did not show any significant difference between RBCs obtained manually or by MCS, indicating that processing differences have no detrimental effects on RBC survival. Similar findings were reported on RBCs and platelet obtained with the Trima device [13,42]. The mean 24-hour recovery values were 83.6 ± 5.4 percent, with a mean percentage of hemolysis on day 42 at 0.46 ± 0.19 percent. 25 patients received platelet components and achieved a mean corrected count increment of 15.1 ± 10.4 x 103 PLTs [42]. Results for the plasma collected concurrently on the Trima showed comparable data with plasma collected on the COBE Spectra [13] meeting the international standards for plasma units [28].

SIDE EFFECTS OF MULTICOMPONENT DONATION In general apheresis procedures have a higher incidence of adverse events compared to whole blood donation. Depotis et al. reported on 19,736 apheresis procedures, of which 159 (0.81 %) were associated with adverse events [43]. In 2,376 first time donations, 26 (1.09 %) developed adverse events compared to 133 (0.77 %) of 17,360 repeat procedures. Seventy (0.35 %) of 159 donation-related adverse events involved hemodynamic or citrate-related complications, of which 2 required subsequent neurologic consultation. The remaining 23 (0.12 %) adverse events involved procedure-related, non-specific complications. Forty-seven (0.24 %) of the 19,736 apheresis procedures were associated with severe adverse events. Seven of these severe adverse events required admission to an emergency department, and 2 required hospitalisation for further evaluation. Multivariate analysis revealed that apheresis

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machine model, donor gender and weight, the concomitant harvesting of plasma, the frequency of donation, and citrate-related symptoms (e.g. paresthesias) were independently associated with severe hypotensive reactions. Rugg et al. reported on six donor incidents in 44 Trima procedures [42]. All were minor in nature. Four involved tingling during the collection procedure and were attributed to citrate. Oral calcium was administered to two of these donors, and the procedures were continued. One donor complained of pain at the needle site; the needle was adjusted and the procedure continued. One donor felt light-headed during the procedure. The apheresis was halted until the episode passed, and then it was continued at a lower flow rate. Another study on 68 Trima collection procedures found minor citrate reactions (n = 13), characterized by tingling sensations or a metallic taste [13]. One donor felt light-headed and four felt tired. Pain at the needle site (n = 2) and vein infiltration (n = 4) were also reported. In 2-unit RBC collection most reactions, actually were immediate and probably attributable to citrate toxicity, rather than symptoms specific to RBC removal. Meyer and coworkers noted chest tightness during eight apheresis collections and hypotension during three collections, but this study used a whole blood/citrate ratio of 8:1 rather then the usual 15:1, these symptoms were likely due to citrate toxicity [37]. Furthermore, these symptoms resolved in all cases within 5 – 10 minutes after saline administration or a decrease in the plasma return rate. Radtke and co-workers reported on adverse events at 36 visits (2.9%), which were related to venipuncture (1.5%), mild citrate toxicity (0.7%), vasovagal reactions (0.6%), and hypertension (0.1%) [44]. In regard to longer-term effects, the question is whether allogeneic RBC apheresis donors do appear to be at greater risk for iron deficiency than whole blood donors. Over a 1-year period, Meyer and colleagues followed serum ferritin, serum iron, total iron-binding capacity, transferring saturation, and zinc protoporhyrin/heme ratios in 40 donors divided between an RBC apheresis group donating 450 ml of RBCs every 4 months and a whole blood group donating 225 ml of RBCs every 2 months [37]. Half of the donors received iron supplementation. No significant difference in any iron balance measurement was found between whole blood and apheresis donors or between male and female donors. There was a significant increase in iron stores from the donor’s baseline level in donors who received iron supplementation, while there was a decrease from baseline level in those who did not, and in three in the whole blood group without iron supplementation developed unacceptable low Hct values and ferritin levels < 12 ng per ml after donating 4, 1, and 4 units, respectively. Therefore, although no measures of iron balance became abnormally low in apheresis donors without iron supplementation, all regular 2-unit RBC apheresis donors should probably be given iron supplementation. The study by Sherman et al. also reports this conclusion, as two of eight 2-unit apheresis donors, both female, without iron supplementation had ferritin levels < 12 ng per ml at 16 weeks after donation [45]. Högler et al. reported on significantly declined ferritin levels from 54.2 ± 33.7 to 23.42 ± 21.94 (predonation vs. day 30), which remained significantly below predonation values, but within the normal range, until the end of the 4 month study period [46]. In contrast, a significant decrease in hemoglobin (15.89 ± 0.82 vs. 14.08± 0.97 mg/dl, baseline vs. day 7) was equalized within 2 months. The authors concluded that a donation interval of 4 months was appropriate in terms of RBC recovery,

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but not in terms of iron store regeneration. Radtke et al. studied 260 blood donors who donated 2-units RBC on each of a total of seven visits at intervals of 8 to 10 weeks [44]. The donors were randomly assigned to receive 100 mg of iron or placebo daily. Using a crossover study design, Group A received iron capsules after the first three donations, and Group B after the second three donations, respectively. Hemoglobin, serum ferritin, and serum iron were measured before each donation. Mean ferritin concentration decreased after each donation in the placebo phase of both treatment groups, but it remained largely constant during the iron phase in Group A, and even increased during the iron phase in Group B. The authors concluded that iron supplementation prevented iron depletion in the majority of donors after 2-unit RBC apheresis within an 8- to 10-week period.

CONCLUSION Blood collection facilities may utilize different apheresis technologies to collect various components from donors based on their demand. These systems improve component collection and make it easier for such collections to be in compliance with good manufacturing practices. They also provide standardized products despite variation in donor parameters. The use of multicomponent devices can reduce the processing and handling of blood components because these products are provided in their final form at the end of the automated collection process. The application of this technology will increase production of blood components and hopefully prevent future large scale blood shortages.

REFERENCES [1] [2] [3]

[4] [5] [6] [7]

[8]

Tullis JL, Eberle WG, Baudanza P et al. Platelet-Pheresis. Description of a new technic. Transfusion 1968;8:154-64. Katz AJ, Genco PV, Blumberg N et al. Platelet collection and transfusion using the Fenwal C-3000 cell separator. Transfusion 1981;21:560-3. Kurtz SR, McMican A, Carciero R et al. Plateletpheresis experience with the Haemonetics blood processor 30, the IBM blood processor 2997, and the Fenwal CS3000 blood processor. Vox Sang 1981;41:212-8. Mintz PD. Comparison of plateletpheresis with two continuous-flow blood cell separators using identical donors. Transfusion 1985;25:330-3. Graw RG, Herzig GP, Eisel RJ et al. Leukocyte and platelet collection from normal donors with the continuous flow blood cell separator. Transfusion 1971;11:94-101. Huestis DW, White RF, Price MJ et al. Use of hydroxyethyl starch to improve granulocyte collection in the Latham blood processor. Transfusion 1975;15:559-64. Zeiler TA, Kretschmer V. Automated blood component collection with the MCS 3p cell separator: evaluation of three protocols for buffy coat-poor and white cell- reduced packed red cells and plasma. Transfusion 1997;37:791-7. Matthes GA. Red cell apheresis: new concepts of blood component processing. Therapeutic Apheresis 1997;1:22-8.

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Rainer Moog Matthes G, Tofote U, Krause KP et al. Improved red cell quality after erythroplasmapheresis with MCS-3p. J Clin Apheresis 1994;9:183-8. Knutson F, Rider J, Franck V et al. A new apheresis procedure for the preparation of high-quality red cells and plasma. Transfusion 1999;39:565-71. Valbonesi M. Multicomponent collection (MCS): a new trend in transfusion medicine. Int J Artif Organs 1994;17:65-9. Valbonesi M, Lercari G, Florio G et al. Erythrothrombocytapheresis and plasmathrombocytapheresis with storage in T-sol of platelets collected by the new Amicus cell separator. Int J Artif Organs 1997;20:272-6. Elfath DM, Whitley P, Jacobson MS et al. Evaluation of an automated system for the collection of packed RBCs, platelets, and plasma. Transfusion 2000;40:1214-22. Leitner, G., Jilma-Stohlawetz, P., Stiegler, G., Weigel, G., Horvath, M., Tanzmann, A., Höcker, P., and Fischer, M. B. Quality of packed red blood cells and platelet concentrates collected by multicomponent collection using the MCS Plus device. J Clin Apheresis 18, 21-25. 2003. Ferrari M, Zia S, Valbonesi M et al. A new technique for hemodilution, preparation of autologous platelet-rich plasma and intraoperative blood salvage in cardiac surgery. Int J Artif Organs 1987;47-50. Valbonesi M, Frisoni R, Capra C et al. Plateletpheresis concentrates produced in 30 minutes along with plasma and packed red cells: prelimary results. J Clin Apheresis 1988;4:152-4. Valbonesi M, Florio G, Ruzzenenti MR et al. Multicomponent collection (MCC) with the latest hemapheresis apparatuses. Int J Artif Organs 1999;22:511-5. Keller, N. C. and Bainbridge, M. A. Model for improved donor utilization by multiple product collection. Transfusion 38(suppl), 7 S J. 1998. Beeler, S. A., Giandelone, J. A., and Axelrod, F. A blood center's motivation toward total apheresis collection. Transfusion 37, 113S. 1997. Gibson JG, Murphy WP, Scheitlin WA et al. The influence of extracellular factors involved in the collection of blood in ACD on maintenance of red cell viability during refrigerated storage. Am J Clin Pathol 2005;26:855-73. Moog R. Implementation of concurrent red blood cell and platelet collection by apheresis in a university haemapheresis unit. Transfusion Med 2004;14:145-50. Moog R, Bartsch R, Müller N. Concurrent collection of in-line filtered platelets and red blood cells by apheresis. Ann Hematol 2002;81:322-5. Moog R. Fuji surge technique and continuous in-line filtration to improve the quality of single donor platelet concentrates. J Clin Apheresis 2002;17:199-203. Holme S, Andres M, Goermar N et al. Improved removal of white cells with minimal platelet loss by filtration of apheresis platelets during collection. Transfusion 1999;39:74-82. Stiegler G, Leitner G, Panzer S et al. Comparison of platelet concentrates obtained with two different plateletpheresis programs of MCS+. Infus Ther Transfus Med 2002;29:12-6.

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[26] Moog R, Müller N. Comparison of different protocols in plateletpheresis with the MCS 3p blood cell separator with respect to parameters of product quality. Infusionsther Transfusionsmed 1995;22:244-8. [27] Kientz D, Laforet M, Isola H et al. Leukodepletion of platelet concentrates and plasma collected with Haemonetics MCS+ apheresis system. Experience of EFS-Alsace. Transfus Apheresis Sci 2001;25:55-9. [28] Council of Europe: Guide to the preparation, use and quality assurance of blood components. 2005. Strassbourg. [29] Riggert J, Humpe A, Simson G et al. Quality and safety of platelet apheresis concentrates produced with a new leukocyte reduction system. Vox Sang 1998;74:1828. [30] Zingsem J, Glaser A, Weisbach V et al. Evaluation of a platelet apheresis technique for the preparation of leukocyte-reduced platelet concentrates. Vox Sang 1998;74:189-92. [31] Zingsem J, Zimmermann R, Weisbach V et al. Comparison of COBE white cellreduction and standard plateletpheresis protocols in the same donors. Transfusion 1997;37:1045-9. [32] Adams, M. R., Dumont, L. J., McCall, M., and Heaton, W. A. Clinical trial and process evaluation of an apheresis system for preparation of white cell-reduced platelet components. Transfusion 38, 966-974. 1998. [33] Zingsem J, Zimmermann R, Weisbach V et al. Comparison of a new WBC-reduction system and the standard plateletpheresis protocol in the same donors. Transfusion 2001;41:396-400. [34] Fournel JJ, Zingsem J, Riggert J et al. A multicenter evaluation of the routine use of a new white cell-reduction apheresis system for the collection of platelets. Transfusion 1997;37:487-92. [35] Moog R, Franck V, Pierce JA et al. Evaluation of a concurrent multicomponent collection system for the collection and storage of WBC-reduced RBC apheresis concentrates. Transfusion 2001;41:1159-64. [36] Shi PA, Ness PM. Two-unit red cell apheresis and its potential advantages over traditional whole-blood donation. Transfusion 1999;39:218-25. [37] Meyer D, Bolgiano DC, Sayers M et al. Red cell collection by apheresis technology. Transfusion 1993;33:819-24. [38] Snyder EL, Elfath DM, Taylor H et al. Collection of two units of leukoreduced RBCs from a single donation with a portable multiple-component collection system. Transfusion 2003;43:1695-705. [39] Schooneman F, Huart JJ, Dernis D et al. Two red blood cell units collected in SAG-M additive solution with the ALYX component collection system. Transfus Apheresis Sci 2005;32:305-13. [40] Smith JW, Gilcher RO. Red blood cells, plasma, and other new apheresis-derived products: improving product quality and donor utilization. Transfus Med Rev 1999;13:118-23. [41] Regan F, Teesdale P, Garner S et al. Comparison of in vivo red cell survival of donations collected by Haemonetics MCS versus conventional collection. Transfusion Medicine 1997;7:25-8.

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[42] Rugg N, Pitman C, Menitove JE et al. A feasibility evaluation of an automated blood component collection system platelets and red cells. Transfusion 1999;39:460-4. [43] Despotis GJ, Goodnough LT, Dynis M et al. Adverse events in platelet apheresis donors: a multivariate analysis in a hospital-based program. Vox Sang 1999;77:24-32. [44] Radtke H, Mayer B, Rocker L et al. Iron supplementation and 2-unit red blood cell apheresis: a randomized, double-blind, placebo-controlled study. Transfusion 2004;44:1463-7. [45] Sherman LA, Lippmann MB, Ahmed P et al. Effect on cardiovascular function and iron metabolism of the acute removal of 2 units of red cells. Transfusion 1994;34:573-7. [46] Högler W, Mayer W, Messmer C et al. Prolonged iron depletion after allogeneic 2-unit RBC apheresis. Transfusion 2001;41:602-5.

In: New Developments in Blood Transfusion Research ISBN 1-59454-962-1 Editor: Brian R. Peterson, pp. 157-169 © 2006 Nova Science Publishers, Inc.

Chapter IX

THE USE OF RECOMBINANT ACTIVATED FACTOR VII IN TRANSFUSION MEDICINE Massimo Franchini∗ Servizio di Immunoematologia e Trasfusione Centro Emofilia, Azienda Ospedaliera di Verona, Italy.

ABSTRACT Recombinant activated factor VII (rFVIIa, NovoSeven® ) is a novel hemostatic agent originally developed to treat bleeding episodes in hemophiliacs with inhibitors against coagulation factors VIII and IX. Successively, rFVIIa has also been employed with benefit for the management of hemorrhages in other congenital and acquired hemostatic abnormalities and nowadays the drug is registered in Europe for the treatment of congenital hemophilia with inhibitors, acquired hemophilia, congenital FVII deficiency and Glanzmann thromboasthenia. More recently, rFVIIa has been utilized to control excessive bleeding, thus reducing the exposure to allogeneic blood, in a wide variety of non-hemophilic bleeding situations unresponsive to conventional therapy including emergency (intracerebral hemorrhage, upper gastro-intestinal bleeds, trauma, oral anticoagulant-induced hemorrhage) or surgery-related (liver resection, orthotopic liver transplantation, neurosurgery, cardiac surgery) bleeds. These latter newer and less wellcharacterized clinical applications of rFVIIa, basing on a literature search including PubMed, references from reviews and abstracts from the most important meetings on this topic, will be discussed in this review.

∗

Correspondence concerning this article should be addressed to Dr. Massimo Franchini, MD Servizio di Immunoematologia e Trasfusione - Centro Emofilia, Ospedale Policlinico, Piazzale Ludovico Scuro, 37134 Verona. Tel: 0039-045-8074321 FAX: 0039-045-8074626 E-mail: [email protected].

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INTRODUCTION Recombinant activated factor VII (rFVIIa, NovoSeven®, Novo Nordisk, Denmark) was originally developed for the treatment of hemophiliacs with inhibitors and then used successfully for treating hemorrhages in patients with acquired hemophilia, or other inherited bleeding disorders such as factor VII deficiency and Glanzmann thromboasthenia [1-7]. In the last few years, along with the improvement of the knowledge of its mechanisms of action, rFVIIa has also been utilized with benefit as a “universal hemostatic agent” in many other non-hemophilic bleeding situations unresponsive to conventional therapy including intracerebral hemorrhage, oral anticoagulant-induced hemorrhage and bleeding associated with hepatic failure, major surgery and trauma (see table 1) [8-13]. These newer, “off-label” clinical applications of rFVIIa, aimed to control excessive bleeding and reduce the exposure to allogeneic blood, will be discussed in this review. Table 1 Approved and potential clinical applications of recombinant activated factorVII. Hemophilia and clotting defects 1) Hemophilia with inhibitors* 2) Acquired hemophilia* 3) Congenital factor VII deficiency* 4) Glanzmann thromboasthenia* 5) Other platelet disorders (qualitative and quantitative) 6) Other coagulation factor defects (factor XI and von Willebrand disease) Emergency bleeds 1) Intracerebral hemorrhage 2) Upper gastro-intestinal bleeds 3) Trauma 4) Oral anticoagulant-induced hemorrhage Surgery 1) Liver resection 2) Orthotopic liver transplantation 3) Neurosurgery 4) Cardiac surgery

Mechanisms of Action of rFVIIa Factor VIIa is an important contributor to the initiation of hemostasis [13]. In fact, according to a cell-based model of coagulation [14,15], following injury to the vessel wall, tissue factor (TF) is exposed to circulating blood and TF-FVIIa complexes are formed on the TF-bearing cells, where they activate factor X (FXa), leading to the conversion of

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prothrombin to thrombin. The limited amount of thrombin formed activates factors V, VIII and XI, as well as platelets, which in turn change shape and expose negatively charged phospholipids, such as phosphatidylserine. These activated platelets provide the template for further FX activation and full thrombin generation with a positive feedback on factors V, VIII and XI [16,17]. The extra thrombin formation results in the activation of thrombin-activable fibrinolysis inhibitor (TAFI), which protects the fibrin clot from premature lysis by downregulating fibrinolysis [18]. In summary, a full thrombin burst is essential for the formation of a stabile fibrin hemostatic plug that is resistant to premature fibrinolysis. In fact, in hemophilia only an initial, limited amount of thrombin dependent on the TF-FVIIa is generated, which is insufficient to consolidate and sustain the fibrin plug [19]. In a cell-based in vitro model, it has been shown that the addition of increasing amounts of rFVIIa (between 50 and 150 nm) to activated platelets in the presence of factor X produces a linear increase of generation of factor Xa independently of the presence of TF on the platelet surface [16,2022]. This dose-response mechanism can lead to the generation of significant amounts of thrombin even in the absence of factors VIII and IX, thus explaining the mechanism of action of rFVIIa in hemophiliacs [13]. The direct activation of FIX on activated platelets in the absence of TF, resulting in improved thrombin generation, may also explain the mechanism of action of rFVIIa in acquired coagulopathy following trauma, surgery or other events [23]. Moreover, the binding of rFVIIa to activated platelets may explain why rFVIIa is localized only to the site of bleeding [13,15]. However, other mechanisms of actions of rFVIIa have been proposed [24]. In fact, ten Cate and colleagues first and van’t Veer and coworkers successively proposed a TF-dependent mechanism of action of rFVIIa [25,26]. This model was more recently strengthened by Butenas and colleagues who reported that the local function of rFVIIa was mediated by the combined effect of TF expression and platelet accumulation at the site of a vascular lesion [27,28]. Lisman and De Groot recently analyzed the experimental data available and concluded that both the proposed mechanisms of actions of rFVIIa (i.e., TF-dependent and TF-independent) are plausible [24]. In fact, if the TF pathway is usually required for the action of rFVIIa, a rFVIIa-mediated thrombin generation can also occur on the activated platelet surface independently of TF. Moreover, the same authors observed that the enhanced thrombin generation from rFVIIa not only accelerates clot formation, but also inhibits fibrinolysis by TAFI activation [29] and enhances platelet adhesion and aggregation under flow conditions [30]. This latter evidence may explain the therapeutic effect of rFVIIa in thrombocytopenic patients. In conclusion, according to the current knowledge, rFVIIa induces hemostasis by enhancing thrombin generation on thrombin-activated platelet surfaces, thereby providing the formation of a stable, tight fibrin clot which is resistant to premature fibrinolysis.

Use of rFVIIa in Liver Disorders Bleeding complications are a common cause of morbidity and mortality in patients with liver disease. Bleeding sources include gastrointestinal, variceal and intracerebral vessels. The coagulopathy of liver disease is multifactorial. Decreased synthesis of vitamin Kdependent coagulation factors (particularly factor VII, protein C and protein S), increased

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fibrinolysis and thrombocytopenia may all play a role [31]. Traditional therapies include vitamin K, fresh-frozen plasma (FFP), desmopressin and platelets [32,33]. Limited data are available in the literature as regards the hemostatic effect of rFVIIa for the treatment of bleeding in patients with liver disease [9]. Moreover, a wide range of dosages (between 5 to 120 µg/Kg) have been applied in the different studies, thus making any comparison of results difficult [32]. A preliminary trial conducted by Bernstein and colleagues in 1997 [33] found that rFVIIa transiently corrected prolonged PT in a group of non-bleeding cirrhotic patients. A multicenter, randomized, double-blind trial investigated 71 patients with advanced liver disease undergoing laparoscopic liver biopsy under the cover of rFVIIa. These patients were randomized to receive one of four doses of rFVIIa (5, 20, 80 or 120 µg/kg); 48 (74%) of 65 patients achieved hemostasis within 10 minutes [34]. The authors concluded that this procedure, otherwise contraindicated due to the coagulopathy, could be performed safely in such patients thanks to the use of rFVIIa. The European Study Group on rFVIIa in Upper Gastrointestinal Haemorrhage recently published [35] the results of a randomized, doubleblind trial on the use of rFVIIa in 245 cirrhotic patients with upper gastrointestinal bleeding in which the patients were randomized to receive 8 doses of 100 µg/kg of rFVIIa or placebo in addition to standard pharmacologic and endoscopic treatment. Although there was no significant difference between the 2 groups for the primary composite endpoint (failure to control bleeding, failure to prevent re-bleeding and death), there was a significant reduction in the composite endpoint among the patients with variceal bleeding and more severe liver disease who received rFVIIa. Other studies have examined the use of rFVIIa in patients with cirrhosis and active variceal bleeding [36-38]. Two single center, open label studies involving small numbers of patients have reported that rFVIIa is effective in controlling variceal bleeding when used as an adjunct to standard treatment [36,37]. In contrast, in a retrospective analysis of the NovoSeven extended-use registry, O’Connell and colleagues [38] found that 6 of the 8 patients who did not respond to rFVIIa had liver disease (3 acute bleeds and 3 liver transplants) with a complex coagulopathy. Recombinant FVIIa was also shown to be more effective than conventional therapy with plasma for treating coagulopathy in fulminant hepatic failure [39]. Another situation in which excessive bleeding can occur and where the administration of rFVIIa might be effective is orthotopic liver transplantation [40]. In fact, there are reports that rFVIIa is safe and reduces transfusion requirements when administered immediately before starting a transplant in patients with severe coagulopathy [41-45]. Kalicinski and colleagues [41] reported on 2 pediatric patients undergoing urgent liver transplantation for fulminant liver failure; conventional therapy with plasma and cryoprecipitate had failed, but the children were successfully treated with 100 µg/kg of rFVIIa prior to the transplant (one child received an additional intra-operative dose). In another small series, Hendriks and colleagues [42] reported on 6 adult patients undergoing liver transplantation for cirrhosis who received a single dose of 80 µg/kg of rFVIIa prior to skin incision. The authors noted that, compared with controls, these patients required significantly fewer red blood cell (RBC) and FFP transfusions. However, one patient developed a post-operative hepatic artery thrombosis. These results were contrasted by the recent randomized multi-center study conducted by Planinsic and colleagues [44,45], who reported no difference in peri-operative red cell or FFP transfusions in 83 patients undergoing OLT and who received a single prophylactic dose of 20-80 µg/kg of rFVIIa or placebo. In

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contrast, another randomized trial [46] conducted in 183 patients and administering repeated perioperative higher doses of rFVIIa (60 or 120 mg/kg of rFVIIa or placebo) found that the use of rFVIIa during OLT significantly reduced the number of patients requiring RBC transfusions.

Use of rFVIIa in Surgery Surgery is another potentially interesting are for rFVIIa [47]. Unfortunately, although a great number of case reports and series are available in the literature, only few randomized placebo-controlled trials have been published so far on this topic [48-51]. A prospective, double-blind randomized trial of rFVIIa (a single dose of 20 µg/kg or 40 µg/kg) versus placebo in 36 patients undergoing radical retropubic prostatectomy found that patients receiving rFVIIa had significantly and dose-dependently less total peri-operative blood loss than did the placebo recipients [52]. Similar conclusions were drawn by Lodge and colleagues [53] in a multicenter, double-blind, placebo-controlled study evaluating the hemostatic efficacy and safety of rFVIIa in 204 patients undergoing partial hepatectomy due to neoplasia who were randomized to receive pre-operative injection of either placebo or rFVIIa (20 µg/kg or 80 µg/kg), followed by a second dose 5 hours after surgery began if the anticipated surgery time exceeded 6 hours. Park and colleagues reported on 9 patients with coagulopathy who required urgent neurosurgery; these patients were treated pre-operatively with rFVIIa (40-90 µg/kg) and had no bleeding or thromboembolic complications [54]. The successful use of rFVIIa was also described by Tobias in two children who developed dilutional coagulopathy during posterior spinal fusion for neuromuscular scoliosis [55]. A number of studies have investigated the role of rFVIIa in cardiac surgery, which is often associated with profuse hemorrhage [56-61]. Aggarwal and coworkers [56] reported on a series of 8 surgical patients with intractable bleeding, 6 of whom underwent cardiopulmonary bypass. Bleeding stopped after 90 µg/kg of rFVIIa in all but one patient who required a further bolus. Al Douri and colleagues [57] and Hendricks and colleagues [58] reported that a single dose of rFVIIa was an effective treatment for severe intractable bleeding in patients undergoing heart surgery. In a recent study, Karkouti and colleagues analyzed the outcomes of 51 cardiac surgery patients who received rFVIIa for intractable blood loss compared with 51 matched control patients and found that rFVIIa, at a dose of 35 to 70 µg/Kg, was effective in reducing intractable hemorrhage after cardiac surgery [59]. Similar positive results were observed by Razon and coworkers [60] and Halkos and colleagues [61] on small series of pediatric and adult patients treated with rFVIIa for excessive blood loss after cardiovascular surgery. Other reports [62,63] have described the efficacy of rFVIIa in controlling severe bleeding following implantation of mechanical cardiac assist devices.

Use of rFVIIa in Trauma A number of hemostastic changes, resulting in defective thrombin generation, occur in patients subjected to extensive surgery with substantial bleeding or in patients with acute,

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severe trauma with profuse bleeding requiring multiple transfusions [48,64]. In 1999, Kenet and colleagues first successfully used rFVIIa infusions to manage acute, life-threatening traumatic bleeding [65]. Since then, many reports have been published on the use of rFVIIa in patients with acquired coagulopathies from both blunt and penetrating injuries [66-75]. Martinowitz and colleagues [66] reported on 7 massively bleeding, multi-transfused, coagulopathic trauma patients successfully treated with a median of 2 doses of rFVIIa ranging from 120 to 212 µg/kg. The same author, in a recent report by the Israeli Multidisciplinary rFVIIa Task Force based on the prospective analysis of the use of rFVIIa in 36 multi-trauma patients, observed a positive effect (cessation of bleeding) in 72 percent (26/36) and a survival rate of 61 percent (22/36) [73]. Recently, Mayo and colleagues [67] observed a reduction of blood transfusion requirements after the use of rFVII (2 doses of 90120 µg/kg) in 13 patients with acute, uncontrolled life-threatening bleeding. Geeraedts and colleagues published a retrospective analysis of 8 blunt trauma patients treated with rFVIIa for uncontrolled bleeding: in all cases the treatment reduced or stopped bleeding thus significant decreasing blood components requirement [74]. A large multicenter, randomized, double-blind trial of rFVIIa (400 µg/kg in three doses) in 277 patients with severe blunt and/or penetrating trauma, aiming to achieve a reduction in transfusion requirements, has recently been completed and preliminary results published [72]. In blunt trauma, rFVIIa significantly reduced the number of RBC, FFP and platelet transfusions and the requirement for massive transfusions (> 20 RBC units) in comparison to the placebo group. A significant decrease in acute respiratory distress syndrome (ARDS) and multiple organ failure (MOF) was also observed. Similar trends on transfusion endpoints, although not statistically significant, were found in penetrating trauma. The use of rFVIIa for blunt trauma is actually under registration by the European Medicines Agency (EMEA). Recombinant FVIIa was also used as a “last chance” in a case of pulmonary hemorrhage after major trauma, associated with coagulopathy, heavy transfusion requirement and multi-organ failure [70]. Bleeding stopped, with resolution of the hemothorax, after 2 doses of 60 µg/Kg of rFVII. In summary, also based mostly on case reports, the experience of rFVIIa use in trauma with excessive bleeding as well as in postoperative profuse bleeding indicates a hemostatic effect of rFVIIa given in one or two doses of 60 to 120 µg/kg.

Use of rFVIIa for Reversal of Anticoagulant Therapy Recombinant FVIIa has also been employed in the reversal of warfarin therapy in cases in which the administration of vitamin K alone was found to be insufficient [76-82]. Warfarin is a coumadin anticoagulant used to treat or prevent primary and secondary venous and arterial thromboembolism. Through vitamin K antagonism, it induces low levels of vitamin K-dependent coagulation factors, in particular factor VII which has been shown to be the earliest and the most sensitive of the coagulation factors to be affected by oral anticoagulant therapy. Spontaneous hemorrhages occur in approximately 10-20 percent of individuals receiving oral anticoagulant therapy [6]. The use of rFVIIa in the reversal of warfarin therapy was first described by Diness and colleagues in 1990 in an animal model [76]. In 1998, a study [77] of 28 healthy volunteers who received warfarin to produce an international

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normalized ratio (INR) > 2, demonstrated that doses from 5 to 320 µg/kg normalized the INR for periods ranging from 12 to 24 hours. A spontaneous nosebleed in a patient on warfarin with an INR of 2.9 was reported to have been successfully treated with 2 doses of rVIIa 80 µg/kg [78]. In 2 uncontrolled case series, one of 13 patients with an elevated INR with or without bleeding [79], the other of 6 patients with central nervous system (CNS) bleeding during warfarin prophylaxis [80], rFVIIa (dose range 10-40 µg/kg and 15-90 µg/kg, respectively) rapidly corrected INR in all cases. In conclusion, rFVIIa at doses between 15 and 90 µg/kg has been shown to markedly shorten PT and improve hemostasis in patients with warfarin intoxication [13]. However, as rFVIIa does not influence the other vitamin Kdependent clotting factors (factors II, IX and X) [78], only clinical assessment can be considered a reliable parameter to assess rFVIIa efficacy.

Use of rFVIIa in Intracerebral Hemorrhage The documented efficacy of rFVIIa in the treatment of CNS bleeding in hemophilic patients with inhibitors [83] led to the extension of its use also to non-hemophilic patients with CNS bleeding. In a recent randomized, double-blind, placebo controlled, dose-escalation trial [84], 48 subjects with intracranial hemorrhage were treated with placebo or rFVIIa (10, 20, 40, 80, 120 or 160 µg/kg). Although no positive effect on hematoma volume was observed with any dose of rFVIIa, there was no biochemical or clinical evidence of increased thromboembolic complications. In a more recent multicenter, double-blind, placebocontrolled trial involving 399 patients who had primary intracerebral hemorrhage without coagulopathy and assigned to receive one of three doses of rFVIIa (40, 80 or 160 µg/kg) or placebo, the group with the highest dose of rFVIIa had a significantly smaller increase in the hematoma volume at 24 hours than placebo group. Moreover, the combined rFVIIa groups had lower mortality at three months than did the placebo group (18 percent versus 29 percent, P = 0.02). However, these positive results were tempered by the fact that the incidence of serious thromboembolic adverse events was three times more common in the rFVIIa groups as in the placebo group (7 percent versus 2 percent) [85].

CONCLUSION The studies reviewed here are very encouraging and suggest that rFVIIa may play a major role not only as a treatment for hemophiliacs with inhibitors but also as a hemostatic agent for the control of emergency- or surgery-related hemorrhage that cannot be managed by conventional means. However, only few randomized, double-blind, placebo-controlled trials have been conducted so far and most of the published studies are reports on single cases or small series. Thus, further randomized, controlled trials are needed in order to assess the efficacy, safety and dosage of rFVIIa in these newer “off-label” clinical applications.

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[77] Erhardtsen E, Nony P, Dechavanne M, Ffrench P, Boissel JP, Hedner U. The effect of recombinant factor VIIa (NovoSeven™) in healthy volunteers receiving acenocoumarol to an International Normalised Ratio above 2.0. Blood Coagul Fibrinolysis 1998; 9:741–748. [78] Berntorp E, Stigendal L, Lethagen S, Olofsson L, Hedner U. NovoSeven in warfarintreated patients. Blood Coagul Fibrinolysis 2000; 11(Suppl 1):S113-S115. [79] Deveras RAE, Kessler CM. Reversal of warfarin-induced excessive anticoagulation with recombinant factor VIIa concentrate. Ann Intern Med 2002; 137:884–888. [80] Sorensen B, Johansen P, Nielsen GL, Sorensen JC, Ingerslev J. Reversal of the International Normalized Ratio with recombinant activated factor VII in central nervous system bleeding during warfarin thromboprophylaxis: clinical and biochemical aspects. Blood Coagul Fibrinolysis 2003;14:469-477. [81] Veshchev I, Elran H, Salame K. Recombinant coagulation factor VIIa for rapid preoperative correction of warfarin-related coagulopathy in patients with acute subdural hematoma. Med Sci Monit 2002; 8:CS98–100. [82] Udvardy M, Telek B, Mezey G, Batar P, Altorjay I. Successful control of massive coumarol-induced acute upper gastrointestinal bleeding and correction of prothrombin time by recombinant active factor VII (Eptacog alpha, NovoSeven) in a patient with a prosthetic aortic valve and two malignancies (chronic lymphoid leukemia and lung cancer). Blood Coagul Fibrinolysis 2004; 15:265–267. [83] Rice KM, Savidge GF. Novoseven® (recombinant factor VIIa) in central nervous system bleeds. Haemostasis 1996; 26(Suppl 1):131-134. [84] Mayer SA, Brun NC, Broderick J, Davis S, Diringer MN, Skolnick BE, Steiner T; Europe/AustralAsia NovoSeven ICH Trial Investigators. Safety and feasibility of recombinant factor VIIa for acute intracerebral hemorrhage. Stroke 2005; 36:74-79. [85] Mayer SA, Brun NC, Begtrup K, Broderick J, Davis S, Diringer MN, Skolnick BE, Steiner T; Recombinant Activated Factor VII Intracerebral Hemorrhage Trial Investigators. Recombinant activated factor VII for acute intracerebral hemorrhage. N Engl J Med 2005; 352:777-785.

INDEX A acceptance, viii, 47, 61, 65, 74, 76 access, 16, 126, 147 accounting, 20 accumulation, 159 acetaminophen, 113 acetylcholinesterase, 102 achievement, 11 acid, 3, 17, 31, 79, 80, 89, 122 acidosis, 8 activation, 90, 108, 113, 114, 119, 159, 164, 165 acute renal failure, 114 acute respiratory distress syndrome, 162 acute stress, 97 adaptation, 28 adenine, 91, 145 adenosine, 93, 145 adenosine triphosphate, 93, 145 adhesion, 56, 159, 165 adjustment, 23 adolescents, 133 adult population, 67 adults, 67, 105, 131, 139, 168 adverse event, vii, 8, 9, 13, 67, 128, 150, 151, 152, 163 affect, 5, 78, 79, 109, 131, 138 age, ix, 6, 7, 11, 26, 69, 82, 94, 103, 105, 107, 109, 130, 132 ageing, 97 agent, xi, 18, 19, 78, 80, 81, 91, 98, 100, 157, 158, 163, 164, 165, 167 aggregates, 79, 80, 89, 90 aggregation, 97, 159, 165 AIDS, 122

alanine, 32 albumin, 8, 9, 119 algorithm, viii, xi, 36, 43, 59, 136, 141 alternative(s), 22, 23, 53, 67, 74, 98, 140 Alzheimer's disease, 99 ambivalent, 5 amino acids, 79 anemia, x, 2, 3, 7, 10, 103, 105, 107, 108, 110, 113, 114, 115, 116, 117, 120, 129, 137 animals, 68, 76, 78, 79, 80, 81, 84, 85, 88, 91, 92, 106, 127 ANOVA, 148 antagonism, 162 antibody, ix, 6, 7, 17, 24, 103, 106, 107, 108, 110, 111, 112, 113, 114, 115, 118 anticoagulant, 9, 92, 143, 146, 162 anticoagulation, 169 antigen, x, 25, 94, 103, 105, 107, 110, 111, 112, 113, 114, 115, 118, 119 antigenicity, 91 antinuclear antibodies, 107 anxiety, 29, 30 aortic stenosis, 127 aortic valve, 169 ARDS, 162 artery, 6, 8, 129, 130, 137, 138, 160 Asia, 67 assessment, 23, 26, 137 assignment, 22 association, 11, 97, 105, 107 asymptomatic, 5, 6, 78, 83, 84, 89, 104, 107 ataxia, 82, 86 ATP, 93, 101, 145, 148, 151 attachment, 114 attacks, 32 attention, 18, 128

Index

172 auditing, 60 autoantibodies, ix, 103, 107, 108, 111, 114, 116, 117, 118, 119 autoantigens, 107 autoimmune hemolytic anemia, 117 autopsy, 84 autosomal dominant, 86 availability, ix, 32, 78, 88, 96, 111, 115 avoidance, x, 29, 104, 108, 112, 115, 134, 135 awareness, 61, 82

B bacteria, vii, 15, 18, 19, 25, 34 bacterial infection, 9, 27 banking, viii, 15, 25, 26, 27, 28, 29, 30, 31 banks, 18, 37 batteries, 43, 46, 47, 59 behavior, 75 Beijing, 32 Belgium, 55, 62 beneficial effect, 134, 139 bilirubin, 2, 6, 7, 8, 12 binding, 9, 90, 152, 159 bioassay, ix, 78, 91 biopsy, 160 biotechnology, 66, 67, 68 birth, 2, 8, 10 birth weight, 2, 8, 10 birthweight, 12 bleeding, xi, 9, 124, 131, 134, 135, 157, 158, 159, 160, 161, 162, 163, 164, 166, 167, 168, 169 blood, v, vi, vii, viii, ix, x, xi, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 39, 40, 41, 42, 43, 44, 45, 46, 47, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 83, 84, 85, 87, 88, 89, 90, 92, 93, 94, 95, 96, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 160, 161, 162, 167 blood flow, 3, 5, 6, 8, 11, 127, 128, 129, 131, 145 blood group, x, 44, 51, 53, 54, 55, 59, 104, 105, 107, 110, 111, 112, 114, 117, 143, 152 blood plasma, 87

blood pressure, 3, 5, 8, 9, 123 blood safety, vii, 15, 16, 17, 19, 20, 22, 23, 24, 25, 27, 28, 30, 32, 85 blood supply, vii, ix, 15, 18, 32, 66, 77, 83, 87, 88, 93, 94 blood transfusion, v, vii, viii, ix, x, 1, 2, 9, 15, 17, 18, 19, 22, 24, 29, 30, 33, 36, 41, 47, 52, 56, 57, 60, 61, 62, 65, 69, 73, 74, 75, 76, 83, 87, 88, 89, 98, 99, 100, 103, 104, 106, 109, 117, 121, 122, 124, 130, 131, 132, 135, 139, 140, 162 blood transfusions, 16, 19, 88, 96, 104, 106, 108, 109, 112, 113, 116, 119, 136 body, 7, 82, 107, 108, 123, 126, 128, 146 body weight, 7, 123, 146 bonding, 89, 90 bowel, 9 brain, 78, 80, 81, 84, 86, 90, 91, 92, 96, 97 Brazil, 105 bypass graft, 130 bystander effect, 114

C calcium, 8, 9, 152 Canada, 85, 89, 99, 110 cancer, 16, 105, 109, 112, 138 candidates, 19, 144 capillary, 3, 4, 127 cardiac arrest, 10 cardiac output, 126, 127 cardiac surgery, xi, 67, 76, 119, 125, 130, 131, 132, 134, 138, 139, 142, 154, 157, 161, 167 cardiopulmonary bypass, 67, 131, 139 cardiovascular function, 156 Caribbean, 117 carrier, 48, 67 case study, 136 catheter, 8, 9 cation, 90 cDNA, 101 cell, ix, x, xi, 2, 3, 4, 6, 9, 10, 12, 18, 20, 56, 74, 75, 76, 78, 79, 89, 91, 92, 93, 94, 101, 102, 104, 107, 108, 112, 114, 115, 116, 117, 118, 119, 120, 121, 122, 124, 126, 130, 132, 133, 136, 139, 141, 142, 144, 145, 146, 151, 153, 154, 155, 156, 158, 160, 164, 165 central nervous system, 3, 5, 81, 163, 169 cerebral blood flow, 5, 11 certificate, 85 Chagas disease, 18

Index channels, 95 chemotherapy, 144 children, 10, 81, 87, 105, 108, 113, 114, 116, 118, 120, 129, 131, 133, 138, 160, 161, 167 China, 17, 27, 28, 31, 34 circulation, 4, 11, 131, 136, 142, 145 cirrhosis, 125, 126, 160, 165, 166 civil service, 88 classification, 99 clinical assessment, 163 clinical trials, 23, 67, 135 cloning, 101 cluster, 43, 46, 60 CNS, 3, 5, 99, 163 CO2, 94 coagulation, xi, 4, 157, 158, 159, 162, 164, 165, 166, 167, 169 coagulation factors, xi, 157, 159, 162 coagulopathy, 159, 161, 163, 166, 167, 168, 169 coding, 58, 62, 82 codon, 79, 82, 98, 99 cognitive impairment, 82 combination therapy, 130 combined effect, 159 commitment, 37, 62, 95 commodity, 66 communication, 72, 73, 74, 75 communication strategies, 74 community, 29, 67, 74, 107 compatibility, 37, 47, 56, 58, 61, 93 compensation, 138 complement, 108, 113, 114 complete blood count, 8 compliance, 21, 36, 37, 42, 153 complications, 5, 9, 19, 20, 104, 111, 114, 119, 131, 132, 135, 159, 161 components, vii, ix, xi, 1, 2, 9, 16, 17, 18, 31, 33, 34, 38, 43, 78, 87, 93, 96, 100, 101, 102, 106, 109, 114, 119, 126, 141, 142, 143, 144, 146, 147, 149, 150, 151, 153, 155, 162 composition, 79, 115 compounds, 30, 67, 68, 75 computation, 123 concentrates, 31, 32, 33, 34, 91, 101, 119, 142, 154, 155 concentration, 6, 7, 91, 123, 128, 129, 131, 138, 143, 153 conditioning, 112 conduct, 91 conduction, 137

173

confidence, 93 consensus, 115, 124 conservation, 19, 122, 123, 126, 129, 132, 133, 134, 135, 138, 139, 145 consumption, 20, 55, 149 contamination, vii, 15, 17, 18, 25, 26, 29, 31, 32, 88, 105, 107, 146, 147 context, 17, 68, 76 contingency, 41, 58 control, xi, 7, 73, 75, 84, 87, 88, 91, 92, 119, 128, 130, 131, 133, 149, 157, 158, 160, 161, 163, 167, 168, 169 controlled trials, x, 7, 122, 128, 134, 161, 163 conversion, 80, 92, 101, 143, 158 cooling, 128 coronary artery disease, 131, 137 correlation, 70 corticosteroids, 112 cost effectiveness, 23 cost saving, 28, 30 cost-benefit analysis, 61 costs, 20, 21, 22, 23, 61 Council of Europe, 92, 93, 94, 101, 155 covering, 112 Creutzfeldt-Jakob disease, ix, 77, 81, 85, 87, 98, 99, 100, 101 culture, 25 cycles, 149 cytokines, 109, 119 cytomegalovirus, 34 cytometry, 102

D damage, 3, 5, 18, 78, 84, 114 death, x, 5, 9, 21, 23, 73, 83, 85, 103, 113, 128, 160 death rate, 85 decision makers, 19, 23, 30 decision making, 73, 95 decision-making process, 25 decisions, 25, 94, 95 defects, 42, 82, 158 deficit, 129, 148 definition, x, 21, 121 delivery, 2, 3, 20, 126, 127, 129, 131, 135, 137 demand, 31, 102, 142, 153 dementia, 82, 83, 86, 99 demographic data, 56 Denmark, 158 Department of Defense, 88

Index

174

Department of Health and Human Services, 99, 101 deposits, 97 derivatives, 87, 100 destruction, 81, 114, 117, 120 detection, 17, 25, 26, 33, 83, 89, 106, 107, 108, 110, 111, 113, 118 detergents, 89 diagnostic criteria, 5 digestion, 89, 91, 92 disability, 21 discipline, 16 discomfort, 21 disorder, 82 dissatisfaction, 21 dissociation, 127, 131 distress, 3, 4, 95, 133 distribution, 24, 79, 89, 100, 101 division, 37, 46 DNA, 24, 33 doctors, 41, 46 dogs, 138 domain, 69, 97 donations, xi, 19, 24, 33, 85, 141, 142, 151, 153, 155 donors, ix, xi, 2, 17, 18, 19, 24, 27, 31, 33, 66, 77, 78, 84, 85, 87, 88, 89, 105, 110, 111, 112, 117, 118, 141, 142, 143, 145, 148, 150, 152, 153, 155, 156 doppler, 104 dosage, 163 double-blind trial, 160, 162 drug therapy, 21 durability, 106 duration, 55, 82

E earnings, 21 eating, 98 economic efficiency, 25 economics, v, 15, 16, 17, 20, 30, 33 EEG, 86 effluent, 145 elaboration, 80 elderly, 83, 129, 138 electrodes, 2 electrolyte, 3 elk, 78, 81, 98 embolism, 9 employees, 88 employment, 21

encephalopathy, ix, 77, 81, 87, 100, 101 end-stage renal disease, 33 England, 100 environment, 24, 29 environmental factors, 37 enzymes, 112 epidemic, 18, 81, 87 equipment, 20, 22, 47, 142, 149 ergonomics, viii, 36 erythrocyte, 3, 4, 108, 115, 118, 119, 124 erythropoietin, 2, 10, 114 estimating, 101 etiology, 4, 6, 106, 113 Europe, ix, xi, 18, 75, 78, 88, 157, 169 European Commission, 74 European Union, 94 evidence, viii, 3, 5, 8, 22, 33, 61, 65, 66, 68, 78, 79, 80, 81, 84, 89, 105, 109, 114, 117, 129, 134, 135, 159, 163 examinations, 83 exchange transfusion, 1, 2, 4, 5, 6, 7, 8, 9, 11, 12, 13, 116 exclusion, 87 expectation, 135 exposure, ix, x, xi, 3, 18, 78, 94, 95, 96, 103, 104, 105, 108, 110, 113, 121, 122, 133, 143, 151, 157, 158 expression, 94, 96, 98, 102, 107, 159 extraction, 126, 127, 129, 137

F failure, x, 3, 37, 42, 55, 103, 104, 108, 113, 125, 126, 135, 158, 160, 162 false positive, 26 family, 20, 21, 73, 83, 88 family members, 20, 21, 88 FDA, 88, 93, 94, 148 febrile reactions, 113 feelings, 74, 75, 76 feet, 149 ferritin, 152 fever, 113 fibers, 90 fibrin, 159, 165 fibrinolysis, 159, 160, 164, 165 filtration, ix, 78, 91, 92, 93, 94, 96, 101, 145, 148, 150, 154 first generation, viii, 36, 43, 45, 59 flexibility, xi, 141

Index fluctuations, 8, 9 fluid, 3, 122, 149 food, viii, 33, 65, 67, 68, 69, 72, 74, 75, 76, 81, 88 food products, 68, 72 framing, 75 France, 18, 56, 62, 85, 87 freezing, 112 fresh frozen plasma, 9, 87 friction, 3 FTIR, 89 fulfillment, 47 funding, 96

G gait, 81, 91, 92 gastrointestinal bleeding, 160, 165, 169 gastrointestinal tract, 67 gender, 38, 146, 152 gene, 76, 79, 80, 86, 96, 98 gene expression, 96 gene promoter, 80 generation, 43, 59, 89, 91, 98, 102, 113, 119, 159, 161, 164, 165 genetic factors, 105 Germany, 17, 31, 88, 141, 148 gestation, 10, 12 glucose, 6, 9, 11 glycol, 112 glycosaminoglycans, 138 glycosylation, 79, 97 gold, 4, 23 goods and services, 22 government, 94, 95 gravitation, 144 gravity, 145 gray matter, 82 Greece, 88, 115 group membership, 75 grouping, 56 groups, 23, 66, 75, 87, 113, 118, 130, 135, 143, 153, 160, 163 growth, 3, 18, 19, 78, 82 growth factor, 19 growth hormone, 78, 82 guidance, 42 guidelines, 2, 3, 8, 10, 20, 46, 92, 93, 94, 100, 124, 129 Guinea, 78, 81, 82

175

H haemostasis, 138, 164 harvesting, 152 hazards, 53, 62, 72, 75 HBV, 17, 24, 31, 33 HBV infection, 31 health, vii, ix, x, 15, 16, 17, 19, 20, 22, 23, 24, 27, 30, 65, 66, 85, 103, 110 health care, vii, 15, 30 health education, ix, 66 health services, 66 heart disease, 125, 167 heart rate, 126, 127 heart valves, 73 height, 91, 92, 149 hematocrit, x, 2, 4, 6, 9, 92, 121, 129, 136, 138, 139, 142 hematoma, 163 heme, 152 hemoglobin, x, xi, 7, 75, 76, 93, 104, 109, 112, 114, 123, 124, 126, 127, 128, 129, 131, 134, 138, 141, 143, 145, 148, 149, 152 hemophilia, xi, 157, 158, 159, 164, 165 hemophiliacs, xi, 157, 158, 159, 163 hemorrhage, 9, 127, 158, 161, 162, 163, 166, 167, 168 hemostasis, 158, 160, 163, 166 hepatic failure, 160, 166 hepatitis, 19, 31, 32, 33, 83 heterogeneity, x, 104, 121 heterozygote, 79 hip, 126, 129, 132, 133, 134, 140 hip arthroplasty, 126 Hispanic population, 18 HIV, 17, 22, 24, 25, 30, 31, 55, 67, 83 HIV infection, 24 HIV-1, 25, 31 HLA, 106, 107, 109, 114, 118, 119 homozygote, 79, 120 Hong Kong, v, viii, 35, 36, 37, 38, 43, 44, 51, 55, 56, 59, 62, 63, 115 host, ix, 3, 9, 57, 103, 105, 107, 109 HTLV, 17 human immunodeficiency virus, 31, 33, 67 human leukocyte antigen, 109 hydrogen, 79, 89, 90, 97 hydrogen peroxide, 79, 97 hydroxyl, 90 hydroxyl groups, 90

Index

176 hyperactivity, 113 hyperbilirubinemia, 2, 7, 10, 12 hypertension, 4, 152 hypertonic saline, 139 hypotension, 128, 139, 152 hypotensive, 133, 152 hypothermia, 131 hypothesis, 97, 98 hypovolemia, 9, 126 hypoxia, 127 hysterectomy, 132, 133, 139

I iatrogenic, 78, 82 icterus, 10, 12 identification, viii, 35, 36, 37, 39, 40, 41, 43, 53, 56, 57, 58, 59, 61, 62, 63, 112, 117, 132 identity, viii, 35, 36, 38, 40, 45, 49, 50, 53, 57, 58, 60 immune response, 104, 106, 107, 115, 118 immune system, 107 immunization, 119, 144 immunogenicity, 105 immunoglobulin, 119 immunomodulation, x, 119, 121, 122 immunomodulatory, 109, 119 immunosuppression, 109 implementation, viii, 19, 26, 28, 29, 31, 36, 42, 58, 59, 62, 89, 94 in vitro, 109, 159 inattention, 37 incentives, 16 incidence, ix, 4, 5, 11, 23, 31, 32, 55, 58, 67, 81, 83, 85, 103, 104, 105, 107, 110, 112, 113, 114, 128, 151, 163 inclusion, xi, 128, 141 income, 21, 22 incubation period, 83 India, 80 Indians, 78 indication, 2, 7, 22, 95, 129 indices, ix, 78, 127 indigenous, 82 induction, 122, 131 industry, 22, 33 infants, 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12 infarction, 128 infection, ix, 9, 16, 17, 18, 24, 31, 32, 55, 77, 81, 82, 83, 84, 86, 100, 114, 125, 126

infectious disease, vii, x, 15, 17, 31, 32, 78, 121, 122 inflammation, 83 influence, 3, 76, 108, 109, 118, 119, 136, 154, 163, 166 information technology, 56 informed consent, 52 infrared spectroscopy, 89 infrastructure, 58 ingestion, ix, 77, 78, 80, 81, 82 inhibitor, 159, 164 initiation, 20, 62, 98, 131, 158 injury, x, 6, 104, 121, 122, 158 input, 20 insight, 78 insomnia, 79, 82, 97 instability, 8, 134 institutions, 111 instruction, 41 instruments, 78, 82 insulin, 9, 88 insurance, 20 integrin, 94, 102 integrity, 60 intelligence, 60 intensive care unit, 8, 22 intentions, 75 interaction, 89, 90 interactions, 93 interest, 20, 142 interface, 145 international standards, 145, 151 interpretation, 20, 56 interval, 148, 152 intervention, vii, 1, 4, 16, 20, 22, 23, 66, 71, 95 intestine, 81 intoxication, 163 intracerebral hemorrhage, 163 intrauterine growth retardation, 4 intravenous immunoglobulins, 108 investment, 26 IQ scores, 11 Ireland, 85 iron, 3, 116, 118, 152, 156 irradiation, 9, 27, 28 ischemia, 126 isolation, 94, 97 Israel, 1, 121 Italy, 85, 88, 142, 157 iteration, 95

Index

J Jamaica, 105, 118 Japan, 17, 27, 28, 29, 31, 33, 34, 85 jaundice, 12 judgment, 94

K kidney, 114, 119 knowledge, 69, 73, 75, 158, 159 Korea, 67

L labeling, 43, 55, 59, 62, 95, 135 labour, 36 laminar, 3 latency, ix, 77, 82, 84 Latin America, 18 layering, 27, 28 lead, 22, 25, 28, 37, 74, 107, 114, 131, 159 leadership, 94 leukemia, 26, 169 life expectancy, 61 lifetime, 104, 113 likelihood, x, 106, 122 limitation, 30, 89 links, 61, 74, 99 lipids, 93, 102 liquids, 67 liver, xi, 134, 135, 138, 140, 157, 158, 159, 165, 166, 167, 168 liver disease, 159, 165, 166 liver failure, 166 liver transplantation, xi, 157, 158, 160, 166 livestock, 80 location, 57 logistics, 27, 59, 62 longevity, 10 long-term memory, 98 Louisiana, 103, 115 low risk, ix, 65, 66, 71 lung cancer, 169 lung disease, 125, 126 lymphocytes, 78, 83 lymphoid, 84, 169 lymphoid tissue, 84 lymphoma, 26

177

lysis, 114, 159

M macrophages, 94 mad cow disease, 81 malaria, 18 males, 106 management, x, xi, 3, 6, 29, 37, 46, 62, 104, 108, 110, 115, 116, 120, 140, 157, 166, 167 mandates, 57 mannitol, 145 manpower, 36, 58, 61 manufacturing, ix, 33, 78, 95, 153, 164 mapping, 101 market, 16, 58 marketing, 30, 67 markets, 17 mass, x, 4, 6, 93, 121, 126 mass loss, x, 121 maternal smoking, 11 mean arterial pressure, 127 measurement, 4, 6, 22, 148, 152 measures, v, vii, 15, 16, 17, 18, 19, 22, 23, 24, 26, 27, 28, 30, 56, 84, 87, 88, 89, 94, 99, 123, 152 meat, 78, 81, 82 media, 73, 80 median, 24, 162 medication, 127 megakaryocyte, 96 membership, 37 memory, 44, 60, 61, 80, 127 men, 69, 81, 105 metabolism, 93, 101, 129, 156 methodology, 4, 5, 22 mice, 79, 80, 97, 119 microcirculation, 3 migration, 82 military, 88 mixing, 148 mode, 5, 33, 82, 145, 148 model system, 75, 165 modeling, 123, 136 models, 29, 84, 98, 100, 132, 134 molecular biology, 97 molecular mass, 79 molecular weight, 167 molecules, 78, 109, 118, 119 money, 16, 21 monitoring, 5, 6, 8, 60, 62, 85, 87, 102, 126

Index

178 monoclonal antibody, 91, 92 monomers, 89 morbidity, 3, 18, 23, 104, 115, 159 morphology, ix, 77 mortality, 3, 9, 18, 23, 27, 83, 115, 159, 163 motivation, 24, 154 movement, 79 mutation, 82 myocardium, 129

N National Institutes of Health, 124 needs, 5, 16, 17, 20, 66, 105, 142, 146 neonates, vii, 1, 2, 3, 6, 7, 8, 9, 10, 12 nerve, 137 nervous system, 91, 99 Netherlands, 26, 32, 88, 119 network, 58, 62 neural function, 79 neurological disease, 83 Nile, 19, 27, 32 nitrous oxide, 136 N-N, 69 Nobel Prize, 81 North America, 105 nucleic acid, 24, 25, 33, 78, 81, 97 nurses, 41, 46 nursing, 20, 37, 42, 46, 56

O objective criteria, 6 obligate, 58 obligation, 95 observations, 106, 107 obstruction, 127 oligomers, 80 operator, 8, 57, 60, 146 optical density, 145 organ, 16, 104, 129, 131, 162 organization, 62 organizations, 59 orientation, 41 output, 21, 126, 127, 137 overload, 3, 116, 118 oxidative stress, 97 oxygen, x, 2, 3, 16, 66, 67, 74, 104, 109, 112, 126, 127, 128, 129, 131, 136, 137, 138

oxygen consumption, 128, 129, 138

P pain, x, 103, 104, 113, 152 pancreatitis, 168 paradigm shift, 30 paralysis, 93 parameter, 10, 123, 163 parasite, 18 paroxysmal cold hemoglobinuria, 108 partial thromboplastin time, 128 particles, 98, 99, 100 pathogenesis, 80, 81 pathogens, vii, 15, 16, 17, 18, 19, 24, 25, 26, 27, 28, 30, 31, 32, 33 pathology, 86 pathophysiology, 115 pathways, viii, 36, 57, 149 PCR, 96 pediatrician, 6 perceptions, 68, 69, 71, 73, 74, 75, 76 perfusion, 9, 137 peripheral blood, 90, 112 permit, xi, 141, 143 perspective, 20, 23, 30, 33, 61 PET, 2, 3, 4, 5, 6 pH, 137, 146, 148 pharmaceuticals, 122 phenotype, 105, 109, 110, 112, 115, 120 phlebotomy, 2, 55 phosphatidylserine, 159 phospholipids, 159, 165 physical properties, 3, 101 physicochemical properties, 91 pilot study, 166 placebo, 153, 156, 160, 161, 162, 163, 167 plants, 68 plasma, xi, 2, 3, 4, 6, 9, 11, 12, 18, 19, 24, 26, 27, 85, 87, 92, 100, 101, 105, 107, 114, 118, 139, 141, 142, 143, 144, 145, 146, 147, 149, 151, 152, 153, 154, 155, 160, 165 plasma proteins, 3, 101, 114 plasmapheresis, 142, 143 plastic surgery, 142 platelets, xi, 18, 19, 26, 27, 28, 29, 31, 32, 33, 34, 78, 106, 120, 128, 141, 142, 144, 146, 154, 156, 159, 160, 165 PLS, 143 PM, 31, 117, 120, 139, 155

Index Poland, 12 policy makers, 19, 24, 96 polycythemia, 1, 3, 4, 5, 6, 11, 12 polymers, x, 104, 109, 112 poor, 3, 21, 109, 127, 142, 144, 153 population, ix, 28, 31, 83, 85, 87, 103, 105, 106, 110, 112, 115, 131, 134, 135 Portugal, 88 positive correlation, 68 positive feedback, 159 potassium, 93, 145, 148, 151 power, 59 prediction, 68 preference, 25 pregnancy, 105, 116 preparation, 91, 142, 147, 154, 155 present value, 22 preservative, 145 pressure, 3, 11, 96, 146 preterm infants, 2 prevention, ix, 6, 8, 10, 18, 27, 28, 30, 94, 95, 96, 103, 104, 112, 115, 120 prices, 23 priming, 146 Principal Components Analysis, 71 principle, 62 prions, ix, 77, 78, 79, 80, 81, 83, 84, 85, 89, 90, 91, 92, 95, 96, 97, 99, 102 probability, 8, 110, 112 production, x, 2, 97, 103, 110, 113, 118, 119, 142, 153 productivity, 22, 30 program, 22, 23, 89, 111, 143, 156 promoter, 98 propagation, 98 prophylactic, 5, 6, 110, 160 prophylaxis, 163 prostatectomy, 132, 133, 161 protein misfolding, 85, 96 proteinase, 80, 89, 91 proteins, ix, 77, 78, 89, 93, 94, 101, 102, 113 prothrombin, 128, 159, 165, 166, 169 protocol, x, 5, 42, 110, 121, 132, 133, 134, 139, 144, 145, 147, 155 psychosis, 82 public health, 88, 96 pulmonary vascular resistance, 5 pumps, 146, 149 purification, 101

179

Q quality assurance, 101, 155 quality of life, 22, 23

R race, ix, 103, 105 radio, 61 radius, 3 range, viii, 3, 18, 65, 71, 80, 108, 125, 130, 131, 135, 147, 149, 152, 160, 163 ratings, ix, 65, 68, 69, 70, 71 RDP, 27 reading, 82 reagents, 111 real estate, 20 reasoning, 96 recall, 25, 26 reception, 75 recognition, 89, 95 recovery, 93, 94, 102, 150, 151, 152 red blood cells, 2, 9, 26, 101, 102, 106, 112, 113, 144, 154 reduction, ix, x, 5, 19, 25, 33, 37, 59, 78, 85, 88, 89, 90, 91, 92, 93, 94, 95, 96, 100, 119, 121, 128, 130, 131, 132, 133, 146, 147, 148, 149, 155, 160, 162 reflection, 62 regenerate, 93 regeneration, 102, 153 regulation, 79 relapses, 21 relationship, 16, 21, 22, 68, 69, 101 relaxation, 19 relevance, 5, 20, 76 religion, 73 renal failure, 167 repair, 167 replacement, x, 12, 121, 122, 127, 139, 140, 167, 168 reproduction, 78 resection, 134, 138, 140 resistance, 3, 59, 89, 91, 126 resolution, 79, 162 resources, x, 20, 21, 22, 24, 30, 62, 111, 121 respiratory, 3, 8, 32 response time, 127 retention, 66

Index

180

returns, 28, 75, 147 risk, vii, ix, 1, 5, 7, 10, 11, 15, 16, 17, 18, 19, 25, 26, 27, 28, 29, 30, 31, 32, 36, 55, 57, 63, 65, 66, 67, 68, 69, 71, 72, 73, 74, 75, 76, 77, 83, 84, 85, 87, 88, 89, 93, 95, 96, 97, 100, 106, 117, 126, 131, 134, 144, 152 risk factors, 7 risk management, 37 risk perception, 74 RNA, 33 room temperature, 32, 91, 92, 122, 128, 145 rural areas, 28

S safety, v, vii, viii, ix, 15, 16, 17, 18, 19, 24, 25, 26, 27, 28, 29, 30, 32, 33, 36, 47, 62, 63, 75, 77, 87, 93, 94, 95, 96, 117, 128, 155, 161, 163, 166, 167 sample, viii, 36, 37, 39, 40, 42, 44, 47, 50, 51, 55, 56, 57, 59, 69, 91, 135 sampling, viii, 4, 11, 26, 35, 36, 39, 42, 44, 51, 55, 56, 57, 58, 60, 61 SARS, vii, 15, 19, 43 satisfaction, 47 saturation, 152 savings, 23, 28, 29, 123, 124, 132, 134 scanning process, 54 school, 11, 21 scoliosis, 140, 161 search, xi, 5, 22, 75, 76, 157 second generation, viii, 36, 59, 61 secretion, 9 security, 34 seed, 80 selecting, 112 self, viii, 36, 38, 39, 41, 50, 54, 94, 107, 115, 118 sensations, 152 sensing, 60 sensitivity, vii, 2, 15, 22, 25, 26, 27, 29, 84 sensors, 149 separation, 142, 145, 147, 149 sepsis, 18, 25, 30, 32 series, 9, 55, 84, 160, 161, 163 serum, 6, 7, 9, 12, 119, 152 services, 16, 20, 21, 24, 66, 143 severity, 73, 108 shape, 159 shear, 4 shear rates, 4 sheep, 78, 80, 81, 84, 91, 96, 98, 100

shoot, 46 sickle cell, v, ix, 103, 104, 106, 111, 113, 115, 116, 117, 118, 119, 120 sickle cell anemia, 116, 117, 120 sign, 51 similarity, 81 sinus, 2, 6 sites, 4, 79 skin, 119, 160 smoking, 4 social relations, 73 software, 47, 62, 147 South Africa, 67 Spain, 15, 17, 27, 31, 88 spastic, 82 species, ix, 25, 77, 78, 79, 80, 81, 91, 106 specificity, 22, 92, 108, 112 spectrum, 26, 34, 66 speculation, 109 speed, 61, 94, 96, 145, 149 spinal cord, 81 spinal fusion, 161, 167 spine, 129, 132, 133, 140 spleen, 81, 94, 108 stability, 132 stages, 44, 83 standard deviation, 149 standardization, xi, 141 standards, ix, 28, 78, 93, 146 starch, 139, 153 stenosis, 127, 129, 131, 137, 138 sterile, 145, 148 steroids, 108 storage, viii, ix, 18, 32, 36, 78, 89, 93, 94, 101, 102, 109, 119, 122, 146, 147, 148, 149, 150, 151, 154, 155 strain, 56, 91 strategies, 19 stress, 75, 79, 136 stroke, 67, 104, 116, 120, 126, 127 stroke volume, 126, 127 structuring, 67 students, viii, 65, 69 subacute, x, 103 sub-Saharan Africa, 22 substitutes, viii, 65, 66, 67, 68, 69, 70, 71, 73, 74, 75, 76 summer, 19 supervision, 42 supply, 19, 31, 42

Index suppression, 112, 113 surface modification, 92 surplus, 126 surprise, 79, 108, 110, 113 surveillance, 83, 88 survival, x, 101, 104, 109, 112, 119, 151, 155, 162 survival rate, 162 survivors, 92 susceptibility, 98 symptom, 3 symptoms, 3, 78, 79, 81, 83, 84, 85, 92, 129, 152 syndrome, 4, 11, 32, 79, 86, 99, 104, 113, 116, 117, 120 synthesis, 159 systems, vii, xi, 15, 16, 25, 26, 29, 30, 31, 37, 55, 58, 59, 94, 95, 105, 141, 142, 143, 153

T technology, vi, viii, ix, xi, 15, 16, 17, 21, 23, 26, 27, 28, 29, 30, 34, 37, 61, 63, 65, 76, 78, 85, 89, 90, 92, 93, 94, 95, 96, 101, 141, 153, 155 telephone, 20 temperature, 8, 18, 93, 101, 108 thalamus, 97 thalassemia, 105, 115 theory, 133 therapeutic approaches, 108, 112 therapeutic process, 20 therapeutics, 74 therapy, vii, xi, 1, 2, 4, 5, 6, 7, 10, 22, 23, 24, 104, 115, 116, 117, 118, 133, 137, 157, 158, 160, 162, 166, 168 thinking, 27, 73 threat(s), 24, 32, 95 threshold(s), 4, 16, 24, 25, 27, 30 thrombin, 159, 161, 164, 165 thrombocytopenia, 118, 160 thrombus, 164 time, vii, ix, x, xi, 4, 8, 9, 11, 18, 19, 20, 25, 29, 38, 39, 44, 50, 51, 54, 55, 56, 57, 58, 59, 60, 61, 77, 78, 84, 85, 87, 88, 96, 103, 105, 106, 108, 111, 112, 113, 123, 128, 141, 143, 145, 147, 148, 149, 151, 161, 165, 166, 169 timing, 136 tin, 152 tissue, 3, 6, 67, 78, 79, 80, 81, 82, 86, 90, 91, 126, 127, 140, 158, 164, 165 tonsils, 81 toxicity, 29, 30, 152

181

tradition, 37 training, 46, 56, 63 transcription, 59 transfusion, vi, vii, viii, ix, x, 1, 2, 3, 5, 6, 7, 8, 9, 10, 12, 15, 16, 17, 18, 19, 20, 22, 24, 25, 27, 28, 29, 30, 32, 33, 34, 35, 36, 37, 39, 41, 42, 43, 44, 46, 47, 54, 55, 56, 57, 59, 60, 61, 62, 63, 65, 66, 67, 70, 71, 73, 74, 75, 76, 78, 83, 84, 87, 88, 89, 95, 96, 97, 100, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 124, 129, 131, 132, 133, 134, 135, 138, 140, 142, 143, 151, 153, 154, 157, 160, 162, 164, 166, 168 transfusion reactions, 29, 54, 62, 113, 115, 117, 120 translation, 98 transmission, 17, 18, 22, 28, 29, 31, 55, 80, 81, 82, 83, 84, 86, 87, 88, 89, 95, 96, 98, 99, 100 transplantation, 70, 73, 82, 112, 119, 120, 160 transport, 67, 129, 136, 137 transportation, 16 trauma, xi, 157, 158, 159, 161, 162, 166, 167, 168 trend, 18, 154 trial, 7, 41, 57, 115, 119, 138, 140, 155, 160, 161, 163, 165, 167 trust, 76 Turkey, 32, 88

U ultrasonography, 116 uncertainty, 5 uniform, 128, 131, 134, 135 United Kingdom (UK), ix, 18, 19, 24, 62, 65, 69, 76, 77, 78, 81, 83, 84, 85, 86, 87, 88, 89, 93, 99, 100, 101, 105, 118 United States, 24, 34, 38, 85, 94, 99, 104, 122, 135, 148

V validation, 47, 91, 136 valine, 79, 82 values, 3, 23, 24, 128, 139, 143, 145, 148, 151, 152 variable(s), x, 22, 26, 75, 105, 106, 121, 151 variance, 71 variation, 4, 55, 111, 153 vascular surgery, 129 vasculature, 129 vasculitis, 129

Index

182 vein, 6, 8, 152 velocity, 5, 11 venipuncture, 149, 152 ventricle, 127 versatility, 142 vessels, 5 viral infection, 17 virus infection, 19, 30 viruses, vii, 15, 19, 29, 81 viscosity, 3, 4, 6, 11, 126, 127, 131 vision, 62 visual field, 82 vitamin K, 162

wear, 61 web, 41, 46, 51, 53, 54, 67 weight gain, 3 weight loss, 91 Western Europe, 28, 87 white blood cells, 87, 113 withdrawal, 8, 135 women, 69, 81, 106, 149 words, 25, 29 work, 6, 21, 22, 69, 72, 74, 80, 115 workers, 79, 81, 145, 152 workload, 56 worry, 74 writing, 60

W Y Wales, 84 war, viii, 36, 41, 60 water, 3, 60, 139 weakness, 82

yield, xi, 24, 28, 141, 144, 145

Z zinc, 152