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This Issue ofImmunohematology

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ImmunohematologyJ O U R N A L O F B L O O D G R O U P S E R O L O G Y A N D E D U C A T I O N

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C O N T E N T S

85Review: the burgeoning history of the complement system 1888–2005

H. CHAPLIN JR.

94On a much higher than reported incidence of anti-c in R1R1 patients with anti-E

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

97Case 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. NESTER

102Is there a relationship between anti-HPA-1a concentration and severity of neonatal alloimmune

thrombocytopenia?H. BESSOS, M.TURNER,AND S.J. URBANIAK

109Review: complement receptor 1 therapeutics for prevention of immune hemolysis

K.YAZDANBAKHSH

119Autoimmune hemolytic anemia and a further example of autoanti-Kpb

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

122The incidence of red cell alloantibodies underlying panreactive warm autoantibodies

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

126Persistent anti-Dra in two pregnancies

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

C O M M U N I C A T I O N S

129 132Letter to the Editors ERRATUM

Inaugural meeting of the consortium for blood Vol. 21, No. 4, 2005, p.60group genes (CBGG): a summary report

G. Denomme and M. Reid

133 136A N N O U N C E M E N T S A D V E R T I S E M E N T S

138I N S T R U C T I O N S F O R A U T H O R S

EDITOR-IN-CHIEF MANAGING EDITORDelores Mallory, MT(ASCP)SBB Cynthia Flickinger, MT(ASCP)SBB

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Philadelphia, Pennsylvania Philadelphia, Pennsylvania Philadelphia, Pennsylvania

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Patricia Arndt, MT(ASCP)SBBPomona, California

James P.AuBuchon, MDLebanon, New Hampshire

Geoffrey Daniels, PhDBristol, United Kingdom

Richard Davey, MDWashington, District of Columbia

Sandra Ellisor, MS, MT(ASCP)SBBAnaheim, California

George Garratty, PhD, FRCPathPomona, California

Brenda J. Grossman, MDSt. Louis, Missouri

W. John Judd, FIBMS, MIBiolAnn Arbor, Michigan

Christine Lomas-Francis, MScNew York, New York

Gary Moroff, PhDRockville, Maryland

Ruth Mougey, MT(ASCP)SBBCarrollton, Kentucky

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Marion E. Reid, PhD, FIBMSNew York, New York

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David F. Stroncek, MDBethesda, Maryland

Marilyn J.Telen, MDDurham, North Carolina

Connie M.Westhoff, SBB, PhDPhiladelphia, Pennsylvania

The complement system first came to the attentionof scientists following work by Koch and Pasteur ontransmissible diseases in the latter half of the 19thcentury. Leading microbiologists such as Paul Ehrlich,Jules Bordet, and George Nuttall were stimulated tolearn more about the human body’s mechanisms ofprotection against infectious disease. In 1888, Nuttallnoted that sheep blood was mildly bactericidal foranthrax bacilli, but that this property disappearedrapidly if the blood was heated to 55°C or kept at roomtemperature. The heat-labile bactericidal activity wasnamed “alexin.”

In 1894, other laboratories demonstrated thatserum from guinea pigs that had recovered fromcholera would protect normal guinea pigs from choleraif it was injected along with the live cholera bacteria.Importantly, it was shown by in vitro studies that thecholera bacteria were only killed by fresh immuneserum, and not by heat-inactivated immune serum.However, it was noted that the heat-inactivatedimmune serum would protect normal guinea pigs fromexposure to cholera bacteria. Bordet subsequentlyshowed that the activity of the heat-inactivated serumcould be restored in vitro by the addition of a smallamount of fresh normal serum that by itself lackedbactericidal activity. These results indicated that thebactericidal activity depended on both a heat-stablesensitizing substance in immune serum and a heat-labile cytotoxic factor present in normal (as well asimmune) serum. Bordet attributed the heat-labileactivity to the alexin bactericidal factor. In 1899,Ehrlich put forward a humoral immunity schemesubstituting the terms “complement” for alexin and“amboceptor” for the heat-stable immune sensitizer.

It soon became evident that complement consistedof more than two serum components. By the 1920s itwas concluded that that there were at least four serum

fractions responsible for the complement activities ofserum. To maintain order in this proliferation ofcomplement components, each factor was assigned anumber in the order in which it was discovered, i.e.,C1,C2,C3,and C4. It later became clear that the factorsreacted sequentially, but not in the order of theirdiscovery. In joint international meetings it wasdecided to not change the numbers already assigned tonewly defined complement factors, but rather todisplay their order of reactivity as this evolved fromongoing research. Thus by the late 1930s, the reactivitysequence for the first four complement factors wasshown as: C1 → C4 → C2 → C3.

The complement story had been developed fromthe perspective of infectious disease, i.e., that it waspart of the safety net that protected humans from theravages of pathogenic microorganisms (bacteria, fungi,viruses). Thus it was postulated that the complementsystem was activated by the body’s response to attackby pathogenic microorganisms, presumably by pro-duction of immune reactants (antibodies) against theattacking microorganisms. The starting point of thecomplement response presupposed an infection-related activator. Because the protective response washypothesized from the infectious disease perspective(earlier research had validated that hypothesis), the C1 → C4 → C2 → C3 sequence was dubbed the“classical pathway” of entry into the complementsystem.

However, it had been noticed that certain strains ofbacteria and yeast could activate the complementsystem without having induced antibodies. In 1913, itwas shown that cobra venom would mediatecomplement-like RBC lysis in the absence of immuneantibodies. These studies suggested that this involvedserum components that differed from those of theclassical pathway. In fact, these findings presaged what

I M M U N O H E M A T O L O G Y, V O L U M E 2 1 , N U M B E R 3 , 2 0 0 5 85

Review: the burgeoning history of the complement system1888–2005H. CHAPLIN JR.

40 years later would be named the “alternativepathway” based on work by other researchers. Theyincluded Dr.Louis Pillemer,who described a previouslyunidentified serum protein named “properdin,” whichwas said to bind to bacteria and yeast, activatingcomplement without requiring the presence of anti-body. In 1957, his claim to have discovered a“properdin pathway” to complement activation wasdenounced by most contemporary complementol-ogists on the grounds of serious technicalinadequacies. Pillemer was deeply hurt by the scathingcriticism. He became distraught over the damage doneto his personal and professional reputation, and tookhis own life several months later. The tragedydeepened when subsequent work proved Pillemer’soriginal findings to have been correct. By 1967,properdin had been reinstated as an important factor inalternative pathway complement activation.

Distinctive Differences Between theClassical and Alternative Pathways ofComplement Activation

As we have seen, the classical pathway depends onactivation by antigen-specific antibodies reacting withthe four principal complement factors (C1, C2, C3, andC4). Table 1 (adapted from Pangburn’s chapter on thealternative pathway) provides numerous examples ofpathogens and particles of microbial origin, as well asnonpathogens, which can activate the six proteincomponents of the alternative pathway without priorimmunization to their specific antigens (Table 2, alsofrom Pangburn). Five of these proteins are unique tothe alternative pathway; only C3 is common to both

pathways. Since none of these activators requires priorstimulation of antigen-specific antibodies, thealternative pathway offers immediate response tocertain serious infections, to amelioration of acutesymptoms of dangerous immune complex syndromes,and to amplification of available C3b to play itsamboceptor role when a rapid response is required.

The exact mechanism by which the first metastablemolecules of C3 are produced is not yet absolutelycertain. According to Pangburn,1 the consensus is thatthere is spontaneous hydrolysis of the thioester groupin native C3, thereby leading to C3b deposition notonly on the target pathogen but also indiscriminatelyon all particles, including the host’s own cells. A systemof control factors rapidly inactivates the C3b moleculesbound to host cells or other nonactivators, but thesecontrol factors work very slowly on C3b bound toparticles recognized by the system as proper sites ofactivation. C3b on such activating particles forms anenzyme that can function like the C3/C5 convertase ofthe classical pathway. It activates C3 and deposits C3bmolecules onto the surface being attacked. An

86 I M M U N O H E M A T O L O G Y, V O L U M E 2 1 , N U M B E R 3 , 2 0 0 5

H. CHAPLIN JR.

Table 1. Activators of the alternative pathway*

Pathogens and particles of microbial origin Nonpathogens

*Reprinted with permission from Pangburn.1

Many strains of gram-negativebacteria

Lipopolysaccharides from gram-negative bacteria

Many strains of gram-positivebacteria

Teichoic acid from gram-positivecell walls

Fungi and yeast cell walls(zymosan)

Some viruses and virus-infectedcells

Some tumor cells (Raji)

Parasites (trypanosomes)

Human IgG, IgA, and IgE incomplexes

Rabbit and guinea pig IgG incomplexes

Cobra venom

Heterologous erythrocytes (rabbit,mouse, chicken)

Anionic polymers (dextran sulfate)

Pure carbohydrates (agarose,insulin)

Table 2. Proteins of the alternative pathway*

Serum Molecular concentration

Symbol Name Functions weight (µg/mL)

C3 C3 Attaches covalently 185,000 1200after proteolytic activation generates metastable C3b. C3b fragment is part of C3/C5 convertase.

B Factor B Binds to C3b forming 93,000 200the precursor of the C3/C5 convertase (C3b,Bb). Bb subunit of this complex is a serine protease.

D Factor D Serine protease that 24,000 1activates factor B when B is in complex with C3b.

H Factor H Regulator (negative) 150,000 560that inactivates the C3/C5 convertase by dissociating its subunits.Also a cofactor for factor 1.

I Factor I Regulator (negative) 88,000 34that is a serine protease.Inactivates C3b with the aid of factor H or the C3b receptor (CR1).

P Properdin Regulator (positive) 224,000 20that enhances activation by binding to and stabilizing the C3/C5 convertase.

*Reprinted with permission from Pangburn.1


History of complement system

amplification process now comes into play. Each newC3b bound to this surface can form C3/C5 convertasethat can deposit more C3b, and the process repeatsitself until the surface is saturated or the supply ofcomplement component is depleted. By thismechanism the alternative pathway of complementactivation is capable of depositing more than twomillion C3b molecules onto the surface of a particle inless than 5 minutes after contact!

The confluence of the classical and alternativepathways is illustrated in Figure 1. This highlysimplified diagram emphasized that once the twopathways reached the point where their respective C3convertases provided C3b to the ongoing system,progression through C5 to C9 activation proceeded ona shared final pathway to completion.

New Technologies Provide FundamentalNew Knowledge

It is not within the province of this review todescribe details of the elegant exploitation of newtechnologies for protein purifications, recombinantDNA technology, identification of genetic location,cloning of genes for various complement components,transfection, and production of purified gene products,use of knock-out animal models to verify functionalcapacities of gene prod-ucts, and many more.

Research in thecomplement field acceler-ated and diversified rapidlyduring the second half ofthe 20th century. Table 3summarizes the Interna-tional Complement Work-shops from 1963 to 1993.The 1963 meeting at theNational Institutes ofHealth in the United Stateswas attended by 25scientists; 15 manuscriptswere eventually published.A meeting in Cambridge inthe United Kingdom in1991 had more than 320attendees; 320 abstractswere published. In Rotherand Till’s book, TheComplement System, pub-lished in 1988,4 one

16-page chapter on complement components cited208 references, while a 39-page chapter on geneticsand polymorphism of complement components cited380 references.

Among other accomplishments during this era wasidentification of individual cells and tissues in whichcomplement components were produced. Theyproved to be more diverse than expected. While themain site of synthesis was the hepatocytes in the liver,another prolific site was the fimbriae of the Fallopiantubes. In vitro studies showed that a great many cellscould make complement components, e.g., myeloidcells, macrophages, intestine, bladder, skin, lungs, andmany others.

As understanding of the complement systemexpanded during the explosion of new knowledgedescribed in an earlier section, an increasing awarenessdeveloped of the important benefits humanity reapsfrom the system as well as the potential for seriousnegative consequences if all, or parts, of the systembreak down or function out of control. Individualsborn with deficiencies of various complementcomponents may become chronic invalids, their lifespans greatly shortened, vulnerable to fulminantinfections, and possibly more susceptible to certainmalignancies. The complexity of the complementsystem, its access to every tissue in the body via its

Table 3. International complement workshops*

Number of published

Workshop Date Location abstracts Reference

1 February 28–March 1, 1963 National Institutes of Health, Summary (44)Bethesda, MD only

2 January 17–19, 1966 La Jolla, CA 30 (45)

3 June 3–5, 1968 Harvard Medical School, 30 (46)Boston, MA

4 January 27–29, 1971 Johns Hopkins University, 39 (47)Baltimore, MD

5 February 23–25, 1973 Hotel Del Coronado, 68 (48)Coronado, CA

6 November 23–25, 1975 Sarasota, FL 101 (49)

7 November 20–22, 1977 St. Petersburg Beach, FL 137 (50)

8 October 14–16, 1979 Key Biscayne, FL 140 (51)

9 November 22–25, 1981 Key Biscayne, FL 177 (52)

10 May 25–27, 1983 Mainz, Germany 214 (53)

11 November 3–5, 1985 Key Biscayne, FL 266 (54)

12 September 18–21, 1987 Chamonix, France 320 (55)

13 September 10–15, 1989 San Diego, CA 308 (56)

14 September 15–20, 1991 Cambridge, UK 320 (57)

15 September 27–October 1, 1993 Kyoto, Japan (planned)

*Reprinted with permission from Weiler (references in table refer to original paper).3

location within the circulatory system, and theextraordinary speed with which it can react toperturbations require clinical investigators to proceedwith great caution in efforts to manipulatecomplement. Well-intentioned efforts to achievedesirable ends must be guided by a full understandingof undesirable consequences that could follow fromupsetting the delicate balance that undergirds its vitalroles on behalf of human survival.

Regulation of the Complement SystemFor every stage of activation of the classical

pathway there are regulatory proteins which bringabout rapid destruction of the activated factors,thereby preventing complete consumption of plasmaC4 and C2 in the fluid phase. Rapid inactivation of C1to prevent uncontrollable activation of complement isbrought about by the nonenzymic protein, C1inhibitor, a heavily glycosylated 100,000-Da proteinnormally present in plasma at a concentration of 100 to150 µg/mL. C1 inhibitor combines with a catalytic siteon the light chains of C1r and C1s, causing them todissociate from C1, leaving C1q bound to the targetmembrane. This sequence of events occurs veryrapidly (in 10–20 sec), thus limiting further C1activation. Bound C42 (the C3/C5 convertase) iseliminated in two phases. First, C2 dissociatesspontaneously from C4, a process which is promotedby C4-binding protein and decay-accelerating factor(DAF; CD55), which is found within the membranes ofRBCs and many different cell types. In the secondphase, residual bound C4b is further degraded intoC4c, which dissociates from the target membrane,leaving only C4d attached to the target cell membrane.This degradation is brought about by the enzymeFactor I, aided by C4BP and the C3 receptor CR1.Factor I also cleaves bound C3b. Once C3 is inactivatedit can no longer bind C5, hence further production ofthe membrane attack complex is prevented. This singleexample of inhibitor-mediated regulation illustrates theintricacy of regulatory processes within thecomplement area.

At the time of activation of C5, the C5aanaphylatoxin fragment is released, with the effects onneutrophils and monocytes shown in Table 4.Activated C5 attaches to the membrane of the targetcell, where it binds stoichiometrically to C6, serving asan acceptor for C7, leading finally to assembly of thecompleted membrane attack complex (MAC). TheMAC inserts itself into the membrane, bringing about

lysis of the target cell via the formation of circular“pores” varying from 20 to 100 Å in diameter.

Complement Mediators of Inflammation—the Anaphylatoxins

Among the many protective roles played by thecomplement system is its mobilization of the body’sinflammatory components in response to infectionsand to tissue damage from any cause. Key players in thisarena are the anaphylatoxins, low-molecular-weightfragments derived from complement factors C3, C4,and C5. All of these polypeptides contribute to spasmo-genicity, i.e., they promote smooth muscle contractionand increase vascular permeability. These biologicalproperties have been shown to induce anaphylaxis incertain test animals. The biochemical compositions ofthese polypeptides are very similar, and one of theanaphylatoxins, C5a, plays a leading role in the host’sinflammatory response. Each of the anaphylatoxins iscleaved from the amino terminus of the alpha chain ofits parent molecule by a specific convertase enzymeformed during complement activation. The fragmentsare designated C3a, C4a, and C5a, and when releasedinto the plasma, C3a and C4a are rapidly converted bythe enzyme serum carboxypeptidase to their inert des-Arg analogs. C5a is largely catabolized afterinternalization by its target cells. Note the complexityof Figure 2 and compare its complexity with thesimplicity of Figure 1. The “lectin pathway” has beenadded to the classical and alternative pathways. Lectinsare nonimmune carbohydrate-recognizing moleculesfound in plants and animals. Two lectins of humanorigin are conglutinin and mannose-binding protein,shown in Figure 2 as mannose-binding lectin (MBL). Forexample, MBL can activate the complement systemwhen it binds to mannose receptors on certain bacterialand virus surfaces. Yet even the pathway depicted inFigure 2 is simpler than reality because it does not


H. CHAPLIN JR.

Table 4. Granulocyte-related activities of human C5a*

Target cells Cellular responses

Neutrophils Augmented adherence

Monocytes Cellular polarization

Chemotactic migration

Degradation and enzyme release

Production of toxic oxygen metabolites

Enhanced arachidonic acid metabolism

Augmented expression of complement receptors

Increased elaboration of IL-1 by monocytoid cells

*Reprinted with permission from Chenoweth.5



include many other complement inhibitors and thereceptors for many complement factors andbyproducts.

The biological activities of the anaphylatoxins areillustrated in Tables 4 and 5. It is clear from Table 5 thatthe spasmogenic properties (smooth muscle contrac-tion and vascular permeability) of C5a are far morepotent than those of C3a and C4a. The samepredominance of C5a over C3a and C4a was observedfor the granulocyte- and monocyte-related activitieslisted in Table 4, justifying the reputation of C5a as amajor anaphylatoxin inflammatory mediator. Asadmirable as C5a’s superiority appears in this generalcontext, Chenoweth5 cautions that it must be rec-ognized that C5a may also act systemically and triggersimilar types of granulocytic responses in blood ordistant organs. For example, when this occurs duringcomplement-induced pulmonary vascular leukoseques-tration, C5a-activated granulocytes release cytotoxicsubstances that destroy normal tissues. In this case,and in certain autoimmune disorders, normal hostdefense mechanisms may actually contribute to thepathogenesis of a specific disease, such as adultrespiratory distress syndrome or rheumatoid arthritis.

C5a initiates a wide variety of responses (Table 4)via binding to receptors that are known to be locatedon target cells (peripheral blood neutrophils andmonocytes, alveolar macrophages, and cultured cells ofmonocytoid origin). Characterization of thesereceptors utilized radio-iodinated (125IC5a) orfluorescein-labeled human C5a (fl-C5a). The C5areceptors of the previously described cells and theplasma membrane fraction isolated from these cells arefunctionally indistinguishable.

At the time Chenoweth was writing, otherinvestigators were attempting to define more preciselythe “recognition domain” determinants of C5a becausepeptide or chemical analogs of the regions couldpossibly act as competitive antagonists to C5a bindingand thus function as anti-inflammatory compounds.Work in progress included similarly directed studies ofreceptors for CR2, CR3, CR4, H-R, C1q-R, C3a/C4a-R,and CR3e-R. Also under way were similar studies ofopsonic IgG, opsonic C3, opsonic C4, and fibronectin.Other studies were being directed at CR1 membranecomplement receptors on human, sheep, rabbit,mouse, rat, and guinea pig RBCs. Identification andcharacterization of complement receptors onneutrophils, monocytes, lymphocytes, Kupfer cells,phagocytes, and platelets goes on apace.

Fig.1. Activation pathways of the complement system—mid-20thcentury*

*Reprinted with permission from Ross.2

Fig. 2. Activation pathways of the complement system—late-20thcentury*

*Reprinted with permission from Sahu et al.6

Relation of Complement Components toBlood Group Serology

Not to be outdone by the flourishing worldwidecommunity of blood group specialists, comple-mentologists became card-carrying blood groupspecialists themselves with the announcement in 1978by O’Neill et al.7 that the Chido/Rodgers blood groupantigens were in fact distinct antigenic components ofhuman complement C4. The subject is well chronicledin Blood Transfusion in Clinical Medicine byMollison, Engelfriet, and Contreras.8 Since the antigensare generally detectable in normal human plasma, it isassumed that their presence on RBC membranesreflects adsorption from the individual’s plasma. TheCh and Rg determinants are localized to chromosome6. Anti-Ch and anti-Rg are found only in Ch- or Rg-negative subjects. RBCs of the relatively rare C4 nullpatients are Ch- and Rg-negative. Fortunately,incompatibility within the Ch/Rg system has notcaused documented hemolytic transfusion reactions orHDN, although there have been rare reports ofanaphylactic symptoms in multi-transfused Ch- or Rg-negative recipients of platelet concentrates or plasmatransfusions.

As a more contemporary example of thecomplement system’s encroachment on the bloodbank serologist’s turf, elucidation of the clinical role ofthe decay-accelerating factor (DAF;CD55) glycoproteinassociated with the Cromer blood group system hasmade important progress in recent years. Like theCh/Rg antigens, the Cromer antigens are widelydetectable on multiple human cells: RBCs, WBCs,platelets,and endothelial and epithelial tissues. Lublin’sgroup9 at Washington University in St. Louis hasrecently completed a detailed definition of themolecular basis of nine Cromer antigens. The Cromersystem antigens have been shown to reside on the DAFprotein, one of the regulators of the complement

system and hence one of the protectors ofautologous cells and tissues from complement-mediated damage. Antibodies to Cromer systemantigens could lead to RBC destruction, buthemolytic transfusion reactions and HDN havenot been reported. However, it is necessary toidentify antibodies in the Cromer system duringpretransfusion testing.

One can expect a major expansion ofcollaborative research in the overlapping areas ofcomplement physiology and transfusionmedicine.

The Role of Complement in ImmuneDestruction of Transfused RBCs and inAutoimmune Hemolytic Anemias

Thus far, this review has focused on the role ofcomplement in protection against infectious diseases.However, it has also shown that there is a downside tothe complement story.

For many decades, the human RBC has been wellknown as a victim of complement activity, asexemplified by complement’s noxious contribution tohemolytic transfusion reactions and its frequent role inautoimmune hemolytic anemias (AIHAs). In thediagnosis and management of both of these life-threatening illnesses, it has challenged the skills oflaboratory technicians, manufacturers of specificantiglobulin reagents, and molecular biologyresearchers. Complement can be activated by naturallyoccurring antibodies (e.g., anti-A and anti-B in normalgroup O, A, and B blood donors) as well as by bloodgroup alloantibodies formed in response to multipletransfusions or pregnancies (e.g.,antibodies to antigenswithin the Kidd, Duffy, and Kell systems). In thesecircumstances, blood group alloantibodies that activatecomplement can provoke more serious hemolyticreactions, sometimes with rapid intravascular RBCdestruction and accompanying hemoglobinuria.

Complement can play an analogous role in manyAIHAs (e.g., the biphasic anti-P Donath-Landsteinerautohemolysin in paroxysmal cold hemoglobinuria).Complement’s role is supported by the frequentfinding of strong reactivity with anti-C3d reagentsaccompanying strong reactivity with anti-IgG reagentson the RBCs of some patients with severe AIHAs.

The extensive literature on the role of complementin immune destruction of human RBCs is wellreviewed in Blood Transfusion in Clinical Medicineby Mollison, Engelfriet, and Contreras.8


H. CHAPLIN JR.

Table 5. Spasmogenic properties of the human anaphylatoxins*

Biologicalresponse C5a des-Arg74-C5a C3a C4a

Smooth muscle contraction

Ileum 5.0 × 10–10 1.2 × 10–6 1 × 10–8 1.5 × 10–6

Uterus 7.5 × 10–10 1 × 10–8

Vascular 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

Human 0.1–1.0 × 10–13 0.01–1.0 × 10–10

* Reprinted with permission from Chenoweth.5

Effective dose (M)



The Role of Complement in the RheumaticDiseases

As noted earlier, the “normal” functions of thecomplement system may sometimes become a double-edged sword. Autoantibodies and circulating immunecomplexes in certain rheumatic diseases may activatethe complement system, thereby enhancing comple-ment’s “good” pro-inflammatory response but leadingto further tissue destruction. The same undesiredeffect may lead to deposition of immune complexes inthe walls of blood vessels (vasculitis), resulting innecrosis of vessels. When immune complex vasculitisoccurs in the kidneys, lungs, skin, or other organs,patients may become seriously ill.

Therapeutic complement inhibitor approacheshave been considered for treatment of bullouspemphigus, rejection of transplanted tissues,Alzheimer’s disease (because plaques contain highlevels of classical and alternative pathway componentsas well as MAC components), immune-based fetal loss, HIV, and a great many other serious medicalconditions. Other disorders that interface importantlywith the complement system (including systemic lupus erythematosus, rheumatoid and relatedarthritides,and cryoglobulinemia) are well reviewed byAtkinson et al.10

Looking ahead, careful attention is being given toimportant questions that need further exploration.Where in the three activation pathways (classical,alternative, lectin) should the various inhibitors beapplied: prior to or coincident with the shared C3bstep? Can local,systemic,or both therapeutic strategiesbe predictably and safely controlled? Should researchemphasis focus on recombinant or nonrecombinantagents? What important proteins outside thecomplement system (e.g., clotting factors) could alsobe inhibited unintentionally? How would theeffectiveness of complement inhibitors stack upagainst other pro-inflammatory measures? There areseveral major concerns regarding all of the aboveissues: what would be the impact of each with respectto increased risks of autoimmunization,11 and howmight each alter the self-tolerance of B cells12?

The Role of Complement in ParoxysmalNocturnal Hemoglobinuria

The rapid broadening of understanding of thecomplement system has promoted improved under-standing of the pathogenesis of many diseases, bothcommon (e.g., rheumatoid arthritis) and rare (e.g.,

paroxysmal nocturnal hemoglobinuria [PNH]). Newunderstanding has given rise to imaginative comple-ment research designed to improve the lives ofindividuals suffering from these ailments. As with anyinnovative development at the start, enthusiasticpredictions and high expectations abound. Whileunanticipated difficulties may delay or even derailsome of these efforts, some important goals have beenachieved.

PNH is an example of a highly successful laboratoryand clinical research effort. A recent article in the NewEngland Journal of Medicine13 describes a study basedin Leeds, England, on eleven transfusion-dependentadult patients (six men and five women) with well-documented PNH, a disorder arising as a result of asomatic mutation of the PIG-A gene in a hematopoieticstem cell. The mutation results in the subsequentproduction of blood cells with a deficiency of the DAFproteins that protect normal RBCs against attack by thecomplement system. In a well-designed, well-standardized, and well-executed 12-week clinical andlaboratory study, the effects of intravenous infusions ofeculizumab, a humanized antibody that inhibits theactivation of terminal complement components C5 toC9, were examined. If effective, the drug would beexpected to reduce RBC hemolysis sufficiently toeliminate the hemoglobinuria, reduce or eliminate thetransfusion dependency, and relieve troublesomesymptoms related to the chronic hemolysis. Well-chosen laboratory tests to quantitate the rate ofhemolysis were performed at regular intervals, as wasquantitation of the numbers of abnormal RBCs in thecirculation and visual daily quantitation of the urine fordetectable hemoglobinuria. The standardized inter-view of the European Organization for Research andTreatment of Cancer (EORTC), titled the QLQ-30, wasemployed periodically to evaluate quality of life.

Patient compliance was excellent. No evidence offormation of antibodies to the eculizumab was found,and no untoward drug-related side effects weredetected. Laboratory results documented significantreduction in the rate of hemolysis (though not totalnormalization, and the Hb level did not rise).Paroxysms of hemoglobinuria were markedlydecreased. Regarding quality of life, there wassignificant improvement in the domains of globalhealth (p = 0.02), physical functioning (p < 0.001),emotional functioning (p < 0.001), cognitivefunctioning (p = 0.002), fatigue (p < 0.001),dyspnea (p= 0.002), and insomnia (p = 0.049).

All the patients enlisted in a 12-month extension ofthe study, with no ill effects noted and with sustainedimprovement in their symptoms and laboratorystudies. The authors conclude: “Eculizumab appears toenhance the survival of type III PNH erythrocytes,improving the quality of life and reducing the extent ofhemolysis, hemoglobinuria (the clinical hallmark ofPNH), and the need for blood transfusions in patientswith PNH. This study confirms that terminalcomplement activation is the key mediator oferythrocyte destruction in PNH.”

The author of the present review strongly encour-ages readers to enjoy this exemplary publication for itslucidity and inspirational value.

The Emerging Role of ComplementInhibitors in Immunohematology

The PNH article cited in the previous sectiondescribes primarily a clinical trial of a candidate drugto add to the therapeutic armamentarium of trans-fusion medicine. As a fitting finale to the presenthistorical review of the complement system, I wouldlike to summarize briefly an ongoing “work inprogress” at the New York Blood Center by a researchgroup under the direction of Karina Yazdanbakhsh.The titles of three recent publications from herlaboratory track the program’s progress during the past3 to 4 years. The title of the first paper, published in2003, is “Complement receptor 1 inhibitors forprevention of immune-mediated red cell destruction:potential use in transfusion therapy.”14 The paperdescribes preparation of recombinant human CR1from which several truncated soluble CR1 proteinswere expressed and tested for their complementinhibitory properties. Two of the derivatives wereselected for in vivo inhibition of complement-mediatedimmune hemolysis in a xenotransfusion mouse modeland for lowering the deposition of C3 and C4 on thetarget RBCs. The second paper, published in 2004 andtitled “Prevention of complement-mediated immunehemolysis by a small-molecule compound,”15 describesthe screening of a commercial 10,000 compoundlibrary enriched in anti-inflammatory molecules, fromwhich a single 549-Da compound was selected on thebasis of its ability to reduce hemolysis induced via theclassical and alternative pathways. In a xenotrans-fusion mouse model, significantly prolonged RBCsurvival was observed, as was reduced C3 depositionon the surviving RBCs. The findings support thepossibility that the small-molecule complement

compound could be useful in a human transfusionsetting, The third publication, “Characterization ofcomplement receptor-1 domains for prevention ofcomplement-mediated red cell destruction,”16 waspublished in 2005. This paper, building on the findingsof the two preceding papers, chronicles further thesearch for even smaller soluble CR1 inhibitors. A250–amino acid region in CR1 that possessedantihemolytic activity and was effective at prolongingsurvival of transfused RBCs was identified. Mutation oftwo critical residues in this 250–amino acid domainresulted in improved complement-inhibitory functionscompared to the unmutated CR1 derivatives, resultingin a more potent inhibition of complement activationin vitro. However, in vivo, the activity of the mutantprotein was comparable to the wild type molecules.These studies indicated the importance of testing CR1inhibitors in vivo.

Taken together, the three careful and sophisticatedstudies indicate the increased precision achievablewhen using today’s scientific technology to explorestructure/function relationships in evaluation ofcomplement inhibitors.

The Index Medicus lists more than 40,000publications under “complement” in the past 39 years.Complement system research continues to grow.Enjoy it!

AcknowledgmentThe author expresses special appreciation to

colleagues Prof. John P. Atkinson and Sir Peter J.Lachmann for their wise advice and welcomeencouragement.

References1. Pangburn MK. The alternative pathway. In: Ross

GD, ed. Immunobiology of the complementsystem. New York: Academic Press, Inc., 1986:46-7, 57.

2. Ross GD. Introduction. In: Ross GD, ed.Immunobiology of the complement system. NewYork:Academic Press, Inc., 1986:13.

3. Weiler JM. Introduction. In: Whaley K, Loos M,Weiler JM. Complement in health and disease. 2nded. Dordrecht: Kluwer Academic Publishers,1993:5.

4. Rother K, Till GO. The complement system.Berlin: Springer-Verlag, 1988:61, 119.

5. Chenoweth DE. Complement mediators ofinflammation. In: Ross GD, ed. Immunobiology of


H. CHAPLIN JR.



the complement system. New York: AcademicPress, Inc., 1986:72-3, 75.

6. Sahu A, Morikis D, Lambris JD. Complementinhibitors targeting C3,C4,and C5. In:Lambris JD,Holers VM. Therapeutic interventions in thecomplement system. Totowa, NJ: Humana Press,2000:76.

7. O’Neill GJ, Yang SY, Tegoli J, et al. Chido andRodgers blood groups are distinctive antigeniccomponents of human complement C4. Nature1978;273:668-70.

8. Mollison PL, Engelfriet CP, Contreras M. Bloodtransfusion in clinical medicine. 10th ed. Oxford:Blackwell Science, 1997:205.

9. Lublin DM, Kompelli S, Storry JR, et al. Molecularbasis of Cromer blood group antigens.Transfusion 2000;40:208-13.

10. Atkinson JP, Kaine JL, Holers VM, et al.Complement and the rheumatic diseases. In: RossGD, ed. Immunobiology of the complementsystem. New York:Academic Press, Inc., 1986:197-211.

11. Boackle SA,Holers VM.Role of complement in thedevelopment of autoimmunity. In:Nemazee D,ed.B cell biology in autoimmunity: current directionsin autoimmunity. Basel: Karger, 2003;6:154-68.

12. Prodeus AP, Georg S, Shen L-M, et al.A critical rolefor complement in maintenance of self-tolerance.Immunity 1998;9:721-31.

13. Hillmen P, Hall C, Marsh JCW, et al. Effect ofeculizumab on hemolysis and transfusionrequirements in patients with paroxysmalnocturnal hemoglobinuria. N Engl J Med2004;350:552-9.

14. Yazdanbakhsh K, Kang S, Tamasauskas D, et al.Complement receptor 1 inhibitors for preventionof immune-mediated red cell destruction:potential use in transfusion therapy. Blood2003;101:5046-52.

15. Mqadmi A, Zheng X, Song J, et al. Prevention ofcomplement-mediated immune hemolysis by asmall molecule compound. Biochem Biophys ResCommun 2004;325:1465-71.

16. Mqadmi A, Abdulla Y, Yazdanbahksh K.Characterization of complement receptor 1domains for prevention of complement-mediatedred cell destruction.Transfusion 2005;45:234-44.

Hugh Chaplin Jr. MD, Emeritus Professor of Medicineand Pathology, Washington University School ofMedicine, St. Louis, Missouri.

A previous study involving tube IATs, untreated RBCs, and a low-ionic-strength additive reagent revealed that approximately one-third of R1R1 patients with anti-E have a concomitant anti-c.However, the current study finds a much higher incidence of anti-cin such patients, using gel technology in conjunction with ficin-pretreated RBCs. Results of antibody identification studies andtransfusion records of 82 R1R1 patients with anti-E were reviewed.Serologic test methods included a LISS wash solution for tube IATs(15 min at 37°C, anti-IgG), ficin-tube IATs (30 min at 37°C, anti-IgG+ anti-C3), and gel IATs (untreated or ficin-treated RBCs or both,anti-IgG gels). LISS-tube or gel IATs with untreated RBCs revealedanti-c in 32 patients with anti-E. When gel-IAT and ficin-pretreatedRBCs were used, 21 additional patients with anti-E were found tohave anti-c. In samples from 26 R1R1 patients with anti-E, anti-c wasnot demonstrable by ficin-gel IATs, and in 3 cases, the ficin-gel testswere inconclusive. In five cases in which E– RBCs not tested for cantigen were transfused to patients found by ficin-gel IAT to bewithout anti-c, all subsequently performed crossmatches with E–, c-untested RBCs were compatible. The incidence of anti-c in R1R1

patients with anti-E in this study was 32 of 82 (39%) with untreatedRBCs and 53 of 82 (65%) when the ficin gel data were included.The latter is significantly higher than the 32 percent incidencepreviously reported (p = 0.0001). Accordingly, all patients at ourfacility with an Rh antibody are now tested for those additional Rhantibodies they can make, as predicted from their Rh phenotype.The data from this study strongly support the selection of R1R1

RBCs for all c– patients with anti-E. Immunohematology2005;21:94–96.

Key Words: anti-c, anti-E

The immune response to RhCE protein is variable.For example, R1R1 (c–, E–) individuals may make anti-c,anti-E, anti-c plus anti-E, or anti-cE (Rh 27), the latterreacting with a determinant encoded by c and E withinthe same haplotype (in cis). Some individuals maymake all three antibodies, as demonstrated byadsorption-elution studies,1 or a combination of anti-cplus anti-E.

Shirey et al.2 found the incidence of anti-c in R1R1

patients with anti-E to be 32 percent when performingIATs by a low-ionic-strength additive technique. Theyalso showed if the RBCs selected for transfusion wereE– but not tested for c antigen, 18.5 percent of

recipients developed anti-c. Since their report, geltechnology has emerged and has been found to havegood sensitivity for Rh antibodies. Further, we haveshown that tests performed with anti-IgG gel cards andficin-pretreated RBCs are exquisitely sensitive for Rhantibodies.3

In this report we present data from ficin-gel teststhat demonstrate a higher than previously publishedincidence of anti-c in R1R1 patients with anti-E. Thesedata support the selection of c– RBCs for this patientpopulation.

Materials and MethodsID-MTS gel technology was from Ortho-Clinical

Diagnostics (Raritan,NJ). Reagent RBCs,both untreatedand ficin-treated, and LISS (Löw and Messeter4

formulation) were from ImmucorGamma, Norcross,Georgia. For gel testing,5 untreated reagent RBCs wereprepared in ID-MTS Diluent 2 at a concentration of 0.8%and tested on anti-IgG cards according to themanufacturer’s product circular. Gel tests with ficin-pretreated RBCs were similarly performed on anti-IgGcards; such testing (previously validated by us3)conflicts with the manufacturer’s product circular,which stipulates the use of buffered gel cards. LISS-tubetests6 were incubated at 37°C for 15 minutes, washedfour times with saline, and tested with anti-IgG (Ortho).Ficin-tube tests6 were incubated at 37°C for 30 minutesand tested with polyspecific (anti-IgG + C3) anti-globulin reagent (Ortho). Negative tube tests werevalidated with IgG-coated RBCs (ImmucorGamma).

Serologic and transfusion records of 82 R1R1

patients with anti-E were reviewed. No further testingwas performed on 32 patients with concomitant anti-cby routine testing (LISS-tube and ficin-tube). Ficin-geltests were used to detect the presence or absence ofanti-c in samples from the remaining 50 patients.


On a much higher than reportedincidence of anti-c in R1R1 patientswith anti-EW.J. JUDD, L.R. DAKE,AND R.D. DAVENPORT


Incidence of anti-c in R1R1 patients with anti-E

ResultsWe 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), thepresence of anti-c was clearly demonstrable only inficin-gel IATs. However, among these 21 cases, weakreactivity was also seen in two cases with someuntreated c+, E– RBCs in gel IATs and suspected fromthe results of ficin-tube tests in five cases. There werethree additional cases in which the presence of anti-ccould not be determined due to panreactivity in ficin-gel tests.

Of the 26 R1R1 patients with anti-E that did not haveanti-c clearly demonstrable by ficin gel, 18 did notrequire transfusion (five were pregnant, and there wererecords of previous transfusions on six). A furthereight patients were transfused with E– RBCs that werenot tested for c antigen. Three of these patients werelost to follow-up. We had the opportunity to test thefive remaining patients for anti-c in subsequentantiglobulin crossmatches with E– donor units that hadnot been selected to be c–; in fact, two patients weretransfused with Rh– RBCs that were undoubtedly c+.These crossmatches were performed between 3 and 20months after anti-E was initially detected in thepatients’ plasma. All units (n = 10) were crossmatchcompatible.

DiscussionThe development of alloantibodies to RBC antigens

through transfusion or pregnancy is not benign.Patients who become alloimmunized are at risk ofhemolytic transfusion reactions and high-riskpregnancies associated with HDN.7,8 Further,alloimmunization is sometimes accompanied byautoantibody formation, which may lead toautoimmune hemolytic anemia.9 These risks promptedsome investigators to recommend the use ofphenotypically matched RBCs, especially for sickle cellanemia patients.10 As shown recently,11,12 thisrecommendation is by no means universally followed.

In the 11th edition of the AABB TechnicalManual,13 it was suggested that R1R1 transfusioncandidates who have made anti-E should be transfusedwith R1R1 RBCs to prevent formation of anti-c, possibleposttransfusion hemolysis, and autoantibody forma-tion. This suggestion prompted Shirey and colleagues2

to determine the incidence of anti-c in 100 R1R1

patients with anti-E. In their study,using LISS-tube IATs,

they found the incidence to be 32 percent. Among the68 R1R1 patients with anti-E alone, 27 were transfusedwith E– RBCs that were not typed for c antigen; five(18.5%) of these patients subsequently formed anti-c.

Given our past experiences with gel technology,14

which is exquisitely sensitive for Rh antibodies, weexpected to find a somewhat higher incidence of anti-c in R1R1 patients with anti-E than was reported byShirey et al.2 However, we did not expect to find atwofold increase (32% vs.65%;p = 0.0001) through theuse of gel technology and ficin-treated RBCs, given thatbefore implementation of gel technology in ourlaboratory our routine antibody identification protocolincluded ficin-tube IATs. We postulate that these anti-cantibodies are, for the most part, low-affinity antibodies

Table 1. Incidence of anti-c in 82 R1R1 patients with anti-E

LISS tube or gel Ficin gel Number of patients

anti-E + anti-c NT* 32 (39%)

anti-E anti-E + anti-c 21 (26%)

anti-E anti-E 26 (32%)

anti-E inconclusive 3 (3%)

*NT = not tested

Fig. 1. A. Anti-IgG gel tests with untreated RBCs reveal presence of anti-E. B. Tests with ficin-pretreated RBCs reveal presence ofconcomitant anti-c.

R1R1

IgG IgG IgG IgG IgG IgG

R1R1 R2R2 r″r rr rr

Anti-EUntreated RBCs PK NO: 44-B REV.DATE 02-02-98

R1R1

IgG IgG IgG IgG IgG IgG

R1R1 R2R2 r″r rr rr

Anti-c+EFicin-Treated RBCs PK NO: 44-B REV.DATE 02-02-98

A

B

that dissociate during the washing phase of tube IATs;gel technology is, of course, a no-wash IAT.

Although we saw no evidence of anti-cdevelopment in five R1R1 patients with anti-E, asdemonstrated by compatible IAT crossmatches withE–, c-untested RBCs, we now select c–blood for R1R1 patients with anti-E,regardless of whether or not anti-c isdetected. However, due to the compar-ative rarity of R2R2 donors, we do notautomatically select R2R2 RBCs forpatients with anti-C who are e–. Rather,we test for anti-e by ficin gel, if it is notevident by routine studies, and, if it is

present, we crossmatch R2R2 donor RBCs. We have asimilar policy for Ro patients with anti-C or anti-E,inasmuch as we select C–, E– RBCs for crossmatchingonly if ficin-gel tests reveal that both anti-C and anti-Eare present (Table 2).

References1. Issitt PD, ed. Serology and genetics of Rh.

Cincinnati, OH: Montgomery Scientific Publi-cations, 1979.

2. Shirey RS, Edwards RE, Ness PM. The risk ofalloimmunization to c (Rh4) in R1R1 patients whopresent with anti-E.Transfusion 1994;34:756-8.

3. Judd WJ,Dake LR.Enzyme tests using anti-IgG gels.Vox Sang 2002;83(S2):154.

4. Löw B, Messeter L. Antiglobulin test in low-ionicstrength salt solution for rapid antibody screeningand crossmatching.Vox Sang 1974;26:53-61.

5. Lapierre Y,Rigal D,Adam J, et al.The gel test: a newway to detect antigen-antibody reactions.Transfusion 1990;30:109-13.

6. Judd WJ. Methods in immunohematology. 2nd ed.Durham, NC: Montgomery Scientific Publications,1994.

7. Ness PM, Shirey RS, Thoman SK, Buck SA. Thedifferentiation of delayed serologic and delayedhemolytic transfusion reactions: incidence, long-term serologic findings, and clinical significance.Transfusion 1990;30:688-93.

8. Garratty G, ed. Hemolytic disease of the newborn.Arlington, VA: American Association of BloodBanks, 1984.

9. Young PP, Uzieblo A, Trulock E, Lublin DM,Goodnough LT. Autoantibody formation afteralloimmunization: are blood transfusions a riskfactor for autoimmune hemolytic anemia?Transfusion 2004;44:67-72.

10. Rosse WF, Gallagher D, Kinney TR, et al.Transfusion and alloimmunization in sickle celldisease. The Cooperative Study of Sickle CellDisease. Blood 1990;76:1431-7.

11. Affenyi-Annan A, Brecher ME. Pre-transfusionphenotype matching for sickle cell diseasepatients (letter).Transfusion 2004;44:619-20.

12. Osby M, Shulman I. Phenotype matching of donorred blood cell units for nonalloimmunized sicklecell disease patients: a survey of 1182 NorthAmerican laboratories. Arch Pathol Lab Med2005;129:190-3.

13. Walker Rh, ed. Technical manual. 11th ed.Bethesda, MD: American Association of BloodBanks, 1993.

14. Judd WJ, Steiner EA, Knafl PC, Masters C. The geltest: use in the identification of unexpectedantibodies to blood group antigens. Immuno-hematology 1998;14:59-62.

W. John Judd, FIBMS, MIBiol; Louann R. Dake, MA,MT(ASCP)SBB, and Robertson D. Davenport, MD,Department of Pathology, UH-2G332, University ofMichigan Medical Center, 1500 East Medical CenterDrive, Ann Arbor, MI 48109-0054.


W.J. JUDD ET AL.

Table 2. Policy for ficin-gel testing and blood selection in D+ patients with Rh alloantibodies

Patient’s RBCs If* ◆ Then† Or, if* ◆ Then†

R1R1 anti-E ◆ issue c–, E– RBCs

R2R2 anti-C ◆ exclude anti-e anti-e ◆ issue C–, e– RBCs

R0 anti-C ◆ exclude anti-E anti-E ◆ exclude anti-C

*Antibody found in serum with untreated RBCs.†Exclusion tests performed with ficin-treated RBCs by gel technology.


Case report: massive postpartumtransfusion of Jr(a+) red cells inthe presence of anti-Jra

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

Jra is a high-prevalence antigen. The rare Jr(a–) individuals can formanti-Jra after exposure to the Jra antigen through transfusion orpregnancy. The clinical significance of anti-Jra is not wellestablished. This study reports a case of a 31-year-old woman witha previously identified anti-Jra who required massive transfusion ofRBCs after developing life-threatening postpartum disseminatedintravascular coagulopathy. Despite the emergent transfusion of 15units of Jra untested RBCs, she did not develop laboratory or clinicalevidence of acute hemolysis. The patient’s anti-Jra had apretransfusion titer of 4 and a monocyte monolayer assay (MMA)reactivity of 68.5% (reactivity > 5% is considered capable ofshortening the survival of incompatible RBCs). The titer increasedfourfold to 64 and the MMA reactivity was 72.5% on Day 10posttransfusion. Review of laboratory data showed evidence of amild delayed hemolytic transfusion reaction by Day 10posttransfusion. Despite rare reports of hemolytic transfusionreactions due to anti-Jra in the literature, most cases, including thisone, report that this antibody is clinically insignificant or causesonly mild delayed hemolysis. Clinicians should be advised tobalance the risks of withholding transfusion with the small chanceof significant hemolysis after transfusion of Jr(a+) RBCs in thepresence of anti-Jra. Immunohematology 2005;21:97–101.

Key Words: anti-Jra, massive transfusion, hemolytictransfusion reaction

Jra is a high-prevalence antigen in all populations.The Japanese population has the highest frequency ofthe Jr(a–) phenotype. However, even in thatpopulation, the incidence is reported to be only 0.03 to0.12 percent.1 Because of its rarity, the clinicalsignificance of anti-Jra is not well established. Reid andLomas-Francis2 describe anti-Jra as being capable ofcausing decreased RBC survival. In the obstetricsetting, the authors note that although the DAT can bepositive, anti-Jra is not associated with HDN.

Jr(a+) RBCs have been transfused to individualswith anti-Jra without incident.3–5 On the other hand,there exist in the literature a dozen or so reportshighlighting the ability of anti-Jra to cause both mild tomoderate HDN and delayed hemolytic transfusionreactions.6 The typical hemolytic reaction associatedwith anti-Jra is delayed, extravascular, and self-limited,

and the decreasing Hct is usually accompanied bytransiently increased antibody titers.7 More recently,Kwon et al.6 reported a case of anti-Jra causing acutehemolysis. In this report, we describe the case of apatient with anti-Jra who developed postpartumdisseminated intravascular coagulopathy (DIC) andreceived 15 units of incompatible Jr(a+) RBCs.Subsequently she had no overt clinical signs ofhemolysis, and laboratory findings suggested only milddelayed hemolysis.

Case ReportA 31-year-old multiparous Vietnamese woman with

history of three previously uncomplicated pregnancies(with a different partner from the current pregnancy)presented for prenatal care at an outside facility in the32nd week of her fourth pregnancy. ABO/D typingshowed the patient’s RBCs to be Group A, D+. Theantibody screen test showed that the patient’s serumreacted weakly with all reagent RBCs in theantiglobulin phase of testing. RBC treatment usingeither DTT or ficin had no effect on reactivity. Theautologous control and DAT were negative; thus anantibody directed at a high-prevalence antigen wassuspected. The patient’s serum was then tested withseveral RBC samples lacking various high-prevalenceantigens, including k, Kpb, and Ch, and RBCs with veryweak U expression. All RBCs tested reacted with thepatient’s serum. The specimen was subsequentlyreferred to our laboratory, where phenotyping of thepatient’s RBCs for high-prevalence antigens showedthat her RBCs possessed Vel,Dib,k,and Kpb antigens butlacked Jra antigens. Jr(a–) RBCs from two differentdonors located through Serum Cells and Rare FluidsExchange (SCARF) were not agglutinated by thepatient’s serum, which was consistent with thepresence of anti-Jra. Allogeneic adsorptions were


performed with W.A.R.M-treated RBCs (W.A.R.M.;Immucor Inc., Norcross, GA) that were alsophenotypically matched for Rh and Kidd antigens. Noadditional alloantibodies were detected in the serum.The anti-Jra was shown to have a titer of 4. Because ofthe difficulty in locating Jr(a–) RBCs,and the possibilityof shortened survival of transfused Jr(a+) RBCs, werecommended autologous donation or screening familymembers in the hope of finding a compatible donor ifthere was a reasonable concern that the patient or thebaby might need transfusion during the pregnancy anddelivery.

The patient went into labor during the 38th weekof her pregnancy and vaginally delivered a healthy maleinfant without evidence of HDN. Her postpartumcourse was complicated by development of DIC andprofuse, life-threatening vaginal bleeding, whichnecessitated an emergent abdominal hysterectomy.Intraoperatively, she received four units of uncross-matched Group O,D– RBCs and 11 units of crossmatch-incompatible Group A, D+ RBCs. Because of the highincidence of Jra, all of the RBC units were presumablyJr(a+). Her hemorrhage was eventually brought undercontrol after the surgery. Clinically, there was noevidence of acute intravascular hemolysis during theresuscitation effort.

Approximately 15 days after the initial surgery, thepatient was found to have developed abdominalabscesses. Because surgery with possible significantblood loss was anticipated, and because of the casereport from Kwon et al.6 reporting acute hemolysisafter repeated exposures to Jr(a+) RBCs, two frozenunits of Jr(a–) RBCs were imported from Blood Bank ofHawaii. Of note, the only other possible source of unitsof Jr(a–) RBCs at that time was from Japan. However,these units would not have been immediately availableand would not have been fully tested for transfusion-transmissible diseases according to FDA guidelines.Initial attempts at computerized tomography–guideddrainage of the abscesses were complicated byperforation of the colon. This necessitated a laparo-tomy for hemicolectomy and abscess drainage. Bothunits of Jr(a–) RBCs were transfused intraoperativelywithout incident. The patient did not require furthertransfusions. Twelve days after the second surgery, herHct was stable at 36.8%.

Materials and MethodsAntibody detection and identification were

performed by tube method. Antibody detection tests

were performed using reagent RBCs (Immucor, Inc.).Antibody identification was performed using panels ofreagent RBCs (Ortho-Clinical Diagnostics, Inc., Raritan,NJ, and Immucor, Inc.). Panels of ficin-treated RBCs(Immucor, Inc.) were also used. DTT-treated RBCswere prepared using 0.2 M solution (DTT, Sigma-Aldrich Corp., St Louis, MO). Alloadsorption wasperformed using W.A.R.M.-treated RBCs (W.A.R.M.;Immucor, Inc.), which were also phenotypicallymatched for Rh and Kidd antigens.

Polyspecific and monospecific DATs wereperformed (Bioclone Anti-IgG, Bioclone Anti-C3d, andBioclone AHG, Ortho-Clinical Diagnostics) by standardtube method. The elution was performed using therapid acid method (Elukit II, Gamma Biologicals, Inc.,Houston,TX). Two different enhancement media (PEG,Gamma Biologicals, and O.A.E.S., Ortho-ClinicalDiagnostics) were used to enhance antibodyagglutination. Antibody titer was determined withserial dilutions of patient’s serum using IAT.

Patient’s RBCs were phenotyped usingconventional methods for the common antigens. Rareantisera and reagent RBCs were prepared in-house orprovided through SCARF. Monocyte monolayer assay(MMA) was performed by the American Red CrossBlood Services of Southern California Region, LosAngeles,California. The method was described in detailelsewhere.8

ResultsAnti-Jra was not detectable in the serum

immediately after the transfusion of the 15 units ofuncrossmatched (presumably Jr[a+]) RBCs. This wasnot surprising because of the large volume of crystalloidsolutions and blood components administered duringthe resuscitation. The DAT using polyspecific anti-human globulin at this time was weakly positive (1+),but the patient’s RBCs were negative in the DAT usinganti-C3d or anti-IgG. An eluate was performed andtested and found to contain anti-Jra. Allogeneicabsorption of the eluate, using W.A.R.M-treated RBCsthat were also phenotypically matched for Rh and Kidd,did not show any additional alloantibodies.

Because of the concern for a significant delayedhemolytic transfusion reaction, we recommended thatthe clinicians give intravenous fluids as necessary tomaintain adequate renal perfusion and monitorlaboratory values, including Hct, bilirubin, LDH, andhaptoglobin. Additionally, a sample was sent daily toour reference laboratory for DATs and evaluation for

S.YUAN ET AL.


Massive transfusion in presence of anti-Jra

hemolysis. The patient’s Hct decreased from 38.6% onDay 5 posttransfusion to 30.9% on Day 10. Of note,during this period the patient also developedsyndrome of inappropriate antidiuretic hormonesecretion and hyponatremia. The resulting fluidretention and dilutional anemia contributed to thedecreasing Hct and made it unlikely that it was solelydue to hemolysis. On Day 10 posttransfusion, the totalbilirubin increased from 0.4 mg/dL at the time oftransfusion to 1.6 mg/dL (normal range 0.2–1.0 mg/dL)and LDH increased from 307 to 431 IU/L (normal range91–180 IU/L). Haptoglobin levels were obtained onlyon Days 4 and 5 posttransfusion;both were well withinnormal range. Anti-Jra became detectable in the serumagain on Day 6 posttransfusion and the titer had risen

from a pretransfusion titer of 4 to 64. Concurrently, thereactivity of the polyspecific DAT increased from 1+ to2+, and both the monospecific DATs (using anti-IgGand anti-C3) reacted 2+ at 6 days posttransfusion. Boththe prenatal pretransfusion serum sample and a serumsample obtained on Day 10 posttransfusion were sentfor MMA testing, which showed 68.5% and 72.5%reactivity respectively (reactivity > 5% is consideredcapable of shortening the survival of incompatibleRBCs). However, visual check of serial serum samplesshowed no evidence of hemolysis, and the patientcontinued to have no overt clinical evidence ofintravascular hemolysis during the posttransfusionperiod. 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 thepresence 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 =138.87.4 Fibrinogen = 72 mg/dL

D-dimer > 10,000 ng/mLPlatelet = 13 × 109/L

1 33.2 139 – 0.9 Polyspecific: 1w – –anti-IgG: 0anti-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 201Direct 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.


S.YUAN ET AL.

DiscussionBecause of the rarity of anti-Jra, its clinical

significance is not clear. Several reports of anti-Jra

causing mild to moderate HDN9,10 exist in the literature,but others have questioned the validity of thediagnoses in such cases.11 Mild to moderate delayedhemolytic transfusion reactions9,12,13 have also beenreported in association with anti-Jra. More recently,Kwon et al.6 reported a concerning case of acutehemolytic transfusion reaction due to this antibody inthe setting of repeated exposures to the antigen withina week. On the other hand, others have reported noclinical evidence of hemolysis after incompatibletransfusions, even in the face of rising antibody titers.4

Studies using monocyte phagocytosis14 and MMA8,15

suggested that most anti-Jra are not clinicallysignificant. The lack of clinical significance in mostcases could be in part accounted for by the low antigendensity of Jra on the RBC membrane.14,16

Additional data are needed to fully establish theclinical significance of anti-Jra. The MMA has beenproposed as a valuable tool for predicting the clinicalsignificance of antibodies to high-prevalence antigenssuch as anti-Jra.8,15 A recent retrospective review of 20years of MMA data by Arndt and Garratty8 showed thatonly results greater than 5% are potentially clinicallysignificant. Furthermore, one-third of patients withMMA results between 5.1% and 20% had clinical signs ofhemolysis (defined as jaundice, fever, chills, change inblood pressure, back pain, vomiting, tachypnea, andhemoglobinuria8), and two-thirds of patients with MMAresults greater than 20% reactivity had such symptoms.When clinical signs of hemolysis were absent, patientsfrom both groups often had laboratory indications ofhemolysis, which included decreased Hb, Hct, andhaptoglobin, increased bilirubin or LDH, hemo-globinemia,or the presence of bilirubin in the urine. Inthe same review, only 5 out of 14 (36%) cases of anti-Jra

examined showed MMA reactivity greater than 5%, twoof which had reactivity greater than 20%. But thepositive predictive value of the MMA result forhemolysis is not established specifically for anti-Jra

because of the small number of cases and the lack ofassociated clinical information. Through a personalcommunication with these authors, we learned thatclinical information was available on 4 of the 15 cases.Two of these cases had MMA results greater than 20%.Of these, one experienced a transfusion reaction withthe infusion of a third unit of Jr(a+) RBCs. This is thesame case reported by Kwon et al.6 In the other case,

flow cytometry showed normal RBC survival comparedwith the calculated expected RBC survival rate.Clinically the patient did not have signs of hemolysis.Clinical information on the other 11 patients isunavailable. In our case, despite the strong MMAreactivity both before and after the transfusion of a totalof 15 units of incompatible RBCs, we found no clinicalevidence of hemolysis and only laboratory evidence ofvery mild, well-tolerated, delayed hemolysis. Thedecrease in Hct was at least in part due to dilutionalanemia and postoperative surgical blood loss.

It is likely that most transfusions of Jr(a+) RBCs willnot result in significant acute or delayed hemolysis inthe presence of anti-Jra. However, because anti-Jra cancause significant acute hemolysis at least in rare casesafter repeated exposures to Jr(a+) RBCs6, it is prudentto make a reasonable attempt to locate Jr(a–) RBC unitswhen anti-Jra is identified,especially in those cases withstrongly positive MMA results. If blood loss can beanticipated, as in the case of an elective surgery ordelivery, autologous donations should be encouraged.It may also be necessary to look into family membersand rare donor registries as potential sources ofcompatible units because of the extreme scarcity of theJr(a–) phenotype in North America. It is important toweigh the availability of Jr(a–) RBCs against the costand the infectious disease risks when importing suchunits from countries outside the United States or olderunits frozen before the availability of the most currentinfectious disease testing. In our opinion, the risks ofacquiring an infectious disease appear to outweigh therisks of transfusing Jr(a+) units, and clinicians shouldbe guided in this way. Additionally, clinicians should beadvised of the available serologic results, which mightinclude antibody titer, DAT reactivity, and MMA results.The limitations of these data and the lack of conclusiveinformation in the current literature regarding theclinical significance of anti-Jra must be made clear aswell. The clinical decision to transfuse Jr(a+) RBCs andthe risks of hemolysis after transfusion of incompatibleRBCs must also be evaluated against the risks ofwithholding transfusion. The recent guidelines of theUK National Blood Service17 recommended transfusingserologically least-incompatible RBCs to patients withanti-Jra and antigen-negative RBCs to patients withstrong anti-Jra when transfusion is necessary. We feelthis approach is reasonable given the rarity of Jr(a–)RBC units, the lack of data we have about anti-Jra, andthe lack of laboratory assays that can reliably predict itsclinical significance.


AcknowledgmentsWe thank Dr. George Garratty and Patricia Arndt of

the American Red Cross, Southern California Region, inLos Angeles, California, for their performance of theMMA, technical expertise, and willingness to openlyshare knowledge gained.

References1. Nakajima H, Ito K. An example of anti-Jra causing

hemolytic disease of the newborn and frequencyof Jra antigen in the Japanese population.Vox Sang1978;35:265-7.

2. Reid ME, Lomas-Francis C. Blood group antigenfactsbook. 2nd ed. San Diego: Academic Press,2003.

3. Azar PM,Kitagawa H,Fukunishi A,et al.Uneventfultransfusion of Jr (a+) red cells in the presence ofanti-Jra. Jpn J Transfus Med 1988;34:406-10.

4. Bacon J, Sherrin D,Wright RG. Case report, anti-Jra.Transfusion 1986;26:543-4.

5. Schanfield MS, Stevens JO, Bauman D. Thedetection of clinically significant erythrocytealloantibodies, using a human mononuclearphagocyte assay.Transfusion 1981;21:571-6.

6. Kwon MY, Su L, Arndt PA, Garratty G, Blackall DP.Clinical significance of anti-Jra: report of two casesand review of the literature.Transfusion 2004;44:197-201.

7. Yoshida H, Yurugi K, Ito K. A case of delayedhemolytic transfusion reaction due to anti-Jra. JpnJ Tranfus Med 1991;34:528-30.

8. Arndt PA, Garratty G. A retrospective analysis ofthe value of monocyte monolayer assay results forpredicting the clinical significance of blood groupalloantibodies.Transfusion 2004;44:1273-81.

9. Takabayashi T, Murakami M, Yajima H, Tsujiei M,Ozawa N,Yajima A. Influence of maternal antibodyanti-Jra on the baby: a case report and pedigreechart.Tohoku J Exp Med 1985 Jan;145(1):97-101.

10. Orrick LR, Golde SH. Jra mediated hemolyticdisease of the newborn infant. Am J ObstetGynecol 1980;137:135-6.

11. Toy P,Reid M,Lewis T,Ellisor S,Avoy DR.Does anti-Jra cause hemolytic disease of the newborn? VoxSang 1981;41(1):40-4.

12. Kendall AG. Clinical importance of the rareerythrocyte antibody anti-Jra. Transfusion1976;16:646-7.

13. Jowitt S, Powell H, Shwe KH, Love EM.Transfusionreaction due to anti-Jra (abstract). Transfus Med1994;4(suppl 1):49.

14. Ogasawara K, Mazuda T. Characterization of Jra

antibodies by monocyte phagocytosis assays andflow cytometry analysis. Acta Haematol Jpn (inJap) 1990;53:1131-7.

15. Garratty G,Arndt P, Nance S.The potential clinicalsignificance of blood group alloantibodies to highfrequency antigens (abstract). Blood 1997;10(suppl 1):473a.

16. Miyazaki T, Kwon K,Yamamoto K, et al. A humanmonoclonal antibody to high-frequency red cellantigen Jra.Vox Sang 1994;66:51-4.

17. National Blood Service.Guidance for the selectionof blood for patients with common and rare redcell antibodies.Blood Matters 2003;13.Available athttp://www.blood.co.uk/hospitals/library/bm/issue13/BM1303.htm. Posted August 13, 2003.

Shan Yuan, MD, Transfusion Medicine Fellow,Department of Laboratory Medicine, University ofWashington and Puget Sound Blood Center, Seattle,WA; Rosalind Armour, BA, BB(ASCP); and AllisonReid, MT(ASCP)SBB, Senior Technologist, Red CellReference Laboratory, Puget Sound Blood Center,Seattle, WA; Khaled F. Abdel-Rahman, MD, AttendingPhysician, Auburn Regional Medical Center, Auburn,WA; Michael Phillips, MT(ASCP)SBB, Supervisor, RedCell Reference Laboratory; Dawn M. Rumsey,ART(CSLT) Director, Transfusion Service; and TheresaNester, MD, Assistant Medical Director, Puget SoundBlood Center, Seattle, WA, and Assistant Professor,Department of Laboratory Medicine, University ofWashington, Seattle, WA 98104.

Massive transfusion in presence of anti-Jra

There is uncertainty about the relationship between anti-HPA-1alevels and severity of neonatal alloimmune thrombocytopenia(NAIT). To investigate this relationship further, the concentration ofanti-HPA-1a in HPA-1b homozygous women was determined, usinga newly developed quantitative ELISA that uses purified anti-HPA-1ato obtain a standard curve. Seventy-eight samples collected from 22HPA-1b homozygous pregnant women at various stages ofpregnancy were tested. These included five women who haddelivered babies with severe NAIT. A national HPA-1a antibodystandard (NIBSC 93/710), designated as 1 arbitrary unit/mL(AU/mL), was used in each ELISA to calibrate the purified anti-HPA-1a, enabling the presentation of results as AU/mL. Moreover,selected samples were also assayed by PAK 12 and their reactivitycompared with quantity of antibody. The use of the purified HPA-1a antibody yielded consistent sigmoid curves, enabling themeasurement of HPA-1a antibody concentration in the test samples.The antibody concentration was significantly correlated with theantibody titer in the 78 samples studied (R = 0.54, p < 0.001).Furthermore, there was a significant correlation between PAK 12and the quantitative ELISA in a selected number of cases, with orwithout NAIT (R = 0.71,n = 10;p < 0.02). On the other hand, therewas no correlation of antibody concentration with NAIT incidence(R = –0.046). This study indicates that there is no relationshipbetween anti-HPA-1a concentration and severity of NAIT whenELISA is used, although the correlation between ELISA and othermethods, such as monoclonal antibody immobilization of plateletantigens (MAIPA) assay, remains to be determined.Immunohematology 2005;21:102–108.

Key Words: platelets, anti-HPA-1a, neonatalalloimmune thrombocytopenia (NAIT)

Neonatal alloimmune thrombocytopenia (NAIT)occurs when the pregnant woman and her fetus differin their platelet glycoprotein (GP) polymorphisms andthe woman develops an antibody to the fetal plateletantigens. In the Caucasian population, most severecases of NAIT occur when the woman and fetus differin their HPA-1 phenotype.1–3 About 10 percent of HPA-1b homozygous women who are pregnant with a fetusexpressing HPA-1a antigens develop antibodies to the

fetal platelets, which leads to NAIT in about 20 percentof these cases.1–3 This can lead to internal hemorrhage,of which the most serious form is intracranialhemorrhage (ICH), which can cause the death orlifelong morbidity of the baby.4 As NAIT is caused bythe passage of IgG across the placenta, it is reasonableto assume that a relationship would exist between theisotype of anti-HPA-1a, its concentration, or both andthe severity of the disease, as has been observed forHDN.5 However, to date, evidence of the relationshipbetween HPA-1 antibody titer and the severity of NAIThas been inconclusive. Most studies have shown thatno such relationship exists,2,6–12 while a few studieshave shown a notable or significant correlationbetween the HPA-1a antibody isotype or concentrationand the severity of NAIT.13–16

In a recent nationwide study in Scotland involving26,506 pregnant women, 8.8 percent (28/327) ofconfirmed HPA-1b homozygous women developedHPA-1a antibody. Of those, eight delivered babies withNAIT, which in five of them was severe (platelet count< 50 × 109/L).17 Antibody titers during pregnancy weremonitored by a direct ELISA,18 and no correlationbetween antibody titer and severity of NAIT wasobserved.

The previously mentioned discrepancy in therelationship between antibody levels and severity ofNAIT could be due to the analytic methods used,because these are, in general, qualitative or semi-quantitative. For example, in ELISA methods, patientsamples are incubated with purified GPIIb/IIIa coatedonto the well,10,18 while in other assays, patient samplesare incubated with intact platelets.19,20 In themonoclonal antibody immobilization of platelet


Is there a relationship betweenanti-HPA-1a concentration andseverity of neonatal alloimmunethrombocytopenia? H. BESSOS, M.TURNER,AND S. URBANIAK


Anti-HPA-1a concentration and severity of NAIT

antigens (MAIPA) assay,platelets are incubated with thepatient’s antibodies before they are washed andincubated with monoclonal antibodies directed againstvarious GPs. The GP, bound with the patient’s antibodyand respective monoclonal antibody, is then extractedand tested further to determine the specificity of thepatient’s antibody.20 Recently we developed an ELISAthat enables quantitative measurement of antibody inpatients’ serum.21 The aim of this study was to use thisassay to confirm whether there is a relationshipbetween the concentration of anti-HPA-1a and severityof NAIT in the cohort of pregnant women with anti-HPA-1a in our study.The correlation between the directELISA titer and the quantitative assay was alsodetermined.

Materials and Methods

ELISAUsing immobilized HPA-1a, anti-HPA-1a was

purified from serum containing a high titer (256) ofthis antibody; the purified anti-HPA-1 was used togenerate a standard curve in the ELISA as previouslydescribed.21 The quantitative antibody ELISA consistedof the following steps, all of which were performed atroom temperature.21,22 Seven serial doubling dilutionsof the standard (purified) anti-HPA-1a and up to threeserial dilutions of the test samples were incubated inHPA-1a–coated wells for 30 minutes. Controlsconsisting of the same constituents were incubated inuncoated wells. After washing, the wells wereincubated with alkaline-phosphatase conjugated anti-human IgG for 30 minutes. The wells were againwashed and incubated with substrate for 30 minutes.Stop solution was added and absorbances wereobtained using an ELISA reader (MRXTC; DynexTechnologies, Chantilly, VA). Specific absorbance wasobtained by subtracting the absorbance in uncoatedwells (i.e., without antigen) from that in wells coatedwith antigen. Normal serum was not included in thetest because a study of 94 normal antenatal sampleshad yielded a mean absorbance plus two SD of lessthan 0.1, indicating no cross-reactivity of normalsamples with coated GPIIb/IIIa.23 All ELISA buffers andreagents were obtained from Axis-Shield plc, Dundee,Scotland. The purified anti-HPA-1a was used in theELISA at a starting dilution of 1 in 4 (added as one partanti-HPA-1a and 3 parts buffer) in buffer containing25% of normal pool plasma, with serial doublingdilutions to 1 in 256. This gave a sigmoid curve againstthe linear section of which test plasma samples could

be measured (see Results section). To obtain universalresults, the purified anti-HPA-1a was calibrated against anational minimum-potency anti-HPA-1a standard(NIBSC 93/710) designated as 1 arbitrary unit per mL(AU/mL).

Test samplesAs previously indicated, 28 of 327 HPA-1b women

who consented to our nationwide study were found tohave anti-HPA-1a. Of these women, eight deliveredbabies with NAIT, five of whom had severethrombocytopenia. At the end of the latter study,samples from 22 women were additionally tested forantibody, using the quantitative ELISA; this studyincluded samples from all five women who had

Fig. 1. Calibration of the standard anti-HPA-1a in each assay using NIBSC93/710 and determination of the anti-HPA-1a concentration inthe test samples from the calibrated standard (AU/mL).A standardcurve is shown with its associated trendline and correspondingequation. In each ELISA, NIBSC 93/710 anti-HPA-1a (at 1 AU/mL)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. Usingthe formula y = –0.1128 ln(x) + 0.7511, we could calculate thecorresponding reciprocal dilution of S on the x-axis as follows(where S = the amount of anti-HPA-1a in the standard in AU/mL):

0.1128 ln(x) = 0.7511 – 0.23 = 0.5211ln(x) = 0.5211/0.1128 = 4.62x = 100 (calculated through EXP on Microsoft Excel math/trig),

This indicates that S at 1 in 100 = 0.25 AU/ml, implying that S =25 AU/mL

With S as 25 AU/mL in this assay, we can then calculate theamount of anti-HPA-1a in test samples. For example, if a testsample at a dilution of 1 in 16 gives a specific absorbance valueon the y-axis of 0.25, the corresponding anti-HPA-1aconcentration in that sample is calculated as follows:

0.1128 ln(x) = 0.7511 – 0.25 = 0.5011ln(x) = 0.5011/0.1128 = 4.44x = 84.8, indicating that anti-HPA-1a in the test sample at adilution of 1 in 16 = 25/84.8 = 0.29 AU/mL; implying that the neatconcentration of anti-HPA-1a in the test sample = 16 × 0.29 = 4.72AU/mL.

Spec

ific

abso

rban

ce(5

40nm

)

Reciprocal of anti-HPA-1a dilution


H. BESSOS ET AL.

delivered babies with severe NAIT. Eight consecutiveELISAs were performed to measure 78 samplescollected from the 22 women at various stages ofpregnancy. In addition,10 selected samples (at deliveryor postdelivery) were tested using PAK 12 (GTI,Waukesha, WI) to determine whether the lattercorrelated in any way with the ELISA. It is worthnoting that PAK 12 also incorporates GPIIb/IIIa-coatedwells, although the GP is attached to the wells via amonoclonal antibody and not directly as in our ELISA.

Statistics The correlation between the titer or absorbance in

the PAK 12 and the antibody concentration wascalculated using the Pearson product momentcorrelation coefficient. The significance in thedifference of antibody quantity between low and hightiter samples was determined using the Wilcoxon t test.

Results

Consistency of the anti-HPA-1a standardAs before,21 consistent sigmoid curves were

obtained by the purified anti-HPA-1a standard in theELISA (data not shown). In each assay, the NIBSC93/710 anti-HPA-1a (designated 1 AU/mL) was assayedat a 1 in 4 dilution and the amount of antibody in thepurified anti-HPA-1a standard was calculated as shownin Figure 1. This yielded an average value of 25 AU/mLfor the purified anti-HPA-1a. Such calibration of thepurified anti-HPA-1a in each ELISA enabledmeasurement of the test samples in standardquantitative units (AU/mL).

Amount of antibody in the test samplesSeventy-eight samples obtained from various stages

of pregnancy of 22 women with anti-HPA-1a wereassayed. Thirty-four weakly reactive antibody samplesof titers less than or equal to 2 gave a mean antibodyquantity of 0.58 AU/mL (± 0.42 SD). In contrast, 16strongly reactive antibody samples with titers between32 and 256 gave a significantly higher mean antibodyconcentration of 4.27 (± 3.44) AU/mL, p < 0.0005.Overall, the antibody concentration was significantlycorrelated with the titer in the 78 samples studied (R =0.54, p < 0.001) (Fig. 2). Furthermore, there was asignificant correlation between the results of PAK 12and the quantitative ELISA (Fig. 3). However, norelationship was found between antibodyconcentration and severity of NAIT (R = –0.046).

Indeed, as shown in Figure 4, antibody concentrationbarely rose above 1 AU/mL during pregnancy in threeof the five severe NAIT cases (charts B, D, and E;respective platelet counts are shown in the figurelegend); in contrast, in two cases of normal deliveryplatelet counts, appreciably higher concentrations of 4to 10 AU/mL were observed in the third trimester(charts H and I). Antibody concentrations of between1 and 6 AU/mL were observed during pregnancy in theremaining two severe NAIT cases (charts A and C). Theselected cases (10/22) in Figure 4 highlight thevariability of the relationship between antibody

Fig. 2. The correlation between anti-HPA-1a titer and concentration.Comparison of the antibody titer (obtained in a previous studyusing a direct ELISA) with the antibody concentration obtainedusing the quantitative ELISA in this study showed a significantcorrelation between the two assays (p < 0.001).

Reci

proc

alof

titer

AU/mL

Fig. 3. The correlation between PAK 12 and the quantitative ELISA. Tenselected samples, including three severe NAIT cases (arrows),were tested using the PAK 12 and quantitative ELISA. Asignificant correlation was obtained between the levels obtainedby both assays.

Quan

titat

ive

ELIS

A-

AU/m

L

GTI ELISA - Absorbance (450 nm)


Anti-HPA-1a concentration and severity of NAITAU

/mL

Week

AU/m

L

Week

AU/m

L

Week

AU/m

L

Week

AU/m

L

Week

AU/m

L

Week

AU/m

L

Week

AU/m

L

Week

AU/m

L

Week

AU/m

L

Week

Fig. 4. Antibody concentration in 10 representative women with anti-HPA-1a during pregnancy.D = delivery, PN = postnatal. All platelet counts at delivery (× 109/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 antibodyduring pregnancy did not appear to be indicative of the severity of NAIT.

C

concentration and severity of NAIT. All five womenwho gave birth to babies with severe NAIT were HLA-DR3*01 positive (data not shown). The thrombocyto-penia resolved without treatment in one of the babies,while those of the other four women received one ortwo transfusions with HPA-1b homozygous plateletsbefore their platelet counts returned to normal.17

DiscussionIn this study, the relationship between anti-HPA-1a

concentration and severity of NAIT was assessed usinga newly developed quantitative ELISA. During ournationwide Scottish study we did not find a correlationbetween antibody titer and severity of NAIT. Our aim,therefore,was to determine whether such a correlationwould be observed upon accurate quantitation ofantibody in samples obtained from the same cohort.Seventy-eight samples collected from 22 antibody-positive women during pregnancy were tested. Asignificant correlation was observed between antibodytiter and concentration (i.e.,between the direct ELISA18

and the quantitative ELISA,21 respectively). Based onthis correlation, it was not surprising that norelationship was observed in the current studybetween antibody concentration and severity of NAIT,since the latter was not related to antibody titer either.

Our findings are in keeping with those of manyother studies where investigators have failed to findsuch a relationship.6–12 In a study by Kaplan et al.,8 onein five mothers who delivered thrombocytopenicbabies did not have detectable antibodies. A similarobservation was made by Bussel et al.9 in a study ofseven pregnant women who had previously deliveredinfants with severe NAIT. Two of these seven women,whose fetuses developed thrombocytopenia duringthe early phase of their subsequent pregnancy, had nodetectable antibody. However, based on their previoushistory, IVIG was infused into these women in anattempt to raise the platelet count of the fetuses beforedelivery. Using an ELISA similar to ours, Proulx et al.10

analyzed the sera from 36 mothers who gave birth tothrombocytopenic babies. Their results indicated thatneither the titer nor the isotype of the antibody couldpredict the severity of NAIT. In contrast, Mawas et al.13

found a significant increase of anti-HPA-1a of the IgG3immunoglobulin class in women who deliveredseverely thrombocytopenic babies, although the levelsof IgG1, IgG2, and IgG4 (and overall IgG levels)remained the same between the two groups. However,the method they used was a modified MAIPA assay

using HPA-1a homozygous platelets and not ELISAplates coated with HPA-1a GPIIb/IIIa GP. Likewise, intwo other recent studies where MAIPA assay was used,a relationship was found between antibody titer andquantification and severity of NAIT.14–15 In the study inEast Anglia, England, by Williamson et al.,14 a significantcorrelation between severity of NAIT and a thirdtrimester anti-HPA-1a titer of less than 32 was found.Moreover, in a smaller study by Jaegtvik et al.15 inNorway, an obvious relationship between antibodylevel at the time of delivery and the severity of NAITwas observed. Nevertheless, two women with barelydetectable antibodies delivered babies with severeNAIT. In addition, one woman with high antibodylevels delivered a baby with mild NAIT (cord plateletcount of around 125 × 109/L),but this was attributed tothe protective effect of anti-D that was also present inher serum. In a recent study by Maslanka et al.,16 where144 of 8013 pregnant women were found to be HPA-1bhomozygous, 12 of 122 of the HPA-1b womenproduced anti-HPA-1a (as determined by MAIPA assay);two of the babies had severe antenatal thrombocyto-penia and two had mild NAIT. Due to the small numberof cases, no definitive conclusion could be drawnregarding the relationship of antibody titer to severityof disease. However, the four thrombocytopenic casesoccurred in mothers who had antibody titers of 4 orhigher, while no thrombocytopenia was observed inthose mothers who had weakly reactive anti-HPA-1adetectable only after delivery.

Although antibodies to other human plateletglycoprotein polymorphisms, such as HPA-5b and HPA-4b, may have mechanisms of action that differ fromthose of anti-HPA-1a, it is worth noting that discrep-ancies have also been noted with such antibodies inrelation to severity of NAIT.24–26 While a study by Kurzet al.24 did not find HPA-5b antibody titer to be apredictor of disease, a study by Ohto et al.25 foundsignificant association between postnatal plateletconcentration (Day 3) and anti-HPA-5b titer in the thirdtrimester of greater than or equal to 64. In addition, norelationship between anti-HPA-4b and severity of NAITwas observed in a large Japanese study.26 (Antibodiesin the latter two studies were determined by means ofa mixed passive hemagglutination assay.)

It is clear that there are no consistent resultsilluminating the relationship of anti-HPA-1a concen-tration and severity of NAIT. The discrepancy betweenthe various studies could be due to the use of differentassays. It appears that all those who have used ELISA


H. BESSOS ET AL.


incorporating GP-coated wells have failed to find acorrelation between anti-HPA-1a levels and severity ofNAIT, whereas all those who have found such arelationship have used a MAIPA assay. However, this isnot to say that the use of a MAIPA assay always yieldssuch a correlation.7,10,11 In this study we calibrated thepurified anti-HPA-1a standard in each assay againstNIBSC 93/710,designated as 1 AU/mL. This enabled thequantitation of anti-HPA-1a in all of our samples asAU/mL. Three of five women who had delivered babieswith severe NAIT exhibited consistently lowconcentrations of anti-HPA-1a throughout theirpregnancies, barely exceeding 1 AU/mL. Although theremaining two women in this group exhibited higherantibody concentrations, the amounts interestinglydeclined in the second trimester, rising again in thethird trimester and postdelivery. In contrast, twowomen with high, albeit fluctuating, antibodyconcentrations from 4 to 10 AU/mL during pregnancydelivered babies with normal platelet counts. Ourresults, therefore, indicate that the antibodyquantification during pregnancy is not a reliablepredictor of severity of NAIT. However, an inverserelationship between antibody levels and plateletcounts in utero cannot be ruled out, nor can the effectof variation of maternal blood volume in our cohort ofpregnant women; it is conceivable that some of thosewith low antibody levels could have had larger bloodvolumes than those with high antibody levels.

Based on the apparent discrepancy between ELISA-based test systems and MAIPA assays, it would be usefulto carry out a comparison between MAIPA assay andGPIIb/IIIa-coated quantitative ELISAs using the samesamples. In the meantime, in the absence of aconsensus on the role of anti-HPA-1a concentration inNAIT, the clinical outcome (antenatal ICH), the degreeof thrombocytopenia (platelet count < 20 × 109/L) inpreviously affected pregnancies, or both appear to bethe predictors of the likely severity of fetomaternalalloimmune thrombocytopenia, though only insubsequently affected pregnancies.27

AcknowledgmentsWe would like to thank Robert Munks and Sheryl

Qader (Sheffield National Blood Service, England) forproviding us with generous amounts of the high-titerHPA-1a antibody from which we purified the anti-HPA-1a for the ELISA standard curve. We would also like tothank Axis-Shield plc (Dundee, Scotland) for providingus with ELISA reagents.

References1. Mueller-Eckhardt C, Kiefel V, Grubert A, et al. 348

cases of suspected neonatal alloimmunethrombocytopenia. Lancet 1989;1:363-6.

2. Blanchette VS, Chen I, de Friedberg ZS, et al.Alloimmunization to the PLA1 platelet antigen:results of a prospective study. Br J Haematol1990;74:209-15.

3. Bussel JB, Zabusky MR, Berkowitz RL, McFarlandJG. Fetal alloimmune thrombocytopenia. N Engl JMed 1997;337:22-6.

4. Bussel J, Kaplan C. The fetal and neonatalconsequences of maternal alloimmune thrombo-cytopenia. Baillieres Clin Haematol 1998;11:391-408.

5. Voak D, Mitchell R, Bowell P, et al. Guidelines forblood grouping and red cell antibody testingduring pregnancy.Transfus Med 1996;6:71-4.

6. von dem Borne AE, van Leeuwen EF, von Riesz LE,et al. Neonatal alloimmune thrombocytopenia:detection and characterization of the responsibleantibodies by the platelet immunoflourescencetest. Blood 1981;57:649-56.

7. McFarland JG, Frenzke M, Aster RH. Testing ofmaternal sera in pregnancies at risk for neonatalalloimmune thrombocytopenia.Transfusion 1988;29:128-33.

8. Kaplan C,Daffos F,Forestier F,et al.Management ofalloimmune thrombocytopenia: antenatal diag-nosis and in utero transfusion of maternalplatelets. Blood 1988;72:340-3.

9. Bussel JB, Berkowitz RL, McFarland JG, et al.Antenatal treatment of neonatal alloimmunethrombocytopenia.N Eng J Med 1988;319:1374-8.

10. Proulx C, Filion M, Goldman M, et al. Analysis ofimmunoglobulin class, IgG subclass and titre ofHPA-1a antibodies in alloimmunized mothersgiving birth to babies with or without neonatalalloimmune thrombocytopenia. Br J Haematol1994;87:813-7.

11. Panzer S, Auerbach L, Cechova E, et al. Maternalalloimmunization against fetal platelet antigens: aprospective study. Br J Haematol 1995;90:655-60.

12. Durand-Zaleski I, Schlegel N, Blum Boisgard C, etal. Screening primiparous women and newbornsfor fetal/neonatal alloimmune thrombocytopenia:a prospective comaprison of effectiveness andcosts.Am J Perinatol 1996;13:423-31.

13. Mawas F, Wiener E, Williamson LM, et al.Immunogolobulin G subclass of anti-human

Anti-HPA-1a concentration and severity of NAIT

platelet antigen 1a in maternal sera: relation to theseverity of neonatal alloimmune thrombocyto-penia. Br J Haematol 1997;59:287-92.

14. Williamson LM, Hackett G, Rennie J, et al. Thenatural history of fetomaternal alloimmunizationto the platelet specific antigen HPA-1a (PLA1,Zwa) as determined by antenatal screening. Blood1998;92:2280-7.

15. Jaegtvik S, Husebekk A, Aune B, et al. Neonatalalloimmune thrombocytopenia due to anti-HPA 1aantibodies; the level of maternal antibodiespredicts the severity of thrombocytopenia in thenewborn. BJOG 2000;107:691-4.

16. Maslanka K, Guz K, Zupanska B. Antenatalscreening of unselected pregnant women for HPA-1a antigen, antibody, and alloimmune thrombo-cytopenia.Vox Sang 2003;85:326-7.

17. Turner M, Bessos H, Fagge T, et al. Prospectiveepidemiological study of the outcome and costeffectiveness of antenatal screening to detectneonatal alloimmune thrombocytopenia (NAIT)due to anti-HPA-1a.Transfusion. In Press.

18. Bessos H, Goldschmeding R, von dem Borne A, etal. The development of a simple and quickenzyme-linked immunosorbent assay for anti-HPA-1a (PLA1) antibodies. Thromb Res 1993;69:395-400.

19. Porcelijn L, von dem Borne AE. Immune-mediatedthrombocytopenias: basic and immunologicalaspects. Baillieres Clin Haematol 1998:331–41.

20. Kiefel V,Santoso S,Weisheit M,Mueller-Eckhardt C.Monoclonal antibody-specific immobilization ofplatelet antigens (MAIPA): a new tool for theidentification of platelet-reactive antibodies.Blood1987;70:1722-6.

21. Bessos H, Perez S, Armstrong-Fisher S, et al. Thedevelopment of a quantitative ELISA forantibodies against human platelet antigen type 1a.Transfusion 2003;43:350-6.

22. Bessos H, Hofner M, Salamat A, et al. Aninternational trial demonstrates suitability of a

newly developed whole-blood ELISA kit formulticentre platelet HPA-1 phenotyping.Vox Sang1999;77:103-6.

23. Sukati H, Bessos H, Barker RN, Urbaniak SJ.Characterization of the alloreactive helper T-cellresponse to the platelet membrane glycproteinIIIa (integrin beta3) in human platelet antigen-1aalloimmunized human platelet antigen-1b1bwomen.Transfusion 2005;45:1165-77.

24. Kurz M, Stockelle E, Eichelberger B, Panzer S. IgGtiter, subclass, and light chain phenotype ofpregnancy-induced HPA-5b antibodies that causeor do not cause neonatal alloimmune thrombo-cytopenia.Transfusion 1999;39:379-82.

25. Ohto H,Yamaguchi T, Takeuchi C, et al. Anti-HPA-5b-induced neonatal alloimmune thrombocyto-penia: antibody titre as a predictor. Br J Haematol2000;110:223-7.

26. Ohto H, Miura S,Ariga H, et al.The natural historyof maternal immunization against fetal plateletalloantigens.Transfus Med 2004;14:399-408.

27. Birchall JE, Murphy MF, Kaplan C, et al. Europeancollaborative study of the antenatal managementof feto-maternal alloimmune thrombocytopenia.Br J Haematol 2003;122:275-88.

Hagop Bessos, PhD, Immunohaematology R&DGroup, Scottish National Blood Transfusion Service,21 Ellen’s Glen Rd, Edinburgh, Scotland EH17 7QT;Marc L. Turner, PhD, FRCP, FRCPath, Cell TherapyR&D Group, Scottish National Blood TransfusionService, 21 Ellen’s Glen Rd, Edinburgh, ScotlandEH17 7QT and Oncology Department, The Universityof Edinburgh, Edinburgh, Scotland; and Stanislaw J.Urbaniak, PhD, FRCP, FRCPath, Immuno-haematology R&D Group, Scottish National BloodTransfusion Service, 21 Ellen’s Glen Rd, Edinburgh,Scotland EH17 7QT and Academic TransfusionMedicine Unit, The University of Aberdeen, Scotland.


H. BESSOS ET AL.


The complement system plays a crucial role in fighting infectionsand is an important link between the innate and adaptive immuneresponses. However, inappropriate complement activation cancause tissue damage, and it underlies the pathology of manydiseases. In the transfusion medicine setting, complementsensitization of RBCs can lead to both intravascular andextravascular destruction. Moreover, complement deficiencies areassociated with autoimmune disorders, including autoimmunehemolytic anemia (AIHA). Complement receptor 1 (CR1) is a largesingle-pass glycoprotein that is expressed on a variety of cell typesin blood, including RBCs and immune cells. Among its multiplefunctions is its ability to inhibit complement activation.Furthermore, gene knockout studies in mice implicate a role forCR1 (along with the alternatively spliced gene product CR2) inprevention of autoimmunity. This review discusses the possibilitythat the CR1 protein may be manipulated to prevent and treatAIHA. In addition, it will be shown in an in vivo mouse model oftransfusion reaction that recombinant soluble forms of CR1 canreduce complement-mediated RBC destruction, thereby prolongingsurvival of transfused RBCs. It is proposed that CR1-basedtherapeutics have potential for effective and safe prophylacticshort-term use and for treatment of hemolytic transfusion reactions.Immunohematology 2005;21:109–118.

Key Words: complement, complement receptor 1,sCR1, inhibitors, LHR, immune hemolysis, autoimmunehemolytic anemia, transfusion medicine, regulatory Tcells, CD4+CD25+

The Complement System and Red CellDestruction

The complement system is an important part of theinnate immune system for fighting infections andforeign molecules before an adaptive response hasdeveloped.1 It is also an important regulator of B-cell,and possibly T-cell, immunity.2,3 However, it can alsocause cellular and tissue damage when activatedinappropriately, contributing to many clinicalconditions, including reperfusion injury (followingsurgery, ischemic disease, and organ transplantation),organ rejection,acute inflammatory injury to the lungs,and autoimmune diseases.1 For the system to exert its

biological activities, it has to be activated. Activationoccurs in a sequence that involves proteolytic cleavageof the complement components, resulting in therelease of active biological mediators and the assemblyof active enzyme molecules that result in cleavage ofthe next downstream complement component.1

Depending on the nature of the activators, threecomplement activation pathways have been described:the antibody-dependent classical pathway and theantibody-independent alternative and lectin pathways(Fig. 1).1 Common to all three pathways are twocritical steps: the assembly of the C3 convertaseenzymes and the activation of C5 convertases.Specifically, the C3 convertases cleave C3 into C3a, apotent anaphylatoxin, which acts as a neutrophilchemotaxin and activator,4 and C3b, which covalentlyattaches to nearby targets, where it directs immuneclearance and antigen selection.5 In addition, the C5convertases cause the release of another potentanaphylatoxin, C5a,4 and lead to the formation of thepore-like membrane attack complex (C5b-9), whichinserts into cell membranes, causing lysis or sublyticdamage to the target cell (Fig. 1).5

In the transfusion medicine setting, complement-mediated RBC destruction plays a critical role, beinginvolved in both intravascular and extravascularhemolysis.6 Generally, in the presence of a potent,complement-binding antibody and large numbers ofclosely situated RBC antigens, complement activationcan proceed to completion, resulting in intravascularhemolysis, which can be fatal.7,8 However, the majorityof blood group antibodies (including both alloanti-bodies and autoantibodies) that can fix complementactivate complement up to the C3 stage but do not goon to act as hemolysins.9 Although antibody-coatedRBCs can also be destroyed extravascularly withoutcomplement activation, RBC removal by tissue

Review: complement receptor 1 therapeutics for prevention ofimmune hemolysisK.YAZDANBAKHSH

macrophages in the spleen and liver is enhancedconsiderably when C3 is present on RBCs in additionto IgG.9,10 Indeed, as many as 50 percent of patientswith autoimmune hemolytic anemia (AIHA) have bothIgG and complement on their RBCs.11

Complement Regulatory Proteins and CR1The complement system can be divided into three

separate pathways (classical, alternative, and lectin),depending on the type of activator. The systemconsists of nearly 30 different serum and membraneproteins which, after activation, interact in a highlyregulated enzymatic cascade to generate reactionproducts that mediate inflammation and hostprotection.1 Because of the direct and indirectpowerful cytolytic activity of complement, there existsa family of structurally and functionally relatedproteins, known as regulators of complementactivation (RCA), that prevent potential host celldamage from complement activation (Fig. 1).5 CR1,also known as CD35, is the most versatile of the RCAfamily because it exhibits decay-accelerating andcofactor properties that can inactivate the two criticalenzymes of the complement activation pathways (Fig.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. Inaddition, by acting as a cofactor for plasma proteasefactor I, CR1 is responsible for the degradation of C4band C3b (Fig. 2), and thus complete inactivation of theconvertase. CR1 has also been shown to function as areceptor for C1q,16 the first component of the classicalpathway as well as the mannan-binding lectin (MBL) ofthe lectin pathway.17

CR1 Expression Pattern and FunctionsCR1 is expressed on a number of cell types, mostly

in the blood, and at low levels in soluble form in theplasma.18 Of the C4 and C3 regulatory proteins, CR1has the widest range of activities and functions,including immune complex clearance, regulation ofcomplement activation, phagocytosis, and antibodyresponse, as well as deletion of autoreactive B andmaybe T cells. Through its ability to bind C3b and C4b,erythrocyte CR1 transports immune complexes to thespleen and liver for their removal19–23; CR1, expressedon macrophages and neutrophils, mediates adherenceand phagocytosis24,25; on B cells, CR1 modulates thethreshold for B-cell activation26,27; and on folliculardendritic cells, it is thought to improve immune

response to antigen.28–36 Through its ability to bind C3band C4b, erythrocyte CR1 transports immunecomplexes to the spleen and liver for their removal,19–23

CR1 expressed on macrophages and neutrophilsmediates adherence and phagocytosis,24,25 thatexpressed on B cells modulates the threshold for B-cellactivation,26,27 and that on follicular dendritic cells isthought to improve the immune response toantigen.28–36 Moreover, CR1 is expressed on 10 to 15percent of human T lymphocytes,37–40 although itsfunction on such cells is not clear. The expressionpattern of murine CR1 is for the most part similar tothat of human CR1 except that mouse RBCs do notexpress CR1.41 To date, no individuals completely


K.YAZDANBAKHSH

Fig. 1. The complement system. The classical pathway is activated byantigen-antibody complex and the alternative and lectinpathways by microbial surfaces. Activation of these pathwaysresults in the generation of the key enzymes, C3 and C5convertases, which in turn results in the release of C3a, C4a, andC5a anaphylatoxins (inflammatory response), C3b (opsonizationof target cells), and the generation of the membrane attackcomplex in the target cell (lysis). The different steps of thecascade where CR1 inhibits complement activation are shown.Abbreviations: MBL, mannan-binding lectin; MASP, MBL-associatedserine protease.

Fig. 2. Regulation of complement by decay-accelerating and cofactoractivitiy of CR1. The classical complement inhibitory activity ofCR1 is depicted. CR1 dissociates C3 convertase of the classicalpathway through its decay-accelerating activity. In addition,through its cofactor activity, it degrades C4b into C4c and C4d.Central to these activities is its ability to bind C4b. (Adapted fromHourcade et al.12)


Complement receptor 1 therapeutics

lacking the CR1 protein have been identified.However, RBCs that express low copy numbers of CR1and its associated Knops blood group antigens, knownas the Helgeson phenotype, have been described andappear to be associated with protection against severemalaria.42

CR1 StructureIn humans, four allotypes of CR1,varying in size,are

known.43 The predicted amino acid sequence of thecommon allotype (CR1*1) is 2039 residues (Fig 3).18,44,45

The extracellular 1930-residue–long domain of CR1,with 25 potential N-glycosylation sites, can be dividedinto 30 short consensus repeats (SCRs), each of 59 to72 amino acids (aa) with sequence homology betweenSCRs ranging from 60 to 90 percent.45 HomologousSCRs with four conserved cysteine residues are alsofound in other RCA members.12 The first 28 SCRs ofCR1 are further arranged into four longer regions ofsimilarity, termed long homologous repeats (LHRs Athrough D), consisting of seven SCRs each18,44 (Fig. 3).In mice, CR1 is expressed along with the alternativelyspliced gene product CR2.46–48 Murine CR1 consists of21 SCRs, with 15 SCRs identical to murine CR2 and 6unique SCRs at the amino terminus.46–48

CR1-Based TherapeuticsThe complement regulatory function of CR1 has

been exploited for development of a potent anti-complement agent. Specifically, a recombinant solubleform of CR1 (sCR1), by binding C3b and C4b and

inactivating the convertases, has successfully inhibitedthe complement activation cascade and preventedcomplement-mediated tissue injury in several animalmodels.49,50 More importantly, sCR1 has been in humanclinical trials for the treatment of acute respiratorydistress syndrome and to reduce tissue damage inmyocardial infarction and lung transplantation,51–53 withpossible favorable outcomes.52 Despite the role of CR1as a global inhibitor of complement activation, theantibacterial defenses of the patients undergoingtreatment have not, thus far, been compromised,underscoring the usefulness of sCR1 as a therapeuticagent.53 It is important to note that there are currentlyno anticomplement therapeutics in the clinic. Indeed,sCR1 represents the best in vivo characterizedanticomplement agent to date.

Our studies are the first to demonstrate a potentialfor sCR1 for inhibiting complement-mediated RBCdestruction following transfusion immunizationevents.53,54 Furthermore, through structure-functionanalysis we have identified a 254-aa domain at the N-terminus of CR1, consisting of four SCRs and one-eighth of sCR1, that has antihemolytic activity in vivo.Previous in vitro studies by us and others demonstratedthat the N-terminal domain of CR1 has barely anycofactor activity, but has decay-accelerating activity forthe C3 convertases and inhibits the classical activationpathway.54,56–61 Moreover, we and others have shownthat the four N-terminal SCRs preferentially bindC4b.45,56,59,62–64 Based on these observations, we believethat fine mapping of the C4b binding and the decay-accelerating activity (C4b.2a) for the classicalactivation pathway in the 254-aa domain will lead tofuture design of nonimmunogenic small moleculeinhibitors to down-regulate complement-mediatedRBC destruction. It is important to note that chronictreatment with complement inhibitors is likely toundermine the body’s ability to fight infections65 andmay lead to development of autoimmune diseases(discussed in a later section). The application for theseinhibitors is thus for short-term prophylactic usebefore transfusion of not fully matched blood inemergency situations and as a therapeutic option inselect patients with complement-mediated immunehemolysis to ameliorate the life-threateningcomplications.

Complement and AIHAAIHA is an autoimmune disease caused by

autoantibodies against RBC self-antigens, causing

Fig. 3. Schematic representation of the common isotype of CR1.The 30SCRs are represented by blocks.Groups of seven SCRs are furthersubdivided into four LHRs. The ligand binding sites for C3b andC4b are shaded. The preferred ligand is in bold; the alternativeligand is in parentheses.The positions of C1q/MBL binding siteshave not yet been defined.


K.YAZDANBAKHSH

shortened RBC survival.6 The underlying mechanismfor breakdown of immunologic tolerance is not wellestablished. Several lines of evidence support the roleof complement in the maintenance of tolerance to self-antigens: (1) Hereditary deficiencies of complementproteins of the classical pathway have been associatedwith autoimmune diseases.66 For example, 90 percentof C1q-deficient patients develop autoimmunedisease,67 and C1q knockout mice have a highincidence of developing autoimmune disease.68 (2)Altered levels of expression of CR1 have been observedin patients with AIHA as well as other autoimmunediseases, such as systemic lupus erythematosus,rheumatoid arthritis, Sjogren’s syndrome, and somediabetic patients.69–77 In a mouse model of severelupus-like disease (MRL/lpr), lower levels of CR1/CR2receptors have been found on B cells before thedevelopment of disease manifestations,78 suggestingthat altered complement receptor expression maycontribute to initiation or progression of autoimmunedisease. Gene knockout studies of mice lacking CR1(along with the alternatively spliced gene productCR2) indicate that CR1 and CR2 control the activationthresholds of B cells to self-antigens,79 although theexact molecular mechanism is not clearly understood.Because CR1/CR2 knockout mice lack both CR1 andCR2, the specific contribution of each receptor cannotbe dissected. However, mice deficient in C4, but notC3, have a phenotype similar to CR1/CR2-deficientmice in studies demonstrating their role in immunetolerance, strongly suggesting that the primary effect inthese mice is mediated by CR1.79 (3) C3b was recentlyshown to induce the development of T-regulatory cells,known to be important for maintenance of peripheraltolerance (discussed in a later section). Specifically, itwas shown that membrane cofactor protein (MCP,CD46), a natural complement regulatory proteinwhose ligands are C3b and C4b,80 can act as acoreceptor for inducing the development of IL-10–secreting CD4+CD25+ regulatory T cells, which areresponsible for active suppression of autoreactive Tcells.81

T-Regulatory CellsT-regulatory cells (Tregs) are a subset of T cells that

function to control immune responses, including thosedirected against self-antigens. Different populations ofTregs have been described, including thymicallyderived naturally occurring cells and those that are

induced in the periphery through exposure toantigen.82,83 Naturally occurring Tregs constitute about1 to 2 percent of peripheral blood mononuclear cells,or about 5 to 10 percent of the CD4+ T cells, and arecharacterized by coexpression of CD25 (α subunit ofIL-2 receptor) and the transcriptional repressor FoxP3(forkhead box P3).84 Their role in maintenance of self-tolerance and their ability to suppress a number ofautoimmune diseases has attracted a great deal ofattention and opened the possibility of developingnovel immunotherapeutic strategies for suppression ofautoimmunity.84,85 Nevertheless, many questionsremain to be answered about the characteristics andbiology of Tregs. For example, it is not known whetherthe suppressive activity of the CD4+CD25+ Tregs can besubdivided to smaller subpopulations or whethersmaller individual CD4+CD25+ Tregs have differentdegrees of suppressive activity.86 Supporting thispossibility, CD103+ T cells within CD4+CD25+ Tregswere shown to have more suppressive activity thanCD103–CD4+CD25+ Tregs and CD62Lhigh expressingCD4+CD25+ Tregs appear more potent in preventingcertain autoimmune diseases in mice.87,88 Preliminarystudies in my laboratory indicate that CR1 is indeedexpressed on a subpopulation of CD4+CD25+ Tregs.The molecular basis of the mechanism of Treg cell-mediated suppression is not fully known, although theconsensus is that they can expand and augment theirsuppressive activity when stimulated.89,90 In vitronaturally occurring CD4+CD25+ mediate suppression ofcocultured CD25- T cells by cell-cell interactions, notby cytokines.91–94 Activation has been shown to occurthrough TCR-specific signals.90,95–97 Interestingly, non-TCR–specific stimuli such as bacterial productsthrough Toll-like receptor 4 have also been shown toactivate CD4+CD25+ Tregs.98 Given that innate immuneresponses such as Toll-like receptors can stimulateTregs, it is conceivable, although as yet untested, thatcomplement activation products, through interactionswith complement receptors such as CR1, may also beinvolved in augmenting or attenuating the activationstate of Tregs, with consequences for their suppressiveactivity. In support of this possibility, C3b-CD46interactions were recently shown to activate a subsetof Tregs, namely the inducible Tregs.81 Preliminarystudies from my laboratory indicate that CR1 can alsomediate the suppressive activity of CD4+CD25+ Tregs,although the exact mechanism of suppression is stillunder investigation.



Possible Role of Complement/CR1 in TregInduction

We speculate that after certain infections,complement is activated and foreign antigens that areopsonized with complement components C3b andC4b, which are known to be the ligands for CR1 andCD46, may induce regulatory T cells. Thus, the initialcomplement activation will help clear the pathogen bydirect lysis, inflammatory response, phagocytosis ofcomplement-sensitized infectious agent, or acombination of these (Fig. 1). In our hypotheticalmodel, complement activation through induction ofTregs will also result in suppression of certain self-reactive lymphocytes as well as effector T cells thatwould normally cause infection-induced immunopa-thology (Fig. 4). Given that complement is so tightlyregulated,1 it is conceivable that many factors,including the nature and strength of the activators(pathogens), will determine the balance amongcomplement activation, Treg induction, and T-cellsuppression. Our model obviously does not precludethe role of other mediators besides complementactivation products for Treg activity,which may explainwhy certain infections persist or become chronic99 andare correlated with lower incidence of auto-immunity.100 Nevertheless, complement deficiencies,which we propose can cause decreased Treg activity,are associated with autoimmune disease.67,101 AIHAappears to be a secondary complication in patientswith a range of diseases, including those with viral ormycoplasmal infections,102 and the presence of cross-reactive foreign antigens may be the underlyingmechanism for breakdown of tolerance in these

patients.103 It is interesting to note that Rh antibodiesdo not fix complement on RBCs. In warm-type AIHA,many of the autoantibodies are directed against the Rhcomplex.6 It may be that antigens such as Rh that arenot initially opsonized with complement componentsmay not be subject to immune tolerance. Thus, uponstimulation with cross-reactive foreign antigens,autoreactive T cells specific to Rh antigens arepreferentially expanded, resulting in AIHA. Futurestudies are needed to further explore the potentialcontribution of CR1 and its ligands in the developmentof AIHA.

AcknowledgmentsThe author was supported in part by grants from

the NIH R01 HL69102 and the American HeartAssociation Grant-in-Aid Heritage Affiliate.

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73. Emancipator SN, Iida K, Nussenzweig V, Gallo GR.Monoclonal antibodies to human complementreceptor (CR1) detect defects in glomerulardiseases. Clin Immunol Immunopathol 1983;27:170-5.

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77. Ruuska PE, Ikaheimo I, Silvennoinen-Kassinen S,Kaar ML,Tiilikainen A.Normal C3b receptor (CR1)genomic polymorphism in patients with insulin-dependent diabetes mellitus (IDDM): is the low



erythrocyte CR1 expression an acquiredphenomenon? Clin Exp Immunol 1992;89:18-21.

78. Takahashi K, Kozono Y, Waldschmidt TJ, et al.Mouse complement receptors type 1 (CR1;CD35)and type 2 (CR2;CD21): expression on normal Bcell subpopulations and decreased levels duringthe development of autoimmunity in MRL/lprmice. J Immunol 1997;159:1557-69.

79. Prodeus AP, Goerg S, Shen LM, et al. A critical rolefor complement in maintenance of self-tolerance.Immunity 1998;9:721-31.

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81. Kemper C, Chan AC, Green JM, et al. Activation ofhuman CD4+ cells with CD3 and CD46 induces aT-regulatory cell 1 phenotype. Nature 2003;421:388-92.

82. Sakaguchi S. Naturally arising CD4+ regulatory tcells for immunologic self-tolerance and negativecontrol of immune responses.Annu Rev Immunol2004;22:531-62.

83. Thompson C, Powrie F. Regulatory T cells. CurrOpin Pharmacol 2004;4:408-14.

84. Sakaguchi S,Sakaguchi N,Asano M,Itoh M,Toda M.Immunologic self-tolerance maintained byactivated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanismof self-tolerance causes various autoimmunediseases. J Immunol 1995;155:1151-64.

85. Shevach EM.Regulatory T cells in autoimmmunity.Annu Rev Immunol 2000;18:423-49.

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87. Lehmann J, Huehn J, de la RM, et al. Expression ofthe integrin alpha Ebeta 7 identifies uniquesubsets of CD25+ as well as CD25-regulatory Tcells. Proc Natl Acad Sci USA 2002;99:13031-6.

88. Szanya V, Ermann J, Taylor C, Holness C, FathmanCG. The subpopulation of CD4+CD25+splenocytes that delays adoptive transfer ofdiabetes expresses L-selectin and high levels ofCCR7. J Immunol 2002;169:2461-5.

89. Thornton AM, Shevach EM. Suppressor effectorfunction of CD4+CD25+ immunoregulatory T

cells is antigen nonspecific. J Immunol 2000;164:183-90.

90. Takahashi T,Kuniyasu Y,Toda M,et al. Immunologicself-tolerance maintained by CD25+CD4+naturally anergic and suppressive T cells: induc-tion of autoimmune disease by breaking theiranergic/suppressive state. Int Immunol 1998;10:1969-80.

91. Jonuleit H, Schmitt E, Stassen M, et al.Identification and functional characterization ofhuman CD4(+)CD25(+) T cells with regulatoryproperties isolated from peripheral blood. J ExpMed 2001;193:1285-94.

92. Dieckmann D, Plottner H, Berchtold S, Berger T,Schuler G. Ex vivo isolation and characterizationof CD4(+)CD25(+) T cells with regulatoryproperties from human blood. J Exp Med2001;193:1303-10.

93. Levings MK,Sangregorio R,Roncarolo MG.Humancd25(+)cd4(+) t regulatory cells suppress naiveand memory T cell proliferation and can beexpanded in vitro without loss of function. J ExpMed 2001;193:1295-1302.

94. Baecher-Allan C,Brown JA,Freeman GJ,Hafler DA.CD4+CD25high regulatory cells in humanperipheral blood. J Immunol 2001;167:1245-53.

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101. Walport MJ. Complement. Second of two parts. NEngl J Med 2001;344:1140-44.

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Karina Yazdanbakhsh, PhD, Associate Member,Complement Biology, New York Blood Center, 310 E67th Street, New York, NY 10021, USA.

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A 65-year-old Caucasian man with myelodysplasia was admittedwith autoimmune hemolytic anemia and a Hb of 5.6 g/dL. Thepatient’s serum contained anti-K; the DAT on the patient’s RBCsreacted 3+ with anti-IgG and 3+ with anti-C3d. K– RBC units weretransfused, but there was no sustained increase in Hb level. Thesamples were referred to the reference laboratory of the NationalBlood Service. The DAT results remained the same, with anti-Kdetected only in the serum. An eluate prepared from the patient’sDAT-positive RBCs revealed anti-Kpb specificity. This study reportsan unusual case of autoanti-Kpb, which is different from previouslypublished cases in that no free anti-Kpb was detectable in theserum. Immunohematology 2005;21:119–121.

Key Words: autoimmune hemolytic anemia, anti-Kpb

Autoantibodies found in patients with warmautoimmune hemolytic anemia (WAIHA) often show aRBC specificity related to the Rh system. The firstexample of this type of autoantibody was described byRace and Sanger.1 Although antibodies to other high-prevalence antigens have been reported, such asautoanti-AnWj, -Co3, -Dib, -Ena, -Ge, -Hro, -Kpb, -K13, -Kx,-LW, -‘N,’ -Rh34, -Sc1, -Sc3, -U, -Vel, and -Wrb, they are rare.We report an unusual case of autoanti-Kpb, which isdifferent from previously published cases2–4 in that nofree anti-Kpb was detectable in the patient’s serum.Kp(b–) RBCs were issued for further transfusionsupport. Transfusion of presumably Kp(b+) RBCs didnot result in a sustained increase in Hb, thereforeKp(b–) RBCs were issued for further transfusionsupport. Subsequent transfusions were uneventful andachieved prolonged Hb increment.

Case ReportA sample from a 65-year-old Caucasian man with

myelodysplasia, on regular transfusion support, wastested to be Group O, R1R2 with anti-K in the serum.Later the patient had colitis and developed acuteautoimmune hemolytic anemia (AIHA) with a positiveDAT. The patient was transfused with K– RBCs but

there was no sustained response in Hb level. Thesamples were submitted to the Reference Laboratory ofthe National Blood Service (NBS), North London, forinvestigation.

Materials and MethodsAntigen typing for ABO, Rh, and K were performed

by standard tube technique. Anti-A and anti-B reagentswere from Diagnostic Scotland, Edinburgh, Scotland;anti-D reagents (RUM1 and BS226) were from NBSReagents, Cambridge, United Kingdom; monoclonalanti-Rh (anti-C, -E, -c, and -e ), and anti-K were fromBIOSCOT (now Serologicals), Ltd., Livingston, Scotland.Antibody identification was performed by standardtube LISS-IAT (LISS from Inverclyde Biologicals,Bellshill, Scotland). The patient’s serum was testedwith an in-house panel of RBCs to determine antibodyspecificities. The manual polybrene technique was alsoused to determine additional antibody specificities.The 0.05% working solution of polybrene, theresuspending solution, and low–ionic-strength mediumwere prepared in house according to Methods inImmunohematology.5 The DAT was performed withpolyspecific AHG and monospecific anti-IgG and anti-C3d reagents (NBS Reagents). An eluate was preparedfrom the patient’s RBCs using chloroform following theprotocol in Methods in Immunohematology5 and wastested with in-house selected-RBC panels and withRBCs from the frozen inventory for antibodyidentification. Chloroquine diphosphate (CDP, 200mg/mL, pH 5.0 ± 0.1) was prepared by NBS Reagents.CDP treatment consisted of adding four volumes ofCDP to one volume of washed packed RBCs, mixing,and incubating at 20°C for a maximum of 2 hours. ADAT using anti-IgG was performed on the CDP-treatedRBCs every 30 minutes during this incubation toensure the effectiveness of the CDP treatment.

Autoimmune hemolytic anemiaand a further example of autoanti-Kpb

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


ResultsA posttransfusion sample from the patient showed

the RBCs to be Group O, R1R2 and to have a positiveDAT, reacting 3+ with both anti-IgG and anti-C3d. Testsby routine LISS-IAT, saline, and manual polybrenetechniques revealed anti-K in the patient’s serum. Aneluate was prepared using a chloroform method andautoanti-Kpb was detected, reacting 3+. After CDPtreatment of the patient’s RBCs, they were typed asKp(b+) (Table 1). The patient was transfused with twounits of K–, Kp(b–) RBCs. Three additional units of K–,Kp(b–) RBCs were transfused two days later. All of theunits were frozen-recovered, deglycerolized RBCs fromthe same donor. The transfusions were well toleratedand the patient recovered from this hemolytic episode.

One month later, a DAT performed on the patient’sRBCs was still positive (anti-IgG 3+, anti-C3d weakpositive), and anti-K was detected by LISS-IAT in thepatient’s serum in addition to a weak autoantibodydetectable by manual polybrene technique only.Autoanti-Kpb was not apparent in the serum or eluate.Although all of the RBCs transfused to the patient wereK–, anti-K was eluted from the patient’s RBCs, probablydue to the Matuhasi-Ogata phenomenon, in whichalloantibodies are nonspecifically bound to the RBCalong with autoantibody (Table 1).

DiscussionAn autoantibody with Kpb specificity has been

reported in AIHA and appears to be rare. In allpreviously reported cases of AIHA with autoanti-Kpb

specificity, free autoantibody was always detectable inthe serum. In the case we describe,no free autoanti-Kpb

was detected, distinguishing it from the other publishedcases. The identification of autoanti-Kpb in this case wasfortuitous, as the routine panel of reagent RBCs used atthe time had one example of Kp(b–) RBCs, whichhelped to define the autoantibody specificity.

Most autoantibody specificities are Rh-related, suchas anti-nl, anti-pdl,and anti-dl,6 and it is reported that upto 50 percent of antibody specificities in WAIHA are inthese groups.7 Usually it is not necessary to honor thespecificity of the autoantibody when selecting RBCsfor transfusion. The use of a high-prevalence antigen-negative panel is often not indicated and the ability toresolve these specificities is limited by the availabilityof suitably typed reagent RBCs, such as Rhnull and Rhdeletion. Extensive investigations are only performedin selected rare circumstances. In our institution, Rh-and K-matched RBCs are selected and issued fortransfusion in cases of AIHA.

The patient in this case had pre-existingmyelodysplasia, developing colitis, and infection.Although disease-associated transient suppression ofKell antigens has been reported,8–10 no suppression ofthe Kpb antigen was observed in this case. A positiveresult was obtained in the Kpb typing after CDPtreatment of the patient’s RBCs. This finding may,however, be due to the timing of the test sample.

There are a few case reports of autoantibody withKpb specificity described in the literature. Marsh et al.11

reported a case of AIHA, due to anti-Kpb, in a patientwho had a severe hemolytic transfusion reaction afterreceiving Kp(b+) RBCs. As previous transfusions withK– RBCs had not resulted in a sustained Hb increment,Kp(b–) RBCs were selected; transfusion was uneventfuland an excellent hematologic response was achieved.

Win et al.3 also reported a rare case of AIHA in a 12-week-old infant. Autoanti-Kpb was identified in theinfant’s serum. Initially the infant was transfused withKp(b+) RBCs and had recurrent hemolytic transfusionreactions. Pronounced jaundice was noted and therewas no sustained increase in Hb level after transfusion.The reference laboratory detected autoanti-Kpb in theserum and transfusion with Kp(b–) RBCs gave anexcellent result with no further reactions.

Table 1. Summary of laboratory findings*

Hb Antibody AntibodyDate (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–

6/4/98 5.6 Anti-K (5+)† Anti-Kpb (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+ (+) – Noneweak 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

DAT

E. LEE ET AL.


AIHA and autoanti-Kpb

The use of Rh- and K-matched RBCs for AIHApatients is standard practice in our organization andonly rarely are RBCs that are negative for antigensagainst which autoantibodies have been detectedselected for transfusion.12 In the case we describehere, because of the unusual nature of theautoantibody, and because of the lack of a response tothe transfusion of Kp(b+) RBCs, we selected frozen-recovered, deglycerolized Kp(a+b–) RBCs fortransfusion during the hemolytic episodes.

AIHA with autoanti-Kpb specificity is rare and ourcase supports previous findings that Kp(b–) RBCsshould be selected for patients with Kpb autoantibodiesfor transfusion support.3,11

References1. Race RR, Sanger R. Blood groups in man. 2nd ed.

Oxford: Blackwell Scientific Publications,1954;361.

2. Marsh WL, Redman CM. The Kell blood groupsystem: a review.Transfusion 1990;30:158-61.

3. Win N, Kaye T, Mir N, et al. Autoimmunehaemolytic anaemia in infancy with anti-Kpb

specificity.Vox Sang 1996;71:187-8.

4. Kohan AI, Reybaud JF, Salamone HJ, et al.Management of a severe transfusional problem ina patient with alloantibody to Kpb (K4).Vox Sang1990;59:216-7.

5. Judd,WJ. Methods in immunohematology. 2nd ed.Durham, NC: Montgomery Scientific Publications,1994.

6. Weiner W,Vos GH. Serology of acquired hemolyticanemias. Blood 1963;22:606.

7. Issitt PD, Pavone BG, Goldfinger D, et al. Anti-Wrb

and other autoantibodies responsible for positivedirect antiglobulin tests in 150 individuals. Br JHaematol 1976; 34-5.

8. Beck ML, Marsh WL, Pierce SR, et al.Auto anti-Kpb

associated with weakened antigenicity in the Kellblood group system: a second example.Transfusion 1979;19:197-202.

9. Seyfried H, Gorsks B, Maj S, et al. Apparentdepression of antigens of the Kell bood groupsystem associated with autoimmune haemolyticanaemia.Vox Sang 1972;23:528-36.

10. Williamson LM, Poole J, Redman C, et al.Transientloss of proteins carrying Kell and Lutheran redcell antigens during consecutive relapses of auto-immune thrombocytopenia. Br J Haematol1994;87:805-12.

11. Marsh WL, Oyen R, Alicea E, Linter M, Horton S.Autoimmune hemolytic anemia and the Kell bloodgroups.Am J Hematol 1979;7(2):155-62.

12. Sokol RJ, Hewitt S, Booker DJ, Morris BM. Patientswith red cell autoantibodies:selection of blood fortransfusion. Clin Lab Haematol, 1988;10:257-64.

Edmond Lee (corresponding author), MSc, SeniorBiomedical Scientist, and Gordon Burgess, ReferenceService Manager, Red Cell Immunohematology,National Blood Service Colindale, Colindale Avenue,London, NW9 5BG, United Kingdom; and Nay Win,Consultant Hematologist, National Blood ServiceTooting, London, United Kingdom.

Table 2. Grading system for hemagglutination

Reactiongrade Score Description

5+ 12 Cell button remains in one clump ordislodges into a few large clumps,macroscopically visible

4+ 10 Cell button dislodges into numerous largeclumps, macroscopically visible

3+ 8 Cell button dislodges into many smallclumps, macroscopically visible

2+ 5 Cell button dislodges into finely granular butdefinite small clumps, macroscopically visible

1+ 3 Cell button dislodges into very smallagglutinates, these are more distinct if thetube is left on its side to allow the cells tosettle, macroscopically visible

w 2 Grainy appearance, agglutinates best seenmicroscopically

0 0 Negative result


A recognized hazard of administering blood transfusions to patientswith panreactive warm autoantibodies is that alloantibodies may bemasked. Studies have shown the incidence of underlyingalloantibodies to be 30 to 40 percent. Adsorption procedures canbe used to remove autoantibodies and allow detection andidentification of underlying alloantibodies. This study contains datafrom 126 patients referred to the Red Cell Immunohaematologylaboratory at the National Blood Service, Newcastle upon Tyne,United Kingdom. These patients were from the northeast ofEngland, a population for which data have not previously beenreported. Samples identified as containing panreactive warmautoantibodies were subjected to adsorption procedures (95 byalloadsorption and 31 by autoadsorption). Absorbed sera were thentested to identify underlying alloantibodies. Of 126 samples, 39(31%) contained a total of 61 RBC alloantibodies; 15 (12%)contained 2 or more antibody specificities; and 14 (11%) containedalloantibodies not found within the Rh or Kell blood group systems.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,and f (ce). This study reinforces the value of adsorption studies,whether using autologous or allogeneic RBCs, when panreactivewarm autoantibodies are present. In addition, this study confirmsthat it is not appropriate in these cases simply to issue blood whichis “least incompatible” or Rh phenotype- and K antigen-matched.Immunohematology 2005;21:122–125.

Key Words: autoantibody, alloantibody, alloadsorption,autoadsorption

One of the recognized hazards of administeringblood transfusions to patients with panreactive warmautoantibodies is the failure to detect clinicallysignificant alloantibodies that may be masked.1,2

Traditionally, some blood banks have had policies ofcrossmatching and issuing blood that is “leastincompatible” or “as compatible” as the patient’s ownRBCs, or they have issued blood that is Rh phenotype-and K antigen-matched.3 Several methods may be usedto remove free autoantibody from the patient’s plasmato allow the detection and identification of anyunderlying alloantibodies.4,5 Adsorption,whether usingautologous or allogeneic enzyme-treated RBCs, is themethod of choice within the National Blood Service

(NBS) in the United Kingdom. British Committee forStandards in Haematology guidelines recommend thatthese techniques be used in such cases.6 This studywas performed over 18 months in the Red CellImmunohaematology laboratory at the NBS inNewcastle. The region has 18 National Health Service(NHS) hospital blood banks, serving a population of 2.9million.

Materials and MethodsOur alloadsorption method uses at least two sets of

strongly papain-treated donor RBCs selected to havecomplementary expression of important RBC antigens.It is our usual practice to use Group O,rr,K– and GroupO, R1R1, K– RBCs, one RBC sample being Jk(a–b+) andthe other being Jk(a+b–). Reagent RBCs for adsorptionwere obtained from the UK NBS central reagents unit.Autoadsorption uses the patient’s papain-treated RBCs.

Alloadsorption was performed on more cases asthere was a limited supply of patients’ RBCs to use toperform autoadsorption, due to severe anemia or lowsample volumes. Serial adsorptions were performed byincubating 1 mL of the patient’s plasma with 1 mL ofthe papain-treated packed RBCs at 37°C; the absorbedplasma was then retrieved by centrifugation. Amaximum of 4 aliquots of allogeneic RBCs, or typically2 aliquots of autologous RBCs, were used for thisprocess. The number of autoadsorption procedureswas dependent on the volume of RBCs available fromthe patient sample. Absorbed plasma was theninvestigated to detect and identify any underlyingalloantibodies, using reagent RBCs suspended in low-ionic-strength red cell preservative solution (RCPS;Inverclyde Biologicals, UK) by tube IAT. Wherepossible, phenotype studies were used to confirm thatthe identified antibodies were alloantibodies.

The incidence of red cellalloantibodies underlyingpanreactive warm autoantibodiesM. MALEY, D.G. BRUCE, R.G. BABB,A.W.WELLS,AND M.WILLIAMS


Alloantibodies underlying autoantibodies

ResultsSamples from 130 individual patients were referred

for investigation of panreactive autoantibodies byautoadsorption or alloadsorption procedures. Datafrom four samples in which we failed to successfullyremove the autoantibody were omitted from this study.Alloadsorption procedures were performed on 95samples and autoadsorption procedures on 31samples. Thirty-nine of 126 samples (31%) contained atotal of 61 RBC alloantibodies (Table 1); 87 samples(69%) contained no underlying alloantibodies. Fifteensamples (12%) contained two or more specificities(Table 2). Fourteen samples (11%) containedalloantibodies with specificities for antigens outsidethe Rh or Kell blood group systems.

Of the 61 alloantibodies identified, all but 4 (anti-Kpa [2], anti-N [1], and anti-Cw [1]) would require theselection of antigen-negative RBCs for crossmatching in

the UK.6 Of 95 alloadsorbed samples, 33 (35%)contained alloantibodies, whereas 6 of 31 (19%)autoadsorbed samples contained alloantibodies.

All but six antibodies (anti-Fya [3], anti-Kpa [2], andanti-Fyb [1]) were confirmed as alloantibodies, usingphenotyping studies. Phenotyping studies were notundertaken for these six patients as their RBC sampleshad strongly positive DATs. With only typing reagentsrequiring the use of AHG available and without a localprocedure for removing autoantibody for typing, testresults would be unreliable. These six antibodies weretherefore assumed to be probable alloantibodies.Autoantibodies of these specificities are rarelyreported.

Discussion

Limitations and advantages of the adsorptionprocedures

1.AlloadsorptionAlloadsorption procedures were preferentially

performed in the cases described, but a number offactors must be borne in mind when interpretingresults. Alloadsorption procedures require a minimumof two individual RBC samples with a complementaryantigenic profile at key antigens, in our study, Rh, K, Jka,and Jkb antigens, and at least 2 mL of available patientplasma or serum. Papain treatment of the adsorptionRBCs removes enzyme-labile antigens, effectivelyrendering the RBCs negative for such antigens. By thisprocedure we ensured that underlying alloantibodiessuch as anti-S, -s, -Fya, -Fyb, -M, and -N remained behindafter adsorption. We acknowledge that many workershave found that the s antigen is not readily destroyed.7

Although not every batch of reagent RBCs isspecifically tested for antigen destruction, the processwas thoroughly validated when introduced by the UKNBS reagents unit. Although no patients in this studywere found to have underlying anti-s at the time ofcollating data, the patient referred to in Table 2 withanti-C, -Fya, and -Jkb did subsequently produce anunderlying anti-s. The enzyme treatment usuallyincreases the uptake and removal of autoantibody,although there were some exceptions to this in ourstudy (four patients) when autoantibody removal wasless effective.

Our standard protocol does not include the use ofe– adsorption RBCs. In our experience the benefits ofincluding RBCs of the R2R2 phenotype are outweighedby the practical difficulties of obtaining suitable RBCs

Table 1. Underlying alloantibody specificities

Antibody specificity Number identified

Anti-E 19

Anti-D 9

Anti-C 7

Anti-c 6

Anti-S 5

Anti-Fya 3

Anti-Jka 2

Anti-Jkb 2

Anti-K 2

Anti-Kpa 2

Anti-Fyb 1

Anti-Cw 1

Anti-N 1

Anti-f (ce) 1

Total 61

Table 2. Multiple alloantibody specificities

Antibody specificity Number of samples

Anti-D, -C 3

Anti-c, -E 3

Anti-E, -Kpa 1

Anti-E, -Fya, -Jka 1

Anti-S, -N 1

Anti-Fyb, -f(ce) 1

Anti-c, -E, -K, -Fya 1

Anti-C, -Fya, -Jkb 1

Anti-D, -C, -E, -S 1

Anti-E, -K, -Jka 1

Anti-C, -Jkb 1


M. MALEY ET AL.

and the increased volume of plasma required.However, RBCs of the R2R2 phenotype are used whenthe initial investigation suggests they may be useful.The policy of matching the Rh phenotype of the donorunits to patients with autoantibodies decreases the riskof an underlying alloanti-e causing a transfusionreaction.

Recently transfused patients may give misleadingor inconclusive Rh phenotyping results. In such casesit is important to establish the transfusion history andmake an individual assessment of the most appropriateRh phenotype to select. Where possible it is goodpractice to prospectively establish the extendedphenotype of patients likely to be multiply transfused.

Alloadsorption procedures carry the risk that analloantibody to a high-prevalence blood group antigencan be adsorbed by the procedure and not detectedwhen screening or matching blood. We believe ourfindings show that it is better to carry out theadsorption procedures, being aware of this risk, thannot to adsorb at all. We have experience of one case(not included in this study) where an underlying anti-Vel was not detected following alloadsorption (data notpublished).

2.AutoadsorptionAutoadsorption uses the patient’s own RBCs to

remove autoantibody so that underlying alloantibodiescan be tested for. The advantages of autoadsorption arethat it does not remove any alloantibodies and itrequires adsorption of only a single aliquot of patient’splasma, considerably reducing the volume required.The number of tests performed is also reduced sincethe tests need not be carried out in duplicate, ortriplicate as in the case of alloadsorptions.

Autoadsorption is not without its problems,however. The patient’s RBCs often are heavily coatedwith antibody, with reduced capacity for furtherantibody uptake. Patients may also be anemic,sometimes very anemic, with few RBCs available forautoadsorption. In our experience, anything less thantwo equal-volume (RBCs:plasma) adsorptions does notremove sufficient autoantibody to be of investigativevalue. The relatively large amount of plasma availablefrom anemic patients lends itself more toalloadsorption.

Autoadsorption is inappropriate for the recentlytransfused patient. If the procedure is inadvertentlyapplied to samples from such patients there is a riskthat alloantibodies may be adsorbed in vitro onto

transfused RBCs. Our protocol for patients transfusedwithin the last 3 months is to perform alloadsorptionand to prepare an eluate to detect alloantibodies boundin vivo.8

Transfusion practices

Transfusion in the presence of panreactive warmautoantibodies can be a complicated and dangerousproposition.9 In our study, adsorption techniquesexcluded the presence of alloantibodies in 69 percentof our patients. In the patients found to havealloantibodies, adsorption studies allowed the selectionof appropriate RBCs for transfusion. In this group, Rhand Kell phenotype matching alone would haveexposed 14 patients (11%) to the risk of a significanttransfusion reaction.

We believe this study reinforces the value ofadsorption studies when panreactive warmautoantibodies are present and concurs with thefindings of similar studies. Our findings also show thatit is not appropriate in these cases simply to issue RBCsthat are “least incompatible” or Rh phenotype- and Kantigen-matched.

References1. Branch DR, Petz LD. Detecting alloantibodies in

patients with autoantibodies.Transfusion 1999;39:6-10.

2. Leger RM, Garratty G. Evaluation of methods fordetecting alloantibodies underlying warmautoantibodies. Transfusion 1999;39:11-6.

3. Petz LD. “Least incompatible”units for transfusionin autoimmune hemolytic anemia: should weeliminate this meaningless term. A commentaryfor clinicians and transfusion medicine pro-fessionals. Transfusion 2003;43:1503-7.

4. James P, Rowe GP, Tozzo GG. Elucidation ofalloantibodies in autoimmune haemolyticanaemia. Vox Sang 1988;54:167-71.

5. Garratty G. Problems associated withcompatibility testing for patients with auto-immune hemolytic anemia. Southeast Asian J TropMed Public Health 1993;24(Suppl 1):76-9.

6. Chapman JF, Elliott C, Knowles SM, Milkins CE,Poole GD;Working Party of the British Committeefor Standards in Haematology Blood TransfusionTask Force. Guidelines for compatibilityprocedures in blood transfusion laboratories.Transfus Med 2004;14:59-73.


Alloantibodies underlying autoantibodies

7. Issitt PD,Anstee DJ. Applied blood group serology.4th ed. Miami: Montgomery Scientific Publi-cations, 1998: 477.

8. Laine EP, Leger RM, Arndt PA, Calhoun L. In vitrostudies of the impact of transfusion on thedetection of alloantibodies after autoadsorption.Transfusion. 2000;40:1384-7.

9. Buetens OW, Ness PM. Red blood cell transfusionin autoimmune hemolytic anemia. Curr OpinHematol 2003;10:429-33.

Martin Maley, MPhil, Section Head, David G. Bruce,MSc, Section Head, Roderick G. Babb, BA, M.Ed,Laboratory Manager, Red Cell ImmunohaematologyLaboratory, and Angus W. Wells, MRCP(UK),MRCPath, Consultant in Transfusion Medicine,Newcastle Blood Service, Barrack Road, Newcastleupon Tyne, NE2 4NQ, UK; and Mark Williams, MSc,Reference Service Manager, Leeds Blood Service,Bridle Path, Seacroft, Leeds, LS15 7TW, UK.

Free Classified Ads and AnnouncementsImmunohematology will publish classified ads and announcements (SBB schools, meetings, symposia, etc.)without charge. Deadlines for receipt of these items are as follows:

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The Drori (Dra) antigen is one of the ten high-prevalence antigensof the Cromer blood system, which are carried on decay-accelerating factor (DAF, CD55). The Dr(a–) phenotype was firstdescribed in a 48-year-old Jewish woman from Bukhara. Her serumcontained an antibody to a high-prevalence antigen named anti-Dra.Most known individuals with the Dr(a–) phenotype are Jews fromthe geographic area of Bukhara, but individuals from Japan havealso been described. Antibodies in the Cromer blood group system,including anti-Dra,have never been reported to cause HDN. In mostof the cases with anti-Dra examined in Israel, the antibodies havebeen subtyped as IgG2 and IgG4. This report is of a woman withDr(a–) phenotype and an anti-Dra titer of 256 to 512 in her serum,observed during two successive pregnancies. At birth, the RBCs ofthe first- and second-born child were negative and positive in theDAT, respectively, and neither manifested clinical signs of HDN. Thedisappearance of Cromer system antibodies, including anti-Dra inmidpregnancy, has been described in a previous study. In thatstudy, it was theorized that the antibodies in the serum of thewomen were adsorbed onto placental DAF. The finding of a highanti-Dra titer in two successive pregnancies in this patient, with apositive DAT for the RBCs of one of the two babies at term, differsfrom published reports, suggesting that a different mechanismmight be involved. Immunohematology 2005;21:126–128.

Key Words: Anti-Dra, titers in pregnancy

The Drori (Dra) antigen is part of the Cromer bloodgroup system, which includes ten high- and three low-prevalence antigens.1 The Cromer antigens are carriedon the complement regulatory glycoprotein decay-accelerating factor (DAF;CD55). The Dr(a–) phenotypewas first described in a 48-year-old Jewish woman ofBukharian origin by Levene et al.2 More cases havebeen published; most of them were of Jewish womenborn in Bukhara, a few were of individuals fromJapan.3,4

Antibodies in the Cromer system have not beenreported to cause clinical HDN.1 There are reports inthe literature of women with Cromer antibodies inwhich the titer fell during pregnancy and the babycarrying a Cromer antigen was born with a negativeDAT.5,6 DAF is expressed strongly on the apical surfaceof trophoblasts, more so in the second and thirdtrimesters.7 The placental trophoblasts possess theDAF polymorphism of the fetus, which is inheritedfrom both parents and is most likely positive for the

high-prevalence Cromer antigens, including Dra. Reidet al.8 suggested that antigen-positive trophoblasts mayabsorb maternal antibodies with DAF specificity suchas anti-Dra, causing their disappearance from maternalblood and explaining the lack of HDN secondary to thisantibody. They reported two cases with Cromerantibodies, one of them anti-Dra, which disappearedduring pregnancy, supporting their hypothesis.

In this report two successive pregnancies in aDr(a–) woman with anti-Dra in her serum are described.The anti-Dra titers did not change significantlythroughout either pregnancy. At birth,a DAT performedon the RBCs of the first baby was negative and thatperformed on those of the second baby was positive.Neither baby had clinical evidence of HDN.

Case ReportA 28-year-old Jewish woman of Bukharian origin in

her second pregnancy was admitted to the AssafHarofeh Medical Center delivery room in labor inMarch 1998. A RBC sample was sent to the blood bankfor blood type and antibody screen. An antibody to ahigh-prevalence antigen was found in her serum andinvestigated. Her RBCs were typed as Group A1,D–,C–,E–, c+, e+, Cw–; M–, N+, S–, s+; P1–; Lu(a–b+); K–, k+,Kp(a–b+); Le(a–b+); Jk(a+b+); Yt(a+), and Dr(a–) andthe DAT was negative. During testing for the Cromerantigens,weaker reactions were observed with anti-IFCand -Cra when compared with the positive controls. Asample from her husband was tested and his RBCswere determined to be Group A1, D+, C+, E–, c–, e+,Cw–; M+, N+, S–, s+; P1+; Lu(a–b+); K–, k+, Kp(a–b+);Fy(a+b+); Jk(a+b–);Yt(a+b–),and Dr(a+). His antibodyscreen and DAT were negative.

The patient was found to have anti-D (low titerfrom passive immunization in pregnancy) and anti-LebH.An antibody to a high-prevalence antigen was found inher serum,which was characterized as anti-Dra, and herRBCs typed as Dr(a–). The titer of the antibody by

Persistent anti-Dra in twopregnanciesN. RAHIMI-LEVENE,A. KORNBERG, G. SIEGEL,V. MOROZOV, E. SHINAR, O.ASHER, C. LEVENE,AND V.YAHALOM


Persistence of anti-Dra in pregnancy

saline indirect antiglobulin test was 512 (Table 1). Thepatient’s serum was examined with two known Dr(a–)RBCs and the presence of anti-K, anti-M, anti-S, and anti-P1 were excluded.

Materials and MethodsStandard tube hemagglutination tests were used for

antigen typing, antibody screening, and panels.Antibodies and rare RBCs used in the testing were fromthe Israeli National Blood Group Reference Laboratoryand SCARF. Column agglutination tests (DiaMed AG,Switzerland) were also used for antibody screening andpanels. The RBCs were examined with anti-DAF(CD55) and anti-MIRL (CD59) in column agglutinationtests (DiaMed AG). The elution was performed usingthe rapid acid method (Elukit II, Gamma Biologicals,Inc., Houston,TX).

ResultsThe RBCs of the newborn were negative in the

DAT and there was no clinical evidence of HDN. InMay 2000, during her next pregnancy, anti-Dra wasidentified with a titer of 256 (Table 1). In June 2000,the anti-Dra titer was 512 and in July 2000 the titer was256. The pregnancy was uncomplicated and RhIG wasagain administered antenatally. The patient gave birthin October 2000 to a full-term healthy baby, the titerremaining stable throughout pregnancy. The infant’sRBCs were typed as Group A, D+; the DAT was positivewith anti-IgG resulting in 2+ to 3+ reactivity in tubesand 4+ by the column agglutination test (DiaMed AG).The positive DAT precluded Dra typing. An elution wasperformed on the baby’s RBCs and anti-Dra wasidentified. The eluate did not react with RBCs from themother, Dr(a–) RBCs, or RBCs treated with alpha-chymotrypsin. There was no clinical evidence of HDN.The titer of the anti-Dra in the mother’s serum was 256.Testing of maternal RBCs by the column agglutinationtest (DiaMed) was negative with anti-DAF (CD55) andpositive with anti-MIRL (CD59).

DiscussionDAF is a protein connected to the membrane via a

glycosylphosphatidylinositol anchor. It protects cellsagainst destruction by autologous complement byinhibiting formation and acceleration of the decay ofC3 and C5 convertases, thus preventing thecomplement cascade from causing hemolysis. DAF is amember of the regulators of complement activationgene family encoded by a gene on the long arm ofchromosome 1.9 It is widely distributed throughoutthe body and expressed on epithelial cells, endothelialcells, blood vessels, and the apical surface oftrophoblasts.7,10 The Cromer antigens are carried onDAF and the different phenotypes provide a basis forbiochemical and functional investigation of alternateforms of DAF. Dr(a–) is a rare Cromer phenotype,lacking expression of the Dra antigen, with reducedlevels of DAF and weak expression of other Cromerantigens, including Cra, Tca, Tcb, Tcc, Esa, IFC, Wesb, andUMC.10 No hematological abnormalities have beendescribed in Dr(a–) individuals. The molecular changeassociated with Dr(a–) has been characterized.9 Fourpregnancies in a woman who had Cr(a–) RBCs andanti-Cra, in which the titer of the anti-Cra declinedduring pregnancy and remained low until term, weredescribed by Sacks and Garratty.6 The decline in thetiter was thought to result from absorption of theantibody by white cells or platelets or be due toneutralization by plasma of the fetus. Another case ofanti-Cra in a pregnant woman in which the titer of theanti-Cra decreased was described by Dickson et al.5

Reid et al.8 described two patients with repeatpregnancies who had Cromer blood group systemantibodies (anti-Cra and anti-Dra) with titers of 128 orgreater at the beginning of pregnancy. Theseantibodies became undetectable in the serum from thesecond trimester onward. RBCs from the babies testedpositive for the relevant Cromer antigens but werenegative by the DAT. The antibodies would reappear inthe serum of the mother after delivery or at thebeginning of a subsequent pregnancy.

Holmes et al.7 showed the preferential expressionof DAF at the feto-maternal interface of the placenta,increasing quantitatively during placental develop-ment. DAF at this site plays a protective role againstmaternal complement-mediated attack. DAF canabsorb the Cromer antibodies produced by the mother,more so in the second and third trimesters when theexpression of DAF increases. This hypothesis issupported by reappearance of these antibodies after

Table 1. Anti-Dra titers

Date Titer

May 1998 512

April 1999 256

May 2000 256

June 2000 512

July 2000 256

August 2000 256

September 2000 256


N. RAHIMI-LEVENE ET AL.

pregnancy when the placenta is no longer present.Most of the patients with anti-Dra found in Israel havebeen multiparous women. Subclasses of IgG examinedin these cases during the years 1989–1991 were IgG2and IgG4. The anti-IgG subtype antibodies used werefrom the Netherlands Red Cross and tests wereperformed in tube or capillary tests. None of theoffspring had HDN (C. Levene, unpublished data,1989–1991). The patient reported here was Dr(a–).She developed anti-Dra and was monitored during twosubsequent pregnancies. The anti-Dra titer remainedconsistently at 256 or above during both pregnancies.RBCs from the first baby typed as Dr(a+) and the DATwas negative.RBCs from the second baby were positivein the DAT; they could not be typed for the Dra antigen,but anti-Dra was identified in the eluate,with no clinicalsigns of HDN. Our findings differ from previous5,6,8,11

reports, where anti-Cromer antibody titer declinedduring pregnancy. Reid et al.8 suggested that the titerdecreased due to Cromer blood group systemantibodies binding to the placental DAF. In the casedescribed here, presumably the anti-Dra was notadsorbed by the placental DAF, which might happen ifthe placental DAF was Dr(a–), if Dra was poorlyexpressed, or due to another unexplained mechanism.

AcknowledgmentThe authors wish to thank Leah Mendelson and

Lydia Yosephi for technical assistance.

References1. Daniels GL, Fletcher A, Garratty G, et al. Blood

group terminology 2004: from the InternationalSociety of Blood Transfusion committee onterminology for red cell surface antigens. VoxSang 2004;87:304-16.

2. Levene C, Harel N, Lavie G, Greenberg S, et al. A“new” phenotype confirming a relationshipbetween Cra and Tca. Transfusion 1984;24:13-5.

3. Levene C,Harel N,Kende G,et al. A second Dr(a–)proposita with anti-Dra and a family with theDr(a–) phenotype in two generations. Transfusion1987;27:64-5.

4. Daniels GL, Green CA, Mollinson G, Okubo Y, et al.Decay-accelerating factor (CD55) deficiencyphenotypes in Japanese. Transfus Med1998;8:141-7.

5. Dickson AC, Guest C, Jordan M, et al. Case report:anti-Cra in pregnancy. Immunohematology1995;11:14-7.

6. Sacks DA, Garratty G. Isoimmunization to Cromerantigen in pregnancy. Am J Obstet Gynecol1989;161:928-9.

7. Holmes CH, Simpson KL, Wainwright SD, et al.Preferential expression of the complementregulatory proteins decay accelerating factor atthe fetomaternal interface during humanpregnancy. J Immunol 1990;144:3099-3105.

8. Reid ME, Chandrasekaran V, Sausais L, et al.Disappearance of antibodies to Cromer bloodgroup system antigens during mid pregnancy. VoxSang 1996;71:48-50.

9. Lublin DM, Thompsen ES, Green AM, et al. Dr(a–)polymorphism of decay accelerating factor.Biochemical, functional and molecularcharacterization and production of allele-specifictransfectants. J Clin Invest 1991;87:1945-52.

10. Daniels G: Human blood groups. 2nd ed. Oxford:Blackwell Science, 2002;444-54.

11. Storry JR, Reid ME. The Cromer blood groupsystem: a review. Immunohematology 2002;18:95-103.

Naomi Rahimi-Levene MD, Director of the BloodBank; Abraham Kornberg MD, Chief of theHematology Institute; Gabriela Siegel, High RiskPregnancy Unit; and Valery Morozov, MD, BloodBank, Assaf–Harofeh Medical Center, Zerifin, 70300,Israel; Eilat Shinar MD, Director; Orna Asher PhD,Director Immunohematology Laboratory; CyrilLevene MD, Consultant National Blood GroupReference Laboratory (NBGRL); and Vered YahalomMD, Deputy Director & Medical Director NBGRLMagen David Adom (MDA)–National Blood Services,Ramat Gan, Israel.


C O M M U N I C A T I O N S

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 clinicaltransfusion laboratory. The evaluation and laboratory investigation of alloimmunization,which occurred as a resultof 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 thedifferences in blood group antigen expression between individuals, and to infer the inheritance of the genes thatexpress these antigens. Of course, a natural progression of the latter included the analysis of the genes and allelesthat are responsible for the expression of the antigens. The ease with which molecular biological techniquesbecame 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 bloodgroup genotyping, although their initial application was not true “genotyping”as it merely confirmed the presenceof the RHD gene. The unique application used DNA derived from cells in amniotic fluid to “infer” the blood groupof fetuses at risk for HDN due to anti-D,1 thus alleviating the risk associated with cordocentesis to establish the Dantigen of a fetus. Today, 50 percent of pregnancies at risk for the common antibodies implicated in HDN, whenthe father is heterozygous for the corresponding allele, are deemed to be antigen-negative and not at risk for thedisease. Clinical management varies, but essentially the mother can return to her primary care physician andnoninvasive 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 groupgenes has expanded to include the following uses:

1) Genotype multi-transfused recipients, patients with severe thrombocytopenia, and those whose RBCs orplatelets are sensitized with antibodies

2) Resolve ABO and Rh D discrepancies3) Identify some of the Rh D variants that are at risk for anti-D alloimmunization4) Determine RHD zygosity5) Confirm the true genotype when an antigen is weakly expressed, e.g., Del6) Identify whether the Fyb– transfusion recipient can safely receive Fyb+ RBCs7) Mass screen for antigen-negative and rare RBCs8) Genotype donors for antibody identification panels

During the 1950s and 1960s people with a common interest in blood group serology formed focus groups todisseminate and exchange information, mentor young scientists, and advance our understanding of the field ofimmunohematology. 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 annualmeeting on October 23, 2004, in Baltimore, Maryland. The meeting was attended by 24 delegates from North andSouth America, with contributions from other colleagues who could not attend. The group was formed out ofthese:

• 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 andembrace the opportunity to discuss and promote scientific inquiry in blood group genes. It is anticipated that


C O M M U N I C A T I O N S C O N T ’ D

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

Consortium for Blood Group Genes1) Language

Group meetings and interactions will be conducted in English.

2) MembershipMembership is open to those with an interest in single nucleotide polymorphisms and other geneticvariations 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) NameThe name of the organization is the Consortium for Blood Group Genes (CBGG).

4) Web SiteA Web site will be established in the near future.

5) Working PartiesWorking parties of volunteers were established to investigate, collate, and disseminate recommendationsto the entire group on the areas listed below.

a. Proficiency ProgramOne role of the CBGG is to contribute to and participate in proficiency testing much likeimmunohematology reference laboratories (IRLs), with two samples exchanged per year. One goal ofproficiency testing is to have a forum for DNA testing verification and results analysis. The workingparty 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 reportswere asked to share copies of their reports to give a feel for the types of disclaimers they use. Thegroup could help unify reports and set the standards for DNA blood group testing forms and reportsand recommended disclaimers. The clinical forms and reports section will be addressed by a workingparty.

c. Reference DNA samples There is a need to establish a source of samples for control and for validation purposes. It is anticipatedthat standard DNA reference samples will be made available as well as a listing of important DNA singlenucleotide polymorphisms, amplification primers, and standard protocols.

d. StandardsThe CBGG has the potential to develop standards for its members and act as a voice to regulatorybodies as the testing becomes more commonplace. An FDA representative has been contacted sincethe inaugural meeting. The group is encouraged to see an open partnership developing that willcommunicate needs and issues and ensure appropriate compliance.


e. FundingSources of funding were discussed. Possibilities included a nominal membership fee (e.g., $100), a feefor shipping DNA from the repository, and a fee for participating in the proficiency program. Otherpotential sources of funding were identified.

f. Structure and Bylaws The structure, bylaws, and mechanism for nominating officers to represent the consortium are in theinitial stages.

1. Bennett PR, LeVan Kim C, Colin Y, Warwick RM, et al. Prenatal determination of fetal RhD type by DNAamplification. N Engl J Med 1993;329:607-10.

Greg Denomme, PhDBlood Transfusion Services, Suite 600

Mount Sinai Hospital600 University Avenue

Toronto, Ontario, Canada M5G [email protected]

Tel: 416-586-8855

Marion Reid, PhDNew York Blood Center

New York, NY



ERRATUM

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

Review: acute Donath-Landsteiner hemolytic anemia

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


Ice Ice 37°Cthen37°C

Reaction conditions

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

1 A B C

Set 1: Patient’s serum

2

Set 2: Patient’s serum + Normal serum

3

Set 3: Normal serum

A B C

A B C

A B C


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

A N N O U N C E M E N T S

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

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


A N N O U N C E M E N T S C O N T ’ 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].


S P E C I A L A N N O U N C E M E N T S

Meetings!

April 28–30 Immunohematology Reference Laboratory (IRL) Conference 2006The American Red Cross Immunohematology Reference Laboratory (IRL) Conference will be held April 28through 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].

Manuscripts: The editorial staff of Immunohematology welcomes manuscripts pertaining to blood groupserology and education for consideration for publication. We are especially interested in case reports, paperson platelet and white cell serology, scientific articles covering original investigations, and papers on newmethods 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 everyissue of Immunohematology or on the Web. Include fax and phone numbers and e-mail address withyour manuscript.

IMMUNOHEMATOLOGY IS ON THE WEB!

www.redcross.org/pubs/immuno

Password “2000”

For more information or to send an e-mail message “To the editor”

[email protected]

Notice to Readers: Immunohematology, Journalof Blood Group Serology and Education, is printed on acid-free paper.

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


NATIONAL REFERENCE LABORATORY FORBLOOD GROUP SEROLOGY

Immunohematology ReferenceLaboratory

AABB,ARC, New York State, and CLIA licensed(215) 451-4901—24-hr. phone number

(215) 451-2538—Fax

American Rare Donor Program(215) 451-4900—24-hr. phone number

(215) 451-2538—[email protected]

Immunohematology(215) 451-4902—Phone, business hours

(215) 451-2538—[email protected]

Quality Control of Cryoprecipitated-AHF(215) 451-4903—Phone, business hours

(215) 451-2538—Fax

Granulocyte Antibody Detection and Typing

• Specializing in granulocyte antibody detectionand granulocyte antigen typing

• Patients with granulocytopenia can be classifiedthrough the following tests for proper therapyand monitoring:—Granulocyte agglutination (GA)—Granulocyte immunofluorescence (GIF)—Monoclonal Antibody Immobilization of

Granulocyte Antigens (MAIGA)

For information regarding services, call Gail Eiberat: (651) 291-6797, e-mail: [email protected],

or write to:Neutrophil Serology Reference Laboratory

American Red CrossSt. Paul Regional Blood Services

100 South Robert StreetSt. Paul, MN 55107

CLIA LICENSED

National Platelet Serology ReferenceLaboratory

Diagnostic testing for:• Neonatal alloimmune thrombocytopenia (NAIT)• Posttransfusion purpura (PTP)• Refractoriness to platelet transfusion• Heparin-induced thrombocytopenia (HIT)• Alloimmune idiopathic thrombocytopenia

purpura (AITP)Medical consultation available

Test methods:• GTI systems tests

—detection of glycoprotein-specific plateletantibodies

—detection of heparin-induced antibodies (PF4ELISA)

• Platelet suspension immunofluorescence test(PSIFT)

• Solid phase red cell adherence (SPRCA) assay• Monoclonal antibody immobilization of platelet

antigens (MAIPA) • Molecular analysis for HPA-1a/1b

For information, e-mail: [email protected] call:

Maryann Keashen-Schnell(215) 451-4041 office

(215) 451-4205 laboratory

Sandra Nance(215) 451-4362

Scott Murphy, MD(215) 451-4877

American Red Cross Blood ServicesMusser Blood Center

700 Spring Garden StreetPhiladelphia, PA 19123-3594

CLIA LICENSED

A D V E R T I S E M E N T S


A D V E R T I S E M E N T S C O N T ’ D

IgA/Anti-IgA Testing

IgA and anti-IgA testing is available to do the following:• Monitor known IgA-deficient patients• Investigate anaphylactic reactions• Confirm IgA-deficient donors

Our ELISA assay for IgA detects antigen to 0.05 mg/dL.

For information on charges and sample requirements, call (215) 451-4909, e-mail:

[email protected],or write to:

American Red Cross Blood ServicesMusser Blood Center

700 Spring Garden StreetPhiladelphia, PA 19123-3594

ATTN: Cindy Flickinger

CLIA LICENSED

Reference and Consultation Services

Antibody identification and problem resolution

HLA-A, B, C, and DR typing

HLA-disease association typing

Paternity testing/DNA

For information regarding our services, contact

Mehdizadeh Kashi at (503) 280-0210, or write to:

Pacific Northwest Regional Blood Services

ATTENTION: Tissue Typing Laboratory

American Red Cross

3131 North Vancouver

Portland, OR 97227

CLIA LICENSED, ASHI ACCREDITED

National Neutrophil Serology ReferenceLaboratory

Our laboratory specializes in granulocyteantibody detection and granulocyte antigentyping.

Indications for granulocyte serology testinginclude:• Alloimmune neonatal neutropenia (ANN)• Autoimmune neutropenia (AIN)• Transfusion related acute lung injury (TRALI)

Methodologies employed:• Granulocyte agglutination (GA)• Granulocyte immunofluorescence by flow

cytometry (GIF)• Monoclonal antibody immobilization of

neutrophil antigens (MAINA)

TRALI investigations also include:• HLA (PRA) Class I and Class II antibody

detection

For further information contact:

Neutrophil Serology Laboratory(651) 291-6797

Randy Schuller(651) 291-6758

[email protected]

American Red Cross Blood ServicesNeutrophil Serology Laboratory

100 South Robert StreetSt. Paul, MN 55107

CLIA LICENSED

Notice to Readers: All articles published,including communications and book reviews,reflect the opinions of the authors and do notnecessarily reflect the official policy of theAmerican Red Cross.


SCIENTIFIC ARTICLES, REVIEWS, AND CASE REPORTSBefore submitting a manuscript, consult current issues of

Immunohematology for style. Type the manuscript on white bondpaper (8.5" × 11") and double-space throughout. Number the pagesconsecutively in the upper right-hand corner, beginning with thetitle page. Each component of the manuscript must start on a newpage in the following order:1. Title page2. Abstract3. Text4. Acknowledgments5. References6. Author information7. Tables—see 7 under Preparation8. Figures—see 8 under Preparation

Preparation of manuscripts1. Title page

A.Full title of manuscript with only first letter of first word capitalized (bold title)

B. Initials and last name of each author (no degrees; all CAPS),e.g., M.T. JONES and J.H. BROWN

C.Running title of ≤ 40 characters, including spacesD.3 to 10 key words

2. AbstractA.One paragraph, no longer than 300 wordsB. Purpose, methods, findings, and conclusions of study

3. Key words—list under abstract4. Text (serial pages)

Most manuscripts can usually, but not necessarily, be divided intosections (as described below). Results of surveys and reviewpapers are examples that may need individualized sections.A. Introduction

Purpose and rationale for study, including pertinent back-ground references.

B. Case Report (if study calls for one)Clinical and/or hematologic data and background serology.

C.Materials and MethodsSelection and number of subjects, samples, items, etc. studiedand description of appropriate controls, procedures, methods,equipment, reagents, etc. Equipment and reagents should beidentified in parentheses by model or lot and manufacturer’sname, city, and state. Do not use patients’ names or hospitalnumbers.

D.ResultsPresentation of concise and sequential results, referring to perti-nent tables and/or figures, if applicable.

E. DiscussionImplications and limitations of the study, links to other studies;if appropriate, link conclusions to purpose of study as stated inintroduction.

5. AcknowledgmentsAcknowledge those who have made substantial contributions tothe study, including secretarial assistance; list any grants.

6. ReferencesA. In text, use superscript, arabic numbers.B. Number references consecutively in the order they occur in

the text.C.Use inclusive pages of cited references, e.g., 1431–7.D.Refer to current issues of Immunohematology for style.

7. TablesA.Head each with a brief title, capitalize first letter of first word

(e.g., Table 1. Results of ...), and use no punctuation at the endof the title.

B. Use short headings for each column needed and capitalize firstletter of first word. Omit vertical lines.

C.Place explanations in footnotes (sequence: *, †, ‡, §, ¶, **, ††).8. Figures

A.Figures can be submitted either by e-mail or as photographs (5″ × 7″ glossy).

B. Place caption for a figure on a separate page (e.g.,Fig.1.Resultsof ...), ending with a period. If figure is submitted as a glossy,place first author’s name and figure number on back of eachglossy submitted.

C.When plotting points on a figure, use the following symbols ifpossible: ●● ● ▲▲ ▲ ■■ ■.

9. Author informationA.List first name, middle initial, last name, highest academic

degree, position held, institution and department, and complete address (including zip code) for all authors. Listcountry when applicable.

SCIENTIFIC ARTICLES AND CASE REPORTS SUBMITTED AS LETTERS TO THE EDITOR

Preparation1. Heading—To the Editor:2. Under heading—title with first letter capitalized.3. Text—write in letter format (paragraphs).4. Author(s)—type flush right; for first author: name, degree,

institution, address (including city, state, ZIP code, and country);for other authors: name, degree, institution, city, and state.

5. References—limited to ten.6. One table and/or figure allowed.

ImmunohematologyJOURNAL OF BLOOD GROUP SEROLOGY AND EDUCATION

Instructions for Authors

Send all manuscripts by e-mail to:

Marge Manigly at [email protected]


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 EducatorsSupervisors of Reference Laboratories Research Scientists Consumer Safety OfficersQuality 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 SBBexam.

Conclusion:The BEST route for obtaining an SBB certification is to attend a CAAHEP-accredited Specialist in Blood BankTechnology Program

Becoming a Specialist in Blood Banking (SBB)

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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