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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

1A review of the Knops blood group: separating fact from fallacy

J.M. MOULDS

9A gel microtyping system for diagnosis of paroxysmal nocturnal hemoglobinuria

B. ZUPANSKA, B. BOGDANIK, AND H. PYL

13Antibody screening in 37°C saline. Is it safe to omit it using the indirect antiglobulin (gel) test?

J. DURAN AND M. FIGUEIREDO

16Autoanti-D in a patient after cladribine treatment for lymphoplasmocytic lymphoma

J. CID, V. BELTRAN, L. ESCODA, E. ELIES, AND C. MARTIN-VEGA

19Warm autoimmune hemolytic anemia with mimicking anti-c and -E specificities

H.-Y. HSIEH, D. MORONEY, D. NAUMANN, J. HATA, N. VOSNIDOU, R. KESSINGER, N. SHAHAB, N. HAKAMI, AND D. SMITH

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Anti-cE (Rh27), a rarely occurring antibodyG. LODI, D. RESCA, R. REVERBERI, AND M. GOVONI

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Letter From the Editors

Thoughts on September 11

24 24A 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

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Geoffrey Daniels, PhDBristol, United Kingdom

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Brenda J. Grossman, MDSt. Louis, Missouri

Christine Lomas-Francis, MScAustin, Texas

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A review of the Knops blood group:separating fact from fallacyJ.M. MOULDS

IntroductionIt has been more than 10 years since the topic of “high-titer, low-avidity” (HTLA) antibodies was reviewed in Immunohematology.We have learned a lot about these antibodies in the past 10 yearsand that knowledge has helped us to understand some of theunusual characteristics of these antibodies. Furthermore, it hashelped us to name and delineate the various associated bloodgroup systems. Although we will begin with a general review ofHTLAs, this manuscript will focus on the recent findings in theKnops blood group system. Immunohematology 2002;18:1–8.

Key Words: Knops, polymorphism, CR1, blood group

Historical Perspective

HTLAsTo really understand some of the fallacies that have

arisen regarding HTLAs, we must delve into theirhistory, i.e., how they were discovered and how theywere named. In the late 1960s and early 1970s, bloodgroup serologists from the American Association ofBlood Banks (AABB) and the American Red Cross (ARC)reference laboratories would gather at wet workshopsand share samples for the classification of antibodiesand the formation of blood group systems. During thistime a number of weak antihuman globulin-reactive(AHG) antibodies that appeared to have similarcharacteristics were studied. When the serum wastitrated and the reactions examined microscopically,positive reactions could be observed at dilutions of 1:64or greater. This was very unusual, as the neat reactionswould only be m+ to 1+. The agglutinates often werefragile and could be easily “shaken out.” Hence, severalreference laboratory technologists began referring tothis group as “high-titer, low avidity” antibodies. Theynever meant this to denote a blood group system, onlyan identifying characteristic (D. Mallory, personalcommunication).

Members of this newly formed group included:Chido, Rodgers, Knops, McCoy, Swain-Langley, Cost,York, JMH, Holley, Gregory, and, sometimes, Cartwright.The latter was already known to be an independent

blood group system, i.e., International Society of BloodTransfusion (ISBT) 011. Since that time, all haveachieved blood group status with the exception of Cost(Csa), which remains a “collection” as defined by theISBT Working Party on Terminology of Red Cell SurfaceAntigens. Chido and Rodgers were the first antigens inthe group to be located on a membrane protein andgiven blood group status (ISBT 017). They were foundto be antigens carried on the C4d fragment of the C4Band C4A isotypes, respectively. The Knops antigenswere identified as polymorphisms of complementreceptor type one (CR1) and accordingly Knopsbecame the 22nd blood group system. Serologic andbiochemical studies resulted in the assignment of bothHolley and Gregory to the Dombrock system (ISBT 014)and, most recently, JMH (CDw108) was named system026.1

KnopsAlthough many of the HTLA antibodies were

simultaneously being investigated in the 1960s, one ofthe first to be reported was anti-Csa. Giles et al.2 foundthat the sera from three patients had the samespecificity and they were mutually compatible. Thus,Csa (Cost) was named after two of the first antibodyproducers, i.e., Copeland (CO) and Stirling (ST).Although anti-Csa appeared to have some associationwith York, it would later be shown not to be part of theKnops blood group system.3,4

The Knops system began to take form when anti-Kna

was described in a transfused Caucasian female whohad a saline-reactive anti-K plus an unidentifiedantiglobulin-reactive antibody to a high-frequencyantigen.5 A blood bank technologist (Helgeson) foundthat her red blood cells (RBCs) were compatible withthe Knops serum. The “Helgeson phenotype” wouldlater be identified as the serologically null phenotypefor the Knops blood group. York (Yka) was the nexthigh-incidence Knops antigen (KN5) to be reported butit was initially believed to be associated with Cost (Csa)

I M M U N O H E M A T O L O G Y , V O L U M E 1 8, N U M B E R 1, 2 0 0 2 1

2 I M M U N O H E M A T O L O G Y , V O L U M E 1 8, N U M B E R 1, 2 0 0 2

J.M. MOULDS

Table 1. Prominent characteristics of Knops system antibodies

• High-titer, low-avidity• Can demonstrate variable reactions• Not neutralized by pooled serum or other body fluids• Difficult to adsorb and elute• IgG, reacting by AHG* technique• Do not bind complement• Reactive with enzyme-treated RBCs†

• Usually not clinically significant

* Antihuman globulin† Red blood cells

Table 2. Prominent characteristics of the Knops antigens

• Inherited as Mendelian dominant traits• High-frequency RBC* antigens• Generally developed on cord RBCs• Not denatured by proteolytic enzymes• Not found on platelets• Not found as soluble antigens• Denatured by reducing antigens, e.g., DTT, AET

* Red blood cell

rather than Kna.6 Several years later, Molthan andMoulds7 described a new antigen, McCa, that seemed tobe related to Kna. Interestingly, a majority of McCa

antibody producers were black, while most of thosemaking anti-Kna were Caucasian, thus suggesting thatethnic differences might exist in their respective genefrequencies. Finally, Sla and Vil were reported in separateabstracts, with one author using the term McCc for Sla.8,9

Antigen/Antibody CharacteristicsThe description of HTLA antibody characteristics and

their corresponding antigens has remained fairlyunchanged over the past 30 years. Prominentcharacteristics of the Knops system antibodies and

antigens are summarized in Tables 1 and 2. Urine orsaliva did not inhibit any of these antibodies; however, itwas found that only anti-Chido or -Rodgers could beinhibited with plasma. Because the antibodies werefound in multiply transfused individuals, the serumoften contained additional antibodies, such as anti-K, -E,and Duffy antibodies. The HTLA specificities were notconsidered “clinically significant” because they did notcause overt hemolytic transfusion reactions orhemolytic disease of the newborn.

One of the problems in studying such serum and cellsamples was that they did not travel well. The antigensvaried greatly in strength and often weakly reactiveRBCs were negative by the time they arrived at asecondary consultation laboratory. Although ficindestroyed the Ch, Rg, and JMH antigens, it had no effecton Knops or McCoy. Later it was shown that chemicals

that could disrupt disulfide bonds, i.e., dithiothreitol(DTT) and 2-aminoethylisothiouronium bromide (AET),could also destroy Knops, McCoy, and York. This wasthe dogma, although we had little insight into whythese antibodies behaved as they did.

Biochemical Identification of KnopsAntigens

In 1991, two groups identified CR1 as the proteincarrying the Kna, McCa, and Sla (McCc) blood groupantigens.3,10 In addition, Moulds et al.3 identified Yka onCR1 and suggested that the Helgeson phenotype wasdue to low CR1 copy numbers on the RBCs (E-CR1).The CR1 gene also exhibited two other polymorphismsbesides the Knops blood group. A structural poly-morphism results from four different alleles that encodefour different molecular-weight kiloDalton (kD)proteins: 190 kD (CR1*3), 220 kD (CR1*1), 250 kD(CR1*2), and 280 kD (CR1*4). The third commonlyrecognized polymorphism is based upon quantitativedifferences in E-CR1. A Hind III restriction fragmentlength polymorphism (RFLP), identified in Caucasians, isdetected by two allelic fragments of 7.4 or 6.9 kilobase(kb) on Southern blots. Homozygotes for the 7.4 kb-fragment (HH) are high CR1 expressors, heterozygotes(HL) express intermediate levels of CR1, andhomozygotes for the 6.9-kb fragment (LL) are lowexpressors of CR1.11 Alternatively, a polymerase chainreaction (PCR)-RFLP can be used, which results inbands of 1.8 kb for H alleles or 1.3 and 0.5 kb for Lalleles.12 Although this RFLP correlates with RBCexpression in Caucasians and Chinese,13 there is norelationship between this polymorphism and CR1expression in African Americans14 or West Africans (J.M.Moulds, unpublished data).

Molecular Identification of Knops AntigensThe CR1 gene resides on chromosome 1 (1q32) and

comprises 39 exons spread out over approximately 133kb pairs of DNA.15 These exons encode regions calledshort consensus repeats (SCRs) of approximately 60amino acids in the functional CR1 protein. Seven SCRsare organized into larger units called long homologousrepeats (LHRs). The most common size proteinproduct, CR1-1, is made up of 4 LHRs (A, B, C, D), a trans-membrane region, and a cytoplasmic tail domain (Fig.1). The binding sites for C3b and C4b have beenlocalized to SCRs 8–9 and 15–16 (LHRs B and C) and toSCRs 1–2 (LHR-A), respectively.


Knops blood group review

Fig. 1. Schematic drawing of the most common form of CR1.Thirty short consensus repeats (SCRs) are extracellular andare arranged into four long homologous regions (LHRs)followed by a transmembrane region (TMB) and acytoplasmic tail (CTY).

Using CR1 deletion constructs, Moulds et al.16 firstlocalized the McCoy and Sla antigens to LHR-D of CR1.By direct DNA sequencing they were then able toidentify two separate mutations in SCR 25 thatcorrelated with these two blood group antigens. TheMcCa/McCb polymorphism is at bp 4795, where an Aencodes proline (McCa) and a G encodes aspartic acid(McCb). The Sla/Vil mutation is only 33 bp (11 aminoacids) away at bp 4828; an A encodes arginine while aG encodes glycine. Accordingly, the ISBT1 has nowassigned these antigens to the Knops system with thefollowing numbers: Sla as KN4, McCb as KN6, and Vil asKN7. Two other mutations have been identified in SCR25, one of which was found in a Caucasian and isrelated to Sla.17 It is unknown at this time if the otherDNA mutation correlates with any of the other McCoyantigens named by Dr. Molthan, e.g., McCe, McCf, or ifthis represents “Kn/McC.”

Functions of CR1 and the Knops AntigensCR1 is a membrane-bound glycoprotein and is found

on most human peripheral RBCs. Depending upon themethods used, RBCs display approximately 300–800CR1 molecules per cell while leukocytes display~10,000–30,000 molecules per cell. Because RBCs arepresent in the peripheral circulation at concentrations103-fold higher than the peripheral blood mononuclearcells (PBMCs), they account for greater than 85 percentof CR1 in the blood. RBC CR1 binds immune complexes(ICs), which are shuttled to the liver or spleen fortransfer to and ingestion by macrophages, leading totheir elimination. IC-free RBCs return to the circulation,where they can continue participating in IC clearance.

In 1997, Rowe et al.18 identified CR1 as a ligand forthe rosetting of Plasmodium falciparum-infected RBCsamong uninfected cells. The ability of RBCs infectedwith P. falciparum to form rosettes is a property shownby only some parasite isolates, but is of importancebecause it has been associated with severe malaria.19

They showed that CR1 on uninfected RBCs was

required for the formation of rosettes in somelaboratory-adapted parasite strains, by demonstratingthat CR1-deficient erythrocytes (Helgeson phenotype)had reduced rosetting and soluble recombinant CR1could inhibit rosetting. RBCs having the Sl(a–) pheno-type (found more frequently among African-derivedpersons)20 showed reduced binding to the parasiterosetting ligand P. falciparum erythrocyte membraneprotein 1 (PfEMP1). Thus, the authors hypothesizedthat this polymorphism may have been selected for inmalarious regions by providing protection againstsevere malaria.

CR1, as well as other complement receptors, hasbeen identified as a receptor facilitating cell entry for avariety of pathogenic organisms. Pathogens utilizingCR1 include Babesia rodhaini (erythrocyte), Leish-mania major (monocyte-macrophage),21 Legionellapneumophila (monocyte-macrophage),22 Mycobacte-rium leprae (monocyte-macrophage),23 and Mycobac-terium tuberculosis (monocyte-macrophage).24

Separating Fact From Fallacy

Titer and avidityClearly we have learned a lot in the past 10 years

regarding the Knops blood group system. We can nowuse that knowledge to explain some of the earlierobservations and clarify some of the misconceptionsthat have arisen over the years. Let’s start with the name“high titer, low avidity (HTLA).” According to the AABBtechnical manual (W.V. Miller, ed., 1974) in use at thetime HTLAs were first described, “titer” was defined as“the reciprocal of the highest dilution at whichmacroscopic agglutination is observed.”25 Most of theKnops antibodies give w+ to 1+ reactions, even usingRBCs having moderate expression of CR1, and wouldnot appear to be of high titer using the technicalmanual definition. However, the term “high titer” wasapplied because weak reactions were observedmicroscopically that could give positive results past adilution of 1:64 and sometimes into the thousands. Buteven determining an antibody titer for any Knopsantibody can be problematic, because the CR1expression polymorphism complicates the choice ofRBCs, which ideally should be homozygous for thecorresponding antigen. As shown in Table 3, antibodytiter is very dependent on the indicator RBC that ischosen. RBCs with low expression may give onlymicroscopically positive reactions, resulting in lowscores, while RBCs with abundant CR1 give both high


Table 3. Titer of Knops system antibodies is dependent on the RBC*CR1 copy number (E-CR1)

Anti- Low E-CR1 Medium E-CR1 High E-CR1

Kna 64 (8) † > 1,024 (34) > 1,024 (62)McCa 32 (5) > 1,024 (33) > 1,024 (45)Sla 08 (3) > 1,064 (19) > 1,024 (48)

*Red blood cell† Score is shown in parenthesis

J.M. MOULDS

titers and high scores. Thus, using titration as the solemeans for classifying these antibodies is notrecommended.

The term “low avidity” may be more accurate in itsdescription of these antibodies, as avidity refers to thespeed and intensity of an antigen-antibody reaction. It isthis author’s experience that the Knops systemantibodies are not very avid and give the strongestresults when using a 60-minute incubation in saline at37°C. This recommendation was also made by someearly investigators but was subsequently lost in the rushto speed up serologic testing by using low-ionic-strength conditions or additives. Low-ionic-strengthsaline (LISS), polyethylene glycol (PEG), and evenalbumin do little to enhance the strength of thereactions for Knops system antibodies.26

Variable reactivityEarly investigations of the Knops blood group system

were hampered by the inability to duplicate reactivitybetween laboratories, especially when the RBCs had totravel long distances. This led to arguments betweenserologists and misidentification of many serum and cellsamples. Although the genetic differences in RBCexpression of CR1 contribute to this variability,27 wenow know that it is even more complex than firstbelieved. The first variable is the method used forantibody detection. As mentioned previously, Knopsantibodies prefer longer incubations, with 1 hour beingoptimal. In fact, when either LISS or PEG is used fortesting, antibody strength may be weakened.26 Thisauthor has also observed reduced strength of reactionswhen saline containing azide is used; this may actuallybe an advantage when doing compatibility testing!Finally, increasing the serum-to-cell ratio when weakreactions are observed may be counterproductive andresult in prozoning if the RBCs have below averageE-CR1.

Moulds et al.27 first reported that the weak andvariable reactions obtained with Knops antibodies weredue to variable expression of CR1. However, thecomplexity of this observation was not fully

appreciated at the time. Black Africans were found tohave higher E-CR1 levels than Caucasians,20 but Mouldset al.16 found that heterozygosity could result in a falselynegative phenotype even when E-CR1 was adequate.Combined phenotyping of fresh RBCs along withgenotyping of the same donor for McCa, McCb, Sla, andVil showed that there may be as much as 20 percentdiscordance between the methods. This had beenpreviously reported in a Caucasian donor who onlyexpressed the Yka antigen on one allele and whoseRBCs gave variable results when typed with severalexamples of anti-Yka.27 To clarify, the total E-CR1 may bein the normal range, i.e., 300 copies, but only one allelewould produce Yka. Consequently, only 50 percent ofthe CR1 molecules (~150 copies) would have Yka, andthis is in the range where variable results would beobtained. Since heterozygosity has been found to affectMcCoy, Sla, and York typings, it is very likely that asimilar situation exists for Kna and Knb.

The above scenario assumes equal expression ofboth CR1 alleles; however, we now know that not allalleles are equally expressed. This variation inexpression can be easily visualized using immuno-blotting techniques for the CR1 protein. Immunoblot-ting, in combination with genotyping, has shown thatfalsely negative serologic typings can be obtained evenwith high E-CR1 if a low-expressing allele is present incombination with heterozygosity for a particulargene.16,20 For example, a person with 500 copies of CR1(high copy number) is shown by genotyping to haveboth Sla and Vil encoding genes. If each gene wasequally expressed the result would be 250 copies for Sla

and 250 copies for Vil, which is sufficient to detectserologically. However, the cells might still type asSl(a–) if the Sla-encoding gene had low expression, e.g.,only 100 copies.

Clearly E-CR1 is important in determining thestrength of the reaction, but there are many otherrelated factors that could also impact the final results. Iftest RBCs being used are several weeks old, e.g.,commercial reagent RBCs near the expiration date,reactions may be weaker than if RBCs from a donor unitjust recently drawn are used. CR1 is lost from the RBCmembrane during storage through vesiculation andbudding.28,29 Thus, RBCs that may have given weakreactions at the time they were drawn due to“borderline E-CR1”may give negative reactions afterprolonged storage. Consequently, if one is trying toperform Knops phenotyping, the RBCs should be asfresh as possible to obtain accurate results.



To summarize, many factors can affect the finalKnops phenotype result, including: antibody titer,detection method, total E-CR1 copy number,heterozygosity, low-expressing or nonexpressing alleles,and prolonged storage of the RBCs. Is it any wonderthat early investigators were unable to duplicate eachother’s work and were frustrated with the Knopssystem?

Reactivity with enzyme-treated cellsAgain, some confusion exists in the early reports of

Knops system antibodies and can be attributed tomisleading statements such as “reactive with enzyme-treated cells.” This, of course, depends not only on theenzyme but also on the length of time used forpremodification of the RBC membrane. There are noexamples of these antibodies that are enhanced byenzyme treatment of RBCs, and most are still reactive(sometimes more weakly) with either ficin- or papain-treated cells.30,31 However, all Knops system antibodiesidentified to date are nonreactive with trypsinizedRBCs. It is known that a trypsin cleavage site exists inSCR 28 of the CR1 protein. Since the blood groupantigens identified to date have been found in SCR 25,they are lost upon trypsin treatment of the RBCs. Thiscan be a useful tool not only in antibody identification,but also for adsorption, to remove other antibodies suchas anti-A or -B from a serum sample.

Other antigen characteristicsThe inherited expression, polymorphism, and

instability of the antigen upon storage have causedmisinterpretation of many test results. In 1986, Danielset al.32 reported that the In(Lu) gene often suppressedKna, McCa, Sla, Yka, and Csa. However, not all Lu(a–b–)families showed suppression and, when present, thesuppression was not as dramatic as that for P1 and Aua.The questionable variability in the results was recentlyreaddressed in light of the CR1 expression polymor-phism. Using samples less than 72 hours old, Mouldsand Shah33 found there was no suppression of the high-frequency Knops antigens. They suggested that theprevious results may have been due to prolongedstorage of the RBCs either before or after frozen storagein glycerol.

Although RBCs from cord blood samples have beenreported to have weakened Knops antigens,26,31 othershave not found any reduction in antigen strength.34 Inour studies of black African children, we have typedinfants less than 1 year old and found no difference in

strength of reactions as compared to older children andadults (J.M. Moulds, unpublished observation).

Other antibody characteristicsTwo other characteristics attributed to Knops

antibodies were they were not neutralized with plasma,saliva, or urine and they were difficult to adsorb andelute. The latter most likely reflects the low density ofthe CR1 protein on the RBC membrane. However, Raceand Sanger35 reported that adsorption performed withbuffy coats (white blood cells [WBCs]) was able toremove anti-Kna from serum. This led to the speculationthat anti-Kna and related specificities were WBCantibodies (see next section).

Although CR1 has not been found in saliva, low levelshave been found in both urine36 and plasma.37 This isbelieved to be the result of proteolytic cleavage of CR1from WBCs.38,39 Serum CR1 is present only in nanogramamounts39 and, therefore, the levels are insufficient toneutralize Knops antibodies using routine serologictechniques. Hence, Moulds and Rowe40 developed aninhibition technique using recombinant, soluble CR1(sCR1). Since their source of sCR1was positive for Kna,McCa, Sla, and Yka, it would not inhibit anti-Knb or -McCb.It must be remembered that the Knops phenotype ofthe sCR1 will be dependent upon the gene chosen forits production. More recently, these investigators haveused mutated CR1 constructs to produce peptidescapable of inhibiting anti-McCb and Vil.16

The name gameBecause antibodies in the Knops system were often

found in sera containing HLA antibodies and becausethey could be adsorbed on WBCs, some investigatorsinitially believed that they were antibodies to WBCs.This concept was eventually proved to be incorrect andconsequently the term “HTLA antibodies” came intovogue. But even then there were some serologists whowere not comfortable with this terminology andpointed out that many examples of these antibodies didnot have a high titer. With the assignment of most ofthese specificities to blood group systems, the termHTLA should be discarded. It now becomes part of ourhistory, along with terms like “non-specific coldagglutinins” (anti-I) and “non-A, non-B hepatitis”(hepatitis C).

But the confusion over terminology is bound toremain with us for at least a few more years. AlthoughMcCc and Slb have now been officially named Sla and Vil,additional specificities described by Molthan41 have yet


J.M. MOULDS

to be identified at the molecular level. These includeMcCe and McCf, along with “Kn/McC,” which have beenused by many laboratories to denote Knops systemantibodies that are nonreactive with the Helgeson RBCs.Finally, the compound specificities, such as anti-Kna/McCa, may actually represent conformational epitopessimilar to Rg2.

Indeed, Moulds et al.17 have reported evidence forthe existence of even more complex Knopsspecificities. They have shown that sera containing Sla

may be heterogeneous. Furthermore, two amino acidsmay be involved, including the arginine at amino acid1601 (Sla) and a new mutation at amino acid 1610,resulting in a total of five Sl epitopes. If the Knops,McCoy, and York antisera prove to be as diverse as Sla,we can expect a rapid increase in the numberedantigens for this system similar to what has recentlyoccurred with the Diego system!

SummaryAlthough this author has tried to clarify some of the

misunderstandings and confusion regarding the Knopsblood group system, interested readers are urged toobtain the original publications to better appreciate thecomplexities of this system. Some of the earlyinvestigators were criticized for their work and theirviewpoints; yet we now know that they were correct(at least in some of their interpretations). In light of ourcurrent knowledge of the Knops system, I would like toend this review with a quote from Dr. Lyndall Molthan,who in 1983 predicted what has now beenscientifically proved regarding CR1 expression and theKnops antigens. She stated “Other difficulties attributedto (Knops) antigens are their variations in strength,partly due to zygosity, or unrelated to zygosity butgenetically determined, partly due to race, or on thebasis of presence or absence of related antigens. All ofthese factors account for unexpected negative typingsin working with patients’ samples, donors’ RBCs, andcommercial panel cells.”26

AcknowledgmentsThe Knops blood group polymorphism research

(JMM) has been supported by grants from the NationalBlood Foundation and the National Institutes of Health(R01 AI 42367). The author thanks John J. Moulds,MT(ASCP)SBB, who saved all the early correspondencewith his colleagues related to HTLA antibodies andwhose guidance has helped in the investigation and

molecular identification of the various Knopsspecificities.

References1. Daniels GL, Anstee DJ, Cartron J-P, et al. International

Society of Blood Transfusion working party onterminology for red cell surface antigens. Vox Sang2001;80:193-6.

2. Giles CM, Huth MC, Wilson TE, Lewis HBM, GroveGEB. Three examples of a new antibody, anti-Csa,which reacts with 98% of red cell samples. VoxSang 1965;10:405-15.

3. Moulds JM, Nickells MW, Moulds JJ, Brown MC,Atkinson JP. The C3b/C4b receptor is recognized bythe Knops, McCoy, Swain-Langley, and York bloodgroup sera. J Exp Med 1991;173:1159-63.

4. Petty AC, Green CA, Poole J, Daniels GL. Analysis ofKnops blood group antigens on CR1 (CD35) by theMAIEA test and by immunoblotting. Transfus Med1997;7:55-62.

5. Helgeson M, Swanson J, Polesky HF. Knops-Helgeson(Kna), a high frequency erythrocyte antigen.Transfusion 1970;10:737-8.

6. Molthan L, Giles CM. A new antigen, Yka, and itsrelationship to Csa (Cost). Vox Sang 29:1975;145-53.

7. Molthan L, Moulds J. A new antigen, McCa (McCoy),and its relationship to Kna (Knops). Transfusion1978;18:566-8.

8. Lacey P, Laird-Fryer B, Block U, Lair J, Guilbeau L,Moulds JJ. A new high incidence blood group factor,Sla, and its hypothetical allele (abstract). Transfusion1980;20:632.

9. Molthan L. Expansion of the York, Cost, McCoy,Knops blood group system: the new McCoyantigens McCc and McCd. Med Lab Sci 1983;40:113-21.

10. Rao N, Ferguson DJ, Lee SF, Telen MJ. Identificationof human erythrocyte blood group antigens on theC3b/C4b receptor. J Immunol 1991;146:3501-7.

11. Wilson JG, Murphy EE, Wong WW, Klickstein LB, WeisJH, Fearon DT. Identification of a restrictionfragment polymorphism by a CR1 cDNA thatcorrelates with the number of CR1 on erythrocytes.J Exp Med 1986;164:50-9.

12. Cornillet P, Philbert F, Kazatchkine MD, Cohen JHM.Genomic determination of the CR1 (CD35) densitypolymorphism on erythrocytes using polymerasechain reaction amplification and Hind III restrictionenzyme digestion. J Immunol Meth 1991;136:193-7.



13. Moulds JM, Brai M, Cohen J, et al. Reference typingreport for complement receptor 1 (CR1). Exp ClinImmunogenet 1998;15:291-4.

14. Herrera AH, Xiang L, Martin SG, Lewis J, Wilson JG.Analysis of complement receptor type 1 (CR1)expression on erythrocytes and of CR1 allelicmarkers in Caucasian and African Americanpopulations. Clin Immunol Immunopathol1998;87:176-83.

15. Vik DP, Wong WW. Structure of the gene for the Fallele of complement receptor type 1 and sequenceof the coding region unique to the S allele. JImmunol 1993;151:6214-24.

16. Moulds JM, Zimmerman PA, Doumbo OK, et al.Molecular identification of Knops blood grouppolymorphisms found in long homologous region Dof complement receptor 1. Blood 2001;97:2879-85.

17. Moulds JM, Zimmerman PA, Doumbo OK, et al.Expansion of the Knops blood group system andsubdivision of Sla. Transfusion (in press: 2001).

18. Rowe JA, Moulds JM, Newbold CI, Miller LH. P.falciparum rosetting mediated by a parasite-varianterythrocyte protein and complement-receptor 1.Nature 1997;388:292-5.

19. Rowe A, Obeiro J, Newbold CI, Marsh K.Plasmodium falciparum rosetting is associatedwith malaria severity in Kenya. Infect Immun1995;63:2323-6.

20. Moulds JM, Kassambara L, Middleton JJ, et al.Identification of complement receptor one (CR1)polymorphisms in West Africa. Genes and Immunity2000;1:325-9.

21. Da Silva RP, Hall BF, Joiner KA, Sacks DL. CR1, theC3b receptor, mediates binding of infectiveLeishmania major metacyclic promastigotes tohuman macrophages. J Immunol 1989;143:617-22.

22. Payne NR, Horwitz MA. Phagocytosis of Legionellapneumophila is mediated by human monocytecomplement receptors. J Exp Med 1987;166:1377-89.

23. Schlesinger LS, Horwitz MA. Phagocytosis of leprosybacilli is mediated by complement receptors CR1and CR3 on human monocytes and complementcomponent C3 in serum. J Clin Invest 1990;85:1304-14.

24. Schlesinger LS, Bellinger-Kawahara CG, Payne NR,Horwitz MA. Phagocytosis of Mycobacteriumtuberculosis is mediated by human monocytecomplement receptors and complement compo-nent C3. J Immunol 1990;144:2771-80.

25. Miller WV. ed. Technical methods and procedures ofthe American Association of Blood Banks.Washington, DC: American Association of BloodBanks, 1974.

26. Molthan L. The serology of the York-Cost-McCoy-Knops red blood cell system. Am J Med Tech1983;49:49-56.

27. Moulds JM, Moulds JJ, Brown MC, Atkinson JP.Antiglobulin testing for CR1-related (Knops/McCoy/Swain-Langley/York) blood group antigens: negativeand weak reactions are caused by variableexpression of CR1. Vox Sang 1992;62:230-5.

28. Pascual M, Lutz HU, Steiger G, Stammler P, SchifferliJA. Release of vesicles enriched in complementreceptor 1 from human erythrocytes. J Immunol1993;151:397-404.

29. Moulds JM, Brown LL. Loss of complement receptorone (CR1) from stored blood and its effect onKnops blood group reactivity. Immunohematology1995;11:46-50.

30. Moulds MK. Serological investigation and clinicalsignificance of high-titer, low-avidity (HTLA)antibodies. Am J Med Tech 1981;10:789-95.

31. Reid ME, Lomas-Francis C. Knops blood groupsystem. The blood group antigen factsbook. NewYork: Harcourt Brace & Company, 1997:332-41.

32. Daniels GL, Shaw MA, Lomas CG, Leak MR, TippettP. The effect of In(Lu) on some high-frequencyantigens. Transfusion 1986;26:171-2.

33. Moulds JM, Shah C. Complement receptor 1 red cellexpression is not controlled by the In(Lu) gene.Transfusion 1999;39:751-5.

34. Ferguson SJ, Blajchman MA, Guzewski H, Taylor CR,Moulds J. Alloantibody-induced impaired neonatalexpression of a red blood cell antigen associatedwith maternal alloimmunization. Vox Sang1982;43:82-6.

35. Race RR, Sanger R. Some very frequent antigens.Blood groups in man. London: Blackwell ScientificPublications, 1975:410-30.

36. Pascual M, Steiger G, Sadallah S, et al. Identificationof membrane-bound CR1 (CD35) in human urine:evidence for its release by glomerular podocytes. JExp Med 1994;179:889-99.

37. Yoon SH, Fearon DT. Characterization of a solubleform of the C3b/C4b receptor (CR1) in humanplasma. J Immunol 1985;134:3332-8.

38. Danielsson C, Pascual M, Larsen B, Steiger G,Schifferli JA. Soluble complement receptor type 1


J.M. MOULDS

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(CD35) is released from leukocytes by surfacecleavage. Eur J Immunol 1994;24:2725-31.

39. Pascual M, Duchosal MA, Steiger G, et al. Circulatingsoluble CR1 (CD35). J Immunol 1993;151:1702-11.

40. Moulds JM, Rowe KE. Neutralization of Knopssystem antibodies using soluble complementreceptor 1. Transfusion 1996;36:517-20.

41. Molthan L. The status of the McCoy/Knopsantigens. Med Lab Sci 1983;40:59-63.

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A gel microtyping system fordiagnosis of paroxysmal nocturnalhemoglobinuriaB. ZUPANSKA, B. BOGDANIK, AND H. PYL

(GPI) anchor.6-10 In patients with PNH, a somaticmutation in an X-linked gene, PIG-A, leads to impairedsynthesis of the GPI anchor. A proportion of RBCs istherefore deficient in GPI-linked proteins, includingMIRL (CD59) and DAF (CD55).

Despite a great deal of work on its molecular basis inrecent years, PNH remains not fully understood. Forinstance, it is not clear how the nonhemolytic sequelaeare related to the deficiency of GPI-anchored proteinsand what other defects associated with clinical eventsseen in PNH (e.g., thrombosis) may be acquired byother than RBCs. One reason why PNH is not fullyunderstood is that it is underdiagnosed, partly due to itsheterogeneity. Also, PNH is underdiagnosed partlybecause many patients have atypical symptoms that donot resemble classic cases of PNH. Moreover, availablelaboratory methods of diagnosing PNH, some of whichare time-consuming and complicated, are not widelyused. The use of the hemagglutinating gel test for thediagnosis of PNH seems to open new possibilities.11-13

This test can detect CD59- and CD55-deficient RBCs.It is based on the antiglobulin test using murine mono-clonal antibodies against CD59 and CD55 proteins.Defective cells are not agglutinated by these antibodies.

The aim of this study was to evaluate the usefulnessof the gel test for the diagnosis of PNH by comparingthe gel test with lytic tests and with flow cytometry(FC) assessment of the expression of CD59 and CD55on RBCs.

Materials and MethodsFifty-one blood samples from 48 patients with

anemia (suspected for PNH, aplastic anemia, or otherhemolytic anemias) and 30 samples from blood donors(control group) were evaluated for this study. Bloodsamples were examined by the gel test, the Ham test,sucrose lysis, and FC on the same day as they were


Paroxysmal nocturnal hemoglobinuria (PNH), an acquired stem celldefect, is underdiagnosed because of its atypical symptoms in somepatients and because available methods, which are time consumingand complicated, are not widely used. The hemolysis of PNH redblood cells (RBCs) is attributed to their enhanced susceptibility tocomplement lysis caused by a deficiency in glycosylsphos-phatidylinositol (GPI)-anchored complement regulatory membraneproteins, especially membrane inhibitor of reactive lysis (MIRL[CD59]). We evaluated the diagnostic value of a simple hemagglu-tination test using the gel microtyping system by comparing it withlytic tests (the Ham test and the sucrose lysis test) and with flowcytometry (FC) assessment of expression of GPI-anchored proteins(CD59 and CD55). Examining 51 blood samples from 48 patients,we found that the gel test is useful as a screening test for PNHdiagnosis and can replace the Ham test and the sucrose lysis test.The threshold of the gel test is about 10 percent of defective RBCsdetected by FC. It should, however, be supplemented with FC so asto analyze precisely the defective RBCs and granulocytes in patientswith positive gel test results, and, in case of negative results, todetect a small clone of defective cells in atypical cases. Due to thesimplicity of the gel test, its wide use can facilitate the diagnosis ofPNH. Immunohematology 2002;18:9–12.

Key Words: paroxysmal nocturnal hemoglobinuria, geltest, Ham test, sucrose lysis test, flow cytometry

Paroxysmal nocturnal hemoglobinuria (PNH) is arare, acquired stem cell disorder of a clonal nature,resulting in intravascular hemolysis, cytopenia ofvariable degrees, and recurrent thrombotic events. PNHhas been described in patients already affected by bonemarrow aplasia and, conversely, in PNH patients wholater develop aplasia and, rarely, leukemia.1-4 Moreover,PNH clones have been observed in patients with otherdiseases.5

Hemolysis of red blood cells (RBCs) is attributed tothe enhanced susceptibility of affected RBCs to lysis bycomplement. The molecular mechanism of thisincreased susceptibility is a deficiency in complementregulatory membrane proteins, such as the decay-accelerating factor (DAF) and the membrane inhibitorof reactive lysis (MIRL), which are covalently attachedto the cell membrane via a glycosylphosphatidylinositol

B. ZUPANSKA ET AL.


Table 1. Patients with positive results in the gel test; comparison withlytic tests and FC analysis

Tests Flow cytometry analysisNo. % of lysis % of red cells

Ham test sucrose lysis CD59* CD59† CD55*

1 8 27 71 522 3 8 33 143 10 28 47 32 594 3 13 31 234a 4 24 52 16 565 6 33 55 22 516 2 14 17 157 3 34 35 25 618 7 43 67 7 609 8 39 83 899a 2 15 22 2410 14 35 52 36 6211 2 10 19 2112 6 33 46 28 6613 2 24 54 3014 11 83 89 8315 0 8 39 3 2816 2 27 66 6 4917 7 26 43 4 3618 6 21 63 6519 15 49 53 5320 nt‡ nt 22 2521 nt nt 8 1422 nt nt 16 3 1623 3 8 23 27

*CD59 and CD55: PNH type III, completely deficient cells†CD59: PNH type II, partially deficient cells‡Not testedControl values: The Ham test = zero lysis; sucrose lysis test = < 5% lysis;FC = < 1% CD59 defective red cells and < 6% CD55 defective red cells.

drawn. Informed consent was obtained from all patientsstudied.

Gel testThe gel test (DiaMed-ID Micro Typing System PNH

test, DiaMed AG, Cressier, Switzerland) was performedaccording to the manufacturer’s instructions. In brief: 50µL of an 0.8% RBC suspension in LISS diluent and 50 µLof murine monoclonal antibody (anti-CD59 or anti-CD55) were pipetted into a microtube that containedan antiglobulin reagent, incubated at 37°C for 15minutes, and then centrifuged and read. Defective cells,i.e., those deficient in CD59, CD55, or both, were foundat the bottom of the microtube (positive result), whilenormal cells were agglutinated and found on the top ofthe gel (negative result). In each card, a microtube withthe patient’s RBCs and without monoclonal antibodieswas included as a negative control. In patients withRBC-bound immunoglobulins, a crossreaction mayoccur between them and the antiglobulin reagentdirected against mouse immunoglobulins. In these casesthe manufacturer suggests repeating the test afterautoantibody elution.

Lytic testsThe acid lysis test (the Ham test) and the sucrose

lysis test were done as part of the routine workup;results are evaluated by spectrophotometry andexpressed as the percentage of lysis. Zero lysis wasregarded as a negative Ham test, and lysis below 5percent as a negative sucrose test.14

Flow cytometryFC was performed with RBCs by the indirect

immunofluorescence technique, using murine mono-clonals anti-CD59 (BRIC 229, IBGRL, Bristol, UK), anti-CD55 (a mixture of BRIC 216, 110, 230, and 220, IBGRL,Bristol, UK), and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse (Ortho-Clinical Diagnostics,Raritan, NJ, or DAKO, Denmark).15,16 FC was performedwith granulocytes (separated by dextran sedimentationand ficolgradient centrifugation with subsequenthemolysis of RBCs), using FITC-conjugated anti-CD59(BRIC 229, IBGRL, Bristol, UK) and anti-CD67 (CLB-B13.9, Ortho-Clinical Diagnostics).17

ResultsIn all 30 samples from blood donors the gel test was

negative with both anti-CD59 and anti-CD55. The Hamtest and the sucrose lysis test were also negative. The

FC analysis in each case consistently showed less than 1percent CD59-negative RBCs and less than 6 percentCD55-negative RBCs. The percentage of negativelystaining granulocytes was always below 2 percent withanti-CD59 and 5 percent with anti-CD67.

In 23 patients (25 blood samples), the gel testshowed GPI-deficient RBCs with both anti-CD59 and-CD55 (Table 1). A parallel examination of 22 of the 25samples by lytic tests demonstrated a positive sucrose

lysis test in all 22 and a positive Ham test in all but onesample (Table 1, No. 15). The FC analysis, together withthe above results, indicated a diagnosis of PNH(suspected from clinical symptoms in most of thepatients), even in the one patient with a negative Hamtest. In addition, we found defective granulocytes in all22 patients (not shown in Table 1). The percentage ofdefective granulocytes was usually higher than that ofdefective RBCs.

In 25 of the remaining 26 samples from patients, theresults of the PNH gel test were negative. These caseswere divided into two groups: (1) six patients (Nos. 1to 6) with a PNH clone found by FC (Table 2) and(2) 20 patients without such a clone.

Gel test for diagnosis of PNH


Table 2. Patients with negative results in the gel test; comparison withlytic tests and FC analysis (remaining 20 patients with anegative gel test are described in the text)

% of lysis Flow cytometry analysis, % of defectivesucrose red cells granulocytes

No.* Ham test lysis test CD59 CD55 CD59 CD67

1 0 4 4 6 12 162 0 4 12 10 54 563 0 1 2 9 15 144 0 3 4 5 9 95 0 2 8 6 7 86 nt nt 8 7 86 91

*Clinical comments: Patients No. 1–5 had bone marrow aplasia/hypoplasia with pancytopenia; Patient No. 4 also had severe aplasticanemia and severe thrombocytopenia; Patient No. 6 was diagnosedpreviously as PNH (see Table 1, Patient No. 9).Control values: Ham test = zero lysis; sucrose lysis test = < 5% lysis; FCanalysis = < 1% CD59 defective red cells and < 6% CD55 defective redcells; < 2% defective granulocytes with anti-CD59 and < 5% defectivegranulocytes with anti-CD67.

In all six cases with a PNH clone detected by FC andwith a negative PNH gel test (Table 2), the results oflytic tests were also negative. The percentage of CD55-deficient RBCs by FC for some of the patients waswithin the range observed in the blood donor samples.However, the percentage of CD59-deficient RBCs anddeficient granulocytes was above the percentage foundin blood donor samples. Thus, all of these patients hadan identifiable PNH clone, although in four cases it wassmall. Review of clinical data revealed that five patientshad bone marrow hypoplasia/aplasia, usually withpancytopenia. One patient (see Table 1, No. 9) had beenpreviously diagnosed with PNH.

In 19 of 20 patients (not included in Table 2) withnegative gel tests and negative lytic tests, defective RBCsdetected by FC were within the range found in donorsamples. In one patient, however, the gel test was onlypositive with anti-CD55. The Ham test was weaklypositive, but low (1%), and FC analysis identified 18percent of CD55-defective RBCs. This patient hadanemia with myelofibrosis. The other 19 patients hadvarious kinds of anemia (e.g., due to differentdeficiencies, other types of hemolysis, accompanyingmyeloproliferative diseases, and bone marrow aplasia).

DiscussionOur data show that the gel test is useful as a

screening test for the diagnosis of PNH and can replacethe lytic tests. When we compared the PNH gel testresults with FC results, we found that the gel test candetect 10 percent of RBCs deficient in CD59 and CD55.This is consistent with previous observations.13

However, others11,12 reported 2 to 5 percent of defectiveRBCs detectable by the gel test. However, they used an

earlier version of the gel test that required the user topipette into the gel the mixture of the antiglobulinreagent with RBCs that had previously been incubatedwith monoclonal antibodies.

Our method also allowed us to define whichprotein(s) (CD59 and/or CD55) was/were lacking fromthe patient’s defective cells, and this was not possibleby lytic tests. In one case, for instance, CD55-deficientRBCs only were detected; this information may beuseful since DAF-defective RBCs alone are lessimportant from a clinical point of view.18 The inclusionof both anti-CD55 and -CD59 monoclonal antibodies inthe gel system is advantageous because the person whoreads the test can be surer about the result. The mainadvantage of the gel test, compared to the lytic tests, isthat it is simple to use, and the results are ready within1⁄2 hour.

Our comparison of a relatively large group of patientswith both positive and negative results in the gel testshowed that the positive results correlated well withthe presence of abnormal cells. In all patients suspectedof having PNH, a preliminary diagnosis by a positive geltest was possible. The FC analysis was, however, moresensitive, which was always useful to evaluate hownumerous the defective cells were, and whether type IIIand type II RBCs were present. This was not possibleusing the gel test. Similar observations have beenpublished but they were based on single cases.12

Negative gel test results were less reliable thanpositive results. In 6 of 26 (23 percent) samples with anegative gel test, CD59-deficient RBCs, as well asgranulocytes deficient in both CD59 and CD67, weredetected by FC. Most of these patients had bonemarrow hypoplasia/aplasia, conditions known to beassociated with the presence of PNH (or PNH-like)clones.1,2 It should be stressed that the detection of adefective clone, in spite of a negative gel test, is notinconsistent with our opinion that this test can replacethe lytic tests, since the Ham and sucrose lysis testswere also negative in this group of patients. It is,however, worth noting that 77 percent of negative geltest results (20 of 26 samples) predicted properly thelack of defective cells confirmed by FC in thepopulation studied.

In summary, the gel test appears to be useful as ascreening test for PNH and can replace lytic tests. Itcannot, however, replace the FC, since only the lattercan diagnose a small clone of defective RBCs and candetect PNH defects on granulocytes in transfusedpatients. Because of the simplicity of the gel test, its


B. ZUPANSKA ET AL.

wide use can result in an increased ability to diagnosePNH. The use of this test would be especially easy inlaboratories that already use the microtyping gel systemfor antibody detection and blood grouping and have anappropriate centrifuge and persons experienced ininterpreting the results.

AcknowledgmentsWe thank Zofia Pijanowska for help in preparing this

manuscript.

References1. Schubert J, Vogt HG, Zielinska-Skowronek M, et al.

Development of the glycosylphosphatidylinositol-anchoring defect characteristic for paroxysmalnocturnal hemoglobinuria in patients with aplasticanemia. Blood 1994;83:2323-8.

2. Griscelli-Bennaceur A, Gluckman E, Scrobohaci ML,et al. Aplastic anemia and paroxysmal nocturnalhemoglobinuria: search for a pathogenetic link.Blood 1995;85:1354-63.

3. Hillmen P, Lewis SM, Bessler M, Luzzatto L, Dacie JV.Natural history of paroxysmal nocturnal hemoglo-binuria. N Engl J Med 1995;333:1253-8.

4. Socie G, Mary JY, Gramont A, et al. Paroxysmalnocturnal haemoglobinuria: long-term follow-upand prognostic factors. Lancet 1996;348:573-7.

5. Zupanska B, Bogdanik I, Fabijanska-Mitek J, Pyl H.Autoimmune haemolytic anaemia with a paroxys-mal nocturnal haemoglobinuria-like defect. Eur JHaematol 1999;62:346-9.

6. Rosse WF. Phosphatidylinositol-linked proteinsand paroxysmal nocturnal hemoglobinuria. Blood1990;75:1595-1601.

7. Wilcox LA, Ezzell JL, Bernshaw NJ, Parker ChJ.Molecular basis of the enhanced susceptibility ofthe erythrocytes of paroxysmal nocturnal hemo-globinuria to hemolysis in acidified serum. Blood1991;78:820-9.

8. Yomtovian R, Prince GM, Medof ME. The molecularbasis for paroxysmal nocturnal hemoglobinuria.Transfusion 1993;33:852-73.

9. Miyata T, Takeda J, Iida Y, et al. Cloning of PIG-A, acomponent in the early step of PGI-anchorbiosynthesis. Science 1993;259:1318-20.

10. Nishimura JI,Smith CA, Phillips KL, Warere EW,Rosse WF. Paroxysmal nocturnal hemoglobinuria:

Molecular pathogenesis and molecular therapeuticapproaches. Hematopathol Molecular Hematol1998;11:119-46.

11. Navenot JM, Bernard D, Petit-Frioux Y, et al.Diagnostic rapide des hemoglobinuries nocturnesparoxystiques par agglutination en gel. Rev FrTransfus Hemobiol 1993;36:135-47.

12. Nilsson B, Hagstrom U, Englund A, Safwenberg J. Asimplified assay for the specific diagnosis ofparoxysmal nocturnal hemoglobinuria: detection ofDAF (CD55)– and HRF20 (CD59)– erythrocytes inmicrotyping cards. Vox Sang 1993;64:43-6.

13. Meletis J, Michali E, Samarkos M, et al. Detection of“PNH red cell” populations in hematologicaldisorders using the sephacryl gel test micro typingsystem. Leukemia Lymphoma 1997;28:177-82

14. Beutler E. Sucrose hemolysis and acidified-serumlysis test. In: Williams Hematology. McGraw-Hill,Inc., 1995;L48-L49.

15. Schubert J, Alvarado M, Uciechowski P, et al.Diagnosis of paroxysmal nocturnal haemoglo-binuria using immunophenotyping of peripheralblood cells. Br J Haematol 1991;79:487-92.

16. Bogdanik I, Pyl H, Zupanska B, Robak T. Flowcytometric analysis of erythrocyte and granulocytedefects in patients with paroxysmal nocturnalhemoglobinuria. Acta Haematologica Polonica1998;29:69-75.

17. van der Schoot CE, Huizinga TWJ, van t’Veer-KorthofET, Wijams R, Pinkster J, von dem Borne AEGKr.Deficiency of glycosyl-phosphatidylinositol-linkedmembrane glycoproteins of leukocytes in paroxys-mal nocturnal haemoglobinuria; description of anew diagnostic cytofluorometric assay. Blood1990;76:1853-9.

18. Marilyn B, Telen J, Green AM. The Inab phenotype:characterization of the membrane protein andcomplement regulatory defect. Blood 1989;74:437-41.

Prof. Barbara Zupanska, MD, PhD, Head of theDepartment of Immunohematology andImmunology for Transfusion Medicine, Institute ofHematology and Blood Transfusion, 5 ChocimskaStreet, Warsaw, Poland; Irena Bogdanik, MS, andHanna Pyl, MS, Institute of Hematology and BloodTransfusion, 5 Chocimska Street, Warsaw, Poland.

Antibody screening in 37°C saline.Is it safe to omit it using the indirectantiglobulin (gel) test?J. DURAN AND M. FIGUEIREDO

Pretransfusion tests must detect antibodies that can shorten the lifeof red blood cells (RBCs). Some studies have demonstrated theexistence of clinically significant antibodies detected at 37°C insaline that are not detected by the indirect antiglobulin test (IAT)when the conventional tube test is used. Our aim was to determinewhether these antibodies, detected with a 37°C saline tube test, arealso detected when a sensitive column gel agglutination method isused. The 2373 pretransfusion samples were tested as they werereceived (from May 1998 to December 1999), in 37°C saline and byIAT using the DiaMed gel system. The screening tests wereperformed using 50 µL of 0.8% low-ionic-strength saline suspendedRBCs and 50 µL of plasma. The tests were examined foragglutination and hemolysis. Two hundred and thirty three samples(9.81%) were reactive by IAT and 88 (3.70%) by 37°C saline. All 88samples reactive by 37°C saline also reacted by IAT. These dataindicate that 37°C saline is not an essential pretransfusionprocedure when the DiaMed gel test is used. Immunohematology2002;18:13–15.

Key Words: antibody screening, pretransfusion tests,gel test

Pretransfusion tests must detect antibodies that cancause hemolysis of red blood cells (RBCs). For manyyears, routine pretransfusion RBC antibody detectionhas been performed using the conventional tubemethod. The tube tests use saline-suspended RBCs, withor without low-ionic-strength saline solution (LISS), thatare mixed with the sera to be tested. A 37°C incubationphase is performed before centrifugation and reading.After this step an indirect antiglobulin test (IAT) isperformed. Several authors have demonstrated thatsome clinically significant antibodies were missed whenthe 37°C reading phase was eliminated.1,2 Theseantibodies likely were predominantly IgM, representinga primary immune response.3,4 Nevertheless, about 20percent of these antibodies could be consideredclinically significant (Rh, K, or Jka specificity).1 Over theyears, new methods, such as column agglutination andsolid phase, have been introduced in an effort toimprove the sensitivity, safety, and ease of pretransfusiontests.5 Our aim was to evaluate whether clinically

significant antibodies would be missed if the 37°Csaline test was omitted when a sensitive IAT column gelagglutination method developed by Lapierre et al.6 (theDiaMed gel system) was used.7-9

Materials and MethodsThe study was performed at Centro Hospitalar de Vila

Nova de Gaia (CHVNG), a general hospital with 600beds, and at Centro Regional de Sangue do Porto(CRSP), the Oporto Regional Blood Center, from May1998 to December 1999. The samples tested at CHVNG(1528) were from patients with orders for bloodtransfusions. Many of the 845 samples tested at CRSPwere sent for antibody identification from smallhospitals where a positive antibody screen had beendetected. The 2373 samples were tested when received,in 37°C saline and by IAT using the DiaMed gel system(DiaMed-ID Micro Typing System, Cressier, Switzerland).The 37°C saline tests were performed using “NaCl/enzyme” cards (neutral gel) and the IAT using “LISS/Coombs” cards (anti-IgG + C3d). Fifty µL of 0.8% LISS-suspended R1R1, R2R2, and rr RBCs (ID-DiaCell I+II+III)and 50 µL of plasma were used. When the screeningwas positive, either in 37°C saline or in IAT, antibodyidentification was performed both in 37°C saline and inIAT again, using 0.8% LISS RBC panels (ID-DiaMedPanel) and 50 µL of the patient’s plasma. The RBCs andthe plasmas were not warmed before being mixed inthe cards. The cards were incubated at 37°C for 15minutes, then centrifuged at 900 rpm for 10 minutes,according to the manufacturer’s instructions (DiaMed’sID-DiaCent centrifuge). The tests were then examinedfor agglutination and hemolysis.

ResultsThe results of antibody screens were analyzed

retrospectively. All positive samples were from different


Table 1. Samples with positive antibody screening tests

Results IAT* 37°C saline

Clinically significant antibodies 176 (7.41%) 57 (2.40%)Clinically insignificant antibodies 35 (1.47%) 20 (0.84%)Undetermined antibodies 22 (0.92%) 11 (0.46%)

Total 233 (9.81%) 88 (3.70%)

*Indirect antiglobulin test

Table 2. Clinically significant antibodies detected by IAT and by 37°Csaline using the DiaMed gel system

Antibodies No. reactive No. reactiveby IAT* by 37°C saline†

Anti-D 74 28Anti-D+C+K 11 0

Anti-D+C+E+Fya 1 1Anti-D+Fya 2 1Anti-D+K 2 1Anti-D+C 14 7

Anti-D+C+E 3 1Anti-D+C+Lea 2 2

Anti-D+Jka 3 2Anti-D+S 1 0

Anti-C 2 2Anti-C+e 3 1

Anti-c 3 0Anti-c+E 6 3

Anti-c+E+Fya 1 0Anti-c+Fya+Jka+M 1 1

Anti-E 15 3Anti-E+K 1 0

Anti-E+K+Fya 1 1Anti-E+Jka+M 1 1

Anti-e 1 0Anti-K 12 0

Anti-K+Fya 1 0Anti-Fya 1 0Anti-Fyb 2 0Anti-Jka 7 0Anti-S 3 0Anti-s 1 1

Anti-S+P1 1 1

Total 176 Total 57

*Indirect antiglobulin test†All antibodies reacting in saline at 37°C also reacted by IAT.

Table 3. Clinically insignificant antibodies detected by IAT and 37°Csaline

Antibodies No. reactive No. reactiveby IAT* by 37°C saline†

Anti-Lea 14 7Anti-Leb 1

Anti-Lea+Lua 1 1Anti-Lua 1 1Anti-M 12 8Anti-P1 6 3

Total 35 20

*Indirect antiglobulin test†All antibodies reacting in saline at 37°C also reacted by IAT.


J. DURAN AND M. FIGUEIREDO

patients. When multiple antibodies were present, eachspecificity active at 37°C was verified. Among the 2373samples, 233 (9.81%) were reactive by IAT and 88(3.70%) by 37°C saline. All 88 samples reactive by 37°Csaline did, in fact, also react by IAT (Table 1). Clinically

significant antibodies (anti-D, anti-C, anti-c, anti-E, anti-e,anti-K, anti-Fy, anti-Jk, anti-S, and anti-s) were detected in176 samples (7.41%) by IAT and 57 samples (2.40%) by37°C saline (Table 2). Antibodies considered clinically

insignificant (anti-Lea, anti-Lua, anti-M, and anti-P1) weredetected in 35 samples (1.47%) by IAT and in 20samples (0.84%) by 37°C saline (Table 3). Antibodies ofundetermined specificity were detected in 22 samples(0.92%) by IAT and in 11 samples (0.46%) by 37°Csaline (Table 1).

DiscussionThe objective of antibody screening is to detect

antibodies that can cause accelerated destruction ofRBCs by transfusion or pregnancy. These clinicallysignificant antibodies can be screened by severalmethods. The IAT and 37°C saline tube tests still remainthe tests of choice for many. Some earlier studiesdemonstrated that, using the tube test, some clinicallysignificant antibodies could remain undetected if the37°C saline reading phase were eliminated.1,2 Morerecently, Judd et al.,10,11 discussing the risk of omitting37°C saline readings, reported that no increase intransfusion reactions or cases of immune hemolysishave been reported since they decided to eliminate thistest in 1996.10,11

The gel test is an RBC-affinity column methodintroduced in 1988 that has several advantages: smallsample size, no cell washing step, and easy-to-read andstable results.12,13 It is a sensitive and specific method,avoiding most falsely positive rouleaux-induced results.When the tube test is used, the reading after 37°C salineincubation is a simple and fast step before performingIAT. It consumes very little time and does not signi-ficantly increase the costs or work time. When usingthe gel test, the 37°C saline screening doubles the workand costs and delays patient care. We decided toevaluate whether we could eliminate it from ourroutine work without risk of missing clinically signi-ficant antibodies. Our data show a high percentage ofpositive antibody screening results (9.81%). This isbecause many of the samples that were tested at CRSPhad been referred from small hospitals whereunexpected antibodies had already been detected. All88 samples positive in saline at 37°C also reacted by theIAT gel test, indicating that a 37°C saline test is not anessential pretransfusion procedure.

Judd et al.11 found that the risk of eliminating 37°Csaline testing was lower than or similar to that ofeliminating other procedures, such as the direct


Saline 37°C antibody screening

antiglobulin test and antihuman globulin crossmatch,and that no increase in reported transfusion reactionsor cases of immune hemolysis has occurred since theyeliminated this test in 1996. Based on these data and ourown, we have decided to omit testing at 37°C in salinefrom our routine pretransfusion tests.

AcknowledgmentsWe thank A. Ferreira, C. Freitas, D. Sousa, H. Rocha, I.

Pires, I. Tavares, J.F. Ferreira, J.L. Ferreira, M. Cardoso,M.J. Couto, M.J. Miguel, P. Costa, R. Pacheco, and R. Silvafor technical help.

References1. Judd WJ, Steiner EA, Obermen HA, Nance SJ. Can the

reading for serological reactivity following 37°Cincubation be omitted? Transfusion 1992;32:304-8.

2. Pestaner JP, Shulman IA. Is it safe to omit 37°Creading from pretransfusion red blood cell antibodydetection testing? Am J Clin Pathol 1994;101:361-4.

3. Issitt PD. Serology and genetics of the Rhesus bloodgroup system. Cincinnati: Montgomery ScientificPublications 1979.

4. Mollison PL, Engelfreit CP, Contreras M. Bloodtransfusion in clinical medicine. 10th ed. Oxford:Blackwell Science, 1997.

5. Voak D. The status of new methods for thedetection of red cell agglutination. Transfusion1999;39:1037-40.

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

7. Lapierre Y. Gel tests and their evolution. TransfusClin Biol 1994;1:115-9.

8. Konig AL, Stuth C, Schabel A, Sugg U. Efficientantibody screening using gel centrifugation. BeitrInfusionsther Transfusionsmed 1994;32:159-61.

9. Weisbach V, Ziener A, Zimmermann R, Glaser A,Zingsem J, Eckstein R. Comparison of the perfor-mance of four microtube column agglutinationsystems in the detection of red cell antibodies.Transfusion 1999;39:1045-50.

10. Judd WJ, Fullen DR, Steiner EA, Davenport RD, KnaflPC. Revisiting the issue: can the reading forserologic reactivity following 37°C incubation beomitted? Transfusion 1999;39:295-9.

11. Judd WJ. Modern approaches to pretransfusiontesting. Immunohematology 1999;15:41-52.

12. Rumsey DH, Ciesielsky DJ. New protocols inserologic testing: a review of techniques to meettoday’s challenges. Immunohematology 2000;16:3-9.

13. de Figueiredo M, Lima M, Morais S, Porto G, JustiçaB. The gel test: some problems and solutions.Transfus Med 1992;2:115-8.

José A. Duran, MD, Laboratory ofImmunohematology, Centro Regional de Sangue doPorto, Estrada da Circunvalação (ao Hospital deMagalhães Lemos) 4149-003 Porto, Portugal; andManuel Figueiredo, MD, Servico deImmunohemoterapia, Centro Hospitalar de VilaNova de Gaia, Rua Conceição Fernandes 4434-502Vila Nova de Gaia, Portugal.

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Autoanti-D in a patient aftercladribine treatment forlymphoplasmocytic lymphomaJ. CID, V. BELTRAN, L. ESCODA, E. ELIES, AND C. MARTIN-VEGA

lymphoma is much rarer and usually involves coldagglutinating IgM antibodies produced by the malignantclone.6 Prior reports documenting cladribine-inducedautoimmune hemolysis in patients with non-Hodgkin’slymphoma are rare. Tetreault and Saven7 reported fourpatients who developed AIHA after cladribine treatmentfor Waldenström’s macroglobulinemia (WM).

We report a case of autoanti-D in a D+ bloodrecipient after cladribine treatment for lymphoplas-mocytic lymphoma without evidence of hemolysis.

Case ReportIn May 2000, a 62-year-old woman underwent

splenectomy. She was transfused with ten units of AB,D+ packed red blood cells (RBCs) because of bleeding.No antibodies were found in her serum at that time. Noother cellular or plasma-containing components nor Rhimmune globulin were transfused. The diagnosis oflymphoplasmocytic lymphoma was established bypathology study. One month later, she received twocycles of cladribine. This drug was administered as a 2-hour intravenous infusion for 5 successive days at adose of 0.12 mg/kg/day, repeated every 28 days.

Materials and MethodsRoutine serologic testing, elution, and antibody

identification were performed by standard methods.1

Briefly, ABO and D antigens were detected by tubetechnique following the manufacturers’ instructions(Diagast, France, Gamma Biologicals, Houston, TX,respectively).

The antibody screen test was performed by low-ionic-strength saline (LISS) indirect antiglobulin test(Ortho-Clinical Diagnostics, Raritan, NJ) and an Rh panelof 11 reagent RBCs was used to identify any antibodyspecificity, using the gel test (Ortho-ClinicalDiagnostics).

We report the case of a 62-year-old woman who developed anautoanti-D after cladribine treatment. In May 2000, the patientunderwent splenectomy for a stage IV-B lymphoplasmocyticlymphoma. She was transfused with ABO- and Rh(D)-matchedblood. A month later, she received chemotherapy with cladribine.In February 2001, blood grouping showed her to be AB, D+ and thedirect antiglobulin test was positive for IgG. An autoanti-D wasidentified in the eluate. Genotypic analysis confirmed the Rhphenotype of the patient as ccDEe. No hemolysis was evident, asjudged by the absence of anemia, a bilirubin of 15.7 µmol/L, andlactic dehydrogenase of 412 IU/L. When an anti-D is identified in aD+ blood recipient, a passive transfer of anti-D, and analloimmunization in a recipient with a weak D phenotype, shouldbe ruled out. Finally, as in our case, an autoantibody is an additionalpossibility. Immunohematology 2002;18:16–18.

Key Words: autoantibody, direct antiglobulin test,cladribine, anti-D in a D+ recipient

The occurrence of anti-D in a D+ blood recipient isunusual and may arise by several potential mecha-nisms.1 The receipt of intravenous immune globulin orplasma-containing components from an immunized D–donor may be the source of passively transferred anti-D.The production of an alloantibody in a D+ subject withweak D expression is also a possibility. Also, in a D+allograft recipient, the production of an alloantibody bythe persistent presence of donor immunocytes, i.e.,microchimerism when the donor is D–, is also possible.2

Finally, an autoantibody is an additional, though rare,possibility.3

The introduction of the purine analogs hasdramatically changed the treatment of lymphopro-liferative diseases, particularly chronic lymphocyticleukemia (CLL) and certain low-grade non-Hodgkin’slymphomas. The use of the purine analogs is associated,however, with a spectrum of toxicities different fromthose seen in patients treated with alkylating agents.4

Among such toxicities, severe autoimmune hemolysishas been reported, even with fatal outcome.5 Incontrast, autoimmune hemolytic anemia (AIHA)occurring in the setting of lymphoplasmocytic


An autoantibody could produce a positive DAT andno signs of hemolysis, as in our case. Warm type IgGautoantibodies can attach to the patient’s RBCs,optimally at 37°C, and may result in splenic sequestra-tion and extravascular hemolysis.10 Although most ofthese autoantibodies appear to be “nonspecific,” manyhave specificity to an Rh antigen, notably to e. Autoanti-D have been described previously, often accompanyingalloimmunization,11 but are uncommon by themselves.12

Other authors have published an incidence < 1 percentin a 5-year period.13

Long before the introduction of treatment usingpurine analogs, lymphoproliferative diseases wereknown to be associated with AIHA. Fludarabine, apurine analog, may induce severe autoimmunehemolysis, even with fatal outcome,14 particularly inpatients with CLL.5 Cladribine (2-chloro-2'-deoxy-β-D-adenosine, Leustatin, Ortho Biotech, Raritan, NJ) is asynthetic purine analog used to evaluate any effect onautoimmune diseases. Interestingly, the first CLL patienttreated with cladribine was included due to AIHA.15,16

More recently, one study shows some evidence thatcladribine may suppress the autoimmune hemolyticprocess in some patients with CLL.17 Cladribine therapyhas been beneficial in patients with a variety of chroniclymphoproliferative disorders and immediate toxicitiesgenerally are mild and consist chiefly of myelosup-pression and infections.18 Few data are available on thelong-term toxicities of cladribine. In this setting, Kong etal.19 reported Sjögren’s syndrome in one patient andsecond malignancies in three patients. Recently,cladribine has also been associated with AIHA in singleCLL patients17,20,21 and Tetreault and Saven7 reportedfour cases of delayed onset of AIHA after cladribinetreatment for WM. On the one hand, it seems thathemolysis could be considered an early complication,occurring generally within 6 weeks, in CLL patientstreated with fludarabine.22 On the other hand, Tetreaultand Saven7 reported a median interval of 40 months(range, 24 to 60 months) from the administration ofcladribine to the onset of hemolysis. In our case,autoantibody was detected at 9 months after cladribinetreatment.

For these reasons, it is recommended that a DAT beperformed at regular intervals during treatment withpurine analogs for CLL,15 and a specific caution iswarranted in patients who develop a positive DATduring such treatment.23 Less caution is required inthe treatment of other disorders, such as folliclelymphoma.15 In our case, cladribine was not given again.

The direct antiglobulin test (DAT) was performed bytube test, using polyvalent antihuman globulin (GammaBiologicals), monospecific anti-IgG (Ortho-ClinicalDiagnostics), and anti-complement (Diagast).

Eluates prepared from the patient’s RBCs using theElu-kitTM II (Gamma Biologicals) according to themanufacturer’s instructions were tested with panels of11 reagent RBCs, to detect any autoantibody specificityand to rule out alloantibodies, using the gel test.

Genotypic analysis by polymerase chain reactionusing 12 sequence-specific primers (PCR-SSP), accord-ing to the technique described by Gassner et al.,8 wasperformed, to confirm the Rh phenotype, in the immu-nohematology laboratory of our center.

ResultsThe DAT and antibody screen were negative before

cladribine treatment and were also negative at 1, 3, and6 months after cladribine treatment. Laboratory testsperformed 9 months after cladribine treatment yieldedthe following results. Blood grouping showed thepatient to be AB, D+. The antibody screen test waspositive and an anti-D was identified. The DAT waspositive (3+) with anti-IgG and an autoanti-D wasidentified in the eluate. Genotypic analysis confirmedthe Rh phenotype as ccDEe. No hemolysis wasdetected, as evidenced by a hemoglobin of 143 g/L(normal range: 120 to 140), a bilirubin of 15.7 µmol/L(normal range: 1.7 to 17.1) and lactic dehydrogenase of412 IU/L (normal range: 210 to 460). Reticulocyte countand haptoglobin level were not determined at that timenor was an adsorption with D– RBCs done to rule in orout a mimicking autoanti-D.9

DiscussionWe report a case of autoanti-D in a D+ woman with

a hematologic malignancy who developed autoanti-Dfollowing cladribine treatment.

In our case, a passive transfer of anti-D is excludedbecause the patient only received ten units of AB, D+packed RBCs. In addition, an antibody acquired bypassive transfer would not be detected nine monthsafter the last transfusion, as the half-life of human anti-Dis approximately 21 days.9 Alloimmunization in a weakD recipient is also excluded in our case because agenotypic analysis using the PCR-SSP techniqueconfirmed the normal D phenotype. Thus, the presenceof an autoanti-D is the most likely explanation for ourcase.


Autoantibody after cladribine treatment


J. CID ET AL.

The last follow-up in July 2001 did not show evidenceof hemolytic anemia. Repeat DAT and antibody screenswere not done.

References1. Vengelen-Tyler V, ed. Technical manual. 13th ed.

Bethesda, MD: American Association of Blood Banks,1999.

2. Saba NF, Sweeney JD, Lawton JC, Yankee RL, HuangCH, Schanfield MS. Anti-D in a D-positive renaltransplant patient. Transfusion 1997;37:321-4.

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

4. Rozman C, Montserrat E. Chronic lymphocyticleukemia. N Engl J Med 1995;333:1052-7.

5. Bergman L. Present status of purine analogs inthe therapy of chronic lymphocytic leukemias.Leukemia 1997;11(Suppl 2):S29-S34.

6. Dimopoulos MA, Panayiotidis P, Moulopoulos LA,Sfikakis P, Dalakas M. Waldenström’s macroglob-ulinemia: clinical features, complications andmanagement. J Clin Oncol 2000;18:214-26.

7. Tetreault SA, Saven A. Delayed onset of autoimmunehemolytic anemia complicating cladribine therapyfor Waldenström’s macroglobulinemia. Leukemiaand Lymphoma 2000;37:125-30.

8. Gassner C, Schmarda A, Kilga-Nogler S, et. al.Rhesus D/CE typing by polymerase chain reactionusing sequence-specific primers. Transfusion 1997;37:1020-26.

9. Callaghan TA, Fleetwood P, Contreras M, ScherrmannJM. Human monoclonal anti-D with a normal half-life. Transfusion 1993;33:784-5.

10. Petz LD, Garratty G. Mechanisms of immunehemolysis. In: Petz LD, Garratty G, eds. Acquiredimmune hemolytic anemias. London: ChurchillLivingston, 1980:110-38.

11. Issitt PD. Serology and genetics of the Rhesus bloodgroup system. Cincinnati, OH: MontgomeryScientific Publications 1979:161-7.

12. Avent ND, Reid ME. The Rh blood group system: areview. Blood 2000;95:375-87.

13. Vagace JM, Bureo JC, Groiss J, et al. Autoanticuerposcon especificidad anti-D. Comunicación de doscasos y revisión del tema. Haematologica2000;85:375-87.

14. Myint H, Copplestone JA, Orchand J, et. al.Fludarabine-related autoimmune hemolytic anemia

in patients with chronic lymphocytic leukemia. Br JHaematol 1995;91:341-4.

15. Juliusson G. Complications in the treatment of CLLwith purine analogues. Haematology Cell Ther1997;39:S41-S44.

16. DiRaimondo F, Giustolisi R, Cacciola E, et al.Autoimmune hemolytic anemia in chroniclymphocytic leukemia patients treated withfludarabine. Leukemia Lymphoma 1993;11:63-8.

17. Robak T, Blasinska-Morawiec M, Kryowski E,Hellman A, Konopa L. Autoimmune hemolyticanemia in patients with chronic lymphocyticleukemia treated with 2-chlorodeoxyadenosine(cladribine). Eur J Haematol 1997;58:109-13.

18. Cheson BD. Infections and immunosuppressivecomplications of purine analog therapy. J ClinOncol 1995;13:28-33.

19. Kong LR, Huang C, Hakimian D, et al. Long termfollow-up and late complications of 2-chlorodeoxyadenosine in previously treated,advanced, indolent non-Hodgkin’s lymphoma.Cancer 1998;82:957-64.

20. Fleischman A, Croy D. Acute onset of severeautoimmune hemolytic anemia after treatment with2-chlorodeoxyadenosine for chronic lymphocyticleukemia (letter). Am J Haematol 1995;48:293.

21. Piro LD, Carrera CJ, Beutler E, Carson DA. 2-chlorodeoxyadenosine: an effective new agent forthe treatment of chronic lymphocytic leukemia.Blood 1988;72:1069-73.

22. Weiss RB, Freeman J, Kweder SL, Diehl LF, Byrd JC.Hemolytic anemia after fludarabine therapy forchronic lymphocytic leukemia. J Clin Oncol1998;16:1885-9.

23. Byrd JC, Hertler AA, Weiss RB, Freimann J, KwederSL, Dieth LF. Fatal recurrence of autoimmunehemolytic anemia following pentostatin therapywith a history of fludarabine-associated hemolyticanemia. Ann Oncol 1995;6:300-1.

Joan Cid, MD (corresponding author), Centre deTransfusió I Banc de Teixits C/. Dr. Mallafré Guasch,4, 43007 Tarragona, Spain; Victor Beltran, MD,Haematology Service, Hospital Joan XXIII, C/Dr.Mallafré Guasch 4, 43007 Tarragona, Spain; L.Escoda, MD, Centre de Transfusió I Banc de Teixits,Tarragona, Spain; Enric Elies, MD, Centre deTransfusió I Banc de Teixits, Tarragona, Spain;and Carmen Martin-Vega, MD, Centre de TransfusióI Banc de Teixits, Tarragona, Spain.

Warm autoimmune hemolyticanemia with mimicking anti-c and -EspecificitiesH.-Y. HSIEH, D. MORONEY, D. NAUMANN, J. HATA, N. VOSNIDOU, R. KESSINGER, N. SHAHAB, N. HAKAMI, AND D. SMITH

An 18-month-old male was admitted to a hospital with ahemoglobin of 4.1 g/dL and a reticulocyte count of 53 percent.There was no history of prior transfusion. Serologic evaluationrevealed the presence of both a positive direct antiglobulin test(DAT) and an indirect antiglobulin test (IAT). The patient’s redblood cells (RBCs) typed as group A, C–D–E–c+e+ (cde/cde).Evaluation of the IAT revealed the presence of anti-c and anti-E. Allother major antibodies were ruled out. Upon adsorption of thepatient’s serum with ficin-treated Cde/Cde RBCs, both antibodyspecificities were adsorbed; however, the antibodies were notadsorbed with native (untreated) Cde/Cde RBCs. Furthermore, theautoantibody was not adsorbed by Rhnull cells, thereby suggestingRh specificity. The serum was incompatible with cde/cde RBCs andcompatible with Cde/Cde RBCs. The patient was successfullytransfused with Cde/Cde RBCs followed by resolution of hisanemia, as evidenced by an increased and stable hemoglobin. It wasconcluded that the autoantibody had mimicking anti-c and -Especificities. This is a report of an unusual case of autoimmunehemolytic anemia because the Rh autoantibody appeared to havedual mimicking specificities, and the patient’s RBCs were antigennegative for one of the antibody specificities, i.e., anti-E.Immunohematology 2002;18:19–22.

Key Words: autoimmune hemolytic anemia, Rhmimicking antibodies

Patients with warm autoimmune hemolytic anemiaoften require transfusion. These transfusions may becomplicated by hemolytic reactions secondary toundetected alloantibodies or the autoantibody proper.1,2

For these reasons it is critical to perform appropriatetests to obtain compatible or most compatible units fortransfusion.

Autoantibodies with Rh specificity commonly causeautoimmune hemolytic anemia.3 Rh antibodies usuallydo not fix complement,4,5 however, one such casedescribing a complement-binding anti-D in a Du variantwoman was reported.6 Some of these antibodies aredetected by routine serologic methods while others aredetected by polybrene or polyethylene glycol tech-niques.7,8 Autoantibodies mimicking alloantibodies havebeen described by others.9–11 Some of these have Rhspecificity12,13 and several examples of patients with

multiple Rh autoantibodies have been reported.14,15 IgAand IgM autoantibodies with Rh (anti-e) specificity havealso been described.16,17

In this report we describe a previously untransfusedinfant with autoimmune hemolytic anemia after a viralillness. The serum antibody had a unique specificityreacting with c and E antigens. Although the serumfailed to react with native (untreated) Cde/Cde redblood cells (RBCs), both specificities could be adsorbedwith ficin-treated Cde/Cde RBCs. These findingssuggest a more complex Rh autoantibody exhibitingmimicking anti-c and -E specificities.

Case ReportAn 18-month-old nontransfused male was admitted to

a hospital with symptoms of fatigue and fever. Thepatient had been exposed to several family memberswith upper respiratory symptoms. Pertinent physicalfindings included pallor, elevated temperature, andjaundice. A blood count was obtained and revealed ahemoglobin of 4.1 g/dL (normal range 10.5 to 14.0g/dL), a hematocrit of 13.4 percent (normal range 32 to42%), a reticulocyte count of 53 percent (normal range1.0 to 3.0%), a white blood cell count of 27.1 × 109/L(normal range 6 to 17.5 × 109/L), and a platelet count of331 × 109/L (normal range 150 to 400 × 109/L). Thetotal bilirubin was elevated at 5.3 mg/dL (normal range0.1 to 1.5 mg/dL) with a direct of 0.3 mg/dL (normalrange 0.0 to 0.3 mg/dL). Both the direct antiglobulintest (DAT) and the indirect antiglobulin test (IAT) were3+. The patient’s RBCs typed as group A, C–D–E–c+e+(cde/cde) with anti-E and -c reactivity detected both inserum and in eluate.

The patient was treated with intravenousprednisolone and then transfused with the leastincompatible RBCs available at that time (group A, cde/cde). There was an improvement in the hemoglobin


Table 1. Evaluation of the serum and eluate in LISS at IS and 37°C andby the indirect antiglobulin test (IAT)

Panel Agglutination ResultsCell # Rh-Hr Phenotype LISS IAT

D C c E e f V Cw IS 37°C Serum Eluate CC1 + + 0 + + 0 0 0 0 0 w+ w+2 + + 0 + + 0 0 0 0 0 w+ +3 + + 0 + + 0 0 0 0 0 w+ w+4 + 0 + + 0 0 0 0 0 0 +++ +++5 + 0 + 0 + + 0 0 0 0 ++ ++6 0 + + 0 + + 0 0 0 0 w+ +7 0 0 + + + + 0 0 0 0 +++ +++8 0 0 + 0 + + 0 0 0 0 ++ ++9 0 0 + 0 + + 0 0 0 0 ++ ++

10 + + 0 0 + 0 0 + 0 0 0 0 ++11 + + 0 0 + 0 0 0 0 0 0 0 ++12 + + 0 0 + 0 0 0 0 0 0 0 ++

Note: Immediate spin (IS) and 37°C reactions were performed in LISS.Cells were washed and polyspecific antihuman globulin was added to theserum and monospecific anti-IgG was added to the eluate. All negativereactions were confirmed using check cells (CC).

Table 2. Results of testing an eluate prepared from Cde/Cde RBCs usedto adsorb patient’s serum

Agglutination ResultsCell # Rh-Hr Phenotype IAT*

D C c E e f V Cw IgG CC†

1 + + 0 + + 0 0 0 0 ++2 + 0 + 0 + + 0 0 0 ++

All adsorptions were performed at 37°C for 30 minutes with native(untreated) Cde/Cde RBCs. An eluate was prepared from the cells andtested for reaction with selected panel cells in LISS at 37°C for 30minutes and by the indirect antiglobulin test* using monospecific anti-IgG. All negative reactions were confirmed using check cells.†


H.-Y. HSIEH ET AL.

concentration. Compatible Cde/Cde RBCs wereobtained within 8 hours of admission. The transfusionof both cde/cde and Cde/Cde packed RBCs occurredwithin 30 hours of the hospitalization. A 1.0g/dLincrement in hemoglobin was obtained with 42 mL ofthe packed cde/cde RBCs; however, a similar incrementin hemoglobin concentration was obtained with every36 mL of Cde/Cde RBCs transfused. The patientexperienced no complications, was converted to oralprednisone, and was discharged thereafter with ahemoglobin of 10.2 g/dL. Follow-up 3 weeks laterrevealed complete resolution of his anemia with ahemoglobin of 13.9 g/dL. An additional serum speci-men was not available for further serologic studies.

Materials and MethodsThe standard immunohematology techniques used

are described in the American Association of BloodBanks (AABB) Technical Manual.18 RBC panels wereobtained from Immucor, Inc. (Norcross, GA).Phenotyping antisera were acquired from eitherImmucor or Gamma Biologicals (Houston, TX).Antibody screens and panels were performed in low-ionic-strength saline (LISS). Anti-C (Gamma) and anti-c,-E, and -e (Immucor) were monoclonal reagentsrequiring incubation at 37°C for 5 to 15 minutes.Positive and negative controls were run with eachmonoclonal Rh typing reagent. Antibody elution wasperformed using the Gamma Elu-Kit II. Polyspecific andmonospecific anti-C3d and anti-IgG were obtained fromGamma, as were ficin and GammaZyme-S. Adsorptionswere performed at 37°C for 30 minutes with ficin-treated or untreated Cde/Cde RBCs. Tests with Rhnull

cells were done by the Gamma Biologicals ReferenceLaboratory, Houston, Texas. All specimens used forserologic studies were obtained from the patient withinthe first days of hospitalization. All other routine patientlaboratory data were obtained by standard laboratoryprocedures using reagents and instruments accordingto the manufacturers’ instructions.

ResultsThe patient typed as C–D–E–c+e+ (cde/cde) with Rh

monoclonal typing sera. The positive and negativecontrols run with each monoclonal Rh reagent reactedas expected. Those results excluded interference withthe patient’s Rh typing by the positive DAT. The initialspecimen had a 3+ DAT with monospecific anti-IgG andwas negative with anti-C3d. The IAT was 3+ with cDE/cDE RBCs and 2+ with cde/cde screening RBCs using a

polyspecific antiglobulin reagent. The CDe/CDescreening cell was nonreactive. Evaluation of the serumwith a polyspecific antiglobulin reagent and an eluatefrom the patient’s RBCs with a monospecific anti-IgGrevealed anti-c and -E reactivities (Table 1). Three

antigen negative and three antigen positive RBCs wereused to confirm each reactivity. In both the serum andthe eluate, anti-E reactivity was weak and anti-c strongin the IAT phase using monospecific anti-IgG.Antibodies to other antigens were ruled out. Since theserum and eluate had identical specificities, thepatient’s serum was first adsorbed with untreated Cde/Cde RBCs (37°C for 30 minutes). An eluate wasprepared from the Cde/Cde RBCs used for adsorption.No antibody was detected in the eluate (Table 2),

illustrating that the autoantibody was not adsorbed byuntreated RBCs lacking the corresponding antigens.Another aliquot of serum was adsorbed × 4 with ficin-treated Cde/Cde RBCs and reactivity of the adsorbedserum determined (Table 3). The weaker anti-Especificity was readily adsorbed. However, anti-creactivity remained after three adsorptions, albeit

Table 3. Results obtained with the indirect antiglobulin test (IAT) afterthe serum was adsorbed × 4 with ficin-treated Cde/Cde redblood cells (RBCs).

IAT ResultsCell # Rh-Hr Phenotype Adsorption #

D C c E e f V Cw #1 #2 #3 #4 CC*1 + + 0 + + 0 0 0 w+ 0 0 0 +2 + 0 + 0 + + 0 0 ++ + w+ 0 +

All serum adsorptions with ficin-treated Cde/Cde RBCs were performedat 37°C for 30 minutes. Adsorbed sera were incubated in LISS with aselected panel of RBCs for 15 minutes, then washed, and monospecificIgG antiglobulin reagent was added. All negative reactions wereconfirmed with check cells.* Reactivity of w+ to ++ denotes residualantibody in serum after the respective serial absorption.


AIHA with mimicking anti-c and -E

markedly decreased, and was removed by the fourth.Furthermore, the unadsorbed serum did not react withRhnull cells nor was reactivity reduced by adsorptionwith Rhnull cells. All crossmatches with Cde/Cde RBCswere compatible.

DiscussionThe anti-c and -E activities could be a single

autoantibody (anti-cE) reacting with both antigens orwith a common determinant expressed on the proteincarrying c and/or E antigen. A less likely explanationwould be two separate autoantibodies produced bydistinct populations of IgG-secreting plasma cells—oneproducing anti-c and the other producing anti-E.Because additional serum specimens were not available,it is difficult to differentiate between the twopossibilities. The data clearly imply that whatever theapparent specificity was, the antibody reacted with anepitope or epitopes exposed on ficin-treated antigen-negative Cde/Cde RBCs. This makes the true nature ofthe antibody difficult to determine. It is perhaps bestdescribed as an autoantibody with mimicking anti-c and-E specificity.

The clinical significance of this autoantibody is notdebatable as it was associated with profound hemolyticanemia. Whether the mimicking specificities properwere clinically significant may be questioned. Since thetransfusions occurred within a short period of time andimmediately after initiation of intravenous prednisolonetherapy, the effect of prednisolone on antibody titer andtransfused RBC clearance is questionable. However,since the Cde/Cde RBCs resulted in only a 117 percentbetter increment than the cde/cde RBCs in hemoglobinconcentration, the clinical significance of the mimickingspecificities is uncertain.

Rh autoantibodies are common, and some that reactwith more than one Rh antigen have been described.Schonitzer and Kilga-Nolger reported an autoanti-DE in

a 16-year-old girl with a cystic ovarian teratoma.19

Fudenberg et al. were the first to report autoantibodiesof apparently the wrong specificity, i.e., to antigensabsent on the subject’s erythrocytes.20 Others have alsoreported autoantibodies to Rh epitopes absent on thepatient’s RBCs. Such was the case described by Issitt etal. in a Cde/Cde patient with myelofibrosis whodeveloped an autoanti-E.21 This may not be anuncommon phenomenon, as others have describedsimilar individuals with RBCs that have a positive DATand yield an eluate that reacts with antigens theyphenotypically lack. In this report, an autoanti-E waseluted from the patient’s cde/cde cells. This antibodywould not react with RBCs with a homozygousexpression for the e antigen; however, the autoantibodyreacted with RBCs with a homozygous expression forthe C antigen and a heterozygous expression for the Eantigen. Unfortunately, reagent RBCs with homozygousexpression for C and E antigens (CDE/CDE) were notavailable to further characterize the autoantibody, butone would expect the anti-E component of theautoantibody to react more strongly with RBCs with ahomozygous expression for the E antigen. Thus, thisautoantibody had dual mimicking specificity with onecomponent of the autoantibody directed to an antigenabsent on the patient’s RBCs. This unusual case ofautoimmune hemolytic anemia exemplifies thecomplexity of the Rh system.

References1. Salama A, Berghofer H, Mueller-Eckhardt C. Red

blood cell transfusion in warm-type autoimmunehemolytic anemia. Lancet 1992;340:1515-7.

2. Habibi B. Autoimmune hemolytic anemia inchildren. Am J Med 1974;56-61.

3. Mollison PL, Engelfriet CP, Contreras M. Red cellantibodies against self-antigens, bound antigens andinduced antigens. In: P. Mollison, C. Engelfriet, M.Contreras, eds. Blood transfusion in clinicalmedicine. 10th ed. Malden, MA: Blackwell Science,1997;226-8.

4. Weiner W, Vos G. Serology of acquired hemolyticanemias. Blood 1963;22:606-13.

5. Garratty G. Target antigens for red-cell-boundautoantibodies. In: S. Nance, ed. Clinical and basicscience aspects of immunohematology. Arlington,VA: American Association of Blood Banks, 1991;33.

6. Ayland J, Horton M, Tippet TP, Waters A.Complement binding anti-D made in a Du variantwoman. Vox Sang 1978;34:40-2.


H.-Y. HSIEH ET AL.

IMPORTANT NOTICE ABOUT MANUSCRIPTS FOR IMMUNOHEMATOLOGYSubmit all manuscripts (original and 2 copies) to Mary H. McGinniss, Managing Editor, 10262 Arizona Circle,Bethesda, MD 20817. Please include the manuscript on a 3 1/2 inch disk, in Microsoft Word 97/6.0 orWordPerfect 6.0/5.1 or e-mail a copy to [email protected]

7. Lin C, Wong K, Mak K, Yuen C, Lee A. Hemolytictransfusion reaction due to Rh antibodies detectableonly by manual polybrene and polyethylene glycoltechnique. Am J Clin Pathol 1995;104:660-2.

8. Owen I, Hows J. Evaluation of the manualhexadimethrine bromide (Polybrene) technique inthe investigation of autoimmune hemolytic anemia.Transfusion 1990;30:814-8.

9. Viggano E, Clary N, Ballas S. Autoanti-K antibodymimicking an alloantibody. Transfusion 1982;22:329-32.

10. Marsh W, Reid M, Scott E. Autoantibodies of U bloodgroup specificity in autoimmune acquiredhaemolytic anemia. Br J Haematol 1972;22:622-9.

11. Harris T, Lukasavage T. Two cases of autoantibodieswhich demonstrate mimicking specificity in theDuffy system (abstract). Transfusion 1989;29:49S.

12. Leddy F, Falany J, Kissel G, et al. Erythrocytemembrane proteins reactive with human (warm-reacting) anti-red cell autoantibodies. J Clin Invest1993;91:1672-80.

13. Barker R, Casswell K, Reid M, Sokol R, Elson C.Identification of autoantigens in autoimmune hemo-lytic anemia by a non-radioisotopic immunoprecip-itation method. Br J Haematol 1992;82:126-32.

14. van’t Veer M, van Wieringen P, van Leeuwen I,Overbeeke M, von dem Borne A, Engelfriet C. Anegative direct antiglobulin test with strong IgG redcell auto-antibodies present in the serum of apatient with autoimmune haemolytic anaemia. Br JHaematol 1981;49:383-6.

15. Bell C, Zwicker H, Sacks H. Autoimmune hemolyticanemia: routine serologic evaluation in a generalhospital population. Am J Clin Pathol 1973;60:903-11.

16. Girelli G, Perrone M, Adorno G, et al. A secondexample of hemolysis due to IgA autoantibody withanti-e specificity. Haematologica 1990;75:182-3.

17. Okubo S, Ishida T, Yasunaga K. Autoimmunehemolytic anemia due to anti-e autoantibody of IgMclass. Jap J Clin Hematol 1990;31:129-30.

18. Vengelen-Tyler V, ed. Technical manual. 13th ed.Bethesda, MD: American Association of Blood Banks,1999:649-703.

19. Schonitzer D, Kilga-Nogler S. Apparent auto-anti-DEof the IgA and IgG classes in a 16 year old girl withmature cystic ovarian teratoma. Vox Sang 1987;53:102-4.

20. Fudenberg H, Rosenfield R, Wasserman L. Unusualspecificity of autoantibody in autoimmunehemolytic disease. J Mt Sinai Hosp 1958;25:324.

21. Issitt P, Zellner D, Rolih S, Duckett J. Autoantibodiesmimicking alloantibodies. Transfusion 1977;17:531-8.

Hsin-Yeh Hsieh, PhD, Department of Pathology andAnatomical Sciences, University of Missouri,Columbia, MO; Diana L. Moroney, MT, AmericanRed Cross, Missouri-Illinois Region, Columbia, MO;Deanne E. Naumann, MT, SBB, American Red Cross,Missouri-Illinois Region, Columbia, MO; D. JaneHata, PhD Candidate, Department of Pathology andAnatomical Sciences, University of Missouri,Columbia, MO; Nancy C. Vosnidou, MS, Division ofBiological Sciences, University of Missouri,Columbia, MO; Rovenna L. Kessinger, MD,Department of Pathology and Anatomical Sciences,University of Missouri, Columbia, MO; NassirShahab, MD, Department of Internal Medicine,University of Missouri, Columbia, MO; NasrollahHakami, MD, Department of Internal Medicine,University of Missouri, Columbia, MO; and Daniel S.Smith, MD (corresponding author), Department ofPathology and Anatomical Sciences, M263 MedicalSciences Building, University of Missouri, Columbia,MO 65212.

Attention: State Blood Bank Meeting Organizers—If you are planning a state meeting and wouldlike copies of Immunohematology for distribution, please contact Mary McGinniss, Managing Editor,4 months in advance, by phone or fax at (301) 299–7443.

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

Letters to the Editors

Anti-cE (Rh27), a rarely occurring antibodyA serum sample from a 73-year-old female was found

to be positive in a pretranfusion antihuman globulincrossmatch. Transfusion was required because ofanemia following a gastrectomy. Past history includedthree pregnancies, and 9 months prior to thegastrectomy she had received a red blood cell (RBC)transfusion. At that time, antibody screening andcrossmatch were negative.

Preliminary serologic investigation of the currentsample suggested an anti-cE. The serum was furthertested vs. group O RBCs of known Rh type using an IgGantihuman globulin test and an enzyme test (bromelin)at room temperature. The serum only agglutinated DcEand dcE (Rh27) RBCs and did not agglutinate DCE, dCE,or dce (Rh27) RBCs, which suggested the presence ofanti-cE as a single antibody. The Rh phenotype of thepatient was D+C+E–c–e+ (DCe).

Anti-cE was first described in 1961 by Gold et al.1 ina serum containing eight antibodies, and only a fewother samples have been described since then.2 Oneexample was of a naturally occurring anti-cE (Rh27)that bound complement, described by Kline et al.3 in1982.1. Gold ER, Gillespie EM, Tovey GH. A serum

containing 8 antibodies. Vox Sang 1961;6:157-63.2. Keith P, Corcoran PA, Caspersen K, Allen ATT. A

new antibody, anti-Rh(27) (cE) in the Rh bloodgroup system. Vox Sang 1965;10:528-35.

3. Kline WE, Sullivan CM, Pope M, Bowman RJ. Anexample of naturally occurring anti-cE (Rh27) thatbinds complement. Vox Sang 1982;43:335-9.

G. Lodi, MD, D. Resca, andR. Reverberi, Blood Transfusion

Service, Arcispedale S. Anna,C. so Giovecca 203, 44100 Ferrara,

Bologna, Italy; M. Govoni, BloodTransfusion Service, Ospedale

Maggiore, Bologna, Italy

September 11The staff of the American Red Cross Southern Region

Reference Laboratory would like to thank our friends atImmucor, Inc., for their support during the events ofSeptember 11. The unprecedented donor response


posed a dilemma for our reference laboratory: so manypotential new rare donors to be found but so few freehands to do the work to find them!

Tama Copeland and her staff at Immucor came to therescue. They volunteered their time, expertise, facilities,and even their rare antisera to help us with this massscreening. One Vel–, one Lan–, one Jsb–, two U–, oneTja–, and numerous multiple-antigen-negative donorswere identified and are now enrolled in our AmericanRare Donor Program. Many of these donors have alreadygiven a second donation.

Thanks, Immucor. You were there when we neededyou.

Debi Long, MT(ASCP)SBBAmerican Red Cross Blood Services

Southern RegionReference Laboratory

Atlanta, Georgia

Letter from the Editors

Thoughts on September 11The events of September 11, 2001, had a terrible

effect on everyone. But, out of the darkness of thisevent, there was much goodness. People helped eachother and we were kind to each other. I am sure thatmany of you have blood-bank-related stories that youwould like to share with the readers of Immu-nohematology. We would like to consider them forpublication.

In this issue, you will find a letter to the editors fromDebi Long of the Reference Laboratory at the SouthernRegion of the American Red Cross in Atlanta, Georgia,that relates one of these special kindnesses. If you havea special story and would like to share it, please fax it toMary McGinniss at (301) 299-7443 or e-mail it [email protected].

Feelings of sadness, anger, patriotism, and kindnesswill be with us forever. Let’s build on our patriotism andkindness.

Delores MalloryEditor-in-Chief

Mary McGinnissManaging Editor

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

NATIONAL REFERENCE LABORATORYFOR BLOOD GROUP SEROLOGY

Immunohematology Reference LaboratoryAABB, ARC, New York State, and CLIA licensed

(215) 451–4901 — 24-hr. phone number(215) 451–2538 — Fax

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

Monoclonal antibodies available. The New YorkBlood Center has developed murine monoclonalantibodies that are useful for donor screening and fortyping red cells with a positive direct antiglobulin test.Anti-Rh:17 is a direct agglutinating monoclonal anti-body. Anti-Fya, anti-K, anti-Jsb, and anti-Kpa are indirectagglutinating antibodies that require anti-mouse IgG fordetection. These antibodies are available in limitedquantities at no charge to anyone who requests them.Contact: Marion Reid, New York Blood Center,310 E. 67th Street, New York, NY 10021; e-mail:[email protected]

Granulocyte Antibody Detection and Typing

• Specializing in granulocyte antibodydetection and granulocyte antigen typing

• Patients with granulocytopenia can beclassified through the following tests forproper therapy and 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:

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Manuscripts: The editorial staff of Immuno-hematology welcomes manuscripts pertaining toblood group serology and education forconsideration for publication. We are especiallyinterested in case reports, papers on platelet andwhite cell serology, scientific articles coveringoriginal investigations, and papers on the use ofcomputers in the blood bank. Deadlines for receipt ofmanuscripts for the March, June, September, andDecember issues are the first weeks in November,February, May, and August, respectively. Instructionsfor scientific articles and case reports can beobtained by phoning or faxing a request to Mary H.McGinnniss, Managing Editor, Immunohematology,at (301) 299–7443, or see “Instructions for Authors” inevery issue of Immunohematology or on the Web.Include fax and phone numbers and e-mail addresswith your manuscript.

Reference and Consultation Services

Red cell antibody identification and problemresolution

HLA-A, B, C, and DR typingHLA-disease association typing

Paternity testing/DNA

For information regarding our services, contactZahra Mehdizadehkashi at (503) 280–0210,

or write to:

Pacific Northwest Regional Blood ServicesATTENTION: Tissue Typing Laboratory

American Red Cross3131 North Vancouver

Portland, OR 97227

CLIA licensed, ASHI accredited

National Platelet SerologyReference Laboratory

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(PF4 ELISA)

• Platelet suspension immunofluorescence test(PSIFT)

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

platelet antigens (MAIPA)

For information, e-mail:[email protected]

or 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 Services

Musser Blood Center

700 Spring Garden Street

Philadelphia, PA 19123–3594

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IgA/Anti-IgA Testing

IgA and anti-IgA testing is available to do thefollowing:

• Monitor known IgA-deficient patients• Investigate anaphylactic reactions• Confirm IgA-deficient donors

Our ELISA assay for IgA detects antigento 0.05 mg/dL.

For information on charges and samplerequirements, call (215) 451–4351,e-mail: [email protected],

or write to:

American Red Cross Blood ServicesMusser Blood Center

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ImmunohematologyJOURNAL OF BLOOD GROUP SEROLOGY AND EDUCATION

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