Immunohematology JOURNAL OF BLOOD GROUP SEROLOGY AND EDUCATION

V OLUME 21, NUMBER 3, 2005 This Issue of Immunohematology Is Supported by a Contribution From

Dedicated to Education in the Field of Blood Banking Immunohematology JOURNAL OF BLOOD GROUP SEROLOGY AND EDUCATION VOLUME 21, NUMBER 3, 2005 CONTENTS

85 Review: the burgeoning history of the complement system 1888–2005 H. CHAPLIN JR. 94

On a much higher than reported incidence of anti-c in R1R1 patients with anti-E W. J. J UDD, L.R. DAKE,AND R.D. DAVENPORT 97 Case report: massive postpartum transfusion of Jr(a+) red cells in the presence of anti-Jra S.YUAN,R.ARMOUR,A.REID, K.F.ABDEL-RAHMAN,M.PHILLIPS,D.M.RUMSEY,AND T. N ESTER 102 Is there a relationship between anti-HPA-1a concentration and severity of neonatal alloimmune thrombocytopenia? H. BESSOS,M.TURNER,AND S.J. URBANIAK 109 Review: therapeutics for prevention of immune hemolysis K.YAZDANBAKHSH 119 Autoimmune and a further example of autoanti-Kpb E. LEE,G.BURGESS,AND N.WIN 122 The incidence of red cell alloantibodies underlying panreactive warm autoantibodies M. MALEY,D.G.BRUCE,R.G.BABB,A.W.WELLS,AND M.WILLIAMS 126 Persistent anti-Dra in two pregnancies N. RAHIMI-LEVENE,A.KORNBERG,G.SIEGEL,V.MOROZOV,E.SHINAR,O.ASHER,C.LEVENE,AND V. Y AHALOM

COMMUNICATIONS 129 132 Letter to the Editors ERRATUM Inaugural meeting of the consortium for blood Vol. 21, No. 4, 2005, p.60 group (CBGG): a summary report G. Denomme and M. Reid 133 136 ANNOUNCEMENTS ADVERTISEMENTS 138 INSTRUCTIONS FOR AUTHORS EDITOR-IN-CHIEF MANAGING EDITOR Delores Mallory, MT(ASCP)SBB Cynthia Flickinger, MT(ASCP)SBB Supply, North Carolina Philadelphia, Pennsylvania TECHNICAL EDITOR SENIOR MEDICAL EDITOR Christine Lomas-Francis, MSc Scott Murphy, MD New York, New York Philadelphia, Pennsylvania ASSOCIATE MEDICAL EDITORS Geralyn Meny, MD David Moolton, MD Ralph Vassallo, MD Philadelphia, Pennsylvania Philadelphia, Pennsylvania Philadelphia, Pennsylvania EDITORIAL BOARD Patricia Arndt, MT(ASCP)SBB Christine Lomas-Francis, MSc Marion E. Reid, PhD, FIBMS Pomona, California New York, New York New York, New York James P.AuBuchon, MD Gary Moroff, PhD Susan Rolih, MS, MT(ASCP)SBB Lebanon, New Hampshire Rockville, Maryland Cincinnati, Ohio Geoffrey Daniels, PhD Ruth Mougey, MT(ASCP)SBB S. Gerald Sandler, MD Bristol, United Kingdom Carrollton, Kentucky Washington, District of Columbia Richard Davey, MD John J. Moulds, MT(ASCP)SBB David F. Stroncek, MD Washington, District of Columbia Shreveport, Louisiana Bethesda, Maryland Sandra Ellisor, MS, MT(ASCP)SBB Marilyn K. Moulds, MT(ASCP)SBB Marilyn J.Telen, MD Anaheim, California Houston, Texas Durham, North Carolina George Garratty, PhD, FRCPath Sandra Nance, MS, MT(ASCP)SBB Connie M.Westhoff, SBB, PhD Pomona, California Philadelphia, Pennsylvania Philadelphia, Pennsylvania Brenda J. Grossman, MD Paul M. Ness, MD St. Louis, Missouri Baltimore, Maryland W. John Judd, FIBMS, MIBiol Mark Popovsky,MD Ann Arbor, Michigan Braintree, Massachusetts

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Copyright 2005 by The American National Red Cross ISSN 0894-203X Review: the burgeoning history of the complement system 1888–2005

H. CHAPLIN JR.

The complement system first came to the attention fractions responsible for the complement activities of of scientists following work by Koch and Pasteur on serum. To maintain order in this proliferation of transmissible diseases in the latter half of the 19th complement components, each factor was assigned a century. Leading microbiologists such as Paul Ehrlich, number in the order in which it was discovered, i.e., Jules Bordet, and George Nuttall were stimulated to C1,C2,C3,and C4. It later became clear that the factors learn more about the human body’s mechanisms of reacted sequentially, but not in the order of their protection against infectious disease. In 1888, Nuttall discovery. In joint international meetings it was noted that sheep blood was mildly bactericidal for decided to not change the numbers already assigned to anthrax bacilli, but that this property disappeared newly defined complement factors, but rather to rapidly if the blood was heated to 55°C or kept at room display their order of reactivity as this evolved from temperature. The heat-labile bactericidal activity was ongoing research. Thus by the late 1930s,the reactivity named “alexin.” sequence for the first four complement factors was In 1894, other laboratories demonstrated that shown as: C1 → C4 → C2 → C3. serum from guinea pigs that had recovered from The complement story had been developed from cholera would protect normal guinea pigs from cholera the perspective of infectious disease, i.e., that it was if it was injected along with the live cholera bacteria. part of the safety net that protected humans from the Importantly, it was shown by in vitro studies that the ravages of pathogenic microorganisms (bacteria, fungi, cholera bacteria were only killed by fresh immune viruses). Thus it was postulated that the complement serum, and not by heat-inactivated immune serum. system was activated by the body’s response to attack However, it was noted that the heat-inactivated by pathogenic microorganisms, presumably by pro- immune serum would protect normal guinea pigs from duction of immune reactants () against the exposure to cholera bacteria. Bordet subsequently attacking microorganisms. The starting point of the showed that the activity of the heat-inactivated serum complement response presupposed an infection- could be restored in vitro by the addition of a small related activator. Because the protective response was amount of fresh normal serum that by itself lacked hypothesized from the infectious disease perspective bactericidal activity. These results indicated that the (earlier research had validated that hypothesis), the bactericidal activity depended on both a heat-stable C1 → C4 → C2 → C3 sequence was dubbed the sensitizing substance in immune serum and a heat- “classical pathway” of entry into the complement labile cytotoxic factor present in normal (as well as system. immune) serum. Bordet attributed the heat-labile However,it had been noticed that certain strains of activity to the alexin bactericidal factor. In 1899, bacteria and yeast could activate the complement Ehrlich put forward a humoral immunity scheme system without having induced antibodies. In 1913, it substituting the terms “complement” for alexin and was shown that cobra venom would mediate “amboceptor” for the heat-stable immune sensitizer. complement-like RBC lysis in the absence of immune It soon became evident that complement consisted antibodies. These studies suggested that this involved of more than two serum components. By the 1920s it serum components that differed from those of the was concluded that that there were at least four serum classical pathway. In fact, these findings presaged what

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40 years later would be named the “alternative Table 2. of the alternative pathway* pathway” based on work by other researchers. They Serum Molecular concentration included Dr.Louis Pillemer,who described a previously Symbol Name Functions weight (µg/mL) unidentified serum named “properdin,” which C3 C3 Attaches covalently 185,000 1200 was said to bind to bacteria and yeast, activating after proteolytic activation generates complement without requiring the presence of anti- metastable C3b. C3b body. In 1957, his claim to have discovered a fragment is part of “properdin pathway” to complement activation was C3/C5 convertase. denounced by most contemporary complementol- B Factor B Binds to C3b forming 93,000 200 the precursor of the ogists on the grounds of serious technical C3/C5 convertase inadequacies. Pillemer was deeply hurt by the scathing (C3b,Bb). Bb subunit of this complex is a criticism. He became distraught over the damage done serine protease. to his personal and professional reputation, and took D Factor D Serine protease that 24,000 1 his own life several months later. The tragedy activates factor B when B is in complex with deepened when subsequent work proved Pillemer’s C3b. original findings to have been correct. By 1967, H Factor H Regulator (negative) 150,000 560 properdin had been reinstated as an important factor in that inactivates the C3/C5 convertase by alternative pathway complement activation. dissociating its subunits. Also a cofactor for factor 1. Distinctive Differences Between the I Factor I Regulator (negative) 88,000 34 that is a serine protease. Classical and Alternative Pathways of Inactivates C3b with the Complement Activation aid of factor H or the C3b receptor (CR1). As we have seen, the classical pathway depends on P Properdin Regulator (positive) 224,000 20 activation by -specific antibodies reacting with that enhances activation by binding to and the four principal complement factors (C1, C2, C3, and stabilizing the C3/C5 C4). Table 1 (adapted from Pangburn’s chapter on the convertase. alternative pathway) provides numerous examples of *Reprinted with permission from Pangburn.1 pathogens and particles of microbial origin, as well as nonpathogens, which can activate the six protein pathways. Since none of these activators requires prior components of the alternative pathway without prior stimulation of antigen-specific antibodies, the immunization to their specific (Table 2, also alternative pathway offers immediate response to from Pangburn). Five of these proteins are unique to certain serious infections, to amelioration of acute the alternative pathway; only C3 is common to both symptoms of dangerous immune complex syndromes, and to amplification of available C3b to play its Table 1. Activators of the alternative pathway* amboceptor role when a rapid response is required. Pathogens and particles The exact mechanism by which the first metastable of microbial origin Nonpathogens molecules of C3 are produced is not yet absolutely Many strains of gram-negative Human IgG, IgA, and IgE in 1 bacteria complexes certain. According to Pangburn, the consensus is that Lipopolysaccharides from gram- Rabbit and guinea pig IgG in there is spontaneous hydrolysis of the thioester group negative bacteria complexes in native C3, thereby leading to C3b deposition not Many strains of gram-positive Cobra venom only on the target pathogen but also indiscriminately bacteria Heterologous erythrocytes (rabbit, on all particles,including the host’s own cells. A system Teichoic acid from gram-positive mouse, chicken) of control factors rapidly inactivates the C3b molecules cell walls Anionic polymers (dextran sulfate) bound to host cells or other nonactivators, but these Fungi and yeast cell walls Pure carbohydrates (agarose, (zymosan) control factors work very slowly on C3b bound to insulin) Some viruses and virus-infected particles recognized by the system as proper sites of cells activation. C3b on such activating particles forms an Some tumor cells (Raji) enzyme that can function like the C3/C5 convertase of Parasites (trypanosomes) the classical pathway. It activates C3 and deposits C3b *Reprinted with permission from Pangburn.1 molecules onto the surface being attacked. An

86 IMMUNOHEMATOLOGY, VOLUME 21, NUMBER 3, 2005 History of complement system amplification process now comes into play. Each new 16-page chapter on complement components cited C3b bound to this surface can form C3/C5 convertase 208 references, while a 39-page chapter on genetics that can deposit more C3b, and the process repeats and polymorphism of complement components cited itself until the surface is saturated or the supply of 380 references. complement component is depleted. By this Among other accomplishments during this era was mechanism the alternative pathway of complement identification of individual cells and tissues in which activation is capable of depositing more than two complement components were produced. They million C3b molecules onto the surface of a particle in proved to be more diverse than expected. While the less than 5 minutes after contact! main site of synthesis was the hepatocytes in the liver, The confluence of the classical and alternative another prolific site was the fimbriae of the Fallopian pathways is illustrated in Figure 1. This highly tubes. In vitro studies showed that a great many cells simplified diagram emphasized that once the two could make complement components, e.g., myeloid pathways reached the point where their respective C3 cells, macrophages, intestine, bladder, skin, lungs, and convertases provided C3b to the ongoing system, many others. progression through C5 to C9 activation proceeded on As understanding of the complement system a shared final pathway to completion. expanded during the explosion of new knowledge described in an earlier section,an increasing awareness New Technologies Provide Fundamental developed of the important benefits humanity reaps New Knowledge from the system as well as the potential for serious It is not within the province of this review to negative consequences if all, or parts, of the system describe details of the elegant exploitation of new break down or function out of control. Individuals technologies for protein purifications, recombinant born with deficiencies of various complement DNA technology, identification of genetic location, components may become chronic invalids, their life cloning of genes for various complement components, spans greatly shortened, vulnerable to fulminant transfection,and production of purified products, infections, and possibly more susceptible to certain use of knock-out animal models to verify functional malignancies. The complexity of the complement capacities of gene prod- system, its access to every tissue in the body via its ucts, and many more. Table 3. Research in the International complement workshops* Number of complement field acceler- published ated and diversified rapidly Workshop Date Location abstracts Reference during the second half of 1 February 28–March 1, 1963 National Institutes of Health, Summary (44) the 20th century. Table 3 Bethesda, MD only summarizes the Interna- 2 January 17–19, 1966 La Jolla, CA 30 (45) tional Complement Work- 3 June 3–5, 1968 Harvard Medical School, 30 (46) Boston, MA shops from 1963 to 1993. 4 January 27–29, 1971 Johns Hopkins University, 39 (47) The 1963 meeting at the Baltimore, MD National Institutes of 5 February 23–25, 1973 Hotel Del Coronado, 68 (48) Health in the United States Coronado, CA was attended by 25 6 November 23–25, 1975 Sarasota, FL 101 (49) scientists; 15 manuscripts 7 November 20–22, 1977 St. Petersburg Beach, FL 137 (50) were eventually published. 8 October 14–16, 1979 Key Biscayne, FL 140 (51) A meeting in Cambridge in 9 November 22–25, 1981 Key Biscayne, FL 177 (52) the United Kingdom in 10 May 25–27, 1983 Mainz, Germany 214 (53) 1991 had more than 320 11 November 3–5, 1985 Key Biscayne, FL 266 (54) attendees; 320 abstracts 12 September 18–21, 1987 Chamonix, France 320 (55) were published. In Rother 13 September 10–15, 1989 San Diego, CA 308 (56) and Till’s book, The 14 September 15–20, 1991 Cambridge, UK 320 (57) Complement System, pub- 15 September 27–October 1, 1993 Kyoto, Japan (planned) 4 lished in 1988, one *Reprinted with permission from Weiler (references in table refer to original paper).3

IMMUNOHEMATOLOGY, VOLUME 21, NUMBER 3, 2005 87 H. CHAPLIN JR. location within the circulatory system, and the Table 4. Granulocyte-related activities of human C5a* extraordinary speed with which it can react to Target cells Cellular responses perturbations require clinical investigators to proceed Neutrophils Augmented adherence with great caution in efforts to manipulate Monocytes Cellular polarization complement. Well-intentioned efforts to achieve Chemotactic migration desirable ends must be guided by a full understanding Degradation and enzyme release of undesirable consequences that could follow from Production of toxic oxygen metabolites upsetting the delicate balance that undergirds its vital Enhanced arachidonic acid metabolism roles on behalf of human survival. Augmented expression of complement receptors Increased elaboration of IL-1 by monocytoid cells Regulation of the Complement System *Reprinted with permission from Chenoweth.5 For every stage of activation of the classical lysis of the target cell via the formation of circular pathway there are regulatory proteins which bring “pores” varying from 20 to 100 Å in diameter. about rapid destruction of the activated factors, thereby preventing complete consumption of plasma C4 and C2 in the fluid phase. Rapid inactivation of C1 Complement Mediators of Inflammation— to prevent uncontrollable activation of complement is the Anaphylatoxins brought about by the nonenzymic protein, C1 Among the many protective roles played by the inhibitor, a heavily glycosylated 100,000-Da protein complement system is its mobilization of the body’s normally present in plasma at a concentration of 100 to inflammatory components in response to infections 150 µg/mL. C1 inhibitor combines with a catalytic site and to tissue damage from any cause. Key players in this on the chains of C1r and C1s, causing them to arena are the anaphylatoxins, low-molecular-weight dissociate from C1, leaving C1q bound to the target fragments derived from complement factors C3, C4, membrane. This sequence of events occurs very and C5. All of these polypeptides contribute to spasmo- rapidly (in 10–20 sec), thus limiting further C1 genicity, i.e., they promote smooth muscle contraction activation. Bound C42 (the C3/C5 convertase) is and increase vascular permeability. These biological eliminated in two phases. First, C2 dissociates properties have been shown to induce anaphylaxis in spontaneously from C4, a process which is promoted certain test animals. The biochemical compositions of by C4-binding protein and decay-accelerating factor these polypeptides are very similar, and one of the (DAF; CD55), which is found within the membranes of anaphylatoxins, C5a, plays a leading role in the host’s RBCs and many different cell types. In the second inflammatory response. Each of the anaphylatoxins is phase, residual bound C4b is further degraded into cleaved from the amino terminus of the alpha chain of C4c, which dissociates from the target membrane, its parent molecule by a specific convertase enzyme leaving only C4d attached to the target . formed during complement activation. The fragments This degradation is brought about by the enzyme are designated C3a, C4a, and C5a, and when released Factor I, aided by C4BP and the C3 receptor CR1. into the plasma, C3a and C4a are rapidly converted by Factor I also cleaves bound C3b. Once C3 is inactivated the enzyme serum carboxypeptidase to their inert it can no longer bind C5, hence further production of des-Arg analogs. C5a is largely catabolized after the membrane attack complex is prevented. This single internalization by its target cells. Note the complexity example of inhibitor-mediated regulation illustrates the of Figure 2 and compare its complexity with the intricacy of regulatory processes within the simplicity of Figure 1. The “lectin pathway” has been complement area. added to the classical and alternative pathways. Lectins At the time of activation of C5, the C5a are nonimmune carbohydrate-recognizing molecules anaphylatoxin fragment is released, with the effects on found in plants and animals. Two lectins of human neutrophils and monocytes shown in Table 4. origin are conglutinin and mannose-binding protein, Activated C5 attaches to the membrane of the target shown in Figure 2 as mannose-binding lectin (MBL). For cell, where it binds stoichiometrically to C6, serving as example, MBL can activate the complement system an acceptor for C7, leading finally to assembly of the when it binds to mannose receptors on certain bacterial completed membrane attack complex (MAC). The and virus surfaces. Yet even the pathway depicted in MAC inserts itself into the membrane, bringing about Figure 2 is simpler than reality because it does not

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include many other complement inhibitors and the receptors for many complement factors and byproducts. The biological activities of the anaphylatoxins are illustrated in Tables 4 and 5. It is clear from Table 5 that the spasmogenic properties (smooth muscle contrac- tion and vascular permeability) of C5a are far more potent than those of C3a and C4a. The same predominance of C5a over C3a and C4a was observed for the granulocyte- and monocyte-related activities listed in Table 4, justifying the reputation of C5a as a major anaphylatoxin inflammatory mediator. As admirable as C5a’s superiority appears in this general context, Chenoweth5 cautions that it must be rec- Fig.1. Activation pathways of the complement system—mid-20th century* ognized that C5a may also act systemically and trigger similar types of granulocytic responses in blood or *Reprinted with permission from Ross.2 distant organs. For example, when this occurs during complement-induced pulmonary vascular leukoseques- tration, C5a-activated granulocytes release cytotoxic substances that destroy normal tissues. In this case, and in certain autoimmune disorders, normal host defense mechanisms may actually contribute to the pathogenesis of a specific disease, such as adult respiratory distress syndrome or rheumatoid arthritis. C5a initiates a wide variety of responses (Table 4) via binding to receptors that are known to be located on target cells (peripheral blood neutrophils and monocytes, alveolar macrophages, and cultured cells of monocytoid origin). Characterization of these receptors utilized radio-iodinated (125IC5a) or fluorescein-labeled human C5a (fl-C5a). The C5a receptors of the previously described cells and the plasma membrane fraction isolated from these cells are functionally indistinguishable. At the time Chenoweth was writing, other investigators were attempting to define more precisely the “recognition domain” determinants of C5a because peptide or chemical analogs of the regions could possibly act as competitive antagonists to C5a binding and thus function as anti-inflammatory compounds. Work in progress included similarly directed studies of receptors for CR2, CR3, CR4, H-R, C1q-R, C3a/C4a-R, and CR3e-R. Also under way were similar studies of opsonic IgG, opsonic C3, opsonic C4, and fibronectin. Other studies were being directed at CR1 membrane complement receptors on human, sheep, rabbit, mouse, rat, and guinea pig RBCs. Identification and Fig. 2. Activation pathways of the complement system—late-20th characterization of complement receptors on century* neutrophils, monocytes, lymphocytes, Kupfer cells, *Reprinted with permission from Sahu et al.6 phagocytes, and platelets goes on apace.

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Table 5. Spasmogenic properties of the human anaphylatoxins* system and hence one of the protectors of Biological Effective dose (M) autologous cells and tissues from complement- response C5a des-Arg74-C5a C3a C4a mediated damage. Antibodies to Cromer system Smooth muscle contraction antigens could lead to RBC destruction, but Ileum 5.0 10–10 1.2 10–6 1 10–8 1.5 10–6 hemolytic transfusion reactions and HDN have × –10 × × –8 × Uterus 7.5 × 10 1 × 10 not been reported. However, it is necessary to Vascular identify antibodies in the Cromer system during permeability Guinea pig 0.1–1.0 10–13 1.0 10–10 0.01–1.0 10–10 0.01–1.0 10–8 pretransfusion testing. × –13 × × –10 × Human 0.1–1.0 × 10 0.01–1.0 × 10 One can expect a major expansion of * Reprinted with permission from Chenoweth.5 collaborative research in the overlapping areas of complement physiology and transfusion medicine. Relation of Complement Components to Blood Group Serology The Role of Complement in Immune Not to be outdone by the flourishing worldwide Destruction of Transfused RBCs and in community of blood group specialists, comple- Autoimmune Hemolytic Anemias mentologists became card-carrying blood group Thus far, this review has focused on the role of specialists themselves with the announcement in 1978 complement in protection against infectious diseases. 7 by O’Neill et al. that the Chido/Rodgers blood group However, it has also shown that there is a downside to antigens were in fact distinct antigenic components of the complement story. human complement C4. The subject is well chronicled For many decades, the human RBC has been well in Blood Transfusion in Clinical Medicine by known as a victim of complement activity, as 8 Mollison, Engelfriet, and Contreras. Since the antigens exemplified by complement’s noxious contribution to are generally detectable in normal human plasma, it is hemolytic transfusion reactions and its frequent role in assumed that their presence on RBC membranes autoimmune hemolytic anemias (AIHAs). In the reflects adsorption from the individual’s plasma. The diagnosis and management of both of these life- Ch and Rg determinants are localized to threatening illnesses, it has challenged the skills of 6. Anti-Ch and anti-Rg are found only in Ch- or Rg- laboratory technicians, manufacturers of specific negative subjects. RBCs of the relatively rare C4 null antiglobulin reagents, and molecular biology patients are Ch- and Rg-negative. Fortunately, researchers. Complement can be activated by naturally incompatibility within the Ch/Rg system has not occurring antibodies (e.g., anti-A and anti-B in normal caused documented hemolytic transfusion reactions or group O, A, and B blood donors) as well as by blood HDN, although there have been rare reports of group alloantibodies formed in response to multiple anaphylactic symptoms in multi-transfused Ch- or Rg- transfusions or pregnancies (e.g.,antibodies to antigens negative recipients of platelet concentrates or plasma within the Kidd, Duffy, and Kell systems). In these transfusions. circumstances, blood group alloantibodies that activate As a more contemporary example of the complement can provoke more serious hemolytic complement system’s encroachment on the blood reactions, sometimes with rapid intravascular RBC bank serologist’s turf, elucidation of the clinical role of destruction and accompanying hemoglobinuria. the decay-accelerating factor (DAF;CD55) glycoprotein Complement can play an analogous role in many associated with the Cromer blood group system has AIHAs (e.g., the biphasic anti-P Donath-Landsteiner made important progress in recent years. Like the autohemolysin in paroxysmal cold hemoglobinuria). Ch/Rg antigens, the Cromer antigens are widely Complement’s role is supported by the frequent detectable on multiple human cells: RBCs, WBCs, finding of strong reactivity with anti-C3d reagents platelets,and endothelial and epithelial tissues. Lublin’s accompanying strong reactivity with anti-IgG reagents group9 at Washington University in St. Louis has on the RBCs of some patients with severe AIHAs. recently completed a detailed definition of the The extensive literature on the role of complement molecular basis of nine Cromer antigens. The Cromer in immune destruction of human RBCs is well reviewed in Blood Transfusion in Clinical Medicine system antigens have been shown to reside on the DAF 8 protein, one of the regulators of the complement by Mollison, Engelfriet, and Contreras.

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The Role of Complement in the Rheumatic paroxysmal nocturnal hemoglobinuria [PNH]). New Diseases understanding has given rise to imaginative comple- As noted earlier, the “normal” functions of the ment research designed to improve the lives of complement system may sometimes become a double- individuals suffering from these ailments. As with any edged sword. Autoantibodies and circulating immune innovative development at the start, enthusiastic complexes in certain rheumatic diseases may activate predictions and high expectations abound. While the complement system, thereby enhancing comple- unanticipated difficulties may delay or even derail ment’s “good” pro-inflammatory response but leading some of these efforts, some important goals have been to further tissue destruction. The same undesired achieved. effect may lead to deposition of immune complexes in PNH is an example of a highly successful laboratory the walls of blood vessels (vasculitis), resulting in and clinical research effort. A recent article in the New necrosis of vessels. When immune complex vasculitis England Journal of Medicine13 describes a study based occurs in the kidneys, lungs, skin, or other organs, in Leeds, England, on eleven transfusion-dependent patients may become seriously ill. adult patients (six men and five women) with well- Therapeutic complement inhibitor approaches documented PNH, a disorder arising as a result of a have been considered for treatment of bullous somatic mutation of the PIG-A gene in a hematopoietic pemphigus, rejection of transplanted tissues, stem cell. The mutation results in the subsequent Alzheimer’s disease (because plaques contain high production of blood cells with a deficiency of the DAF levels of classical and alternative pathway components proteins that protect normal RBCs against attack by the as well as MAC components), immune-based fetal complement system. In a well-designed, well- loss, HIV, and a great many other serious medical standardized, and well-executed 12-week clinical and conditions. Other disorders that interface importantly laboratory study,the effects of intravenous infusions of with the complement system (including systemic eculizumab, a humanized that inhibits the lupus erythematosus, rheumatoid and related activation of terminal complement components C5 to arthritides,and cryoglobulinemia) are well reviewed by C9, were examined. If effective, the drug would be Atkinson et al.10 expected to reduce RBC hemolysis sufficiently to Looking ahead, careful attention is being given to eliminate the hemoglobinuria, reduce or eliminate the important questions that need further exploration. transfusion dependency, and relieve troublesome Where in the three activation pathways (classical, symptoms related to the chronic hemolysis. Well- alternative, lectin) should the various inhibitors be chosen laboratory tests to quantitate the rate of applied: prior to or coincident with the shared C3b hemolysis were performed at regular intervals, as was step? Can local,systemic,or both therapeutic strategies quantitation of the numbers of abnormal RBCs in the be predictably and safely controlled? Should research circulation and visual daily quantitation of the urine for emphasis focus on recombinant or nonrecombinant detectable hemoglobinuria. The standardized inter- agents? What important proteins outside the view of the European Organization for Research and complement system (e.g., clotting factors) could also Treatment of Cancer (EORTC), titled the QLQ-30, was be inhibited unintentionally? How would the employed periodically to evaluate quality of life. effectiveness of complement inhibitors stack up Patient compliance was excellent. No evidence of against other pro-inflammatory measures? There are formation of antibodies to the eculizumab was found, several major concerns regarding all of the above and no untoward drug-related side effects were issues: what would be the impact of each with respect detected. Laboratory results documented significant 11 to increased risks of autoimmunization, and how reduction in the rate of hemolysis (though not total 12 might each alter the self-tolerance of B cells ? normalization, and the Hb level did not rise). Paroxysms of hemoglobinuria were markedly The Role of Complement in Paroxysmal decreased. Regarding quality of life, there was Nocturnal Hemoglobinuria significant improvement in the domains of global The rapid broadening of understanding of the health (p = 0.02), physical functioning (p < 0.001), complement system has promoted improved under- emotional functioning (p < 0.001), cognitive standing of the pathogenesis of many diseases, both functioning (p = 0.002),fatigue (p < 0.001), dyspnea (p common (e.g., rheumatoid arthritis) and rare (e.g., = 0.002), and insomnia (p = 0.049).

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All the patients enlisted in a 12-month extension of compound could be useful in a human transfusion the study, with no ill effects noted and with sustained setting, The third publication, “Characterization of improvement in their symptoms and laboratory complement receptor-1 domains for prevention of studies. The authors conclude: “Eculizumab appears to complement-mediated red cell destruction,”16 was enhance the survival of type III PNH erythrocytes, published in 2005. This paper, building on the findings improving the quality of life and reducing the extent of of the two preceding papers, chronicles further the hemolysis, hemoglobinuria (the clinical hallmark of search for even smaller soluble CR1 inhibitors. A PNH), and the need for blood transfusions in patients 250–amino acid region in CR1 that possessed with PNH. This study confirms that terminal antihemolytic activity and was effective at prolonging complement activation is the key mediator of survival of transfused RBCs was identified. Mutation of erythrocyte destruction in PNH.” two critical residues in this 250–amino acid domain The author of the present review strongly encour- resulted in improved complement-inhibitory functions ages readers to enjoy this exemplary publication for its compared to the unmutated CR1 derivatives, resulting lucidity and inspirational value. in a more potent inhibition of complement activation in vitro. However, in vivo, the activity of the mutant The Emerging Role of Complement protein was comparable to the wild type molecules. Inhibitors in Immunohematology These studies indicated the importance of testing CR1 inhibitors in vivo. The PNH article cited in the previous section Taken together, the three careful and sophisticated describes primarily a clinical trial of a candidate drug studies indicate the increased precision achievable to add to the therapeutic armamentarium of trans- when using today’s scientific technology to explore fusion medicine. As a fitting finale to the present structure/function relationships in evaluation of historical review of the complement system, I would complement inhibitors. like to summarize briefly an ongoing “work in The Index Medicus lists more than 40,000 progress” at the New York Blood Center by a research publications under “complement”in the past 39 years. group under the direction of Karina Yazdanbakhsh. Complement system research continues to grow. The titles of three recent publications from her Enjoy it! laboratory track the program’s progress during the past 3 to 4 years. The title of the first paper, published in 2003, is “Complement receptor 1 inhibitors for Acknowledgment prevention of immune-mediated red cell destruction: The author expresses special appreciation to potential use in transfusion therapy.”14 The paper colleagues Prof. John P. Atkinson and Sir Peter J. describes preparation of recombinant human CR1 Lachmann for their wise advice and welcome from which several truncated soluble CR1 proteins encouragement. were expressed and tested for their complement inhibitory properties. Two of the derivatives were References selected for in vivo inhibition of complement-mediated 1. Pangburn MK. The alternative pathway. In: Ross immune hemolysis in a xenotransfusion mouse model GD, ed. Immunobiology of the complement and for lowering the deposition of C3 and C4 on the system. New York: Academic Press, Inc., 1986: target RBCs. The second paper, published in 2004 and 46-7, 57. titled “Prevention of complement-mediated immune 2. Ross GD. Introduction. In: Ross GD, ed. hemolysis by a small-molecule compound,”15 describes Immunobiology of the complement system. New the screening of a commercial 10,000 compound York:Academic Press, Inc., 1986:13. library enriched in anti-inflammatory molecules, from 3. Weiler JM. Introduction. In: Whaley K, Loos M, which a single 549-Da compound was selected on the Weiler JM.Complement in health and disease. 2nd basis of its ability to reduce hemolysis induced via the ed. Dordrecht: Kluwer Academic Publishers, classical and alternative pathways. In a xenotrans- 1993:5. fusion mouse model, significantly prolonged RBC 4. Rother K, Till GO. The complement system. survival was observed, as was reduced C3 deposition Berlin: Springer-Verlag, 1988:61, 119. on the surviving RBCs. The findings support the 5. Chenoweth DE. Complement mediators of possibility that the small-molecule complement inflammation. In: Ross GD, ed. Immunobiology of

92 IMMUNOHEMATOLOGY, VOLUME 21, NUMBER 3, 2005 History of complement system

the complement system. New York: Academic 12. Prodeus AP,Georg S, Shen L-M, et al.A critical role Press, Inc., 1986:72-3, 75. for complement in maintenance of self-tolerance. 6. Sahu A, Morikis D, Lambris JD. Complement Immunity 1998;9:721-31. inhibitors targeting C3,C4,and C5. In:Lambris JD, 13. Hillmen P, Hall C, Marsh JCW, et al. Effect of Holers VM. Therapeutic interventions in the eculizumab on hemolysis and transfusion complement system. Totowa, NJ: Humana Press, requirements in patients with paroxysmal 2000:76. nocturnal hemoglobinuria. N Engl J Med 7. O’Neill GJ, Yang SY, Tegoli J, et al. Chido and 2004;350:552-9. Rodgers blood groups are distinctive antigenic 14. Yazdanbakhsh K, Kang S, Tamasauskas D, et al. components of human complement C4. Nature Complement receptor 1 inhibitors for prevention 1978;273:668-70. of immune-mediated red cell destruction: 8. Mollison PL, Engelfriet CP, Contreras M. Blood potential use in transfusion therapy. Blood transfusion in clinical medicine. 10th ed. Oxford: 2003;101:5046-52. Blackwell Science, 1997:205. 15. Mqadmi A, Zheng X, Song J, et al. Prevention of 9. Lublin DM, Kompelli S, Storry JR, et al. Molecular complement-mediated immune hemolysis by a basis of Cromer blood group antigens. small molecule compound. Biochem Biophys Res Transfusion 2000;40:208-13. Commun 2004;325:1465-71. 10. Atkinson JP, Kaine JL, Holers VM, et al. 16. Mqadmi A, Abdulla Y, Yazdanbahksh K. Complement and the rheumatic diseases. In: Ross Characterization of complement receptor 1 GD, ed. Immunobiology of the complement domains for prevention of complement-mediated system. New York:Academic Press, Inc., 1986:197- red cell destruction.Transfusion 2005;45:234-44. 211. 11. Boackle SA,Holers VM.Role of complement in the Hugh Chaplin Jr. MD, Emeritus Professor of Medicine development of autoimmunity. In:Nemazee D,ed. and Pathology, Washington University School of B cell biology in autoimmunity: current directions Medicine, St. Louis, Missouri. in autoimmunity. Basel: Karger, 2003;6:154-68.

IMMUNOHEMATOLOGY, VOLUME 21, NUMBER 3, 2005 93 On a much higher than reported incidence of anti-c in R1R1 patients with anti-E

W. J. J UDD, L.R. DAKE,AND R.D. DAVENPORT

A previous study involving tube IATs, untreated RBCs, and a low- recipients developed anti-c. Since their report, gel ionic-strength additive reagent revealed that approximately one- technology has emerged and has been found to have third of R1R1 patients with anti-E have a concomitant anti-c. However, the current study finds a much higher incidence of anti-c good sensitivity for Rh antibodies. Further, we have in such patients, using gel technology in conjunction with ficin- shown that tests performed with anti-IgG gel cards and pretreated RBCs. Results of antibody identification studies and ficin-pretreated RBCs are exquisitely sensitive for Rh transfusion records of 82 R1R1 patients with anti-E were reviewed. 3 Serologic test methods included a LISS wash solution for tube IATs antibodies. (15 min at 37°C, anti-IgG), ficin-tube IATs (30 min at 37°C, anti-IgG In this report we present data from ficin-gel tests + anti-C3), and gel IATs (untreated or ficin-treated RBCs or both, that demonstrate a higher than previously published anti-IgG gels). LISS-tube or gel IATs with untreated RBCs revealed incidence of anti-c in R R patients with anti-E. These anti-c in 32 patients with anti-E. When gel-IAT and ficin-pretreated 1 1 RBCs were used, 21 additional patients with anti-E were found to data support the selection of c– RBCs for this patient have anti-c. In samples from 26 R1R1 patients with anti-E, anti-c was population. not demonstrable by ficin-gel IATs, and in 3 cases, the ficin-gel tests were inconclusive. In five cases in which E– RBCs not tested for c antigen were transfused to patients found by ficin-gel IAT to be Materials and Methods without anti-c, all subsequently performed crossmatches with E–, c- ID-MTS gel technology was from Ortho-Clinical untested RBCs were compatible. The incidence of anti-c in R1R1 patients with anti-E in this study was 32 of 82 (39%) with untreated Diagnostics (Raritan,NJ). Reagent RBCs,both untreated RBCs and 53 of 82 (65%) when the ficin gel data were included. and ficin-treated, and LISS (Löw and Messeter4 The latter is significantly higher than the 32 percent incidence formulation) were from ImmucorGamma, Norcross, previously reported (p = 0.0001). Accordingly, all patients at our Georgia. For gel testing,5 untreated reagent RBCs were facility with an Rh antibody are now tested for those additional Rh antibodies they can make, as predicted from their Rh phenotype. prepared in ID-MTS Diluent 2 at a concentration of 0.8%

The data from this study strongly support the selection of R1R1 and tested on anti-IgG cards according to the RBCs for all c– patients with anti-E. Immunohematology manufacturer’s product circular. Gel tests with ficin- 2005;21:94–96. pretreated RBCs were similarly performed on anti-IgG Key Words: anti-c, anti-E cards; such testing (previously validated by us3) conflicts with the manufacturer’s product circular, The immune response to RhCE protein is variable. which stipulates the use of buffered gel cards. LISS-tube 6 For example, R1R1 (c–, E–) individuals may make anti-c, tests were incubated at 37°C for 15 minutes, washed anti-E, anti-c plus anti-E, or anti-cE (Rh 27), the latter four times with saline, and tested with anti-IgG (Ortho). reacting with a determinant encoded by c and E within Ficin-tube tests6 were incubated at 37°C for 30 minutes the same haplotype (in cis). Some individuals may and tested with polyspecific (anti-IgG + C3) anti- make all three antibodies, as demonstrated by globulin reagent (Ortho). Negative tube tests were adsorption-elution studies,1 or a combination of anti-c validated with IgG-coated RBCs (ImmucorGamma). plus anti-E. Serologic and transfusion records of 82 R1R1 2 Shirey et al. found the incidence of anti-c in R1R1 patients with anti-E were reviewed. No further testing patients with anti-E to be 32 percent when performing was performed on 32 patients with concomitant anti-c IATs by a low-ionic-strength additive technique. They by routine testing (LISS-tube and ficin-tube). Ficin-gel also showed if the RBCs selected for transfusion were tests were used to detect the presence or absence of E– but not tested for c antigen, 18.5 percent of anti-c in samples from the remaining 50 patients.

94 IMMUNOHEMATOLOGY, VOLUME 21, NUMBER 3, 2005 Incidence of anti-c in R1R1 patients with anti-E

Results

We found anti-c in 53 out of 82 (65%) R1R1 patients with anti-E (Table 1). In 32 cases, the presence of anti- c was evident from the results of LISS-tube or gel IATs. In the other 21 cases (see Fig. 1 for an example), the A presence of anti-c was clearly demonstrable only in ficin-gel IATs. However, among these 21 cases, weak reactivity was also seen in two cases with some R R R R R R r r rr rr untreated c+, E– RBCs in gel IATs and suspected from 1 1 1 1 2 2 ″ IgG IgG IgG IgG IgG IgG the results of ficin-tube tests in five cases. There were three additional cases in which the presence of anti-c Untreated RBCsAnti-E PK NO: 44-B REV.DATE 02-02-98 could not be determined due to panreactivity in ficin- gel tests.

Of the 26 R1R1 patients with anti-E that did not have anti-c clearly demonstrable by ficin gel, 18 did not require transfusion (five were pregnant,and there were B records of previous transfusions on six). A further eight patients were transfused with E– RBCs that were not tested for c antigen. Three of these patients were lost to follow-up. We had the opportunity to test the R R R R R R r r rr rr 1 1 1 1 2 2 ″ five remaining patients for anti-c in subsequent IgG IgG IgG IgG IgG IgG antiglobulin crossmatches with E– donor units that had not been selected to be c–; in fact, two patients were Ficin-Treated RBCsAnti-c+E PK NO: 44-B REV.DATE 02-02-98 transfused with Rh– RBCs that were undoubtedly c+. Fig. 1. A. Anti-IgG gel tests with untreated RBCs reveal presence of These crossmatches were performed between 3 and 20 anti-E. B. Tests with ficin-pretreated RBCs reveal presence of months after anti-E was initially detected in the concomitant anti-c. patients’ plasma. All units (n = 10) were crossmatch compatible. Table 1. Incidence of anti-c in 82 R1R1 patients with anti-E LISS tube or gel Ficin gel Number of patients Discussion anti-E + anti-c NT* 32 (39%) The development of alloantibodies to RBC antigens anti-E anti-E + anti-c 21 (26%) through transfusion or pregnancy is not benign. anti-E anti-E 26 (32%) Patients who become alloimmunized are at risk of anti-E inconclusive 3 (3%) hemolytic transfusion reactions and high-risk *NT = not tested pregnancies associated with HDN.7,8 Further, alloimmunization is sometimes accompanied by they found the incidence to be 32 percent. Among the autoantibody formation, which may lead to 68 R1R1 patients with anti-E alone, 27 were transfused autoimmune hemolytic anemia.9 These risks prompted with E– RBCs that were not typed for c antigen; five some investigators to recommend the use of (18.5%) of these patients subsequently formed anti-c. phenotypically matched RBCs, especially for sickle cell Given our past experiences with gel technology,14 anemia patients.10 As shown recently,11,12 this which is exquisitely sensitive for Rh antibodies, we recommendation is by no means universally followed. expected to find a somewhat higher incidence of anti-

In the 11th edition of the AABB Technical c in R1R1 patients with anti-E than was reported by 13 2 Manual, it was suggested that R1R1 transfusion Shirey et al. However, we did not expect to find a candidates who have made anti-E should be transfused twofold increase (32% vs. 65%; p = 0.0001) through the with R1R1 RBCs to prevent formation of anti-c, possible use of gel technology and ficin-treated RBCs, given that posttransfusion hemolysis, and autoantibody forma- before implementation of gel technology in our tion. This suggestion prompted Shirey and colleagues2 laboratory our routine antibody identification protocol to determine the incidence of anti-c in 100 R1R1 included ficin-tube IATs. We postulate that these anti-c patients with anti-E. In their study,using LISS-tube IATs, antibodies are, for the most part, low-affinity antibodies

IMMUNOHEMATOLOGY, VOLUME 21, NUMBER 3, 2005 95 W. J. J UDD ET AL.

that dissociate during the washing phase of tube IATs; present, we crossmatch R2R2 donor RBCs. We have a gel technology is, of course, a no-wash IAT. similar policy for Ro patients with anti-C or anti-E, Although we saw no evidence of anti-c inasmuch as we select C–, E– RBCs for crossmatching development in five R1R1 patients with anti-E, as only if ficin-gel tests reveal that both anti-C and anti-E demonstrated by compatible IAT crossmatches with are present (Table 2). E–, c-untested RBCs, we now select c– blood for R1R1 patients with anti-E, regardless of whether or not anti-c is Table 2. Policy for ficin-gel testing and blood selection in D+ patients with Rh alloantibodies detected. However, due to the compar- Patient’s RBCs If* ◆ Then† Or, if* ◆ Then† ◆ ative rarity of R2R2 donors, we do not R1R1 anti-E issue c–, E– RBCs automatically select R R RBCs for ◆ ◆ 2 2 R2R2 anti-C exclude anti-e anti-e issue C–, e– RBCs patients with anti-C who are e–. Rather, ◆ ◆ R0 anti-C exclude anti-E anti-E exclude anti-C we test for anti-e by ficin gel, if it is not *Antibody found in serum with untreated RBCs. evident by routine studies, and, if it is †Exclusion tests performed with ficin-treated RBCs by gel technology.

References 10. Rosse WF, Gallagher D, Kinney TR, et al. 1. Issitt PD, ed. Serology and genetics of Rh. Transfusion and alloimmunization in sickle cell Cincinnati, OH: Montgomery Scientific Publi- disease. The Cooperative Study of Sickle Cell cations, 1979. Disease. Blood 1990;76:1431-7. 2. Shirey RS, Edwards RE, Ness PM. The risk of 11. Affenyi-Annan A, Brecher ME. Pre-transfusion alloimmunization to c (Rh4) in R1R1 patients who phenotype matching for sickle cell disease present with anti-E.Transfusion 1994;34:756-8. patients (letter).Transfusion 2004;44:619-20. 3. Judd WJ,Dake LR.Enzyme tests using anti-IgG gels. 12. Osby M, Shulman I. Phenotype matching of donor Vox Sang 2002;83(S2):154. units for nonalloimmunized sickle 4. Löw B, Messeter L. Antiglobulin test in low-ionic cell disease patients: a survey of 1182 North strength salt solution for rapid antibody screening American laboratories. Arch Pathol Lab Med and crossmatching.Vox Sang 1974;26:53-61. 2005;129:190-3. 5. Lapierre Y,Rigal D,Adam J,et al.The gel test:a new 13. Walker Rh, ed. Technical manual. 11th ed. way to detect antigen-antibody reactions. Bethesda, MD: American Association of Blood Transfusion 1990;30:109-13. Banks, 1993. 6. Judd WJ. Methods in immunohematology. 2nd ed. 14. Judd WJ, Steiner EA, Knafl PC, Masters C. The gel Durham, NC: Montgomery Scientific Publications, test: use in the identification of unexpected 1994. antibodies to blood group antigens. Immuno- 7. Ness PM, Shirey RS, Thoman SK, Buck SA. The 1998;14:59-62. differentiation of delayed serologic and delayed hemolytic transfusion reactions: incidence, long- W. John Judd, FIBMS, MIBiol; Louann R. Dake, MA, term serologic findings, and clinical significance. MT(ASCP)SBB, and Robertson D. Davenport, MD, Transfusion 1990;30:688-93. Department of Pathology, UH-2G332, University of 8. Garratty G, ed. Hemolytic disease of the newborn. Michigan Medical Center, 1500 East Medical Center Arlington, VA: American Association of Blood Drive, Ann Arbor, MI 48109-0054. Banks, 1984. 9. Young PP, Uzieblo A, Trulock E, Lublin DM, Goodnough LT. Autoantibody formation after alloimmunization: are blood transfusions a risk factor for autoimmune hemolytic anemia? Transfusion 2004;44:67-72.

96 IMMUNOHEMATOLOGY, VOLUME 21, NUMBER 3, 2005 Case report: massive postpartum transfusion of Jr(a+) red cells in the presence of anti-Jra

S.YUAN,R.ARMOUR,A.REID, K.F.ABDEL-RAHMAN,M.PHILLIPS,D.M.RUMSEY,AND T. N ESTER

Jra is a high-prevalence antigen. The rare Jr(a–) individuals can form and the decreasing Hct is usually accompanied by anti-Jra after exposure to the Jra antigen through transfusion or 7 a transiently increased antibody titers. More recently, pregnancy. The clinical significance of anti-Jr is not well 6 a established. This study reports a case of a 31-year-old woman with Kwon et al. reported a case of anti-Jr causing acute a previously identified anti-Jra who required massive transfusion of hemolysis. In this report, we describe the case of a RBCs after developing life-threatening postpartum disseminated patient with anti-Jra who developed postpartum intravascular coagulopathy. Despite the emergent transfusion of 15 units of Jra untested RBCs,she did not develop laboratory or clinical disseminated intravascular coagulopathy (DIC) and evidence of acute hemolysis. The patient’s anti-Jra had a received 15 units of incompatible Jr(a+) RBCs. pretransfusion titer of 4 and a monocyte monolayer assay (MMA) Subsequently she had no overt clinical signs of reactivity of 68.5% (reactivity > 5% is considered capable of hemolysis, and laboratory findings suggested only mild shortening the survival of incompatible RBCs). The titer increased fourfold to 64 and the MMA reactivity was 72.5% on Day 10 delayed hemolysis. posttransfusion. Review of laboratory data showed evidence of a mild delayed hemolytic transfusion reaction by Day 10 posttransfusion. Despite rare reports of hemolytic transfusion Case Report reactions due to anti-Jra in the literature, most cases, including this A 31-year-old multiparous Vietnamese woman with one, report that this antibody is clinically insignificant or causes history of three previously uncomplicated pregnancies only mild delayed hemolysis. Clinicians should be advised to balance the risks of withholding transfusion with the small chance (with a different partner from the current pregnancy) of significant hemolysis after transfusion of Jr(a+) RBCs in the presented for prenatal care at an outside facility in the a presence of anti-Jr . Immunohematology 2005;21:97–101. 32nd week of her fourth pregnancy. ABO/D typing Key Words: anti-Jra, massive transfusion, hemolytic showed the patient’s RBCs to be Group A, D+. The transfusion reaction antibody screen test showed that the patient’s serum reacted weakly with all reagent RBCs in the Jra is a high-prevalence antigen in all populations. antiglobulin phase of testing. RBC treatment using The Japanese population has the highest frequency of either DTT or ficin had no effect on reactivity. The the Jr(a–) phenotype. However, even in that autologous control and DAT were negative; thus an population,the incidence is reported to be only 0.03 to antibody directed at a high-prevalence antigen was 0.12 percent.1 Because of its rarity, the clinical suspected. The patient’s serum was then tested with significance of anti-Jra is not well established. Reid and several RBC samples lacking various high-prevalence Lomas-Francis2 describe anti-Jra as being capable of antigens, including k, Kpb, and Ch, and RBCs with very causing decreased RBC survival. In the obstetric weak U expression. All RBCs tested reacted with the setting, the authors note that although the DAT can be patient’s serum. The specimen was subsequently positive, anti-Jra is not associated with HDN. referred to our laboratory, where phenotyping of the Jr(a+) RBCs have been transfused to individuals patient’s RBCs for high-prevalence antigens showed with anti-Jra without incident.3–5 On the other hand, that her RBCs possessed Vel,Dib,k,and Kpb antigens but there exist in the literature a dozen or so reports lacked Jra antigens. Jr(a–) RBCs from two different highlighting the ability of anti-Jra to cause both mild to donors located through Serum Cells and Rare Fluids moderate HDN and delayed hemolytic transfusion Exchange (SCARF) were not agglutinated by the reactions.6 The typical hemolytic reaction associated patient’s serum, which was consistent with the with anti-Jra is delayed, extravascular, and self-limited, presence of anti-Jra. Allogeneic adsorptions were

IMMUNOHEMATOLOGY, VOLUME 21, NUMBER 3, 2005 97 S.YUAN ET AL. performed with W.A.R.M-treated RBCs (W.A.R.M.; were performed using reagent RBCs (Immucor, Inc.). Immucor Inc., Norcross, GA) that were also Antibody identification was performed using panels of phenotypically matched for Rh and Kidd antigens. No reagent RBCs (Ortho-Clinical Diagnostics, Inc., Raritan, additional alloantibodies were detected in the serum. NJ, and Immucor, Inc.). Panels of ficin-treated RBCs The anti-Jra was shown to have a titer of 4. Because of (Immucor, Inc.) were also used. DTT-treated RBCs the difficulty in locating Jr(a–) RBCs,and the possibility were prepared using 0.2 M solution (DTT, Sigma- of shortened survival of transfused Jr(a+) RBCs, we Aldrich Corp., St Louis, MO). Alloadsorption was recommended autologous donation or screening family performed using W.A.R.M.-treated RBCs (W.A.R.M.; members in the hope of finding a compatible donor if Immucor, Inc.), which were also phenotypically there was a reasonable concern that the patient or the matched for Rh and Kidd antigens. baby might need transfusion during the pregnancy and Polyspecific and monospecific DATs were delivery. performed (Bioclone Anti-IgG, Bioclone Anti-C3d, and The patient went into labor during the 38th week Bioclone AHG, Ortho-Clinical Diagnostics) by standard of her pregnancy and vaginally delivered a healthy male tube method. The elution was performed using the infant without evidence of HDN. Her postpartum rapid acid method (Elukit II, Gamma Biologicals, Inc., course was complicated by development of DIC and Houston,TX). Two different enhancement media (PEG, profuse, life-threatening vaginal bleeding, which Gamma Biologicals, and O.A.E.S., Ortho-Clinical necessitated an emergent abdominal hysterectomy. Diagnostics) were used to enhance antibody Intraoperatively, she received four units of uncross- agglutination. Antibody titer was determined with matched Group O,D– RBCs and 11 units of crossmatch- serial dilutions of patient’s serum using IAT. incompatible Group A, D+ RBCs. Because of the high Patient’s RBCs were phenotyped using incidence of Jra, all of the RBC units were presumably conventional methods for the common antigens. Rare Jr(a+). Her hemorrhage was eventually brought under antisera and reagent RBCs were prepared in-house or control after the surgery. Clinically, there was no provided through SCARF. Monocyte monolayer assay evidence of acute intravascular hemolysis during the (MMA) was performed by the American Red Cross resuscitation effort. Blood Services of Southern California Region, Los Approximately 15 days after the initial surgery, the Angeles,California. The method was described in detail patient was found to have developed abdominal elsewhere.8 abscesses. Because surgery with possible significant blood loss was anticipated, and because of the case Results 6 report from Kwon et al. reporting acute hemolysis Anti-Jra was not detectable in the serum after repeated exposures to Jr(a+) RBCs, two frozen immediately after the transfusion of the 15 units of units of Jr(a–) RBCs were imported from Blood Bank of uncrossmatched (presumably Jr[a+]) RBCs. This was Hawaii. Of note,the only other possible source of units not surprising because of the large volume of crystalloid of Jr(a–) RBCs at that time was from Japan. However, solutions and blood components administered during these units would not have been immediately available the resuscitation. The DAT using polyspecific anti- and would not have been fully tested for transfusion- human globulin at this time was weakly positive (1+), transmissible diseases according to FDA guidelines. but the patient’s RBCs were negative in the DAT using Initial attempts at computerized tomography–guided anti-C3d or anti-IgG. An eluate was performed and drainage of the abscesses were complicated by tested and found to contain anti-Jra. Allogeneic perforation of the colon. This necessitated a laparo- absorption of the eluate, using W.A.R.M-treated RBCs tomy for hemicolectomy and abscess drainage. Both that were also phenotypically matched for Rh and Kidd, units of Jr(a–) RBCs were transfused intraoperatively did not show any additional alloantibodies. without incident. The patient did not require further Because of the concern for a significant delayed transfusions. Twelve days after the second surgery, her hemolytic transfusion reaction, we recommended that Hct was stable at 36.8%. the clinicians give intravenous fluids as necessary to maintain adequate renal perfusion and monitor Materials and Methods laboratory values, including Hct, bilirubin, LDH, and Antibody detection and identification were haptoglobin. Additionally, a sample was sent daily to performed by tube method. Antibody detection tests our reference laboratory for DATs and evaluation for

98 IMMUNOHEMATOLOGY, VOLUME 21, NUMBER 3, 2005 Massive transfusion in presence of anti-Jra hemolysis. The patient’s Hct decreased from 38.6% on from a pretransfusion titer of 4 to 64. Concurrently,the Day 5 posttransfusion to 30.9% on Day 10. Of note, reactivity of the polyspecific DAT increased from 1+ to during this period the patient also developed 2+, and both the monospecific DATs (using anti-IgG syndrome of inappropriate antidiuretic hormone and anti-C3) reacted 2+ at 6 days posttransfusion. Both secretion and hyponatremia. The resulting fluid the prenatal pretransfusion serum sample and a serum retention and dilutional anemia contributed to the sample obtained on Day 10 posttransfusion were sent decreasing Hct and made it unlikely that it was solely for MMA testing, which showed 68.5% and 72.5% due to hemolysis. On Day 10 posttransfusion, the total reactivity respectively (reactivity > 5% is considered bilirubin increased from 0.4 mg/dL at the time of transfusion to 1.6 mg/dL (normal range 0.2–1.0 mg/dL) capable of shortening the survival of incompatible and LDH increased from 307 to 431 IU/L (normal range RBCs). However, visual check of serial serum samples 91–180 IU/L). Haptoglobin levels were obtained only showed no evidence of hemolysis, and the patient on Days 4 and 5 posttransfusion;both were well within continued to have no overt clinical evidence of normal range. Anti-Jra became detectable in the serum intravascular hemolysis during the posttransfusion again on Day 6 posttransfusion and the titer had risen period. Select laboratory values are shown in Table 1.

Table 1. Results of select pertinent laboratory tests pretransfusion and in the days after massive transfusion of 15 units of presumably Jr(a+) RBCs in the presence of anti-Jra. Day Hct Na LDH Total bilirubin Anti-Jra (posttransfusion) (%) (135–145 mmol/L)* (91–180 IU/L)* (0.2–1.0 mg/dL)* DAT titer Other lab values –30 – – – – Negative 4 MMA reactivity: (pretransfusion) 68.5% (≤ 5%)* 0 24 131 307 0.4 – – Prothrombin time = 36.5, International normalized ratio = 3.53, 50.7 Partial prothrombin time =138.8 7.4 Fibrinogen = 72 mg/dL D-dimer > 10,000 ng/mL 9 Platelet = 13 × 10 /L 1 33.2 139 – 0.9 Polyspecific: 1w – – anti-IgG: 0 anti-C3: 0 4 33.4 138 261 – – – Haptoglobin 121 (43–212 mg/dL)* 5 38.6 125 333 – Polyspecific: 2+ Haptoglobin 101 118 anti-IgG: 2+ (43–212 mg/dl)* 123 anti-C3: weak – Urine bilirubin negative 6† 38.2 123 391 – Polyspecific: 2+ 64 – anti-IgG: 2+ anti-C3: 2+ 8† 33.6 132 386 2.2 Polyspecific: 1+ 64 – Direct 0.5 anti-IgG: 1+ anti-C3: 1+ 10† 30.9 132 431 1.6 Polyspecific: 2+mf 64 MMA reactivity: anti-IgG: 1+mf 72.5% (≤ 5%)* anti-C3: 1w mf 15‡ 28.8 133 – – Polyspecific: 2+ – – anti-IgG: 1+ anti-C3: 2+ 17 25.2 132 372 0.7 – – Haptoglobin 201 Direct 0.1 (43–212 mg/dl)* (0.0–0.2 mg/dL)* Urine bilirubin negative 20 30.0 128 – 0.6 – – – 27 36.8 134 – – – – – *Normal range values †Results from Days 6 to 10 show evidence of onset of mild delayed hemolysis. Visual checks of serum showed no evidence of hemolysis through this period. ‡On Day 15, patient underwent abdominal surgery and received two units of Jr(a–) RBCs intraoperatively.

IMMUNOHEMATOLOGY, VOLUME 21, NUMBER 3, 2005 99 S.YUAN ET AL.

Discussion flow cytometry showed normal RBC survival compared Because of the rarity of anti-Jra, its clinical with the calculated expected RBC survival rate. significance is not clear. Several reports of anti-Jra Clinically the patient did not have signs of hemolysis. causing mild to moderate HDN9,10 exist in the literature, Clinical information on the other 11 patients is but others have questioned the validity of the unavailable. In our case, despite the strong MMA diagnoses in such cases.11 Mild to moderate delayed reactivity both before and after the transfusion of a total hemolytic transfusion reactions9,12,13 have also been of 15 units of incompatible RBCs, we found no clinical reported in association with anti-Jra. More recently, evidence of hemolysis and only laboratory evidence of Kwon et al.6 reported a concerning case of acute very mild, well-tolerated, delayed hemolysis. The hemolytic transfusion reaction due to this antibody in decrease in Hct was at least in part due to dilutional the setting of repeated exposures to the antigen within anemia and postoperative surgical blood loss. a week. On the other hand, others have reported no It is likely that most transfusions of Jr(a+) RBCs will clinical evidence of hemolysis after incompatible not result in significant acute or delayed hemolysis in transfusions, even in the face of rising antibody titers.4 the presence of anti-Jra. However, because anti-Jra can Studies using monocyte phagocytosis14 and MMA8,15 cause significant acute hemolysis at least in rare cases suggested that most anti-Jra are not clinically after repeated exposures to Jr(a+) RBCs6, it is prudent significant. The lack of clinical significance in most to make a reasonable attempt to locate Jr(a–) RBC units cases could be in part accounted for by the low antigen when anti-Jra is identified,especially in those cases with density of Jra on the RBC membrane.14,16 strongly positive MMA results. If blood loss can be Additional data are needed to fully establish the anticipated, as in the case of an elective surgery or clinical significance of anti-Jra. The MMA has been delivery, autologous donations should be encouraged. proposed as a valuable tool for predicting the clinical It may also be necessary to look into family members significance of antibodies to high-prevalence antigens and rare donor registries as potential sources of such as anti-Jra.8,15 A recent retrospective review of 20 compatible units because of the extreme scarcity of the years of MMA data by Arndt and Garratty8 showed that Jr(a–) phenotype in North America. It is important to only results greater than 5% are potentially clinically weigh the availability of Jr(a–) RBCs against the cost significant. Furthermore, one-third of patients with and the infectious disease risks when importing such MMA results between 5.1% and 20% had clinical signs of units from countries outside the United States or older hemolysis (defined as jaundice, fever, chills, change in units frozen before the availability of the most current blood pressure, back pain, vomiting, tachypnea, and infectious disease testing. In our opinion, the risks of hemoglobinuria8), and two-thirds of patients with MMA acquiring an infectious disease appear to outweigh the results greater than 20% reactivity had such symptoms. risks of transfusing Jr(a+) units, and clinicians should When clinical signs of hemolysis were absent, patients be guided in this way. Additionally,clinicians should be from both groups often had laboratory indications of advised of the available serologic results, which might hemolysis, which included decreased Hb, Hct, and include antibody titer, DAT reactivity, and MMA results. haptoglobin, increased bilirubin or LDH, hemo- The limitations of these data and the lack of conclusive globinemia, or the presence of bilirubin in the urine. In information in the current literature regarding the the same review,only 5 out of 14 (36%) cases of anti-Jra clinical significance of anti-Jra must be made clear as examined showed MMA reactivity greater than 5%, two well. The clinical decision to transfuse Jr(a+) RBCs and of which had reactivity greater than 20%. But the the risks of hemolysis after transfusion of incompatible positive predictive value of the MMA result for RBCs must also be evaluated against the risks of hemolysis is not established specifically for anti-Jra withholding transfusion. The recent guidelines of the because of the small number of cases and the lack of UK National Blood Service17 recommended transfusing associated clinical information. Through a personal serologically least-incompatible RBCs to patients with communication with these authors, we learned that anti-Jra and antigen-negative RBCs to patients with clinical information was available on 4 of the 15 cases. strong anti-Jra when transfusion is necessary. We feel Two of these cases had MMA results greater than 20%. this approach is reasonable given the rarity of Jr(a–) Of these, one experienced a transfusion reaction with RBC units, the lack of data we have about anti-Jra, and the infusion of a third unit of Jr(a+) RBCs. This is the the lack of laboratory assays that can reliably predict its same case reported by Kwon et al.6 In the other case, clinical significance.

100 IMMUNOHEMATOLOGY, VOLUME 21, NUMBER 3, 2005 Massive transfusion in presence of anti-Jra

Acknowledgments 11. Toy P,Reid M,Lewis T,Ellisor S,Avoy DR.Does anti- We thank Dr. George Garratty and Patricia Arndt of Jra cause hemolytic disease of the newborn? Vox the American Red Cross, Southern California Region, in Sang 1981;41(1):40-4. Los Angeles, California, for their performance of the 12. Kendall AG. Clinical importance of the rare MMA, technical expertise, and willingness to openly erythrocyte antibody anti-Jra. Transfusion share knowledge gained. 1976;16:646-7. 13. Jowitt S, Powell H, Shwe KH, Love EM.Transfusion a References reaction due to anti-Jr (abstract). Transfus Med 1994;4(suppl 1):49. 1. Nakajima H, Ito K. An example of anti-Jra causing 14. Ogasawara K, Mazuda T. Characterization of Jra hemolytic disease of the newborn and frequency antibodies by monocyte phagocytosis assays and of Jra antigen in the Japanese population.Vox Sang flow cytometry analysis. Acta Haematol Jpn (in 1978;35:265-7. Jap) 1990;53:1131-7. 2. Reid ME, Lomas-Francis C. Blood group antigen 15. Garratty G,Arndt P,Nance S.The potential clinical factsbook. 2nd ed. San Diego: Academic Press, significance of blood group alloantibodies to high 2003. frequency antigens (abstract). Blood 1997; 3. Azar PM,Kitagawa H,Fukunishi A,et al.Uneventful 10(suppl 1):473a. transfusion of Jr (a+) red cells in the presence of 16. Miyazaki T, Kwon K,Yamamoto K, et al. A human anti-Jra. Jpn J Transfus Med 1988;34:406-10. monoclonal antibody to high-frequency red cell 4. Bacon J, Sherrin D,Wright RG. Case report, anti-Jra. antigen Jra.Vox Sang 1994;66:51-4. Transfusion 1986;26:543-4. 17. National Blood Service.Guidance for the selection 5. Schanfield MS, Stevens JO, Bauman D. The of blood for patients with common and rare red detection of clinically significant erythrocyte cell antibodies.Blood Matters 2003;13.Available at alloantibodies, using a human mononuclear http://www.blood.co.uk/hospitals/library/bm/iss phagocyte assay.Transfusion 1981;21:571-6. ue13/BM1303.htm. Posted August 13, 2003. 6. Kwon MY,Su L, Arndt PA, Garratty G, Blackall DP. a Clinical significance of anti-Jr : report of two cases Shan Yuan, MD, Transfusion Medicine Fellow, and review of the literature.Transfusion 2004;44: Department of Laboratory Medicine, University of 197-201. Washington and Puget Sound Blood Center, Seattle, 7. Yoshida H, Yurugi K, Ito K. A case of delayed WA; Rosalind Armour, BA, BB(ASCP); and Allison a hemolytic transfusion reaction due to anti-Jr . Jpn Reid, MT(ASCP)SBB, Senior Technologist, Red Cell J Tranfus Med 1991;34:528-30. Reference Laboratory, Puget Sound Blood Center, 8. Arndt PA, Garratty G. A retrospective analysis of Seattle, WA; Khaled F. Abdel-Rahman, MD, Attending the value of monocyte monolayer assay results for Physician, Auburn Regional Medical Center, Auburn, predicting the clinical significance of blood group WA; Michael Phillips, MT(ASCP)SBB, Supervisor, Red alloantibodies.Transfusion 2004;44:1273-81. Cell Reference Laboratory; Dawn M. Rumsey, 9. Takabayashi T, Murakami M, Yajima H, Tsujiei M, ART(CSLT) Director, Transfusion Service; and Theresa Ozawa N,Yajima A.Influence of maternal antibody Nester, MD, Assistant Medical Director, Puget Sound a anti-Jr on the baby: a case report and pedigree Blood Center, Seattle, WA, and Assistant Professor, chart.Tohoku J Exp Med 1985 Jan;145(1):97-101. Department of Laboratory Medicine, University of a 10. Orrick LR, Golde SH. Jr mediated hemolytic Washington, Seattle, WA 98104. disease of the newborn infant. Am J Obstet Gynecol 1980;137:135-6.

IMMUNOHEMATOLOGY, VOLUME 21, NUMBER 3, 2005 101 Is there a relationship between anti-HPA-1a concentration and severity of neonatal alloimmune thrombocytopenia?

H. BESSOS,M.TURNER,AND S. URBANIAK

There is uncertainty about the relationship between anti-HPA-1a fetal platelets, which leads to NAIT in about 20 percent levels and severity of neonatal alloimmune thrombocytopenia of these cases.1–3 This can lead to internal hemorrhage, (NAIT). To investigate this relationship further,the concentration of anti-HPA-1a in HPA-1b homozygous women was determined, using of which the most serious form is intracranial a newly developed quantitative ELISA that uses purified anti-HPA-1a hemorrhage (ICH), which can cause the death or to obtain a standard curve. Seventy-eight samples collected from 22 lifelong morbidity of the baby.4 As NAIT is caused by HPA-1b homozygous pregnant women at various stages of pregnancy were tested. These included five women who had the passage of IgG across the placenta, it is reasonable delivered babies with severe NAIT. A national HPA-1a antibody to assume that a relationship would exist between the standard (NIBSC 93/710), designated as 1 arbitrary unit/mL isotype of anti-HPA-1a, its concentration, or both and (AU/mL), was used in each ELISA to calibrate the purified anti-HPA- the severity of the disease, as has been observed for 1a, enabling the presentation of results as AU/mL. Moreover, 5 selected samples were also assayed by PAK 12 and their reactivity HDN. However, to date, evidence of the relationship compared with quantity of antibody. The use of the purified HPA- between HPA-1 antibody titer and the severity of NAIT 1a antibody yielded consistent sigmoid curves, enabling the has been inconclusive. Most studies have shown that measurement of HPA-1a antibody concentration in the test samples. 2,6–12 The antibody concentration was significantly correlated with the no such relationship exists, while a few studies antibody titer in the 78 samples studied (R = 0.54, p < 0.001). have shown a notable or significant correlation Furthermore, there was a significant correlation between PAK 12 between the HPA-1a antibody isotype or concentration and the quantitative ELISA in a selected number of cases, with or 13–16 without NAIT (R = 0.71,n = 10;p < 0.02). On the other hand,there and the severity of NAIT. was no correlation of antibody concentration with NAIT incidence In a recent nationwide study in Scotland involving (R = –0.046). This study indicates that there is no relationship 26,506 pregnant women, 8.8 percent (28/327) of between anti-HPA-1a concentration and severity of NAIT when confirmed HPA-1b homozygous women developed ELISA is used, although the correlation between ELISA and other methods, such as monoclonal antibody immobilization of platelet HPA-1a antibody. Of those, eight delivered babies with antigens (MAIPA) assay, remains to be determined. NAIT,which in five of them was severe (platelet count Immunohematology 2005;21:102–108. 9 17 < 50 × 10 /L). Antibody titers during pregnancy were 18 Key Words: platelets, anti-HPA-1a, neonatal monitored by a direct ELISA, and no correlation alloimmune thrombocytopenia (NAIT) between antibody titer and severity of NAIT was observed. Neonatal alloimmune thrombocytopenia (NAIT) The previously mentioned discrepancy in the occurs when the pregnant woman and her fetus differ relationship between antibody levels and severity of in their platelet glycoprotein (GP) polymorphisms and NAIT could be due to the analytic methods used, the woman develops an antibody to the fetal platelet because these are, in general, qualitative or semi- antigens. In the Caucasian population, most severe quantitative. For example, in ELISA methods, patient cases of NAIT occur when the woman and fetus differ samples are incubated with purified GPIIb/IIIa coated in their HPA-1 phenotype.1–3 About 10 percent of HPA- onto the well,10,18 while in other assays, patient samples 1b homozygous women who are pregnant with a fetus are incubated with intact platelets.19,20 In the expressing HPA-1a antigens develop antibodies to the monoclonal antibody immobilization of platelet

102 IMMUNOHEMATOLOGY, VOLUME 21, NUMBER 3, 2005 Anti-HPA-1a concentration and severity of NAIT antigens (MAIPA) assay,platelets are incubated with the be measured (see Results section). To obtain universal patient’s antibodies before they are washed and results,the purified anti-HPA-1a was calibrated against a incubated with monoclonal antibodies directed against national minimum-potency anti-HPA-1a standard various GPs. The GP, bound with the patient’s antibody (NIBSC 93/710) designated as 1 arbitrary unit per mL and respective monoclonal antibody, is then extracted (AU/mL). and tested further to determine the specificity of the patient’s antibody.20 Recently we developed an ELISA Test samples that enables quantitative measurement of antibody in As previously indicated, 28 of 327 HPA-1b women patients’ serum.21 The aim of this study was to use this who consented to our nationwide study were found to assay to confirm whether there is a relationship have anti-HPA-1a. Of these women, eight delivered between the concentration of anti-HPA-1a and severity babies with NAIT, five of whom had severe of NAIT in the cohort of pregnant women with anti- thrombocytopenia. At the end of the latter study, HPA-1a in our study.The correlation between the direct samples from 22 women were additionally tested for ELISA titer and the quantitative assay was also antibody, using the quantitative ELISA; this study determined. included samples from all five women who had Materials and Methods ELISA

Using immobilized HPA-1a, anti-HPA-1a was ) m n purified from serum containing a high titer (256) of 0 4 5 this antibody; the purified anti-HPA-1 was used to ( e c n generate a standard curve in the ELISA as previously a b 21 r o

described. The quantitative antibody ELISA consisted s b a

of the following steps, all of which were performed at c i f 21,22 i room temperature. Seven serial doubling dilutions c e p of the standard (purified) anti-HPA-1a and up to three S serial dilutions of the test samples were incubated in HPA-1a–coated wells for 30 minutes. Controls consisting of the same constituents were incubated in uncoated wells. After washing, the wells were Reciprocal of anti-HPA-1a dilution incubated with alkaline-phosphatase conjugated anti- Fig. 1. Calibration of the standard anti-HPA-1a in each assay using NIBSC human IgG for 30 minutes. The wells were again 93/710 and determination of the anti-HPA-1a concentration in the test samples from the calibrated standard (AU/mL).A standard washed and incubated with substrate for 30 minutes. curve is shown with its associated trendline and corresponding Stop solution was added and absorbances were equation. In each ELISA, NIBSC 93/710 anti-HPA-1a (at 1 AU/mL) obtained using an ELISA reader (MRXTC; Dynex was included at a dilution of 1 in 4 (i.e.,0.25 AU/mL).In this assay, the latter gave a specific absorbance of 0.23 on the y-axis. Using Technologies, Chantilly, VA). Specific absorbance was the formula y = –0.1128 ln(x) + 0.7511, we could calculate the obtained by subtracting the absorbance in uncoated corresponding reciprocal dilution of S on the x-axis as follows (where S = the amount of anti-HPA-1a in the standard in AU/mL): wells (i.e., without antigen) from that in wells coated 0.1128 ln(x) = 0.7511 – 0.23 = 0.5211 with antigen. Normal serum was not included in the ln(x) = 0.5211/0.1128 = 4.62 test because a study of 94 normal antenatal samples x = 100 (calculated through EXP on Microsoft Excel math/trig), had yielded a mean absorbance plus two SD of less This indicates that S at 1 in 100 = 0.25 AU/ml, implying that S = than 0.1, indicating no cross-reactivity of normal 25 AU/mL 23 With S as 25 AU/mL in this assay, we can then calculate the samples with coated GPIIb/IIIa. All ELISA buffers and amount of anti-HPA-1a in test samples. For example, if a test reagents were obtained from Axis-Shield plc, Dundee, sample at a dilution of 1 in 16 gives a specific absorbance value on the y-axis of 0.25, the corresponding anti-HPA-1a Scotland. The purified anti-HPA-1a was used in the concentration in that sample is calculated as follows: ELISA at a starting dilution of 1 in 4 (added as one part 0.1128 ln(x) = 0.7511 – 0.25 = 0.5011 anti-HPA-1a and 3 parts buffer) in buffer containing ln(x) = 0.5011/0.1128 = 4.44 x = 84.8, indicating that anti-HPA-1a in the test sample at a 25% of normal pool plasma, with serial doubling dilution of 1 in 16 = 25/84.8 = 0.29 AU/mL;implying that the neat dilutions to 1 in 256. This gave a sigmoid curve against concentration of anti-HPA-1a in the test sample = 16 × 0.29 = 4.72 the linear section of which test plasma samples could AU/mL.

IMMUNOHEMATOLOGY, VOLUME 21, NUMBER 3, 2005 103 H. BESSOS ET AL. delivered babies with severe NAIT. Eight consecutive ELISAs were performed to measure 78 samples collected from the 22 women at various stages of pregnancy. In addition,10 selected samples (at delivery or postdelivery) were tested using PAK 12 (GTI, r e t

Waukesha, WI) to determine whether the latter i t f o

correlated in any way with the ELISA. It is worth l a c noting that PAK 12 also incorporates GPIIb/IIIa-coated o r p i wells, although the GP is attached to the wells via a c e monoclonal antibody and not directly as in our ELISA. R

Statistics The correlation between the titer or absorbance in the PAK 12 and the antibody concentration was calculated using the Pearson product moment correlation coefficient. The significance in the AU/mL difference of antibody quantity between low and high Fig. 2. The correlation between anti-HPA-1a titer and concentration. Comparison of the antibody titer (obtained in a previous study titer samples was determined using the Wilcoxon t test. using a direct ELISA) with the antibody concentration obtained using the quantitative ELISA in this study showed a significant correlation between the two assays (p < 0.001). Results Consistency of the anti-HPA-1a standard 21 As before, consistent sigmoid curves were L m / obtained by the purified anti-HPA-1a standard in the U A -

ELISA (data not shown). In each assay, the NIBSC A S I L

93/710 anti-HPA-1a (designated 1 AU/mL) was assayed E e v at a 1 in 4 dilution and the amount of antibody in the i t a t i purified anti-HPA-1a standard was calculated as shown t n a in Figure 1. This yielded an average value of 25 AU/mL u Q for the purified anti-HPA-1a. Such calibration of the purified anti-HPA-1a in each ELISA enabled measurement of the test samples in standard quantitative units (AU/mL). GTI ELISA - Absorbance (450 nm)

Fig. 3. The correlation between PAK 12 and the quantitative ELISA. Ten Amount of antibody in the test samples selected samples, including three severe NAIT cases (arrows), Seventy-eight samples obtained from various stages were tested using the PAK 12 and quantitative ELISA. A significant correlation was obtained between the levels obtained of pregnancy of 22 women with anti-HPA-1a were by both assays. assayed. Thirty-four weakly reactive antibody samples of titers less than or equal to 2 gave a mean antibody Indeed, as shown in Figure 4, antibody concentration quantity of 0.58 AU/mL (± 0.42 SD). In contrast, 16 barely rose above 1 AU/mL during pregnancy in three strongly reactive antibody samples with titers between of the five severe NAIT cases (charts B, D, and E; 32 and 256 gave a significantly higher mean antibody respective platelet counts are shown in the figure concentration of 4.27 (± 3.44) AU/mL, p < 0.0005. legend); in contrast, in two cases of normal delivery Overall, the antibody concentration was significantly platelet counts, appreciably higher concentrations of 4 correlated with the titer in the 78 samples studied (R = to 10 AU/mL were observed in the third trimester 0.54, p < 0.001) (Fig. 2). Furthermore, there was a (charts H and I). Antibody concentrations of between significant correlation between the results of PAK 12 1 and 6 AU/mL were observed during pregnancy in the and the quantitative ELISA (Fig. 3). However, no remaining two severe NAIT cases (charts A and C). The relationship was found between antibody selected cases (10/22) in Figure 4 highlight the concentration and severity of NAIT (R = –0.046). variability of the relationship between antibody

104 IMMUNOHEMATOLOGY, VOLUME 21, NUMBER 3, 2005 Anti-HPA-1a concentration and severity of NAIT L L m m / / U U A A

Week Week

C L L m m / / U U A A

Week Week L L m m / / U U A A

Week Week L L m m / / U U A A

Week Week L L m m / / U U A A

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Fig. 4. Antibody concentration in 10 representative women with anti-HPA-1a during pregnancy. 9 D = delivery, PN = postnatal. All platelet counts at delivery (× 10 /L) A to E: Severe NAIT.Respective platelet counts: 21, 13, 14, 8, and 42. F: Mild NAIT.Platelet count: 87. G to J: No NAIT.The concentration of antibody during pregnancy did not appear to be indicative of the severity of NAIT.

IMMUNOHEMATOLOGY, VOLUME 21, NUMBER 3, 2005 105 H. BESSOS ET AL. concentration and severity of NAIT. All five women using HPA-1a homozygous platelets and not ELISA who gave birth to babies with severe NAIT were HLA- plates coated with HPA-1a GPIIb/IIIa GP. Likewise, in DR3*01 positive (data not shown). The thrombocyto- two other recent studies where MAIPA assay was used, penia resolved without treatment in one of the babies, a relationship was found between antibody titer and while those of the other four women received one or quantification and severity of NAIT.14–15 In the study in two transfusions with HPA-1b homozygous platelets East Anglia, England, by Williamson et al.,14 a significant before their platelet counts returned to normal.17 correlation between severity of NAIT and a third trimester anti-HPA-1a titer of less than 32 was found. Discussion Moreover, in a smaller study by Jaegtvik et al.15 in In this study, the relationship between anti-HPA-1a Norway, an obvious relationship between antibody concentration and severity of NAIT was assessed using level at the time of delivery and the severity of NAIT a newly developed quantitative ELISA. During our was observed. Nevertheless, two women with barely nationwide Scottish study we did not find a correlation detectable antibodies delivered babies with severe between antibody titer and severity of NAIT. Our aim, NAIT. In addition, one woman with high antibody therefore,was to determine whether such a correlation levels delivered a baby with mild NAIT (cord platelet 9 would be observed upon accurate quantitation of count of around 125 × 10 /L),but this was attributed to antibody in samples obtained from the same cohort. the protective effect of anti-D that was also present in 16 Seventy-eight samples collected from 22 antibody- her serum. In a recent study by Maslanka et al., where positive women during pregnancy were tested. A 144 of 8013 pregnant women were found to be HPA-1b significant correlation was observed between antibody homozygous, 12 of 122 of the HPA-1b women titer and concentration (i.e.,between the direct ELISA18 produced anti-HPA-1a (as determined by MAIPA assay); and the quantitative ELISA,21 respectively). Based on two of the babies had severe antenatal thrombocyto- this correlation, it was not surprising that no penia and two had mild NAIT. Due to the small number relationship was observed in the current study of cases, no definitive conclusion could be drawn between antibody concentration and severity of NAIT, regarding the relationship of antibody titer to severity since the latter was not related to antibody titer either. of disease. However, the four thrombocytopenic cases Our findings are in keeping with those of many occurred in mothers who had antibody titers of 4 or other studies where investigators have failed to find higher, while no thrombocytopenia was observed in such a relationship.6–12 In a study by Kaplan et al.,8 one those mothers who had weakly reactive anti-HPA-1a in five mothers who delivered thrombocytopenic detectable only after delivery. babies did not have detectable antibodies. A similar Although antibodies to other human platelet observation was made by Bussel et al.9 in a study of glycoprotein polymorphisms, such as HPA-5b and HPA- seven pregnant women who had previously delivered 4b, may have mechanisms of action that differ from infants with severe NAIT. Two of these seven women, those of anti-HPA-1a, it is worth noting that discrep- whose fetuses developed thrombocytopenia during ancies have also been noted with such antibodies in the early phase of their subsequent pregnancy, had no relation to severity of NAIT.24–26 While a study by Kurz detectable antibody. However, based on their previous et al.24 did not find HPA-5b antibody titer to be a history, IVIG was infused into these women in an predictor of disease, a study by Ohto et al.25 found attempt to raise the platelet count of the fetuses before significant association between postnatal platelet delivery. Using an ELISA similar to ours, Proulx et al.10 concentration (Day 3) and anti-HPA-5b titer in the third analyzed the sera from 36 mothers who gave birth to trimester of greater than or equal to 64. In addition, no thrombocytopenic babies. Their results indicated that relationship between anti-HPA-4b and severity of NAIT neither the titer nor the isotype of the antibody could was observed in a large Japanese study.26 (Antibodies predict the severity of NAIT. In contrast, Mawas et al.13 in the latter two studies were determined by means of found a significant increase of anti-HPA-1a of the IgG3 a mixed passive hemagglutination assay.) immunoglobulin class in women who delivered It is clear that there are no consistent results severely thrombocytopenic babies, although the levels illuminating the relationship of anti-HPA-1a concen- of IgG1, IgG2, and IgG4 (and overall IgG levels) tration and severity of NAIT. The discrepancy between remained the same between the two groups. However, the various studies could be due to the use of different the method they used was a modified MAIPA assay assays. It appears that all those who have used ELISA

106 IMMUNOHEMATOLOGY, VOLUME 21, NUMBER 3, 2005 Anti-HPA-1a concentration and severity of NAIT incorporating GP-coated wells have failed to find a References correlation between anti-HPA-1a levels and severity of 1. Mueller-Eckhardt C, Kiefel V,Grubert A, et al. 348 NAIT, whereas all those who have found such a cases of suspected neonatal alloimmune relationship have used a MAIPA assay. However, this is thrombocytopenia. Lancet 1989;1:363-6. not to say that the use of a MAIPA assay always yields 2. Blanchette VS, Chen I, de Friedberg ZS, et al. such a correlation.7,10,11 In this study we calibrated the Alloimmunization to the PLA1 platelet antigen: purified anti-HPA-1a standard in each assay against results of a prospective study. Br J Haematol NIBSC 93/710,designated as 1 AU/mL. This enabled the 1990;74:209-15. quantitation of anti-HPA-1a in all of our samples as 3. Bussel JB, Zabusky MR, Berkowitz RL, McFarland AU/mL. Three of five women who had delivered babies JG. Fetal alloimmune thrombocytopenia. N Engl J with severe NAIT exhibited consistently low Med 1997;337:22-6. concentrations of anti-HPA-1a throughout their 4. Bussel J, Kaplan C. The fetal and neonatal pregnancies, barely exceeding 1 AU/mL. Although the consequences of maternal alloimmune thrombo- remaining two women in this group exhibited higher cytopenia. Baillieres Clin Haematol 1998;11:391- antibody concentrations, the amounts interestingly 408. declined in the second trimester, rising again in the 5. Voak D, Mitchell R, Bowell P,et al. Guidelines for third trimester and postdelivery. In contrast, two blood grouping and red cell antibody testing women with high, albeit fluctuating, antibody during pregnancy.Transfus Med 1996;6:71-4. concentrations from 4 to 10 AU/mL during pregnancy 6. von dem Borne AE, van Leeuwen EF,von Riesz LE, delivered babies with normal platelet counts. Our et al. Neonatal alloimmune thrombocytopenia: results, therefore, indicate that the antibody detection and characterization of the responsible quantification during pregnancy is not a reliable antibodies by the platelet immunoflourescence predictor of severity of NAIT. However, an inverse test. Blood 1981;57:649-56. relationship between antibody levels and platelet 7. McFarland JG, Frenzke M, Aster RH. Testing of counts in utero cannot be ruled out, nor can the effect maternal sera in pregnancies at risk for neonatal of variation of maternal blood volume in our cohort of alloimmune thrombocytopenia.Transfusion 1988; pregnant women; it is conceivable that some of those 29:128-33. with low antibody levels could have had larger blood 8. Kaplan C,Daffos F,Forestier F,et al.Management of volumes than those with high antibody levels. alloimmune thrombocytopenia: antenatal diag- Based on the apparent discrepancy between ELISA- nosis and in utero transfusion of maternal based test systems and MAIPA assays,it would be useful platelets. Blood 1988;72:340-3. to carry out a comparison between MAIPA assay and 9. Bussel JB, Berkowitz RL, McFarland JG, et al. GPIIb/IIIa-coated quantitative ELISAs using the same Antenatal treatment of neonatal alloimmune samples. In the meantime, in the absence of a thrombocytopenia. N Eng J Med 1988;319:1374-8. consensus on the role of anti-HPA-1a concentration in 10. Proulx C, Filion M, Goldman M, et al. Analysis of NAIT, the clinical outcome (antenatal ICH), the degree immunoglobulin class, IgG subclass and titre of 9 of thrombocytopenia (platelet count < 20 × 10 /L) in HPA-1a antibodies in alloimmunized mothers previously affected pregnancies, or both appear to be giving birth to babies with or without neonatal the predictors of the likely severity of fetomaternal alloimmune thrombocytopenia. Br J Haematol alloimmune thrombocytopenia, though only in 1994;87:813-7. 27 subsequently affected pregnancies. 11. Panzer S, Auerbach L, Cechova E, et al. Maternal alloimmunization against fetal platelet antigens: a Acknowledgments prospective study. Br J Haematol 1995;90:655-60. We would like to thank Robert Munks and Sheryl 12. Durand-Zaleski I, Schlegel N, Blum Boisgard C, et Qader (Sheffield National Blood Service, England) for al. Screening primiparous women and newborns providing us with generous amounts of the high-titer for fetal/neonatal alloimmune thrombocytopenia: HPA-1a antibody from which we purified the anti-HPA- a prospective comaprison of effectiveness and 1a for the ELISA standard curve. We would also like to costs.Am J Perinatol 1996;13:423-31. thank Axis-Shield plc (Dundee, Scotland) for providing 13. Mawas F, Wiener E, Williamson LM, et al. us with ELISA reagents. Immunogolobulin G subclass of anti-human

IMMUNOHEMATOLOGY, VOLUME 21, NUMBER 3, 2005 107 H. BESSOS ET AL.

platelet antigen 1a in maternal sera:relation to the newly developed whole-blood ELISA for severity of neonatal alloimmune thrombocyto- multicentre platelet HPA-1 phenotyping.Vox Sang penia. Br J Haematol 1997;59:287-92. 1999;77:103-6. 14. Williamson LM, Hackett G, Rennie J, et al. The 23. Sukati H, Bessos H, Barker RN, Urbaniak SJ. natural history of fetomaternal alloimmunization Characterization of the alloreactive helper T-cell to the platelet specific antigen HPA-1a (PLA1, response to the platelet membrane glycprotein Zwa) as determined by antenatal screening. Blood IIIa (integrin beta3) in human platelet antigen-1a 1998;92:2280-7. alloimmunized human platelet antigen-1b1b 15. Jaegtvik S, Husebekk A, Aune B, et al. Neonatal women.Transfusion 2005;45:1165-77. alloimmune thrombocytopenia due to anti-HPA 1a 24. Kurz M, Stockelle E, Eichelberger B, Panzer S. IgG antibodies; the level of maternal antibodies titer, subclass, and light chain phenotype of predicts the severity of thrombocytopenia in the pregnancy-induced HPA-5b antibodies that cause newborn. BJOG 2000;107:691-4. or do not cause neonatal alloimmune thrombo- 16. Maslanka K, Guz K, Zupanska B. Antenatal cytopenia.Transfusion 1999;39:379-82. screening of unselected pregnant women for HPA- 25. Ohto H,Yamaguchi T,Takeuchi C, et al. Anti-HPA- 1a antigen, antibody, and alloimmune thrombo- 5b-induced neonatal alloimmune thrombocyto- cytopenia.Vox Sang 2003;85:326-7. penia: antibody titre as a predictor. Br J Haematol 17. Turner M, Bessos H, Fagge T, et al. Prospective 2000;110:223-7. epidemiological study of the outcome and cost 26. Ohto H, Miura S,Ariga H, et al.The natural history effectiveness of antenatal screening to detect of maternal immunization against fetal platelet neonatal alloimmune thrombocytopenia (NAIT) alloantigens.Transfus Med 2004;14:399-408. due to anti-HPA-1a.Transfusion. In Press. 27. Birchall JE, Murphy MF,Kaplan C, et al. European 18. Bessos H, Goldschmeding R, von dem Borne A, et collaborative study of the antenatal management al. The development of a simple and quick of feto-maternal alloimmune thrombocytopenia. enzyme-linked immunosorbent assay for anti-HPA- Br J Haematol 2003;122:275-88. 1a (PLA1) antibodies. Thromb Res 1993;69:395- 400. Hagop Bessos, PhD, Immunohaematology R&D 19. Porcelijn L, von dem Borne AE. Immune-mediated Group, Scottish National Blood Transfusion Service, thrombocytopenias: basic and immunological 21 Ellen’s Glen Rd, Edinburgh, Scotland EH17 7QT; aspects. Baillieres Clin Haematol 1998:331–41. Marc L. Turner, PhD, FRCP, FRCPath, Cell Therapy 20. Kiefel V,Santoso S,Weisheit M,Mueller-Eckhardt C. R&D Group, Scottish National Blood Transfusion Monoclonal antibody-specific immobilization of Service, 21 Ellen’s Glen Rd, Edinburgh, Scotland platelet antigens (MAIPA): a new tool for the EH17 7QT and Oncology Department, The University identification of platelet-reactive antibodies.Blood of Edinburgh, Edinburgh, Scotland; and Stanislaw J. 1987;70:1722-6. Urbaniak, PhD, FRCP, FRCPath, Immuno- 21. Bessos H, Perez S, Armstrong-Fisher S, et al. The haematology R&D Group, Scottish National Blood development of a quantitative ELISA for Transfusion Service, 21 Ellen’s Glen Rd, Edinburgh, antibodies against human platelet antigen type 1a. Scotland EH17 7QT and Academic Transfusion Transfusion 2003;43:350-6. Medicine Unit, The University of Aberdeen, Scotland. 22. Bessos H, Hofner M, Salamat A, et al. An international trial demonstrates suitability of a

108 IMMUNOHEMATOLOGY, VOLUME 21, NUMBER 3, 2005 Review: complement receptor 1 therapeutics for prevention of immune hemolysis

K.YAZDANBAKHSH

The complement system plays a crucial role in fighting infections biological activities, it has to be activated. Activation and is an important link between the innate and adaptive immune occurs in a sequence that involves proteolytic cleavage responses. However, inappropriate complement activation can cause tissue damage, and it underlies the pathology of many of the complement components, resulting in the diseases. In the transfusion medicine setting, complement release of active biological mediators and the assembly sensitization of RBCs can lead to both intravascular and of active enzyme molecules that result in cleavage of extravascular destruction. Moreover, complement deficiencies are 1 associated with autoimmune disorders, including autoimmune the next downstream complement component. hemolytic anemia (AIHA). Complement receptor 1 (CR1) is a large Depending on the nature of the activators, three single-pass glycoprotein that is expressed on a variety of cell types complement activation pathways have been described: in blood, including RBCs and immune cells. Among its multiple the antibody-dependent classical pathway and the functions is its ability to inhibit complement activation. Furthermore, gene knockout studies in mice implicate a role for antibody-independent alternative and lectin pathways CR1 (along with the alternatively spliced gene product CR2) in (Fig. 1).1 Common to all three pathways are two prevention of autoimmunity. This review discusses the possibility critical steps: the assembly of the C3 convertase that the CR1 protein may be manipulated to prevent and treat AIHA. In addition, it will be shown in an in vivo mouse model of enzymes and the activation of C5 convertases. transfusion reaction that recombinant soluble forms of CR1 can Specifically, the C3 convertases cleave C3 into C3a, a reduce complement-mediated RBC destruction,thereby prolonging potent anaphylatoxin, which acts as a neutrophil survival of transfused RBCs. It is proposed that CR1-based chemotaxin and activator,4 and C3b, which covalently therapeutics have potential for effective and safe prophylactic short-term use and for treatment of hemolytic transfusion reactions. attaches to nearby targets, where it directs immune Immunohematology 2005;21:109–118. clearance and antigen selection.5 In addition, the C5 convertases cause the release of another potent Key Words: complement, complement receptor 1, anaphylatoxin, C5a,4 and lead to the formation of the sCR1, inhibitors, LHR, immune hemolysis, autoimmune pore-like membrane attack complex (C5b-9), which hemolytic anemia, transfusion medicine, regulatory T inserts into cell membranes, causing lysis or sublytic + + cells, CD4 CD25 damage to the target cell (Fig. 1).5 In the transfusion medicine setting, complement- The Complement System and Red Cell mediated RBC destruction plays a critical role, being Destruction involved in both intravascular and extravascular The complement system is an important part of the hemolysis.6 Generally, in the presence of a potent, innate immune system for fighting infections and complement-binding antibody and large numbers of foreign molecules before an adaptive response has closely situated RBC antigens, complement activation developed.1 It is also an important regulator of B-cell, can proceed to completion, resulting in intravascular and possibly T-cell, immunity.2,3 However, it can also hemolysis, which can be fatal.7,8 However, the majority cause cellular and tissue damage when activated of blood group antibodies (including both alloanti- inappropriately, contributing to many clinical bodies and autoantibodies) that can fix complement conditions, including reperfusion injury (following activate complement up to the C3 stage but do not go surgery, ischemic disease, and organ transplantation), on to act as hemolysins.9 Although antibody-coated organ rejection,acute inflammatory injury to the lungs, RBCs can also be destroyed extravascularly without and autoimmune diseases.1 For the system to exert its complement activation, RBC removal by tissue

IMMUNOHEMATOLOGY, VOLUME 21, NUMBER 3, 2005 109 K.YAZDANBAKHSH macrophages in the spleen and liver is enhanced considerably when C3 is present on RBCs in addition to IgG.9,10 Indeed, as many as 50 percent of patients with autoimmune hemolytic anemia (AIHA) have both IgG and complement on their RBCs.11

Complement Regulatory Proteins and CR1 The complement system can be divided into three separate pathways (classical, alternative, and lectin), depending on the type of activator. The system consists of nearly 30 different serum and membrane proteins which, after activation, interact in a highly regulated enzymatic cascade to generate reaction products that mediate inflammation and host protection.1 Because of the direct and indirect powerful cytolytic activity of complement, there exists a family of structurally and functionally related Fig. 1. The complement system. The classical pathway is activated by proteins, known as regulators of complement antigen-antibody complex and the alternative and lectin pathways by microbial surfaces. Activation of these pathways activation (RCA), that prevent potential host cell results in the generation of the key enzymes, C3 and C5 damage from complement activation (Fig. 1).5 CR1, convertases, which in turn results in the release of C3a, C4a, and also known as CD35, is the most versatile of the RCA C5a anaphylatoxins (inflammatory response), C3b (opsonization of target cells), and the generation of the membrane attack family because it exhibits decay-accelerating and complex in the target cell (lysis). The different steps of the cofactor properties that can inactivate the two critical cascade where CR1 inhibits complement activation are shown. Abbreviations: MBL, mannan-binding lectin; MASP,MBL-associated enzymes of the complement activation pathways (Fig. serine protease. 1 and Fig.2).12–15 Specifically,by binding to C4b or C3b, CR1 can displace the catalytic subunits (decay- accelerating activity [Fig. 2]) of the convertases. In addition, by acting as a cofactor for plasma protease factor I, CR1 is responsible for the degradation of C4b and C3b (Fig. 2), and thus complete inactivation of the convertase. CR1 has also been shown to function as a Fig. 2. Regulation of complement by decay-accelerating and cofactor receptor for C1q,16 the first component of the classical activitiy of CR1. The classical complement inhibitory activity of CR1 is depicted. CR1 dissociates C3 convertase of the classical pathway as well as the mannan-binding lectin (MBL) of pathway through its decay-accelerating activity. In addition, the lectin pathway.17 through its cofactor activity, it degrades C4b into C4c and C4d. Central to these activities is its ability to bind C4b.(Adapted from Hourcade et al.12) CR1 Expression Pattern and Functions CR1 is expressed on a number of cell types, mostly response to antigen.28–36 Through its ability to bind C3b in the blood, and at low levels in soluble form in the and C4b, erythrocyte CR1 transports immune plasma.18 Of the C4 and C3 regulatory proteins, CR1 complexes to the spleen and liver for their removal,19–23 has the widest range of activities and functions, CR1 expressed on macrophages and neutrophils including immune complex clearance, regulation of mediates adherence and phagocytosis,24,25 that complement activation, phagocytosis, and antibody expressed on B cells modulates the threshold for B-cell response, as well as deletion of autoreactive B and activation,26,27 and that on follicular dendritic cells is maybe T cells. Through its ability to bind C3b and C4b, thought to improve the immune response to erythrocyte CR1 transports immune complexes to the antigen.28–36 Moreover, CR1 is expressed on 10 to 15 spleen and liver for their removal19–23; CR1, expressed percent of human T lymphocytes,37–40 although its on macrophages and neutrophils, mediates adherence function on such cells is not clear. The expression and phagocytosis24,25; on B cells, CR1 modulates the pattern of murine CR1 is for the most part similar to threshold for B-cell activation26,27; and on follicular that of human CR1 except that mouse RBCs do not dendritic cells, it is thought to improve immune express CR1.41 To date, no individuals completely

110 IMMUNOHEMATOLOGY, VOLUME 21, NUMBER 3, 2005 Complement receptor 1 therapeutics lacking the CR1 protein have been identified. inactivating the convertases, has successfully inhibited However, RBCs that express low copy numbers of CR1 the complement activation cascade and prevented and its associated Knops blood group antigens, known complement-mediated tissue injury in several animal as the Helgeson phenotype, have been described and models.49,50 More importantly,sCR1 has been in human appear to be associated with protection against severe clinical trials for the treatment of acute respiratory malaria.42 distress syndrome and to reduce tissue damage in myocardial infarction and lung transplantation,51–53 with CR1 Structure possible favorable outcomes.52 Despite the role of CR1 In humans,four allotypes of CR1,varying in size,are as a global inhibitor of complement activation, the known.43 The predicted amino acid sequence of the antibacterial defenses of the patients undergoing common allotype (CR1*1) is 2039 residues (Fig 3).18,44,45 treatment have not, thus far, been compromised, The extracellular 1930-residue–long domain of CR1, underscoring the usefulness of sCR1 as a therapeutic 53 with 25 potential N-glycosylation sites, can be divided agent. It is important to note that there are currently into 30 short consensus repeats (SCRs), each of 59 to no anticomplement therapeutics in the clinic. Indeed, 72 amino acids (aa) with between sCR1 represents the best in vivo characterized SCRs ranging from 60 to 90 percent.45 Homologous anticomplement agent to date. SCRs with four conserved cysteine residues are also Our studies are the first to demonstrate a potential found in other RCA members.12 The first 28 SCRs of for sCR1 for inhibiting complement-mediated RBC CR1 are further arranged into four longer regions of destruction following transfusion immunization similarity, termed long homologous repeats (LHRs A events.53,54 Furthermore, through structure-function through D), consisting of seven SCRs each18,44 (Fig. 3). analysis we have identified a 254-aa domain at the N- In mice, CR1 is expressed along with the alternatively terminus of CR1, consisting of four SCRs and one- spliced gene product CR2.46–48 Murine CR1 consists of eighth of sCR1, that has antihemolytic activity in vivo. 21 SCRs, with 15 SCRs identical to murine CR2 and 6 Previous in vitro studies by us and others demonstrated unique SCRs at the amino terminus.46–48 that the N-terminal domain of CR1 has barely any cofactor activity, but has decay-accelerating activity for the C3 convertases and inhibits the classical activation pathway.54,56–61 Moreover, we and others have shown that the four N-terminal SCRs preferentially bind C4b.45,56,59,62–64 Based on these observations, we believe that fine mapping of the C4b binding and the decay- accelerating activity (C4b.2a) for the classical activation pathway in the 254-aa domain will lead to future design of nonimmunogenic small molecule inhibitors to down-regulate complement-mediated RBC destruction. It is important to note that chronic treatment with complement inhibitors is likely to undermine the body’s ability to fight infections65 and may lead to development of autoimmune diseases (discussed in a later section). The application for these Fig. 3. Schematic representation of the common isotype of CR1.The 30 SCRs are represented by blocks.Groups of seven SCRs are further inhibitors is thus for short-term prophylactic use subdivided into four LHRs. The ligand binding sites for C3b and before transfusion of not fully matched blood in C4b are shaded. The preferred ligand is in bold; the alternative ligand is in parentheses.The positions of C1q/MBL binding sites emergency situations and as a therapeutic option in have not yet been defined. select patients with complement-mediated immune hemolysis to ameliorate the life-threatening CR1-Based Therapeutics complications. The complement regulatory function of CR1 has been exploited for development of a potent anti- Complement and AIHA complement agent. Specifically, a recombinant soluble AIHA is an autoimmune disease caused by form of CR1 (sCR1), by binding C3b and C4b and autoantibodies against RBC self-antigens, causing

IMMUNOHEMATOLOGY, VOLUME 21, NUMBER 3, 2005 111 K.YAZDANBAKHSH shortened RBC survival.6 The underlying mechanism induced in the periphery through exposure to for breakdown of immunologic tolerance is not well antigen.82,83 Naturally occurring Tregs constitute about established. Several lines of evidence support the role 1 to 2 percent of peripheral blood mononuclear cells, of complement in the maintenance of tolerance to self- or about 5 to 10 percent of the CD4+ T cells, and are antigens: (1) Hereditary deficiencies of complement characterized by coexpression of CD25 (α subunit of proteins of the classical pathway have been associated IL-2 receptor) and the transcriptional repressor FoxP3 with autoimmune diseases.66 For example, 90 percent (forkhead box P3).84 Their role in maintenance of self- of C1q-deficient patients develop autoimmune tolerance and their ability to suppress a number of disease,67 and C1q knockout mice have a high autoimmune diseases has attracted a great deal of incidence of developing autoimmune disease.68 (2) attention and opened the possibility of developing Altered levels of expression of CR1 have been observed novel immunotherapeutic strategies for suppression of in patients with AIHA as well as other autoimmune autoimmunity.84,85 Nevertheless, many questions diseases, such as systemic lupus erythematosus, remain to be answered about the characteristics and rheumatoid arthritis, Sjogren’s syndrome, and some biology of Tregs. For example, it is not known whether diabetic patients.69–77 In a mouse model of severe the suppressive activity of the CD4+CD25+ Tregs can be lupus-like disease (MRL/lpr), lower levels of CR1/CR2 subdivided to smaller subpopulations or whether receptors have been found on B cells before the smaller individual CD4+CD25+ Tregs have different development of disease manifestations,78 suggesting degrees of suppressive activity.86 Supporting this that altered complement receptor expression may possibility, CD103+ T cells within CD4+CD25+ Tregs contribute to initiation or progression of autoimmune were shown to have more suppressive activity than disease. Gene knockout studies of mice lacking CR1 CD103–CD4+CD25+ Tregs and CD62Lhigh expressing (along with the alternatively spliced gene product CD4+CD25+ Tregs appear more potent in preventing CR2) indicate that CR1 and CR2 control the activation certain autoimmune diseases in mice.87,88 Preliminary thresholds of B cells to self-antigens,79 although the studies in my laboratory indicate that CR1 is indeed exact molecular mechanism is not clearly understood. expressed on a subpopulation of CD4+CD25+ Tregs. Because CR1/CR2 knockout mice lack both CR1 and The molecular basis of the mechanism of Treg cell- CR2, the specific contribution of each receptor cannot mediated suppression is not fully known, although the be dissected. However, mice deficient in C4, but not consensus is that they can expand and augment their C3, have a phenotype similar to CR1/CR2-deficient suppressive activity when stimulated.89,90 In vitro mice in studies demonstrating their role in immune naturally occurring CD4+CD25+ mediate suppression of tolerance,strongly suggesting that the primary effect in cocultured CD25- T cells by cell-cell interactions, not these mice is mediated by CR1.79 (3) C3b was recently by cytokines.91–94 Activation has been shown to occur shown to induce the development of T-regulatory cells, through TCR-specific signals.90,95–97 Interestingly, non- known to be important for maintenance of peripheral TCR–specific stimuli such as bacterial products tolerance (discussed in a later section). Specifically, it through Toll-like receptor 4 have also been shown to was shown that membrane cofactor protein (MCP, activate CD4+CD25+ Tregs.98 Given that innate immune CD46), a natural complement regulatory protein responses such as Toll-like receptors can stimulate 80 whose ligands are C3b and C4b, can act as a Tregs, it is conceivable, although as yet untested, that coreceptor for inducing the development of IL- complement activation products, through interactions + + 10–secreting CD4 CD25 regulatory T cells, which are with complement receptors such as CR1, may also be responsible for active suppression of autoreactive T involved in augmenting or attenuating the activation 81 cells. state of Tregs, with consequences for their suppressive activity. In support of this possibility, C3b-CD46 T-Regulatory Cells interactions were recently shown to activate a subset T-regulatory cells (Tregs) are a subset of T cells that of Tregs, namely the inducible Tregs.81 Preliminary function to control immune responses, including those studies from my laboratory indicate that CR1 can also directed against self-antigens. Different populations of mediate the suppressive activity of CD4+CD25+ Tregs, Tregs have been described, including thymically although the exact mechanism of suppression is still derived naturally occurring cells and those that are under investigation.

112 IMMUNOHEMATOLOGY, VOLUME 21, NUMBER 3, 2005 Complement receptor 1 therapeutics

Possible Role of Complement/CR1 in Treg patients.103 It is interesting to note that Rh antibodies Induction do not fix complement on RBCs. In warm-type AIHA, We speculate that after certain infections, many of the autoantibodies are directed against the Rh complement is activated and foreign antigens that are complex.6 It may be that antigens such as Rh that are opsonized with complement components C3b and not initially opsonized with complement components C4b, which are known to be the ligands for CR1 and may not be subject to immune tolerance. Thus, upon CD46, may induce regulatory T cells. Thus, the initial stimulation with cross-reactive foreign antigens, complement activation will help clear the pathogen by autoreactive T cells specific to Rh antigens are direct lysis, inflammatory response, phagocytosis of preferentially expanded, resulting in AIHA. Future complement-sensitized infectious agent, or a studies are needed to further explore the potential combination of these (Fig. 1). In our hypothetical contribution of CR1 and its ligands in the development model, complement activation through induction of of AIHA. Tregs will also result in suppression of certain self- reactive lymphocytes as well as effector T cells that Acknowledgments would normally cause infection-induced immunopa- thology (Fig. 4). Given that complement is so tightly The author was supported in part by grants from regulated,1 it is conceivable that many factors, the NIH R01 HL69102 and the American Heart including the nature and strength of the activators Association Grant-in-Aid Heritage Affiliate. (pathogens), will determine the balance among complement activation, Treg induction, and T-cell References suppression. Our model obviously does not preclude 1. Volanakis JE, Frank MM. The human complement the role of other mediators besides complement system in health and disease. New York: Marcel activation products for Treg activity,which may explain Dekker, Inc., 1998. 99 why certain infections persist or become chronic and 2. Carroll MC.The complement system in regulation are correlated with lower incidence of auto- of adaptive immunity. Nat Immunol 2004;5:981-6. 100 immunity. Nevertheless, complement deficiencies, 3. O'Shea JJ, Brown EJ, Gaither TA,Takahashi T,Frank which we propose can cause decreased Treg activity, MM. Tumor-promoting phorbol esters induce 67,101 are associated with autoimmune disease. AIHA rapid internalization of the C3b receptor via a appears to be a secondary complication in patients cytoskeleton-dependent mechanism. J Immunol with a range of diseases, including those with viral or 1985;135:1325-30. mycoplasmal infections,102 and the presence of cross- 4. Hugli TE, Muller-Eberhard HJ.Anaphylatoxins: C3a reactive foreign antigens may be the underlying and C5a.Adv Immunol 1978;26:1-53. mechanism for breakdown of tolerance in these 5. Liszewski MK, Farries TC, Lublin DM, Rooney IA, Atkinson JP. Control of the complement system. Adv Immunol 1996;61:201-83. 6. Petz LD, Garratty G. Immune hemolytic anemias. 2nd ed. Philadelphia: Churchill Livingstone, 2004. 7. Sazama K. Reports of 355 transfusion-associated deaths: 1976 through 1985. Transfusion 1990;30: 583-90. 8. Sazama K. Transfusion errors: scope of the problem, consequences, and solutions. Curr Hematol Rep 2003;2:518-21. 9. Schreiber AD, Frank MM. Role of antibody and complement in the immune clearance and Fig. 4. Hypothetical role of CR1 in suppression of autoreactive T cells. destruction of erythrocytes.II.Molecular nature of Stimulation of Tregs with CR1 ligands such as complement IgG and IgM complement-fixing sites and effects opsonized RBCs results in suppression of Tresp (autoreactive or effector T cells). CR1-mediated activation signals described in of their interaction with serum.J Clin Invest 1972; 39,40 different cell types may be involved in Treg activation. 51:583-9.

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10. Mollison PL, Engelfriet CP, Contreras M. Blood 23. Cornacoff JB, Hebert LA, Smead WL, et al. Primate transfusion in clinical medicine. 10th ed. Oxford, erythrocyte-immune complex-clearing mechan- England: Blackwell Science, 1997. ism. J Clin Invest 1983;71:236-47. 11. Fischer JT, Petz LD, Garratty G, Cooper NR. 24. Gigli I, Nelson RA, Jr. Complement dependent Correlations between quantitative assay of red immune phagocytosis. I. Requirements for C'1, cell-bound C3, serologic reactions, and hemolytic C'4, C'2, C'3. Exp Cell Res 1968;51:45-67. anemia. Blood 1974;44:359-73. 25. Brown EJ. Complement receptors and phagocy- 12. Hourcade D, Liszewski MK, Krych-Goldberg M, tosis. Curr Opin Immunol 1991;3:76-82. Atkinson JP. Functional domains, structural 26. Molina H, Holers VM,Li B, et al. Markedly impaired variations and pathogen interactions of MCP,DAF humoral immune response in mice deficient in complement receptors 1 and 2.Proc Natl Acad Sci and CR1. Immunopharmacology 2000;49:103-16. USA 1996;93:3357-61. 13. Iida K, Nussenzweig V.Complement receptor is an 27. Kozono Y, Abe R, Kozono H, et al. Cross-linking inhibitor of the complement cascade. J Exp Med CD21/CD35 or CD19 increases both B7-1 and B7- 1981;153:1138-50. 2 expression on murine splenic B cells.J Immunol 14. Fearon DT. Regulation of the amplification C3 1998;160:1565-72. convertase of human complement by an 28. Fearon DT, Carroll MC. Regulation of B inhibitory protein isolated from human lymphocyte responses to foreign and self-antigens erythrocyte membrane. Proc Natl Acad Sci USA by the CD19/CD21 complex. Ann Rev Immunol 1979;76:5867-71. 2000;18:393-422. 15. Ross GD. Complement receptor type 1. Curr Top 29. Gray D, Kumararatne DS, Lortan J, Khan M, Microbiol Immunol 1992;178:31-44. MacLennan IC. Relation of intra-splenic migration 16. Klickstein LB, Barbashov SF, Liu T, Jack RM, of marginal zone B cells to antigen localization on Nicholson-Weller A. Complement receptor type 1 follicular dendritic cells. Immunology 1984;52: (CR1, CD35) is a receptor for C1q. Immunity 659-69. 1997;7:345-55. 30. Enriquez-Rincon F,Andrew E,Parkhouse RM,Klaus 17. Ghiran I, Barbashov SF, Klickstein LB, et al. GG. Suppression of follicular trapping of antigen- Complement receptor 1/CD35 is a receptor for antibody complexes in mice treated with anti-IgM mannan-binding lectin. J Exp Med 2000;192:1797- or anti-IgD antibodies from birth. Immunology 1808. 1984;53:713-9. 18. Ahearn JM, Fearon DT. Structure and function of 31. Pozdnyakova O,Guttormsen HK,Lalani FN,Carroll the complement receptors, CR1 (CD35) and CR2 MC, Kasper DL. Impaired antibody response to (CD21).Adv Immunol 1989;46:183-219. group B streptococcal type III capsular 19. Nelson RA.The immune adherence phenomenon: polysaccharide in C3- and complement receptor an immunologically specific reaction between 2-deficient mice. J Immunol 2003;170:84-90. 32. Brown JC, Harris G, Papamichail M, Sljivic VS, microorganisms and erythrocytes leading to Holborow EJ. The localization of aggregated enhanced phagocytosis. Science 1953;118:733-7. human-globulin in the spleens of normal mice. 20. Cooper NR. Immune adherence by the fourth Immunology 1973;24:955-68. component of complement. Science 1969;165: 33. Kroese FG,Wubbena AS, Nieuwenhuis P.Germinal 396-8. centre formation and follicular antigen trapping in 21. Fearon DT. Identification of the membrane the spleen of lethally X-irradiated and reconsti- glycoprotein that is the C3b receptor of the tuted rats. Immunology 1986;57:99-104. human erythrocyte, polymorphonuclear leuko- 34. Van den Berg TK, Yoshida K, Dijkstra CD. cyte, B lymphocyte, and monocyte. J Exp Med Mechanism of immune complex trapping by 1980;152:20-30. follicular dendritic cells. Curr Top Microbiol 22. Paccaud JP, Carpentier JL, Schifferli JA. Direct Immunol 1995;201:49-67. evidence for the clustered nature of complement 35. Fischer MB, Ma M, Hsu NC, Carroll MC. Local receptors type 1 on the erythrocyte membrane. J synthesis of C3 within the splenic lymphoid Immunol 1988;141:3889-94. compartment can reconstitute the impaired

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118 IMMUNOHEMATOLOGY, VOLUME 21, NUMBER 3, 2005 Autoimmune hemolytic anemia and a further example of autoanti-Kpb

E. LEE,G.BURGESS,AND N.WIN

A 65-year-old Caucasian man with myelodysplasia was admitted there was no sustained response in Hb level. The with autoimmune hemolytic anemia and a Hb of 5.6 g/dL. The samples were submitted to the Reference Laboratory of patient’s serum contained anti-K; the DAT on the patient’s RBCs reacted 3+ with anti-IgG and 3+ with anti-C3d. K– RBC units were the National Blood Service (NBS), North London, for transfused, but there was no sustained increase in Hb level. The investigation. samples were referred to the reference laboratory of the National Blood Service. The DAT results remained the same, with anti-K detected only in the serum. An eluate prepared from the patient’s Materials and Methods DAT-positive RBCs revealed anti-Kpb specificity. This study reports Antigen typing for ABO, Rh, and K were performed an unusual case of autoanti-Kpb, which is different from previously published cases in that no free anti-Kpb was detectable in the by standard tube technique. Anti-A and anti-B reagents serum. Immunohematology 2005;21:119–121. were from Diagnostic Scotland, Edinburgh, Scotland; anti-D reagents (RUM1 and BS226) were from NBS b Key Words: autoimmune hemolytic anemia, anti-Kp Reagents, Cambridge, United Kingdom; monoclonal anti-Rh (anti-C, -E, -c, and -e ), and anti-K were from Autoantibodies found in patients with warm BIOSCOT (now Serologicals), Ltd., Livingston, Scotland. autoimmune hemolytic anemia (WAIHA) often show a Antibody identification was performed by standard RBC specificity related to the Rh system. The first tube LISS-IAT (LISS from Inverclyde Biologicals, example of this type of autoantibody was described by Bellshill, Scotland). The patient’s serum was tested Race and Sanger.1 Although antibodies to other high- with an in-house panel of RBCs to determine antibody prevalence antigens have been reported, such as specificities. The manual polybrene technique was also autoanti-AnWj, -Co3, -Dib, -Ena, -Ge, -Hro, -Kpb, -K13, -Kx, used to determine additional antibody specificities. -LW,-‘N,’-Rh34,-Sc1,-Sc3,-U,-Vel,and -Wrb,they are rare. The 0.05% working solution of polybrene, the We report an unusual case of autoanti-Kpb, which is resuspending solution, and low–ionic-strength medium different from previously published cases2–4 in that no were prepared in house according to Methods in free anti-Kpb was detectable in the patient’s serum. Immunohematology.5 The DAT was performed with Kp(b–) RBCs were issued for further transfusion polyspecific AHG and monospecific anti-IgG and anti- support. Transfusion of presumably Kp(b+) RBCs did C3d reagents (NBS Reagents). An eluate was prepared not result in a sustained increase in Hb, therefore from the patient’s RBCs using chloroform following the Kp(b–) RBCs were issued for further transfusion protocol in Methods in Immunohematology5 and was support. Subsequent transfusions were uneventful and tested with in-house selected-RBC panels and with achieved prolonged Hb increment. RBCs from the frozen inventory for antibody identification. Chloroquine diphosphate (CDP, 200 Case Report mg/mL, pH 5.0 ± 0.1) was prepared by NBS Reagents. A sample from a 65-year-old Caucasian man with CDP treatment consisted of adding four volumes of myelodysplasia, on regular transfusion support, was CDP to one volume of washed packed RBCs, mixing, tested to be Group O, R1R2 with anti-K in the serum. and incubating at 20°C for a maximum of 2 hours. A Later the patient had colitis and developed acute DAT using anti-IgG was performed on the CDP-treated autoimmune hemolytic anemia (AIHA) with a positive RBCs every 30 minutes during this incubation to DAT. The patient was transfused with K– RBCs but ensure the effectiveness of the CDP treatment.

IMMUNOHEMATOLOGY, VOLUME 21, NUMBER 3, 2005 119 E. LEE ET AL.

Results Most autoantibody specificities are Rh-related, such A posttransfusion sample from the patient showed as anti-nl,anti-pdl,and anti-dl,6 and it is reported that up the RBCs to be Group O, R1R2 and to have a positive to 50 percent of antibody specificities in WAIHA are in DAT,reacting 3+ with both anti-IgG and anti-C3d. Tests these groups.7 Usually it is not necessary to honor the by routine LISS-IAT, saline, and manual polybrene specificity of the autoantibody when selecting RBCs techniques revealed anti-K in the patient’s serum. An for transfusion. The use of a high-prevalence antigen- eluate was prepared using a chloroform method and negative panel is often not indicated and the ability to autoanti-Kpb was detected, reacting 3+. After CDP resolve these specificities is limited by the availability treatment of the patient’s RBCs, they were typed as of suitably typed reagent RBCs, such as Rhnull and Rh Kp(b+) (Table 1). The patient was transfused with two deletion. Extensive investigations are only performed units of K–, Kp(b–) RBCs. Three additional units of K–, in selected rare circumstances. In our institution, Rh- Kp(b–) RBCs were transfused two days later. All of the and K-matched RBCs are selected and issued for units were frozen-recovered, deglycerolized RBCs from transfusion in cases of AIHA. the same donor. The transfusions were well tolerated The patient in this case had pre-existing and the patient recovered from this hemolytic episode. myelodysplasia, developing colitis, and infection. One month later, a DAT performed on the patient’s Although disease-associated transient suppression of RBCs was still positive (anti-IgG 3+, anti-C3d weak Kell antigens has been reported,8–10 no suppression of positive), and anti-K was detected by LISS-IAT in the the Kpb antigen was observed in this case. A positive patient’s serum in addition to a weak autoantibody result was obtained in the Kpb typing after CDP detectable by manual polybrene technique only. treatment of the patient’s RBCs. This finding may, Autoanti-Kpb was not apparent in the serum or eluate. however, be due to the timing of the test sample. Although all of the RBCs transfused to the patient were There are a few case reports of autoantibody with K–, anti-K was eluted from the patient’s RBCs, probably Kpb specificity described in the literature. Marsh et al.11 due to the Matuhasi-Ogata phenomenon, in which reported a case of AIHA, due to anti-Kpb, in a patient alloantibodies are nonspecifically bound to the RBC who had a severe hemolytic transfusion reaction after along with autoantibody (Table 1). receiving Kp(b+) RBCs. As previous transfusions with K– RBCs had not resulted in a sustained Hb increment, Discussion Kp(b–) RBCs were selected;transfusion was uneventful An autoantibody with Kpb specificity has been and an excellent hematologic response was achieved. reported in AIHA and appears to be rare. In all Win et al.3 also reported a rare case of AIHA in a 12- previously reported cases of AIHA with autoanti-Kpb week-old infant. Autoanti-Kpb was identified in the specificity, free autoantibody was always detectable in infant’s serum. Initially the infant was transfused with the serum. In the case we describe, no free autoanti-Kpb Kp(b+) RBCs and had recurrent hemolytic transfusion was detected, distinguishing it from the other published reactions. Pronounced jaundice was noted and there cases. The identification of autoanti-Kpb in this case was was no sustained increase in Hb level after transfusion. fortuitous, as the routine panel of reagent RBCs used at The reference laboratory detected autoanti-Kpb in the the time had one example of Kp(b–) RBCs, which serum and transfusion with Kp(b–) RBCs gave an helped to define the autoantibody specificity. excellent result with no further reactions.

Table 1. Summary of laboratory findings* Hb Antibody Antibody DAT Date (g/dL) (serum) (eluate) Anti-IgG Anti-C3d Phenotype of patient RBC units provided 5/15/98 NA Anti-K NT NT NT K– 2 units K–

† b 6/4/98 5.6 Anti-K (5+) Anti-Kp (3+) 3+ 3+ K–, Kp(b+) 2 units R2r, K–, Kp(b–)

6/11/98 7.0 NT NT NT NT – 3 units R2r, K–, Kp(b–) 7/10/98 NA Anti-K (5+)† Anti-K (2+)§ 3+ (+) – None weak autoantibody by LIP‡ *NA = not available, NT = not tested †Refer to Table 2 ‡LIP = manual polybrene technique §Anti-K probably due to Matuhasi-Ogata phenomenon

120 IMMUNOHEMATOLOGY, VOLUME 21, NUMBER 3, 2005 AIHA and autoanti-Kpb

Table 2. Grading system for hemagglutination 4. Kohan AI, Reybaud JF, Salamone HJ, et al. Reaction Management of a severe transfusional problem in grade Score Description a patient with alloantibody to Kpb (K4).Vox Sang 5+ 12 Cell button remains in one clump or dislodges into a few large clumps, 1990;59:216-7. macroscopically visible 5. Judd,WJ. Methods in immunohematology. 2nd ed. 4+ 10 Cell button dislodges into numerous large Durham, NC: Montgomery Scientific Publications, clumps, macroscopically visible 1994. 3+ 8 Cell button dislodges into many small 6. Weiner W,Vos GH. Serology of acquired hemolytic clumps, macroscopically visible anemias. Blood 1963;22:606. 2+ 5 Cell button dislodges into finely granular but b definite small clumps, macroscopically visible 7. Issitt PD, Pavone BG, Goldfinger D, et al. Anti-Wr 1+ 3 Cell button dislodges into very small and other autoantibodies responsible for positive agglutinates, these are more distinct if the direct antiglobulin tests in 150 individuals. Br J tube is left on its side to allow the cells to settle, macroscopically visible Haematol 1976; 34-5. 8. Beck ML, Marsh WL, Pierce SR, et al.Auto anti-Kpb w 2 Grainy appearance, agglutinates best seen microscopically associated with weakened antigenicity in the Kell 0 0 Negative result blood group system: a second example. Transfusion 1979;19:197-202. The use of Rh- and K-matched RBCs for AIHA 9. Seyfried H, Gorsks B, Maj S, et al. Apparent patients is standard practice in our organization and depression of antigens of the Kell bood group only rarely are RBCs that are negative for antigens system associated with autoimmune haemolytic against which autoantibodies have been detected anaemia.Vox Sang 1972;23:528-36. selected for transfusion.12 In the case we describe 10. Williamson LM, Poole J, Redman C, et al.Transient here, because of the unusual nature of the loss of proteins carrying Kell and Lutheran red autoantibody, and because of the lack of a response to cell antigens during consecutive relapses of auto- the transfusion of Kp(b+) RBCs, we selected frozen- immune thrombocytopenia. Br J Haematol recovered, deglycerolized Kp(a+b–) RBCs for 1994;87:805-12. transfusion during the hemolytic episodes. 11. Marsh WL, Oyen R, Alicea E, Linter M, Horton S. AIHA with autoanti-Kpb specificity is rare and our Autoimmune hemolytic anemia and the Kell blood case supports previous findings that Kp(b–) RBCs groups.Am J Hematol 1979;7(2):155-62. should be selected for patients with Kpb autoantibodies 12. Sokol RJ, Hewitt S, Booker DJ, Morris BM. Patients for transfusion support.3,11 with red cell autoantibodies:selection of blood for transfusion. Clin Lab Haematol, 1988;10:257-64.

References Edmond Lee (corresponding author), MSc, Senior 1. Race RR, Sanger R. Blood groups in man. 2nd ed. Biomedical Scientist, and Gordon Burgess, Reference Oxford: Blackwell Scientific Publications, Service Manager, Red Cell Immunohematology, 1954;361. National Blood Service Colindale, Colindale Avenue, 2. Marsh WL, Redman CM. The Kell blood group London, NW9 5BG, United Kingdom; and Nay Win, system: a review.Transfusion 1990;30:158-61. Consultant Hematologist, National Blood Service 3. Win N, Kaye T, Mir N, et al. Autoimmune Tooting, London, United Kingdom. haemolytic anaemia in infancy with anti-Kpb specificity.Vox Sang 1996;71:187-8.

IMMUNOHEMATOLOGY, VOLUME 21, NUMBER 3, 2005 121 The incidence of red cell alloantibodies underlying panreactive warm autoantibodies

M. MALEY,D.G.BRUCE,R.G.BABB,A.W.WELLS,AND M.WILLIAMS

A recognized hazard of administering blood transfusions to patients (NBS) in the United Kingdom. British Committee for with panreactive warm autoantibodies is that alloantibodies may be Standards in Haematology guidelines recommend that masked. Studies have shown the incidence of underlying 6 alloantibodies to be 30 to 40 percent. Adsorption procedures can these techniques be used in such cases. This study be used to remove autoantibodies and allow detection and was performed over 18 months in the Red Cell identification of underlying alloantibodies. This study contains data Immunohaematology laboratory at the NBS in from 126 patients referred to the Red Cell Immunohaematology laboratory at the National Blood Service, Newcastle upon Tyne, Newcastle. The region has 18 National Health Service United Kingdom. These patients were from the northeast of (NHS) hospital blood banks,serving a population of 2.9 England, a population for which data have not previously been million. reported. Samples identified as containing panreactive warm autoantibodies were subjected to adsorption procedures (95 by alloadsorption and 31 by autoadsorption). Absorbed sera were then Materials and Methods tested to identify underlying alloantibodies. Of 126 samples, 39 (31%) contained a total of 61 RBC alloantibodies; 15 (12%) Our alloadsorption method uses at least two sets of contained 2 or more antibody specificities; and 14 (11%) contained strongly papain-treated donor RBCs selected to have alloantibodies not found within the Rh or Kell blood group systems. complementary expression of important RBC antigens. Antibodies identified included the following specificities: E (19), D (9), c (7), C (6), S (5), Fya (3), Jka (2), Jkb (2), K (2), Kpa (2), Fyb,Cw,N, It is our usual practice to use Group O,rr,K– and Group and f (ce). This study reinforces the value of adsorption studies, O, R1R1, K– RBCs, one RBC sample being Jk(a–b+) and whether using autologous or allogeneic RBCs, when panreactive the other being Jk(a+b–). Reagent RBCs for adsorption warm autoantibodies are present. In addition, this study confirms that it is not appropriate in these cases simply to issue blood which were obtained from the UK NBS central reagents unit. is “least incompatible” or Rh phenotype- and K antigen-matched. Autoadsorption uses the patient’s papain-treated RBCs. Immunohematology 2005;21:122–125. Alloadsorption was performed on more cases as there was a limited supply of patients’ RBCs to use to Key Words: autoantibody,alloantibody,alloadsorption, perform autoadsorption, due to severe anemia or low autoadsorption sample volumes. Serial adsorptions were performed by One of the recognized hazards of administering incubating 1 mL of the patient’s plasma with 1 mL of blood transfusions to patients with panreactive warm the papain-treated packed RBCs at 37°C; the absorbed autoantibodies is the failure to detect clinically plasma was then retrieved by centrifugation. A significant alloantibodies that may be masked.1,2 maximum of 4 aliquots of allogeneic RBCs, or typically Traditionally, some blood banks have had policies of 2 aliquots of autologous RBCs, were used for this crossmatching and issuing blood that is “least process. The number of autoadsorption procedures incompatible” or “as compatible” as the patient’s own was dependent on the volume of RBCs available from RBCs, or they have issued blood that is Rh phenotype- the patient sample. Absorbed plasma was then and K antigen-matched.3 Several methods may be used investigated to detect and identify any underlying to remove free autoantibody from the patient’s plasma alloantibodies, using reagent RBCs suspended in low- to allow the detection and identification of any ionic-strength red cell preservative solution (RCPS; underlying alloantibodies.4,5 Adsorption,whether using Inverclyde Biologicals, UK) by tube IAT. Where autologous or allogeneic enzyme-treated RBCs, is the possible, phenotype studies were used to confirm that method of choice within the National Blood Service the identified antibodies were alloantibodies.

122 IMMUNOHEMATOLOGY, VOLUME 21, NUMBER 3, 2005 Alloantibodies underlying autoantibodies

Results the UK.6 Of 95 alloadsorbed samples, 33 (35%) Samples from 130 individual patients were referred contained alloantibodies, whereas 6 of 31 (19%) for investigation of panreactive autoantibodies by autoadsorbed samples contained alloantibodies. autoadsorption or alloadsorption procedures. Data All but six antibodies (anti-Fya [3], anti-Kpa [2], and from four samples in which we failed to successfully anti-Fyb [1]) were confirmed as alloantibodies, using remove the autoantibody were omitted from this study. phenotyping studies. Phenotyping studies were not Alloadsorption procedures were performed on 95 undertaken for these six patients as their RBC samples samples and autoadsorption procedures on 31 had strongly positive DATs. With only typing reagents samples. Thirty-nine of 126 samples (31%) contained a requiring the use of AHG available and without a local total of 61 RBC alloantibodies (Table 1); 87 samples procedure for removing autoantibody for typing, test (69%) contained no underlying alloantibodies. Fifteen results would be unreliable. These six antibodies were samples (12%) contained two or more specificities therefore assumed to be probable alloantibodies. (Table 2). Fourteen samples (11%) contained Autoantibodies of these specificities are rarely alloantibodies with specificities for antigens outside reported. the Rh or Kell blood group systems. Of the 61 alloantibodies identified, all but 4 (anti- Discussion Kpa [2], anti-N [1], and anti-Cw [1]) would require the selection of antigen-negative RBCs for crossmatching in Limitations and advantages of the adsorption procedures Table 1. Underlying alloantibody specificities 1.Alloadsorption Antibody specificity Number identified Alloadsorption procedures were preferentially Anti-E 19 performed in the cases described, but a number of Anti-D 9 factors must be borne in mind when interpreting Anti-C 7 results. Alloadsorption procedures require a minimum Anti-c 6 of two individual RBC samples with a complementary Anti-S 5 antigenic profile at key antigens, in our study,Rh, K, Jka, Anti-Fya 3 and Jkb antigens, and at least 2 mL of available patient Anti-Jka 2 plasma or serum. Papain treatment of the adsorption Anti-Jkb 2 RBCs removes enzyme-labile antigens, effectively Anti-K 2 rendering the RBCs negative for such antigens. By this Anti-Kpa 2 procedure we ensured that underlying alloantibodies Anti-Fyb 1 such as anti-S, -s, -Fya, -Fyb, -M, and -N remained behind Anti-Cw 1 after adsorption. We acknowledge that many workers Anti-N 1 have found that the s antigen is not readily destroyed.7 Anti-f (ce) 1 Although not every batch of reagent RBCs is Total 61 specifically tested for antigen destruction, the process was thoroughly validated when introduced by the UK Table 2. Multiple alloantibody specificities NBS reagents unit. Although no patients in this study Antibody specificity Number of samples were found to have underlying anti-s at the time of Anti-D, -C 3 collating data, the patient referred to in Table 2 with Anti-c, -E 3 anti-C, -Fya, and -Jkb did subsequently produce an Anti-E, -Kpa 1 underlying anti-s. The enzyme treatment usually Anti-E, -Fya, -Jka 1 increases the uptake and removal of autoantibody, Anti-S, -N 1 although there were some exceptions to this in our Anti-Fyb, -f(ce) 1 study (four patients) when autoantibody removal was Anti-c, -E, -K, -Fya 1 less effective. Anti-C, -Fya, -Jkb 1 Our standard protocol does not include the use of Anti-D, -C, -E, -S 1 e– adsorption RBCs. In our experience the benefits of Anti-E, -K, -Jka 1 including RBCs of the R2R2 phenotype are outweighed Anti-C, -Jkb 1 by the practical difficulties of obtaining suitable RBCs

IMMUNOHEMATOLOGY, VOLUME 21, NUMBER 3, 2005 123 M. MALEY ET AL. and the increased volume of plasma required. transfused RBCs. Our protocol for patients transfused

However, RBCs of the R2R2 phenotype are used when within the last 3 months is to perform alloadsorption the initial investigation suggests they may be useful. and to prepare an eluate to detect alloantibodies bound The policy of matching the Rh phenotype of the donor in vivo.8 units to patients with autoantibodies decreases the risk of an underlying alloanti-e causing a transfusion Transfusion practices reaction. Transfusion in the presence of panreactive warm Recently transfused patients may give misleading autoantibodies can be a complicated and dangerous or inconclusive Rh phenotyping results. In such cases proposition.9 In our study, adsorption techniques it is important to establish the transfusion history and excluded the presence of alloantibodies in 69 percent make an individual assessment of the most appropriate of our patients. In the patients found to have Rh phenotype to select. Where possible it is good alloantibodies,adsorption studies allowed the selection practice to prospectively establish the extended of appropriate RBCs for transfusion. In this group, Rh phenotype of patients likely to be multiply transfused. and Kell phenotype matching alone would have Alloadsorption procedures carry the risk that an exposed 14 patients (11%) to the risk of a significant alloantibody to a high-prevalence blood group antigen transfusion reaction. can be adsorbed by the procedure and not detected We believe this study reinforces the value of when screening or matching blood. We believe our adsorption studies when panreactive warm findings show that it is better to carry out the autoantibodies are present and concurs with the adsorption procedures, being aware of this risk, than findings of similar studies. Our findings also show that not to adsorb at all. We have experience of one case it is not appropriate in these cases simply to issue RBCs (not included in this study) where an underlying anti- that are “least incompatible” or Rh phenotype- and K Vel was not detected following alloadsorption (data not antigen-matched. published).

2.Autoadsorption References Autoadsorption uses the patient’s own RBCs to 1. Branch DR, Petz LD. Detecting alloantibodies in remove autoantibody so that underlying alloantibodies patients with autoantibodies.Transfusion 1999;39: can be tested for. The advantages of autoadsorption are 6-10. that it does not remove any alloantibodies and it 2. Leger RM, Garratty G. Evaluation of methods for requires adsorption of only a single aliquot of patient’s detecting alloantibodies underlying warm plasma, considerably reducing the volume required. autoantibodies. Transfusion 1999;39:11-6. The number of tests performed is also reduced since 3. Petz LD. “Least incompatible”units for transfusion the tests need not be carried out in duplicate, or in autoimmune hemolytic anemia: should we triplicate as in the case of alloadsorptions. eliminate this meaningless term. A commentary Autoadsorption is not without its problems, for clinicians and transfusion medicine pro- however. The patient’s RBCs often are heavily coated fessionals. Transfusion 2003;43:1503-7. with antibody, with reduced capacity for further 4. James P, Rowe GP, Tozzo GG. Elucidation of antibody uptake. Patients may also be anemic, alloantibodies in autoimmune haemolytic sometimes very anemic, with few RBCs available for anaemia. Vox Sang 1988;54:167-71. autoadsorption. In our experience, anything less than 5. Garratty G. Problems associated with two equal-volume (RBCs:plasma) adsorptions does not compatibility testing for patients with auto- remove sufficient autoantibody to be of investigative immune hemolytic anemia. Southeast Asian J Trop value. The relatively large amount of plasma available Med Public Health 1993;24(Suppl 1):76-9. from anemic patients lends itself more to 6. Chapman JF, Elliott C, Knowles SM, Milkins CE, alloadsorption. Poole GD;Working Party of the British Committee Autoadsorption is inappropriate for the recently for Standards in Haematology Blood Transfusion transfused patient. If the procedure is inadvertently Task Force. Guidelines for compatibility applied to samples from such patients there is a risk procedures in blood transfusion laboratories. that alloantibodies may be adsorbed in vitro onto Transfus Med 2004;14:59-73.

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7. Issitt PD,Anstee DJ. Applied blood group serology. Martin Maley, MPhil, Section Head, David G. Bruce, 4th ed. Miami: Montgomery Scientific Publi- MSc, Section Head, Roderick G. Babb, BA, M.Ed, cations, 1998: 477. Laboratory Manager, Red Cell Immunohaematology 8. Laine EP,Leger RM, Arndt PA, Calhoun L. In vitro Laboratory, and Angus W. Wells, MRCP(UK), studies of the impact of transfusion on the MRCPath, Consultant in Transfusion Medicine, detection of alloantibodies after autoadsorption. Newcastle Blood Service, Barrack Road, Newcastle Transfusion. 2000;40:1384-7. upon Tyne, NE2 4NQ, UK; and Mark Williams, MSc, 9. Buetens OW,Ness PM. Red blood cell transfusion Reference Service Manager, Leeds Blood Service, in autoimmune hemolytic anemia. Curr Opin Bridle Path, Seacroft, Leeds, LS15 7TW, UK. Hematol 2003;10:429-33.

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IMMUNOHEMATOLOGY, VOLUME 21, NUMBER 3, 2005 125 Persistent anti-Dra in two pregnancies

N. RAHIMI-LEVENE,A.KORNBERG,G.SIEGEL,V.MOROZOV,E.SHINAR,O.ASHER,C.LEVENE,AND V. Y AHALOM

The Drori (Dra) antigen is one of the ten high-prevalence antigens high-prevalence Cromer antigens, including Dra. Reid of the Cromer blood system, which are carried on decay- et al.8 suggested that antigen-positive trophoblasts may accelerating factor (DAF, CD55). The Dr(a–) phenotype was first described in a 48-year-old Jewish woman from Bukhara. Her serum absorb maternal antibodies with DAF specificity such contained an antibody to a high-prevalence antigen named anti-Dra. as anti-Dra, causing their disappearance from maternal Most known individuals with the Dr(a–) phenotype are Jews from blood and explaining the lack of HDN secondary to this the geographic area of Bukhara, but individuals from Japan have also been described. Antibodies in the Cromer blood group system, antibody. They reported two cases with Cromer including anti-Dra,have never been reported to cause HDN. In most antibodies, one of them anti-Dra, which disappeared a of the cases with anti-Dr examined in Israel, the antibodies have during pregnancy, supporting their hypothesis. been subtyped as IgG2 and IgG4. This report is of a woman with Dr(a–) phenotype and an anti-Dra titer of 256 to 512 in her serum, In this report two successive pregnancies in a observed during two successive pregnancies. At birth, the RBCs of Dr(a–) woman with anti-Dra in her serum are described. the first- and second-born child were negative and positive in the The anti-Dra titers did not change significantly DAT,respectively,and neither manifested clinical signs of HDN. The disappearance of Cromer system antibodies, including anti-Dra in throughout either pregnancy. At birth,a DAT performed midpregnancy, has been described in a previous study. In that on the RBCs of the first baby was negative and that study, it was theorized that the antibodies in the serum of the performed on those of the second baby was positive. women were adsorbed onto placental DAF. The finding of a high anti-Dra titer in two successive pregnancies in this patient, with a Neither baby had clinical evidence of HDN. positive DAT for the RBCs of one of the two babies at term, differs from published reports, suggesting that a different mechanism might be involved. Immunohematology 2005;21:126–128. Case Report A 28-year-old Jewish woman of Bukharian origin in a Key Words: Anti-Dr , titers in pregnancy her second pregnancy was admitted to the Assaf Harofeh Medical Center delivery room in labor in The Drori (Dra) antigen is part of the Cromer blood group system, which includes ten high- and three low- March 1998. A RBC sample was sent to the blood bank prevalence antigens.1 The Cromer antigens are carried for blood type and antibody screen. An antibody to a on the complement regulatory glycoprotein decay- high-prevalence antigen was found in her serum and 1 accelerating factor (DAF;CD55). The Dr(a–) phenotype investigated. Her RBCs were typed as Group A ,D–,C–, w was first described in a 48-year-old Jewish woman of E–, c+, e+, C –; M–, N+, S–, s+; P1–; Lu(a–b+); K–, k+, Bukharian origin by Levene et al.2 More cases have Kp(a–b+); Le(a–b+); Jk(a+b+); Yt(a+), and Dr(a–) and been published; most of them were of Jewish women the DAT was negative. During testing for the Cromer born in Bukhara, a few were of individuals from antigens,weaker reactions were observed with anti-IFC a Japan.3,4 and -Cr when compared with the positive controls. A Antibodies in the Cromer system have not been sample from her husband was tested and his RBCs reported to cause clinical HDN.1 There are reports in were determined to be Group A1, D+, C+, E–, c–, e+, the literature of women with Cromer antibodies in Cw–; M+, N+, S–, s+; P1+; Lu(a–b+); K–, k+, Kp(a–b+); which the titer fell during pregnancy and the baby Fy(a+b+); Jk(a+b–);Yt(a+b–),and Dr(a+). His antibody carrying a Cromer antigen was born with a negative screen and DAT were negative. DAT.5,6 DAF is expressed strongly on the apical surface The patient was found to have anti-D (low titer of trophoblasts, more so in the second and third from passive immunization in pregnancy) and anti-LebH. trimesters.7 The placental trophoblasts possess the An antibody to a high-prevalence antigen was found in DAF polymorphism of the fetus, which is inherited her serum,which was characterized as anti-Dra,and her from both parents and is most likely positive for the RBCs typed as Dr(a–). The titer of the antibody by

126 IMMUNOHEMATOLOGY, VOLUME 21, NUMBER 3, 2005 Persistence of anti-Dra in pregnancy saline indirect antiglobulin test was 512 (Table 1). The Discussion patient’s serum was examined with two known Dr(a–) DAF is a protein connected to the membrane via a RBCs and the presence of anti-K, anti-M, anti-S, and anti- glycosylphosphatidylinositol anchor. It protects cells P1 were excluded. against destruction by autologous complement by inhibiting formation and acceleration of the decay of Table 1. Anti-Dra titers C3 and C5 convertases, thus preventing the Date Titer complement cascade from causing hemolysis. DAF is a May 1998 512 member of the regulators of complement activation April 1999 256 gene family encoded by a gene on the long arm of May 2000 256 .9 It is widely distributed throughout June 2000 512 the body and expressed on epithelial cells, endothelial July 2000 256 cells, blood vessels, and the apical surface of August 2000 256 trophoblasts.7,10 The Cromer antigens are carried on September 2000 256 DAF and the different phenotypes provide a basis for biochemical and functional investigation of alternate Materials and Methods forms of DAF. Dr(a–) is a rare Cromer phenotype, Standard tube hemagglutination tests were used for lacking expression of the Dra antigen, with reduced antigen typing, antibody screening, and panels. levels of DAF and weak expression of other Cromer Antibodies and rare RBCs used in the testing were from antigens, including Cra,Tca,Tcb,Tcc,Esa, IFC, Wesb, and the Israeli National Blood Group Reference Laboratory UMC.10 No hematological abnormalities have been and SCARF. Column agglutination tests (DiaMed AG, described in Dr(a–) individuals. The molecular change Switzerland) were also used for antibody screening and associated with Dr(a–) has been characterized.9 Four panels. The RBCs were examined with anti-DAF pregnancies in a woman who had Cr(a–) RBCs and (CD55) and anti-MIRL (CD59) in column agglutination anti-Cra, in which the titer of the anti-Cra declined tests (DiaMed AG). The elution was performed using during pregnancy and remained low until term, were the rapid acid method (Elukit II, Gamma Biologicals, described by Sacks and Garratty.6 The decline in the Inc., Houston,TX). titer was thought to result from absorption of the antibody by white cells or platelets or be due to Results neutralization by plasma of the fetus. Another case of a The RBCs of the newborn were negative in the anti-Cr in a pregnant woman in which the titer of the a 5 DAT and there was no clinical evidence of HDN. In anti-Cr decreased was described by Dickson et al. 8 May 2000, during her next pregnancy, anti-Dra was Reid et al. described two patients with repeat identified with a titer of 256 (Table 1). In June 2000, pregnancies who had Cromer blood group system the anti-Dra titer was 512 and in July 2000 the titer was antibodies (anti-Cra and anti-Dra) with titers of 128 or 256. The pregnancy was uncomplicated and RhIG was greater at the beginning of pregnancy. These again administered antenatally. The patient gave birth antibodies became undetectable in the serum from the in October 2000 to a full-term healthy baby, the titer second trimester onward. RBCs from the babies tested remaining stable throughout pregnancy. The infant’s positive for the relevant Cromer antigens but were RBCs were typed as Group A, D+; the DAT was positive negative by the DAT. The antibodies would reappear in with anti-IgG resulting in 2+ to 3+ reactivity in tubes the serum of the mother after delivery or at the and 4+ by the column agglutination test (DiaMed AG). beginning of a subsequent pregnancy. The positive DAT precluded Dra typing. An elution was Holmes et al.7 showed the preferential expression performed on the baby’s RBCs and anti-Dra was of DAF at the feto-maternal interface of the placenta, identified. The eluate did not react with RBCs from the increasing quantitatively during placental develop- mother, Dr(a–) RBCs, or RBCs treated with alpha- ment. DAF at this site plays a protective role against chymotrypsin. There was no clinical evidence of HDN. maternal complement-mediated attack. DAF can The titer of the anti-Dra in the mother’s serum was 256. absorb the Cromer antibodies produced by the mother, Testing of maternal RBCs by the column agglutination more so in the second and third trimesters when the test (DiaMed) was negative with anti-DAF (CD55) and expression of DAF increases. This hypothesis is positive with anti-MIRL (CD59). supported by reappearance of these antibodies after

IMMUNOHEMATOLOGY, VOLUME 21, NUMBER 3, 2005 127 N. RAHIMI-LEVENE ET AL. pregnancy when the placenta is no longer present. 4. Daniels GL, Green CA, Mollinson G, Okubo Y,et al. Most of the patients with anti-Dra found in Israel have Decay-accelerating factor (CD55) deficiency been multiparous women. Subclasses of IgG examined phenotypes in Japanese. Transfus Med in these cases during the years 1989–1991 were IgG2 1998;8:141-7. and IgG4. The anti-IgG subtype antibodies used were 5. Dickson AC, Guest C, Jordan M, et al. Case report: from the Netherlands Red Cross and tests were anti-Cra in pregnancy. Immunohematology performed in tube or capillary tests. None of the 1995;11:14-7. offspring had HDN (C. Levene, unpublished data, 6. Sacks DA, Garratty G. Isoimmunization to Cromer 1989–1991). The patient reported here was Dr(a–). antigen in pregnancy. Am J Obstet Gynecol She developed anti-Dra and was monitored during two 1989;161:928-9. subsequent pregnancies. The anti-Dra titer remained 7. Holmes CH, Simpson KL, Wainwright SD, et al. consistently at 256 or above during both pregnancies. Preferential expression of the complement RBCs from the first baby typed as Dr(a+) and the DAT regulatory proteins decay accelerating factor at was negative.RBCs from the second baby were positive the fetomaternal interface during human in the DAT;they could not be typed for the Dra antigen, pregnancy. J Immunol 1990;144:3099-3105. but anti-Dra was identified in the eluate,with no clinical 8. Reid ME, Chandrasekaran V, Sausais L, et al. signs of HDN. Our findings differ from previous5,6,8,11 Disappearance of antibodies to Cromer blood reports, where anti-Cromer antibody titer declined group system antigens during mid pregnancy. Vox during pregnancy. Reid et al.8 suggested that the titer Sang 1996;71:48-50. decreased due to Cromer blood group system 9. Lublin DM, Thompsen ES, Green AM, et al. Dr(a–) antibodies binding to the placental DAF. In the case polymorphism of decay accelerating factor. described here, presumably the anti-Dra was not Biochemical, functional and molecular adsorbed by the placental DAF, which might happen if characterization and production of allele-specific the placental DAF was Dr(a–), if Dra was poorly transfectants. J Clin Invest 1991;87:1945-52. expressed, or due to another unexplained mechanism. 10. Daniels G: Human blood groups. 2nd ed. Oxford: Blackwell Science, 2002;444-54. Acknowledgment 11. Storry JR, Reid ME. The Cromer blood group The authors wish to thank Leah Mendelson and system: a review. Immunohematology 2002;18: Lydia Yosephi for technical assistance. 95-103.

References Naomi Rahimi-Levene MD, Director of the Blood 1. Daniels GL, Fletcher A, Garratty G, et al. Blood Bank; Abraham Kornberg MD, Chief of the group terminology 2004: from the International Hematology Institute; Gabriela Siegel, High Risk Society of Blood Transfusion committee on Pregnancy Unit; and Valery Morozov, MD, Blood terminology for red cell surface antigens. Vox Bank, Assaf–Harofeh Medical Center, Zerifin, 70300, Sang 2004;87:304-16. Israel; Eilat Shinar MD, Director; Orna Asher PhD, 2. Levene C, Harel N, Lavie G, Greenberg S, et al. A Director Immunohematology Laboratory; Cyril “new” phenotype confirming a relationship Levene MD, Consultant National Blood Group between Cra and Tca. Transfusion 1984;24:13-5. Reference Laboratory (NBGRL); and Vered Yahalom 3. Levene C,Harel N,Kende G,et al. A second Dr(a–) MD, Deputy Director & Medical Director NBGRL proposita with anti-Dra and a family with the Magen David Adom (MDA)–National Blood Services, Dr(a–) phenotype in two generations. Transfusion Ramat Gan, Israel. 1987;27:64-5.

128 IMMUNOHEMATOLOGY, VOLUME 21, NUMBER 3, 2005 COMMUNICATIONS

Letter to the Editors:

Inaugural meeting of the Consortium for Blood Group Genes (CBGG): a summary report

Over half a century ago the serologic analysis of blood group antigens established its roots in the clinical transfusion laboratory. The evaluation and laboratory investigation of alloimmunization,which occurred as a result of pregnancy or after RBC and platelet transfusion, was quickly developing into the field of immunohematology. Moreover, blood group phenotyping became a powerful tool to provide antigen-matched RBCs, to evaluate the differences in blood group antigen expression between individuals, and to infer the inheritance of the genes that express these antigens. Of course, a natural progression of the latter included the analysis of the genes and alleles that are responsible for the expression of the antigens. The ease with which molecular biological techniques became user friendly and adaptable to the clinical laboratories made this progression possible. In 1993, Bennett and associates published a landmark paper on the clinical application of molecular blood group genotyping, although their initial application was not true “genotyping”as it merely confirmed the presence of the RHD gene. The unique application used DNA derived from cells in amniotic fluid to “infer”the blood group of fetuses at risk for HDN due to anti-D,1 thus alleviating the risk associated with cordocentesis to establish the D antigen of a fetus. Today, 50 percent of pregnancies at risk for the common antibodies implicated in HDN, when the father is heterozygous for the corresponding allele, are deemed to be antigen-negative and not at risk for the disease. Clinical management varies, but essentially the mother can return to her primary care physician and noninvasive procedures can be used to monitor for fetal anemia. A decade after the publication of Bennett et al., other molecular applications are being published with unprecedented speed. The DNA analysis of blood group genes has expanded to include the following uses: 1) Genotype multi-transfused recipients, patients with severe thrombocytopenia, and those whose RBCs or platelets are sensitized with antibodies 2) Resolve ABO and Rh D discrepancies 3) Identify some of the Rh D variants that are at risk for anti-D alloimmunization 4) Determine RHD zygosity 5) Confirm the true genotype when an antigen is weakly expressed, e.g., Del 6) Identify whether the Fyb– transfusion recipient can safely receive Fyb+ RBCs 7) Mass screen for antigen-negative and rare RBCs 8) Genotype donors for antibody identification panels During the 1950s and 1960s people with a common interest in blood group serology formed focus groups to disseminate and exchange information, mentor young scientists, and advance our understanding of the field of immunohematology. So too has the study of the genes that express blood group antigens become an entity itself. The inaugural meeting of the Consortium for Blood Group Genes (CBGG) was held during the AABB annual meeting on October 23, 2004, in Baltimore, Maryland. The meeting was attended by 24 delegates from North and South America, with contributions from other colleagues who could not attend. The group was formed out of these: • A common interest in the genes that express RBC, platelet, and neutrophil antigens • A desire to promote the use of molecular technology in the clinical lab • The need for DNA reference samples • The establishment of a proficiency program and standards of practice. A summary of the meeting is provided in this report. All interested readers are encouraged to get involved and embrace the opportunity to discuss and promote scientific inquiry in blood group genes. It is anticipated that

IMMUNOHEMATOLOGY, VOLUME 21, NUMBER 3, 2005 129 COMMUNICATIONS CONT’D

clinical and scientific advancements in the study of the genes that express blood group antigens will continue as a result of global interaction. The next meeting will be held prior to the October 2005 AABB Annual Meeting in Seattle.

Consortium for Blood Group Genes 1) Language Group meetings and interactions will be conducted in English. 2) Membership Membership is open to those with an interest in single nucleotide polymorphisms and other genetic variations that are of interest to transfusion medicine, i.e., RBC, platelet, and neutrophil antigens. Interaction with other existing networking formats is encouraged, e.g.,AABB Special Interest Groups. 3) Name The name of the organization is the Consortium for Blood Group Genes (CBGG). 4) Web Site A Web site will be established in the near future. 5) Working Parties Working parties of volunteers were established to investigate, collate, and disseminate recommendations to the entire group on the areas listed below. a. Proficiency Program One role of the CBGG is to contribute to and participate in proficiency testing much like immunohematology reference laboratories (IRLs), with two samples exchanged per year. One goal of proficiency testing is to have a forum for DNA testing verification and results analysis. The working party will recommend a practical, low-cost proficiency program. b. Forms and Disclaimers Blood group genetic clinical reports were discussed and those members who provide clinical reports were asked to share copies of their reports to give a feel for the types of disclaimers they use. The group could help unify reports and set the standards for DNA blood group testing forms and reports and recommended disclaimers. The clinical forms and reports section will be addressed by a working party. c. Reference DNA samples There is a need to establish a source of samples for control and for validation purposes. It is anticipated that standard DNA reference samples will be made available as well as a listing of important DNA single nucleotide polymorphisms, amplification primers, and standard protocols. d. Standards The CBGG has the potential to develop standards for its members and act as a voice to regulatory bodies as the testing becomes more commonplace. An FDA representative has been contacted since the inaugural meeting. The group is encouraged to see an open partnership developing that will communicate needs and issues and ensure appropriate compliance.

130 IMMUNOHEMATOLOGY, VOLUME 21, NUMBER 3, 2005 COMMUNICATIONS CONT’D

e. Funding Sources of funding were discussed. Possibilities included a nominal membership fee (e.g., $100), a fee for shipping DNA from the repository, and a fee for participating in the proficiency program. Other potential sources of funding were identified. f. Structure and Bylaws The structure, bylaws, and mechanism for nominating officers to represent the consortium are in the initial stages. 1. Bennett PR, LeVan Kim C, Colin Y,Warwick RM, et al. Prenatal determination of fetal RhD type by DNA amplification. N Engl J Med 1993;329:607-10.

Greg Denomme, PhD Blood Transfusion Services, Suite 600 Mount Sinai Hospital 600 University Avenue Toronto, Ontario, Canada M5G 1X5 [email protected] Tel: 416-586-8855

Marion Reid, PhD New York Blood Center New York, NY

IMMUNOHEMATOLOGY, VOLUME 21, NUMBER 3, 2005 131 COMMUNICATIONS CONT’D

ERRATUM 1ABC

Vol. 21, No.2, 2005; page 60

Review: acute Donath-Landsteiner hemolytic anemia Set 1: Patient’s serum

The author has informed the editors of 2 ABC Immunohematology that there is an error on page 60, Fig.2. Set 3 in Fig. 2 should read “Normal serum.”

Set 2: Patient’s serum + Normal serum 3 ABC

Set 3: Normal serum ABC Ice Ice 37°C then 37°C

Reaction conditions

Fig. 2. Indirect Donath-Landsteiner test: sample conditions.

132 IMMUNOHEMATOLOGY, VOLUME 21, NUMBER 3, 2005 ANNOUNCEMENTS

Monoclonal antibodies available at no cost. The Laboratory of Immunochemistry at the New York Blood Center has developed a wide range of monoclonal antibodies (both murine and humanized) that are useful for screening for antigen–negative donors and for typing patients’ RBCs with a positive DAT. Monoclonal antibodies available include anti-M, -Fya, -Fyb, -K, -k, -Kpa, -Jsb, -Dob, -Wrb, and –Rh17. For a complete list of available monoclonal antibodies, please see our Web site at http://www.nybloodcenter.org/framesets/FS-4C7.htm. Most of those antibodies are murine IgG and, thus, require the use of anti-mouse IgG for detection, i.e, anti-K, -k, and -Kpa. Some are directly agglutinating (anti-M, -Wrb, and -Rh17), and a few have been humanized into the IgM isoform and are directly agglutinating (anti-Jsb and -Fya). The monoclonal antibodies are available at no charge to anyone who requests them. Contact: Marion Reid ([email protected]) or Gregory Halverson (ghalverson@ nybloodcenter.org), New York Blood Center, 310 East 67th Street, New York, NY 10021.

Phone, Fax, and Internet Information: If you have any questions concerning Immunohematology, Journal of Blood Group Serology and Education, or the Immunohematology Methods and Procedures manual,contact us by e-mail at [email protected] information concerning the National Reference Laboratory for Blood Group Serology, including the American Rare Donor Program, please contact Sandra Nance, by phone at (215) 451-4362, by fax at (215) 451-2538, or by e-mail at [email protected]

Attention SBB and BB Students: You are eligible for a free 1-year subscription to Immunohematology. Ask your education supervisor to submit the name and complete address for each student and the inclusive dates of the training period to Immunohematology, P.O. Box 40325, Philadelphia, PA 19106.

IMMUNOHEMATOLOGY, VOLUME 21, NUMBER 3, 2005 133 ANNOUNCEMENTS CONT’D

Masters (MSc) in Transfusion and Transplantation Sciences At The University of Bristol, England

Applications are invited from medical or science graduates for the Master of Science (MSc) degree in Transfusion and Transplantation Sciences at the University of Bristol. The course starts in October 2006 and will last for 1 year. A part-time option lasting 2 or 3 years is also available. There may also be opportunities to continue studies for PhD or MD following MSc. The syllabus is organized jointly by The Bristol Institute for Transfusion Sciences and the University of Bristol, Department of Cellular and Molecular Medicine. It includes:

• Scientific principles of transfusion and transplantation • Clinical applications of these principles • Practical techniques in transfusion and transplantation • Principles of study design and biostatistics • An original research project

Applications can also be made for Diploma in Transfusion and Transplantation Science or a Certificate in Transfusion and Transplantation Science.

The course is accredited by the Institute of Biomedical Sciences.

Further information can be obtained from the Web site:

http://www.blood.co.uk/ibgrl/MSc/MScHome.htm

For further details and application forms please contact:

Dr. Patricia Denning-Kendall University of Bristol Paul O’Gorman Lifeline Centre, Department of Pathology and Microbiology, Southmead Hospital Westbury-on-Trym, Bristol BS10 5NB, England Fax +44 1179 595 342, Telephone +44 1779 595 455, e-mail: [email protected].

134 IMMUNOHEMATOLOGY, VOLUME 21, NUMBER 3, 2005 SPECIAL ANNOUNCEMENTS

Meetings!

April 28–30 Immunohematology Reference Laboratory (IRL) Conference 2006 The American Red Cross Immunohematology Reference Laboratory (IRL) Conference will be held April 28 through 30, 2006, in Orlando, Florida. Registration forms will be available in early 2006. For more information, contact Marge Manigly at 215-451-4162, [email protected], or Cindy Flickinger at 215-451-4909, [email protected].

IMMUNOHEMATOLOGY IS ON THE WEB! www.redcross.org/pubs/immuno

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For more information or to send an e-mail Notice to Readers: Immunohematology, Journal message “To the editor” of Blood Group Serology and Education, is printed on acid-free paper. [email protected]

Attention: State Blood Bank Meeting Organizers If you are planning a state meeting and would like copies of Immunohematology for distribution, please contact Cindy Flickinger, Managing Editor, 4 months in advance, by fax or e-mail at (215) 451-2538 or [email protected].

Manuscripts: The editorial staff of Immunohematology welcomes manuscripts pertaining to blood group serology and education for consideration for publication. We are especially interested in case reports, papers on platelet and white cell serology, scientific articles covering original investigations, and papers on new methods for use in the blood bank. Deadlines for receipt of manuscripts for consideration for the March, June,September,and December issues are the first weeks in November,February,May,and August,respectively. For instructions for scientific articles, case reports, and review articles, see “Instructions for Authors” in every issue of Immunohematology or on the Web. Include fax and phone numbers and e-mail address with your manuscript.

IMMUNOHEMATOLOGY, VOLUME 21, NUMBER 3, 2005 135 ADVERTISEMENTS

NATIONAL REFERENCE LABORATORY FOR National Platelet Serology Reference BLOOD GROUP SEROLOGY Laboratory

Immunohematology Reference Diagnostic testing for: Laboratory • Neonatal alloimmune thrombocytopenia (NAIT) AABB,ARC, New York State, and CLIA licensed • Posttransfusion purpura (PTP) (215) 451-4901—24-hr. phone number • Refractoriness to platelet transfusion (215) 451-2538—Fax • Heparin-induced thrombocytopenia (HIT) American Rare Donor Program • Alloimmune idiopathic thrombocytopenia purpura (AITP) (215) 451-4900—24-hr. phone number (215) 451-2538—Fax Medical consultation available [email protected] Test methods: Immunohematology • GTI systems tests (215) 451-4902—Phone, business hours —detection of glycoprotein-specific platelet (215) 451-2538—Fax antibodies [email protected] —detection of heparin-induced antibodies (PF4 ELISA) Quality Control of Cryoprecipitated-AHF • Platelet suspension immunofluorescence test (215) 451-4903—Phone, business hours (PSIFT) (215) 451-2538—Fax • Solid phase red cell adherence (SPRCA) assay • Monoclonal antibody immobilization of platelet Granulocyte Antibody Detection and Typing antigens (MAIPA) • Molecular analysis for HPA-1a/1b • Specializing in granulocyte antibody detection and granulocyte antigen typing For information, e-mail: [email protected] • Patients with granulocytopenia can be classified or call: through the following tests for proper therapy and monitoring: Maryann Keashen-Schnell —Granulocyte agglutination (GA) (215) 451-4041 office —Granulocyte immunofluorescence (GIF) (215) 451-4205 laboratory —Monoclonal Antibody Immobilization of Granulocyte Antigens (MAIGA) Sandra Nance (215) 451-4362 For information regarding services, call Gail Eiber at: (651) 291-6797, e-mail: [email protected], Scott Murphy, MD or write to: (215) 451-4877 Neutrophil Serology Reference Laboratory American Red Cross American Red Cross Blood Services St. Paul Regional Blood Services Musser Blood Center 100 South Robert Street 700 Spring Garden Street St. Paul, MN 55107 Philadelphia, PA 19123-3594

CLIA LICENSED CLIA LICENSED

136 IMMUNOHEMATOLOGY, VOLUME 21, NUMBER 3, 2005 ADVERTISEMENTS CONT’D

IgA/Anti-IgA Testing National Neutrophil Serology Reference Laboratory IgA and anti-IgA testing is available to do the following: Our laboratory specializes in granulocyte • Monitor known IgA-deficient patients antibody detection and granulocyte antigen • Investigate anaphylactic reactions typing. • Confirm IgA-deficient donors Indications for granulocyte serology testing Our ELISA assay for IgA detects antigen to include: 0.05 mg/dL. • Alloimmune neonatal neutropenia (ANN) • Autoimmune neutropenia (AIN) For information on charges and sample • Transfusion related acute lung injury (TRALI) requirements, call (215) 451-4909, e-mail: [email protected], Methodologies employed: or write to: • Granulocyte agglutination (GA) • Granulocyte immunofluorescence by flow American Red Cross Blood Services cytometry (GIF) Musser Blood Center • Monoclonal antibody immobilization of 700 Spring Garden Street neutrophil antigens (MAINA) Philadelphia, PA 19123-3594 ATTN: Cindy Flickinger TRALI investigations also include: • HLA (PRA) Class I and Class II antibody CLIA LICENSED detection For further information contact: Reference and Consultation Services Neutrophil Serology Laboratory (651) 291-6797 Antibody identification and problem resolution Randy Schuller HLA-A, B, C, and DR typing (651) 291-6758 [email protected] HLA-disease association typing American Red Cross Blood Services Paternity testing/DNA Neutrophil Serology Laboratory 100 South Robert Street For information regarding our services, contact St. Paul, MN 55107 Mehdizadeh Kashi at (503) 280-0210, or write to: CLIA LICENSED Pacific Northwest Regional Blood Services ATTENTION: Tissue Typing Laboratory American Red Cross Notice to Readers: 3131 North Vancouver All articles published, including communications and book reviews, Portland, OR 97227 reflect the opinions of the authors and do not necessarily reflect the official policy of the CLIA LICENSED, ASHI ACCREDITED American Red Cross.

IMMUNOHEMATOLOGY, VOLUME 21, NUMBER 3, 2005 137 Immunohematology

JOURNAL OF BLOOD GROUP SEROLOGY AND EDUCATION Instructions for Authors

SCIENTIFIC ARTICLES, REVIEWS, AND CASE REPORTS 5. Acknowledgments Before submitting a manuscript, consult current issues of Acknowledge those who have made substantial contributions to Immunohematology for style. Type the manuscript on white bond the study, including secretarial assistance; list any grants. paper (8.5" × 11") and double-space throughout. Number the pages 6. References consecutively in the upper right-hand corner, beginning with the A.In text, use superscript, arabic numbers. title page. Each component of the manuscript must start on a new B. Number references consecutively in the order they occur in page in the following order: the text. 1. Title page C.Use inclusive pages of cited references, e.g., 1431–7. 2. Abstract D.Refer to current issues of Immunohematology for style. 3. Text 7. Tables 4. Acknowledgments A.Head each with a brief title, capitalize first letter of first word 5. References (e.g., Table 1. Results of ...), and use no punctuation at the end 6. Author information of the title. 7. Tables—see 7 under Preparation B. Use short headings for each column needed and capitalize first 8. Figures—see 8 under Preparation letter of first word. Omit vertical lines. C.Place explanations in footnotes (sequence: *, †, ‡, §, ¶, **, ††). Preparation of manuscripts 8. Figures 1. Title page A.Figures can be submitted either by e-mail or as photographs A.Full title of manuscript with only first letter of first word (5″×7″ glossy). capitalized (bold title) B. Place caption for a figure on a separate page (e.g.,Fig.1.Results B. Initials and last name of each author (no degrees; all CAPS), of ...), ending with a period. If figure is submitted as a glossy, e.g., M.T.JONES and J.H. BROWN place first author’s name and figure number on back of each C.Running title of ≤ 40 characters, including spaces glossy submitted. D.3 to 10 key words C.When plotting points on a figure, use the following symbols if 2. Abstract possible: ● ● ▲ ▲ ■ ■. A.One paragraph, no longer than 300 words 9. Author information B. Purpose, methods, findings, and conclusions of study A.List first name, middle initial, last name, highest academic 3. Key words—list under abstract degree, position held, institution and department, and 4. Text (serial pages) complete address (including zip code) for all authors. List Most manuscripts can usually,but not necessarily,be divided into country when applicable. sections (as described below). Results of surveys and review papers are examples that may need individualized sections. SCIENTIFIC ARTICLES AND CASE REPORTS SUBMITTED A.Introduction AS LETTERS TO THE EDITOR Purpose and rationale for study, including pertinent back- ground references. Preparation B. Case Report (if study calls for one) 1. Heading—To the Editor: Clinical and/or hematologic data and background serology. 2. Under heading—title with first letter capitalized. C.Materials and Methods 3. Text—write in letter format (paragraphs). Selection and number of subjects, samples, items, etc. studied 4. Author(s)—type flush right; for first author: name, degree, and description of appropriate controls, procedures, methods, institution, address (including city, state, ZIP code, and country); equipment, reagents, etc. Equipment and reagents should be for other authors: name, degree, institution, city, and state. identified in parentheses by model or lot and manufacturer’s 5. References—limited to ten. name, city, and state. Do not use patients’ names or hospital 6. One table and/or figure allowed. numbers. D.Results Presentation of concise and sequential results, referring to perti- nent tables and/or figures, if applicable. E. Discussion Implications and limitations of the study,links to other studies; Send all manuscripts by e-mail to: if appropriate,link conclusions to purpose of study as stated in Marge Manigly at [email protected] introduction.

138 IMMUNOHEMATOLOGY, VOLUME 21, NUMBER 3, 2005 Becoming a Specialist in Blood Banking (SBB)

What is a certified Specialist in Blood Banking (SBB)? • Someone with educational and work experience qualifications who successfully passes the American Society for Clinical Pathology (ASCP) board of registry (BOR) examination for the Specialist in Blood Banking. • This person will have advanced knowledge, skills, and abilities in the field of transfusion medicine and blood banking.

Individuals who have an SBB certification serve in many areas of transfusion medicine: • Serve as regulatory, technical, procedural, and research advisors • Perform and direct administrative functions • Develop, validate, implement, and perform laboratory procedures • Analyze quality issues, preparing and implementing corrective actions to prevent and document issues • Design and present educational programs • Provide technical and scientific training in blood transfusion medicine • Conduct research in transfusion medicine

Who are SBBs? Supervisors of Transfusion Services Managers of Blood Centers LIS Coordinators Educators Supervisors of Reference Laboratories Research Scientists Consumer Safety Officers Quality Assurance Officers Technical Representatives Reference Lab Specialist

Why be an SBB? Professional growth Job placement Job satisfaction Career advancement

How does one become an SBB? • Attend a CAAHEP-accredited Specialist in Blood Bank Technology Program OR • Sit for the examination based on criteria established by ASCP for education and experience Fact #1: In recent years, the average SBB exam pass rate is only 38%. Fact #2: In recent years, greater than 73% of people who graduate from CAAHEP-accredited programs pass the SBB exam.

Conclusion: The BEST route for obtaining an SBB certification is to attend a CAAHEP-accredited Specialist in Blood Bank Technology Program Contact the following programs for more information: PROGRAM CONTACT NAME CONTACT INFORMATION Walter Reed Army Medical Center William Turcan 202-782-6210; [email protected] Transfusion Medicine Center at Florida Blood Services Marjorie Doty 727-568-5433 x 1514; [email protected] Univ. of Illinois at Chicago Veronica Lewis 312-996-6721; [email protected] Medical Center of Louisiana Karen Kirkley 504-903-2466; [email protected] NIH Clinical Center Department of Transfusion Medicine Karen Byrne 301-496-8335; [email protected] Johns Hopkins Hospital Christine Beritela 410-955-6580; [email protected] ARC-Central OH Region, OSU Medical Center Joanne Kosanke 614-253-2740 x 2270; [email protected] Hoxworth Blood Center/Univ. of Cincinnati Medical Center Catherine Beiting 513-558-1275; [email protected] Gulf Coast School of Blood Bank Technology Clare Wong 713-791-6201; [email protected] Univ. of Texas SW Medical Center Barbara Laird-Fryer 214-648-1785; [email protected] Univ. of Texas Medical Branch at Galveston Janet Vincent 409-772-4866; [email protected] Univ. of Texas Health Science Center at San Antonio Bonnie Fodermaier SBB Program: 210-358-2807, Linda Smith [email protected] MS Program: 210-567-8869; [email protected] Blood Center of Southeastern Wisconsin Lynne LeMense 414-937-6403; [email protected] Additional information can be found by visiting the following Web sites: www.ascp.org, www.caahep.org, and www.aabb.org

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