vol. 26, no. 1, 2010

Journal of Blood Group Serology and Education

Volume 26, Number 1, 2010

Immunohematology

25 C e l e b r a t i n g

Y e a r s

ImmunohematologyJournal of Blood Group Serology and Education

Volume 26, Number 1, 2010

Contents

Letter to the ReadersG.M. Meny

ReviewThe Knops blood group system: a reviewJ.M. Moulds

Case ReportRole for serial prenatal anti-Vel quantitative serologic monitoring with 2-ME serum treatmentW.J. Linz, J.T. Fueger, S. Allen, and S.T. Johnson

ReviewGranulocyte serology: current concepts and clinical significanceM.E. Clay, R.M. Schuller, and G.J. Bachowski

ReviewA review of the Colton blood group systemG.R. Halverson and T. Peyrard

Case ReportAlloimmunization to the D antigen by a patient with weak D type 21H. McGann and R.E. Wenk

ReviewA review of the Chido/Rodgers blood groupR. Mougey

In Memoriam Marie Cutbush Crookston (1920–2009)D. Mallory

In MemoriamEloise Giblett (1921–2009)D. Mallory

Announcements

Advertisements

Instructions for Authors

1

2

8

11

22

27

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39

40

414246

Immunohematology is published quarterly (March, June, September, and December) by the American Red Cross, National Headquarters, Washington, DC 20006.

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Copyright 2010 by The American National Red CrossISSN 0894-203X

Editors-in-ChiefSandra Nance, MS, MT(ASCP)SBB

Philadelphia, Pennsylvania

Connie M. Westhoff, PhD, MT(ASCP)SBBPhiladelphia, Pennsylvania

Senior Medical EditorGeralyn M. Meny, MD


Associate Medical Editors

David Moolten, MDPhiladelphia, Pennsylvania

Ralph R. Vassallo, MD Philadelphia, Pennsylvania

Emeritus Editorial BoardDelores Mallory, MT(ASCP)SBB

Supply, North Carolina

Patricia Arndt, MT(ASCP)SBBPomona, California

James P. AuBuchon, MDSeattle, Washington

Martha R. Combs, MT(ASCP)SBBDurham, North Carolina

Geoffrey Daniels, PhDBristol, United Kingdom

Anne F. Eder, MDWashington, District of Columbia

George Garratty, PhD, FRCPathPomona, California

Brenda J. Grossman, MDSt. Louis, Missouri

Christine Lomas-Francis, MScNew York City, New York

Gary Moroff, PhDRockville, Maryland

John J. Moulds, MT(ASCP)SBBShreveport, Louisiana

Paul M. Ness, MDBaltimore, Maryland

Joyce Poole, FIBMSBristol, United Kingdom

Mark Popovsky, MDBraintree, Massachusetts

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

S. Gerald Sandler, MDWashington, District of Columbia

Jill R. Storry, PhD Lund, Sweden

David F. Stroncek, MDBethesda, Maryland

Editorial Board

Managing EditorCynthia Flickinger, MT(ASCP)SBB


Technical EditorsChristine Lomas-Francis, MSc

New York City, New York

Dawn M. Rumsey, ART (CSMLT)Glen Allen, Virginia

Editorial AssistantPatti A. Brenner

Copy EditorMary L. Tod

ProofreaderLucy Oppenheim Electronic Publisher

Wilson Tang

Production AssistantMarge Manigly

IMMUNOHEMATOLOGY, Volume 26, Number 1, 2010 1

Letter to the Readers

This issue introduces an exciting change to the cover of Immunohematology. Images depicting a visual interpretation of a journal article or another timely aspect of blood banking will grace the cover of selected issues. I am pleased to announce that David Moolten, MD, award-winning poet and associate medical editor of Immunohematology, will provide a descrip-tion of the cover art from both an artistic and a scientific perspective. I hope these images remind us of the art as well as the science of transfusion medicine. Your feedback is welcome.

Geralyn M. Meny, MDSenior Medical Editor

Immunohematology

On Our CoverEl Acueducto de las Ferreras, an aqueduct also known as Puente del Diablo (Devil’s Bridge), spans a ravine approximately 4 km north of the town to Tarragona in the Autonomous Community of Catalonia, Spain. The aqueduct has a maximum height of 26 m and a length of 249 m. Built by the Roman Empire to supply water to the ancient city of Tarraco, the aqueduct features a superimposed series of arches, and may represent the first aqueduct of its kind. The Colton blood group antigen (discussed in this issue) is of course found on aquaporin-1, a widely expressed water channel protein.

——David Moolten, MD

Communication

G.M. Meny

IMMUNOHEMATOLOGY, Volume 26, Number 1, 20102

The Knops blood group system: a review

The Knops blood group system finds its roots in the group of antibodies that were previously called high-titer, low-avidity, or HTLA, antibodies. This term was used to de-scribe a group of alloantibodies that appeared to have many of the same characteristics, including very weak reactions at the anti-human globulin (AHG) phase of testing. Howev-er, as their specificities were more clearly defined, each was placed into a blood group system, and Knops became the 22nd system recognized by the ISBT Committee on Termi-nology for Red Cell Surface Antigens.1 Since that time, the protein bearing the Knops antigens has been identified, the gene has been cloned, and all of the known Knops antigens have been characterized at the molecular level. This has al-lowed investigators to more precisely study the role of the Knops blood group system in disease.

History In 1965, the sera from three unrelated patients (Cope-land, Stirling, and Wainright) were reported as having an antibody with similar specificity.2 The antibody was named after the first two antibody producers, Co and St (Cost), and given the designation Csa. Ten years later a new anti-gen, York (Yka) was reported.3 The York serum was initially believed to be an example of anti-Csa until the RBCs of the donor were found to be Cs(a+). This same study found that 12 of 1,246 Caucasian donors were both Cs(a–) and Yk(a–), suggesting that these antigens were at least phenotypically re-lated. Neither, however, was given blood group status, and they became part of the HTLA group of antibodies. Even though Yka was thought to be associated with Csa at this time, it would later be assigned to the Knops system. Another similar antibody being inves-tigated in the 1960s was anti-Kna. Anti-Kna was described in a transfused Caucasian woman who had a saline-reactive anti-K plus an unidentified antiglobulin-reactive antibody to a high-frequency antigen.4 The antithetical Knb was later reported in the Hall serum, which contained a potent anti-Kpb and was being used to screen Australian blood donors.5

The Knops blood group system began to expand when Molthan and Moulds6 described a new antigen, McCa, which seemed to be related to Kna. They reported that 53 percent of McC(a–) samples were also Kn(a–). Interestingly, a ma-jority of McCa antibody producers were Black whereas most of those making anti-Kna were Caucasian, thus suggesting that ethnic differences might exist in their respective gene frequencies. The next pair of alleles, Sla and Vil, was reported in separate abstracts with one author using the term McCc for Sla and McCd for Vil.7,8 The Sl terminology came from the names of the first two antibody producers, i.e., Swain and Langley. It was preferred by these authors because they be-lieved that this antigen was independent from McCoy. After identification of the protein bearing the Knops antigens, Sla was renamed S11 and Vil became S12.9

The last high-incidence antigen identified in the Knops system was KAM,10 later renamed KCAM by the ISBT Com-mittee on Terminology for Red Cell Surface Antigens.11 The antibody producer was a Caucasian man who exhibited the Helgeson phenotype, i.e., serologic Kn null. Like Sla, this antigen showed a widely diverse frequency in Blacks vs. Caucasians (Table 1). Although KCAM is a high-frequency antigen in Caucasians, only 20 percent of African Blacks were KCAM+.

Review

J.M. Moulds

Table 1. Frequency of the Knops antigens in several ethnic groups

Population Kna Knb McCa McCb S11 S12 Yka KCAM Refs.

Caucasians 98% 4% 98% 1% 99% <1% 90% 98% 3, 5, 8, 12, 13

West Africans

100% NT 89–92% 49–54% 30–38% 95% NT 20% 10, 14

African Americans

99% <1%* 90% 44% 51–61% 80%* 98% NT 8, 13, 14

African Brazilians

98% 2% 93% 42% 70% 86% NT 53% 12

Asian Brazilians

100% 0% 100% 0% 100% 0% NT 95% 12

*Marilyn Moulds, personal communication.Note: The publication by Covas et al.12 incorrectly lists 4870A (isoleucine) as being KCAM negative.NT = not tested.


Knops blood group system

Knops Protein In 1991, two groups identified complement receptor one (CR1) as the protein carrying the Kna, McCa, and Sla blood group antigens.15,16 In addition, Moulds et al.15 also located Yka to CR1 and suggested that the Helgeson phenotype was attributable to low CR1 copy numbers on the erythrocytes (E-CR1). Several years later, Petty et al.17 confirmed these reports using monoclonal antibody-specific immobiliza-tion of erythrocyte antigens (MAIEA) analyses and also confirmed that Csa was not on CR1. Thus, Csa and Csb have remained a collection as defined by ISBT. CR1 is a membrane-bound glycoprotein and, with the exception of platelets, is found on most human periph-eral blood cells. Depending on the methods used, erythro-cytes display approximately 300 to 800 CR1 molecules per cell whereas leukocytes display approximately 10,000 to 30,000 molecules per cell. Because erythrocytes are pres-ent in the peripheral circulation at concentrations 103-fold higher than the peripheral blood mononuclear cells (PB-MCs), they account for greater than 85 percent of CR1 in the blood. E-CR1 binds immune complexes (ICs) that are shuttled to the liver or spleen for transfer to and ingestion by macrophages, leading to their elimination. IC-free eryth-rocytes return to the circulation, where they may continue participating in IC clearance. We have noted that individu-als with high CR1 copy numbers may exhibit a weak false-positive DAT in the gel technique, most likely as a result of increased binding of ICs.

Knops Antigen Characteristics The Knops antigens can vary greatly in strength (Table 2), and weakly reactive cells can be falsely phenotyped as negative if the cells are stored for some duration or have low E-CR1 numbers. Chemicals that can disrupt disulfide bonds, i.e., DTT and AET, can destroy Knops, McCoy, Swain-Langley, and York antigens because they destroy the conformational structure of the short consensus repeats (SCRs). There are no examples of these antibodies that are enhanced by enzyme treatment of RBCs, and most are still reactive (sometimes weaker) with either ficin- or papain-treated cells.18,19 However, all Knops system antibodies cur-rently identified are nonreactive with trypsin-treated RBCs. It is known that a trypsin cleavage site exists in SCR 28 of the CR1 protein. Because the blood group antigens identi-fied to date have been found in SCR 25, they are lost on trypsin treatment of the cells. This fact can be a useful tool not only in antibody identification but also for absorption to remove other antibodies from a sample. Although the variability in antigen strength can be in-herited, it may also be acquired. Diseases that have high levels of ICs being processed, e.g., systemic lupus erythe-matosus or HIV infection, can cause an increased loss of E-CR1, resulting in weakened Knops antigen strength.20 In

Knops Antibodies The Knops antibody characteristics are shown in Table 3. Two characteristics that were often attributed to Knops antibodies were (1) they were not neutralized with plasma, saliva, or urine, and (2) they were difficult to adsorb and elute. The latter most likely reflects the low density of the CR1 protein on the RBC membrane. However, Race and Sanger23 reported that adsorption performed with buffy coats (WBCs) were able to remove anti-Kna from serum. This led to the speculation that anti-Kna and related speci-ficities were WBC antibodies. The ability of antigen-positive WBCs to adsorb Knops antibodies most likely relates to the fact that the CR1 copy number on WBCs is in the tens of thousands compared with a few hundred on RBCs.

Although CR1 has not been found in the saliva, low levels have been found in both urine24 and plasma.25 These are believed to be the result of proteolytic cleavage of CR1 from leukocytes.26 Serum CR1 is present only in nano-gram amounts,27 and therefore the levels are insufficient to neutralize Knops antibodies using routine serologic tech-niques. Hence, Moulds and Rowe28 developed an inhibition technique using recombinant, soluble CR1 (sCR1). Because their source of sCR1 was genetically positive for Kna, McCa, Sla, and Yka, it would only inhibit these antibodies and not inhibit anti-Knb or McCb. It must be remembered that the Knops phenotype of the sCR1 will be dependent on the gene chosen for its production. More recently, these investigators

addition, when RBCs are stored, vesicles are budded from the membrane, and these vesicles contain CR1.21 Therefore, the longer RBCs are stored, the weaker the Knops antigens become. This may explain why In(Lu) RBCs were initially reported to have weakened Knops antigens.22

Table 2. Characteristics of the Knops antigensInherited as Mendelian-dominant traitsHigh-frequency RBC antigens (except Knb and McCb)Developed on cord blood cells but may be weakerWeaker on stored RBCsNot destroyed by ficin or papainDestroyed by trypsin treatmentNot found on plateletsFound in low levels in plasma but not in urine or saliva

Table 3. Characteristics of Knops system antibodiesTiter is dependent on E-CR1 levelsCan demonstrate variable reactionsNot neutralized by pooled serum or other body fluidsDifficult to adsorb and elute from RBCsIgG reacting by AHG techniqueDo not bind complementUsually not clinically significant


J.M. Moulds

have used mutated CR1 constructs to produce peptides ca-pable of inhibiting anti-McCb and S12 (Vil).29

Clinical Importance Because the Knops antibodies are found in the serum of multiply transfused individuals, these sera often contain additional alloantibodies directed at (for example) K, E, and Duffy antigens. Most of the Knops antibodies are not considered clinically significant because they do not cause overt hemolytic transfusion reactions or hemolytic disease of the fetus and newborn. Some examples of Knops sys-tem antibodies have been studied using in vitro tests such as the monocyte monolayer assay (MMA). Arndt and Gar-ratty30 studied three anti-Kna, three anti-Yka, and five anti-Kn/McC and found that only one had a macrophage index of greater than 20, suggesting increased RBC destruction. Other MMA studies of two anti-Kna, two anti-Sla, one anti-KCAM, and one anti-Yk/Cs all gave indexes of less than 0, indicating no enhanced RBC destruction (J.J. Moulds, unpublished data).

The CR1 Gene The CR1 gene resides on chromosome 1 (1q32) and comprises 39 exons spread out over approximately 133 kilobase (kb) pairs of DNA.31 These exons encode SCRs of approximately 60 amino acids in the functional CR1 protein. Seven SCRs are organized into larger units called long homologous repeats (LHRs). The most common size protein product, CR1-1, is made up of four LHRs (A, B, C, D), a transmembrane region (TM), and a cytoplasmic tail (CYT), as shown in Figure 1. The binding sites for C3b and C4b have been localized to SCRs 8-9 and 15-16 (LHRs B and C) and to SCRs 1-2 (LHR-A), respectively.

The CR1 gene also exhibits two other poly-morphisms besides the Knops blood group. A structural, or size, polymorphism results from four different genes encoding four different molecular weight proteins: 190 kDa (CR1*3), 220 kDa (CR1*1), 250 kDa (CR1*2), and 280 kDa (CR1*4). Owing to the looping structure imparted by multiple disulfide bonds, the molecular weight shifts downward by approximately 30 kDa when the proteins are separated on SDS-PAGE using nonreducing conditions. It is now known that the molecu-lar weight polymorphism is independent from the blood group polymorphism.

The third commonly recognized polymorphism is based on quantitative differences in E-CR1. A HindIII RFLP is detected by two allelic fragments of 7.4 or 6.9 kb on South-ern blots. Homozygotes for the 7.4-kb fragment are high CR1 expressors (H), and homozygotes for the 6.9-kb frag-ment are low expressors (L) of CR1.32 Alternatively, a PCR-RFLP can be used that results in a band of 1.8 kb for H or 1.3 and 0.5 kb for L alleles.33 Although this RFLP correlates with RBC expression in Caucasian and Chinese individu-als,34 there is no relationship between this polymorphism and CR1 expression in African Americans35 or West Afri-cans.36 The exact molecular mechanism regulating CR1 RBC expression has been elusive but does not appear to be part of the promoter or 3′ untranslated portions of the CR1 gene.37

Molecular Basis of Knops Antigens Using CR1 deletion constructs, Moulds et al.29 first lo-calized the McCoy and S11 (Sla) antigens to LHR-D of CR1. By direct DNA sequencing they were then able to identify two separate mutations in SCR 25 that correlated with these two blood group antigens. The McCa/McCb polymorphism is at base pair 4795, where an A encodes proline (McCa) and a G encodes aspartic acid (McCb). The S11/S12 muta-tion is only 11 amino acids away; at base pair 4828, an A encodes arginine and a G encodes glycine. Accordingly, the ISBT has now assigned these antigens to the Knops system with the numbers shown in Table 4.38

Two other mutations have been identified in SCR 25, one of which was found in a Caucasian and is related to S11. KMW was initially believed to have produced anti-Sla. However, after gene sequencing it was found that she was not only homozygous for S11 but was homozygous for an-other SNP at position 4855 that substituted threonine for serine at amino acid 1610. After extensive molecular stud-ies, Moulds et al.39 proposed a model in which S13 was

Table 4. Molecular basis of the Knops antigens

ISBT number Antigen Nucleotide Nucleotide† Amino acid

KN1 Kna 4708G 4681G Va11561

KN2 Knb 4708A 4681A Met1561

KN3 McCa 4795A 4768A Lys1590

KN4 S11 4828A 4801A Arg1601

KN5 Yka Unknown Unknown Unknown

KN6 McCb 4795G 4768G Glu1590

KN7 S12 4828G 4801G Gly1601

KN8 S13 (provisional) 4828A, 4855T* 4801A, 4828T* Arg1601, Ser1610

KN9 KCAM 4870A 4843A Ile1615

*Note: The publication by Moulds et al.39 incorrectly lists the 4855 mutation as A>G. The correct substitution is T>A.†Nucleotide number when nucleotides are counted beginning with the A of the AUG start codon

LHR-A LHR-B LHR-C LHR-D TM CYT●●●●●●●●●●●●●●●●●●●●●●●●●●●● ╫□SCR 1–7 SCR 8–14 SCR 15–21 SCR 22–27

Fig. 1. Schematic of the CR1-1 protein bearing the Knops antigens in SCR 25.


a conformational epitope needing S11 (1601R) and S14 (1610S) to react. S14 and the antithetical antigen S15 are proposed epitopes awaiting the identification of the appro-priate antibodies before they can be officially recognized. However, the related SNPs can be identified molecularly. Limited population studies of Caucasians, Blacks, and Asians predict that S14 may be a high-frequency antigen in all populations and that S15 predominantly occurs in Cau-casians.12,39

The final antigen assigned to the Knops system is KCAM (initially reported as KAM). Interestingly, the SNP produc-ing KCAM was already known but had not been associated with a blood group specificity until a McCoy-like antibody was found in a Caucasian blood donor. On DNA sequenc-ing, it was discovered that he was homozygous for an SNP at bp 4870 that substituted valine for isoleucine.10 Hence, it was assigned the number KN9, and the antigen was re-named KCAM (KC for the city where the donor was found and AM for the donor’s initials).

Disease Associations In 1997, Rowe et al.40 identified CR1 as a ligand for the rosetting of Plasmodium falciparum–infected RBCs with uninfected cells. The ability of erythrocytes infected with P. falciparum to form rosettes is a property shown by only some parasite isolates, but is of importance because it has been associated with severe malaria.41 These authors showed that CR1 on uninfected erythrocytes was required for the formation of rosettes by demonstrating that CR1-deficient erythrocytes (Helgeson phenotype) had reduced rosetting and soluble recombinant CR1 could inhibit ro-setting. RBCs having the Sl:–1 phenotype showed reduced binding to the parasite rosetting ligand PfEMP1.14 Thus, the authors hypothesized that this polymorphism may have been selected for in malaria-endemic regions by provid-ing protection against severe malaria. The hypothesis was then tested by studies of Africans in Mali and Kenya, where it was found that the combined Knops haplotype Kn(a+)/McC(a–)/Sl:–1 appeared to be protective. CR1, as well as other complement receptors, has been identified as a receptor facilitating cell entry for a variety of pathogenic organisms. Pathogens using CR1 include Leishmania major (monocyte-macrophage),42 Legion-ella pneumophila (monocyte-macrophage),43 Leishmania panamensis,44 and Mycobacterium tuberculosis (mono-cyte-macrophage).45 In an AABB abstract, Noumsi et al.46 reported that individuals heterozygous for McCa and McCb were less likely to be infected by M. tuberculosis (odds ra-tio, 0.42; 95% confidence interval, 0.22 to 0.81; P = 0.007). These results suggested that McCb may have evolved among African populations in the context of M. tuberculosis pres-sure, and conferred a survival advantage in its heterozygous form.

Conclusions The molecular identification of the Knops blood group polymorphisms holds the promise for a better means of typ-ing for these antigens, the only exception to this being the yet unidentified Yka. Because of the inherited or acquired changes in RBC expression of CR1, i.e., Knops antigens, genotyping may become the method of choice for identify-ing these antigens, as it is for those of the Dombrock sys-tem. It is interesting to note that all of the known Knops polymorphisms are in SCR 25 of the CR1 protein. However, additional SNPs have been found in the CR1 gene, which suggests that the identification of new Knops blood group antigens is not yet at an end and will surely provide a chal-lenge to serologists in the future.

ReferencesDaniels GL, Anstee DJ, Cartron JP, et al. Blood group 1. terminology 1995. Vox Sang 1995;69:265–79.Giles CM, Huth MC, Wilson TE, Lewis HBM, Grove 2. GEB. Three examples of a new antibody, anti-Csa, which reacts with 98% of red cell samples. Vox Sang 1965;10:405–15.Molthan L, Giles CM. A new antigen, Yk3. a, and its rela-tionship to Csa (Cost). Vox Sang 1975;29:145–53.Helgeson M, Swanson J, Polesky HF. Knops-Helgeson 4. (Kna), a high frequency erythrocyte antigen. Transfu-sion 1970;10:737–8.Mallan MT, Grimm W, Hindley L, Knighton G, Moulds 5. MK, Moulds JJ. The Hall serum: detecting Knb, the antithetical allele to Kna (abstract). Transfusion 1980;20:630.Molthan L, Moulds J. A new antigen, McC6. a (McCoy), and its relationship to Kna (Knops). Transfusion 1978;18:566–8.Lacey P, Laird-Fryer B, Block U, Lair J, Guilbeau L. 7. Moulds JJ. A new high incidence blood group factor, Sla; and its hypothetical allele. (abstract).Transfusion 1980;20:632.Molthan L. Expansion of the York, Cost, McCoy, Knops 8. blood group system: the new McCoy antigens McCc and McCd. Med Lab Sci 1983;40:113–21.Daniels GL, Cartron JP, Fletcher A, et al. International 9. Society of Blood Transfusion Committee on terminol-ogy for red cell surface antigens: Vancouver report. Vox Sang 2003;84:244–7.Moulds JM, Pierce S, Peck KB, Tulley ML, Doumbo O, 10. Moulds JJ. KAM: a new allele in the Knops Blood Group System (abstract). Transfusion 2005;45(Suppl):27A.Daniels G, Castilho L, Flegel WA. International Society 11. of Blood Transfusion Committee on terminology for red cell surface antigens: Cape Town report. Vox Sang 2007;92:250–3.



Covas DT, de Oliveira FS, Rodrigues ES, Abe-Sandes 12. K, Silva WA Jr, Fontes AM. Knops blood group hap-lotypes among distinct Brazilian populations. Transfu-sion 2007;47:147–53.Molthan L. The serology of the York-Cost-McCoy-Knops 13. red blood cell system. Am J Med Technol 1983;49:49–56.Moulds JM, Kassambara L, Middleton JJ. Identifica-14. tion of complement receptor one (CR1) polymorphisms in west Africa. Genes Immun 2000;1:325–9.Moulds JM, Nickells MW, Moulds JJ, Brown M, At-15. kinson JP. The C3b/C4b receptor is recognized by the Knops, McCoy, Swain-Langley, and York blood group sera. J Exp Med 1991;173:1159–63.Rao N, Ferguson DJ, Lee SF, Telen MJ. Identification 16. of human erythrocyte blood group antigens on the C3b/C4b receptor. J Immunol 1991;146:3501–7.Petty AC, Green CA, Poole J, Daniels GL. Analysis of 17. Knops blood group antigens on CR1 (CD35) by the MAIEA test and by immunoblotting. Transfus Med 1997;7:55–62.Moulds MK. Serological investigation and clinical sig-18. nificance of high-titer, low-avidity (HTLA) antibodies. Am J Med Technol 1981;10:789–95.Reid ME, Lomas-Francis C. Knops blood group system. 19. In: The blood group antigen factsbook. 2nd ed. San Di-ego, CA: Academic Press, 2004:439–54.Holme E, Fyfe A, Zoma A, Veitch J, Hunter J, Whaley 20. K. Decreased C3b receptors (CR1) on erythrocytes from patients with systemic lupus erythematosus. Clin Exp Immunol 1986;63:41–8.Pascual M, Lutz HU, Steiger G, Stammler P, Schifferli 21. JA. Release of vesicles enriched in complement receptor 1 from human erythrocytes. J Immunol 1993;151:397–404.Daniels GL, Shaw MA, Lomas CG, Leak MR, Tippett P. 22. The effect of In(Lu) on some high-frequency antigens. Transfusion 1986;26:171–2.Race RR, Sanger R. Some very frequent antigens. In: 23. Blood groups in man. 6th ed. Oxford: Blackwell Scien-tific Publications, 1975:410–30.Pascual M, Steiger G, Sadallah S, et al. Identification 24. of membrane-bound CR1 (CD35) in human urine: ev-idence for its release by glomerular podocytes. J Exp Med 1994;179:889–99.Yoon SH, Fearon DT. Characterization of a soluble form 25. of the C3b/C4b receptor (CR1) in human plasma. J Im-munol 1985;134:3332–8.Danielsson C, Pascual M, Larsen B, Steiger G, Schifferli 26. JA. Soluble complement receptor type 1 (CD35) is re-leased from leukocytes by surface cleavage. Eur J Im-munol 1994;24:2725–31.Pascual M, Duchosal MA, Steiger G, et al. Circulating 27. soluble CR1 (CD35). J Immunol 1993;151:1702–11.

Moulds JM, Rowe KE. Neutralization of Knops system 28. antibodies using soluble complement receptor 1. Trans-fusion 1996;36:517–20.Moulds JM, Zimmerman PA, Doumbo OK, et al. Molecu-29. lar identification of Knops blood group polymorphisms found in long homologous region D of complement re-ceptor 1. Blood 2001;97:2879–85.Arndt PA, Garratty G. A retrospective analysis of the 30. value of monocyte monolayer assay results for predict-ing the clinical significance of blood group alloantibod-ies. Transfusion 2004;44:1273–81.Vik DP, Wong WW. Structure of the gene for the F al-31. lele of complement receptor type 1 and sequence of the coding region unique to the S allele. J Immunol 1993;151:6214–24.Wilson JG, Murphy EE, Wong WW, Klickstein LB, 32. Weis JH, Fearon DT. Identification of a restriction fragment polymorphism by a CR1 cDNA that correlates with the number of CR1 on erythrocytes. J Exp Med 1986;164:50–9.Cornillet P, Philbert F, Kazatchkine MD, Cohen JHM. 33. Genomic determination of the CR1 (CD35) density polymorphism on erythrocytes using polymerase chain reaction amplification and HindIII restriction enzyme digestion. J Immunol Methods 1991;136:193–7.Moulds JM, Brai M, Cohen JHM, et al. Reference typ-34. ing report for complement receptor 1 (CR1). Exp Clin Immunogenet 1998;15:291–4.Herrera AH, Xiang L, Martin SG, Lewis J, Wilson JG. 35. Analysis of complement receptor type 1 (CR1) expression on erythrocytes and of CR1 allelic markers in Caucasian and African American populations. Clin Immunol Immunopathol 1998;87:176–83.Rowe JA, Plowe CV, Diallo DA, et al. Erythrocyte com-36. plement receptor 1 expression level does not correlate with HindIII restriction fragment length polymorphism in ; implications for studies on malaria susceptibility. Genes Immun 2002;8:497–500.Cockburn IA, Rowe JA. Erythrocyte complement recep-37. tor 1 (CR1) expression level is not associated with poly-morphisms in the promoter or 3′ untranslated regions of the CR1 gene. Int J Immunogenet 2006;33:17–20.Daniels GL, Anstee DJ, Cartron JP, et al. Interna-38. tional Society of Blood Transfusion working party on terminology for red cell surface antigens. Vox Sang 2001;80:193–6.Moulds JM, Zimmerman PA, Doumbo OK, et al. Ex-39. pansion of the Knops blood group system and subdivi-sion of Sla. Transfusion 2002;42:251–6.Rowe JA, Moulds JM, Newbold CI, Miller LH. 40. P. falci-parum rosetting mediated by a parasite-variant eryth-rocyte protein and complement-receptor 1. Nature 1997;388:292–5.

J.M. Moulds


Rowe JA, Obeiro J, Newbold CI, Marsh K. 41. Plasmodium falciparum rosetting is associated with malaria severity in Kenya. Infect Immun 1995;63:2323–6.Da Silva RP, Hall BF, Joiner KA, Sacks DL. CR1, the 42. C3b receptor, mediates binding of infective Leishmania major metacyclic promastigotes to human macrophag-es. J Immunol 1989;143:617–22.Payne NR, Horwitz MA. Phagocytosis of 43. legionella pneumophila is mediated by human monocyte comple-ment receptors. J Exp Med 1987;166:1377–89.Robledo S, Wozencraft A, Valencia AZ, Saravia N. Hu-44. man monocyte infection by Leishmania (viannia) pan-amensis. J Immunol 1994;152:1265–75.

Schlesinger LS, Bellinger-Kawahara CG, Payne NR, 45. Horwitz MA. Phagocytosis of Mycobacterium tuber-culosis is mediated by human monocyte complement receptors and complement component C3. J Immunol 1990;144:2771–80.Noumsi GT, Tounkara A, Diallo D, Moulds JM. Knops 46. blood group polymorphism and protection from My-cobacterium tuberculosis (abstract). Transfusion 2006;46(Suppl):19A.

Joann M. Moulds, PhD, MT(ASCP)SBB, Director, Clinical Immunogenetics, LifeShare Blood Centers, 8910 Linwood Avenue, Shreveport, LA 71106.


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Role for serial prenatal anti-Vel quantitative serologic monitoring with 2-ME serum treatment during pregnancy: case report

W.J. Linz, J.T. Fueger, S. Allen, and S.T. Johnson

Anti-Vel is an uncommon antibody to a high-prevalence antigen. Its clinical significance and management in the prenatal setting are not well characterized. We present a case that demonstrates the utility of serial prenatal anti-Vel quantitative serologic monitoring with 2-ME serum treatment during pregnancy. The patient is a 23-year-old Hispanic woman with history of prior pregnancy and prior transfusion who was discovered to have an antibody to the high-prevalence Vel antigen in the first trimester (week 7) of her second pregnancy. Interval measurements of the serologic antibody titers were performed during the next 26 weeks. The untreated se-rum (IgM and IgG) titer increased from a baseline of 4 to 16 during that interval, while the 2-ME (presumed IgG component) titer re-mained stable at 4. Responding to ultrasound findings suspicious for fetal anemia, the child was delivered without complications at 34 weeks’ gestation. At birth, the DAT was negative and there was no evidence of HDN. Placed in the context of other similar reports, this case demonstrates the importance of separately reporting the IgG fraction (after either DTT treatment or 2-ME treatment) from the untreated (IgM and IgG) fraction and the importance of cor-relating the treated serum titer with potential clinical significance. Immunohematology 2010;26:08–10.

Key Words: Vel antigen collection, anti-Vel, prenatal serologic monitoring, 2-ME-treated serum, pregnancy

Vel is a high-prevalence antigen first described by Sussman et al. in 1952 in association with a trans-fusion reaction.1 Although several additional cases

quickly confirmed these authors’ findings,2 our knowledge of the Vel antigen (collection) remains incomplete. The presence of the Vel-negative phenotype in the general pop-ulation is estimated to be 1 in 4000 individuals.3 Vel alloim-munization can be mediated through IgG or IgM. Anti-Vel is thought to be associated rarely with hemolytic disease of the fetus and newborn (HDFN) because many examples of anti-Vel are mostly IgM and Vel is weakly expressed on the surface of fetal RBCs. Although the risk may be low, it is rea-sonable to monitor prenatal serologic titers serially during pregnancy.4 The findings published in a recent case report by van Gammeren et al.,5 of severe HDFN from anti-Vel, support this approach. The van Gammeren case report de-scribes discovery in a prenatal patient of an anti-Vel, which at initial presentation was determined to be composed ex-clusively of IgM. At the end of the pregnancy, an anti-Vel titer of 64 was demonstrated in untreated serum, whereas the DTT-treated serum (presumed IgG content) titer rose to 16. In their case, the infant exhibited severe jaundice and

reticulocytosis. As a corollary to the van Gammeren case re-port, we present a case of an anti-Vel for which the IgG titer as determined by 2-ME treatment was stable through preg-nancy despite a significant rise in the titer of the anti-Vel in the untreated serum. At birth, the child showed no evidence of HDFN. This case report supports the importance of serial determination of the prenatal IgG titer to help assess risk of HDFN.

Case Report A 23-year-old Hispanic woman, with a history of prior blood transfusion and one successful pregnancy, presented initially early in the first trimester of the index pregnancy. Her RBCs were typed as group O, D+. Routine serologic testing demonstrated variable reactivity using gel technolo-gy. The autocontrol was negative. Further testing using tube technology with PEG as enhancement also demonstrated variable weak reactivity, again with no specificity. No reac-tivity was demonstrated when using tube technology with LISS. A repeat serologic evaluation was performed 4 weeks later to help clarify the nature of this reactivity. The subse-quent serologic evaluation demonstrated reactivity with all RBCs tested whether using gel or test tube technology with LISS or PEG methods. The autocontrol was consistently negative. As an antibody to a high-prevalence antigen was suspected, the sample was referred to a large nationally rec-ognized immunohematology reference laboratory. With use of rare RBCs lacking various high-prevalence antigens, re-activity was determined to be that of anti-Vel. The patient’s RBCs were shown to lack the Vel antigen. The initial quan-titative serologic titer was 4 when tested against Vel+ RBCs using both 2-ME-treated and untreated serum. The Marsh score ranged from 14 with untreated serum to 4 with 2-ME-treated serum. The developing baby’s RBCs were presumed to be Vel+ as the father’s RBCs were demonstrated to ex-press the Vel antigen. Subsequent quantitative serologic monitoring was performed in approximately 4- to 6-week intervals for the next 20 weeks of pregnancy (Table 1). At 33 2/7 weeks’ gestation, Doppler ultrasonography showed an increase in middle cerebral artery velocity (>1.5 multiples of the median [MoM] for gestational age) suspi-cious for fetal anemia. This finding was a significant change from previous Doppler ultrasound studies and correspond-ed with the change in quantitative serologic titer of the

Case Report


untreated serum from 4 at presentation to 16 by week 26 (Table 1). A multidisciplinary risk-benefit analysis, includ-ing consideration of potential maternal and fetal need for transfusion with an extremely limited blood supply (two available units), the unpredictable timing issues encoun-tered with induction, and the limited time-availability for transfusion after thawing the available units, prompted the decision to move toward delivery in a manner that op-timized the availability of blood for the newborn, if it was indeed anemic. The patient underwent cesarean delivery at 34 3/7 weeks’ gestational age, 2 days after the initial admin-istration of Celestone to promote fetal pulmonary maturity. A 3088-g male newborn with Apgar scores of 7 at 1 min-ute and 9 at 5 minutes was born without complication. The child’s RBCs typed as group A, D+, and the antibody screen was negative. Initial laboratory data were also remarkable for a Hb level of 16.6 g/dL (normal range), reticulocyte count of 3.5% (normal range), and negative DAT. Approximately 1 week after delivery, a mild rise in bilirubin was observed, reaching a peak of 10 mg/dL. Given that the DAT and the initial screen were negative, the mild hyperbilirubinemia was thought not likely to have resulted from transplacen-tal anti-Vel or a possible anti-A from a group O mother. Moreover, although laboratory data are a bit limited, there was no evidence of anemia or hemolysis. Importantly, the mild hyperbilirubinemia quickly resolved, and the newborn was released to home and is doing well approximately 10 months after birth.

performed at the immunohematology reference laboratory used tube method. The patient’s serum was diluted in 6% albumin. A 30-minute 37°C incubation was performed, fol-lowed by a saline IAT (Anti-IgG, ImmucorGamma Inc.).6 Reaction scoring was performed using the system originally described by Marsh.7 The patient’s serum was treated with 2-ME (Fisher Scientific Co., Hanover Park, IL) and dialyzed overnight using dialysis membranes (Spectra/Por2, 12,000 to 14,000 dalton molecular weight cut-off [MWCO], Spec-trum Laboratories, Inc., Rancho Domingtuez, CA) in PBS, pH 7.3. Dialyzed serum was tested using the methods previ-ously described.

Discussion This case adds to the limited published information regarding the natural history of anti-Vel in the prenatal setting, and it supports the value of sorting immunoglobu-lin classes as an aid to proper prenatal management. It is worth noting that at initial presentation, weak, variable se-rologic reactivity was demonstrated. Despite use of several standard methods, specificity could not be assigned to the serologic reactivity. In the initial serologic evaluation, reac-tivity was present using gel technology and tube technology with PEG as enhancement. Tube testing with LISS showed no reactivity. Although a LISS tube method is considered an appropriate antibody detection method in the prenatal setting,8 this method would not have detected the antibody. A repeat study approximately 7 weeks after the initial study did demonstrate the presence of strong serologic reactivity using tube testing with LISS and PEG as well as with gel. The titer of the untreated serum and 2-ME–treated serum was 4. Presumably, late in the first trimester of pregnancy the mother was exposed to fetal RBCs expressing the Vel antigen because by week 14 she demonstrated a specific se-rologic immune response. Interestingly, for the remainder of the pregnancy, the titer of the anti-Vel in the 2-ME (pre-sumed IgG component)–treated serum remained stable al-though the titer in the untreated serum (presumed IgM and IgG) increased by two dilutions. This observation may be useful to laboratories without the capacity to treat patient serum with 2-ME or DTT. Namely, in this case anti-Vel titer in the untreated serum appeared to rise before the anti-Vel IgG titer in the 2-ME–treated serum. The rise in antibody titer in the untreated serum com-bined with the abnormal Doppler velocimetry prompted the preterm delivery. At birth, the DAT and antibody screen were negative, and the hemoglobin and reticulocyte count were in the normal range. As the anti-Vel present in the ma-ternal serum contained an IgG component, the negative DAT suggests that Vel antigen on the fetal RBCs may either be weakly or partially expressed9 or the antibody avidity to the antigen was poor. Regardless, in this case, an anti-Vel titer of 4 does not appear to be independently sufficient for HDFN, whereas a titer of 16 in the case reported by van Gammeren et al.5 was associated with HDFN. The cause of the rise in

Methods ABO and D testing and DAT were performed using commercially available reagents according to manufactur-ers’ protocols using tube method (Immucor Gamma Inc., Norcross, GA). Antibody detection and identification were performed using either gel technology according to the man-ufacturer’s protocol using reagent cells (RBCs 0.8% Surgis-creen and reagent RBCs 0.8% Resolve Antigram, ID-MTS Gel Test; Ortho-Clinical Diagnostics, Raritan, NJ) or tube method with commercially available cells and reagents (PEG and LISS, Immucor Gamma Inc.). Titration studies

Table 1. Quantitative serologic monitoring

Approxi-mate ges-tational age (wk)

Sero-logic

findings

Untreated serum titer

Untreated serum score

2-ME-treated serum titer

2-ME-treated serum score

7 Weak, variable reactivity

N/A N/A N/A N/A

14 Anti-Vel 4 24 4 24

18 Anti-Vel 4 27 4 14

22 Anti-Vel 8 N/T N/T N/T

26 Anti-Vel 16 25 4 18

34 Anti-Vel 16 30 4 14

N/A = not applicable; N/T = not tested.

Role for anti-Vel prenatal monitoring


W.J. Linz et al.

the bilirubin level after birth remains uncertain, although liver immaturity is the most likely explanation. Finally, the abnormal Doppler velocimetry in the absence of true fetal anemia reflects the inherent limitations of a screening test that is known to have a 12 percent rate of false-positive re-sults.10

The Vel antigen (collection) and anti-Vel remain enig-matic and are in need of further study. To date, it is known that the Vel-negative phenotype is universally uncommon. The incidence of the Vel-negative phenotype in the Hispanic population is not known with certainty. Anti-Vel is famous-ly associated with in vitro hemolysis, severe transfusion re-actions, and shortened RBC survival,11 although there is at least one case report of Vel+ blood being successfully trans-fused to a recipient possessing anti-Vel.12 An auto-anti-Vel13 has been described, although it is uncertain to what degree the autoantibody contributed to the patient’s RBC destruc-tion. Anti-Vel can be difficult to detect, and it can simulate the serologic profile of a cold-reactive autoantibody, with disastrous consequences if improper analytic methods are used.14 The Vel antigen appears to have a variable antigenic expression and appears to be best expressed on adult cells. When viewed in concert with the recent case report by van Gammeren et al.,5 our case report further supports the need not only to properly identify anti-Vel in the prenatal setting but also to perform serial quantitative serologic monitoring using methods to clearly separate the IgG frac-tion (DTT treatment or 2-ME treatment) from the untreat-ed (IgM and IgG) fraction. Although both the untreated and treated serum titer and score results may be reported, changes in the treated serum fraction, representing the IgG component, may be a better trigger to initiate additional clinical investigation. Additional cases may help identify the most appropriate critical 2-ME or DTT titer for anti-Vel.

ReferencesSussman LN, Miller EB. New blood factor: Vel. Rev He-1. matol 1952;7:368–71.Levine P, Robinson EA, Herrington LB, Sussman LN. 2. Second example of the antibody for the high-incidence blood factor Vel. Am J Clin Pathol 1955;25:751–4.Issitt PD, Oyen R, Reihart JK, Adebahr ME, Allen 3. FH, Kuhns WJ. Anti-Vel 2, a new antibody show-ing heterogeneity of Vel system antibodies. Vox Sang 1968;15:125–32.Tarsa M, Kelly TF. Managing a pregnancy with anti-4. bodies: a clinician’s perspective. Immunohematology 2008;24:52–7.van Gammeren AJ, Overbeeke MA, Idema RN, van 5. Beek RH, Ten Kate-Booij MJ, Ermens AA. Haemolytic disease of the newborn because of rare anti-Vel. Trans-fus Med 2008;18:197–8.Roback JD. Technical manual. 16th ed. Bethesda, MD: 6. AABB 2008: 900.

Marsh WL. Scoring of hemagglutination reactions. 7. Transfusion 1972;12:352–3.Judd WJ; Scientific Section Coordinating Commit-8. tee of the AABB. Practice guidelines for prenatal and perinatal immunohematology, revisited. Transfusion 2001;41:1445–52.Reid ME, Lomas-Francis C. The blood group antigen 9. factsbook. 2nd ed. San Diego, CA: Academia Press, 2004:503-4.Giancarlo M. Noninvasive diagnosis by Doppler ultra-10. sonography of fetal anemia due to maternal red cell al-loimmunization. N Engl J Med 2000;42:9–14.Tilley CA, Crookston MC, Haddad SA, Shumak KN. Red 11. blood cell survival studies in patients with anti-Cha, anti-Yka, anti-Ge and anti-Vel. Transfusion 1977;17:169–72.Issitt PD, Anstee DJ. Applied blood group serology. 4th 12. ed. Durham, NC: Montgomery Scientific Publications, 1998: 802.Becton DL, Kinney TR. An infant girl with severe auto-13. immune hemolytic anemia: apparent anti-Vel specific-ity. Vox Sang 1986;51:108–11.Storry JR, Mallory D. Misidentification of anti-Vel due 14. to inappropriate use of prewarming and adsorption techniques. Immunohematology 1994;10:83–6.

Walter J. Linz, MD, MBA, Medical Director, Scott and White Transfusion Service and Donor Center, Assistant Professor of Pathology, Texas A&M UHSC, 2401 South 31st Street, Temple, TX 76508; Judith T. Fueger, MT(ASCP)SBB, Immunohematology Reference Laboratory, Diagnostic Laboratories, BloodCenter of Wisconsin, Milwaukee, WI; Steven Allen, MD, Associate Professor, Texas A&M UHSC/Scott and White Memorial Hospital, Chair, Department of Ob/Gyn, Director, Division of Obstetrics and Gynecol-ogy, Scott & White Healthcare and Texas A&M University Health Science Center College of Medicine, Temple, TX; and Susan T. Johnson, MSTM, MT(ASCP)SBB, Immuno-hematology Reference Laboratory, Diagnostic Laborato-ries, BloodCenter of Wisconsin, Milwaukee, WI.

Notice to Readers

Immunohematology, Journal of Blood Group Serology and Education, is printed on acid-free paper.


Review

Granulocyte serology: current concepts and clinical signifcanceM.E. Clay, R.M. Schuller, and G.J. Bachowski

Applying serologic procedures to the detection of RBC and lym-phocyte antigens has facilitated the identification of granulo-cyte antigens with established clinical significance, which are now classified in the human neutrophil antigen system. Granu-locyte alloantibodies and autoantibodies have been implicated in a variety of clinical conditions including alloimmune neutro-penia, autoimmune neutropenia, febrile and severe pulmonary transfusion reactions, drug-induced neutropenia, refractori-ness to granulocyte transfusions, and immune neutropenia after hematopoietic stem cell transplantation. Although the intrinsically fragile nature of granulocytes contributes to the in-herent challenges of granulocyte serology, several advances in laboratory procedures have improved detection of granulocyte antibodies. This review will provide a current perspective about the importance and use of granulocyte serology for detection of granulocyte antibodies that have significant medical effects. Immunohematology 2010;26:11–21.

Key Words: granulocyte, neutrophil, antigens, antibodies, HNA, alloimmune neonatal neutropenia, autoimmune neutropenia, transfusion-related acute lung injury, TRALI

It has now been half a century since Lalezari1 described the first case of neonatal neutropenia attributable to transplacental transfer of granulocyte-specific antibod-

ies, thus initiating the field of granulocyte serology. This event fostered several decades of work directed at the iden-tification of granulocyte-specific antigens (and antibodies), methods for their detection, and an appreciation for their clinical significance. In many ways, granulocyte serology developed along the laboratory footprints of RBC and hu-man lymphocyte antigen (HLA) serology with one major exception—granulocytes could not be preserved for testing purposes. The requirement for fresh cells was and contin-ues to be a major impediment for wide-scale implementa-tion of granulocyte immunobiology studies. Although the number of laboratories worldwide that specialize in this technology is limited, the clinical significance of granulo-cyte antibodies has not diminished and is now supporting the development of new methodologies that have the poten-tial to enhance the utility of testing and growth of this field. Therefore, as we move ahead, it is important to (1) focus on the clinical events that support the need for granulocyte serology, (2) understand the benefits and limitations of the current serologic assays, and (3) evaluate the application of new technologies for the detection of granulocyte antigens and antibodies.

Clinical Significance Alloimmune Neonatal Neutropenia Neutropenia observed in the neonate is primarily the result of neutrophil-specific alloantibodies transplacentally transferred to the fetus from the maternal circulation. The presence of these human neutrophil antigen (HNA) anti-bodies most likely is the result of prenatal maternal sensi-tization by fetal neutrophils crossing the placental barrier, although the antibodies may have also arisen in association with autoimmune diseases such as systemic lupus erythematosus or rheumatoid arthritis.2,3 Mater-nal alloimmunization can occur anytime after the first trimester of pregnancy. Once present in the fetal circula-tion, maternal HNA antibodies will bind to the mature fetal cells expressing the corresponding neutrophil alloantigens, as the neutrophil precursor cells are spared.4 Neonates af-fected by alloimmune neonatal neutropenia (ANN) are al-most always neutropenic at birth, although cases have been reported in which the neutropenia has been delayed by 1 to 3 days.4 ANN is often asymptomatic and goes undiscovered unless the deficit of the neutrophils is profound enough (absolute neutrophil count < 1.5 × 109/L) that the newborn becomes susceptible and succumbs to infection caused by bacterial or fungal pathogens. These children commonly present with fever, malaise, lethargy, skin infections such as cellulitis and omphalitis, mucosal and respiratory infec-tions (stomatitis, otitis media, and pneumonia), urinary infections, and very rarely septicemia. These infections are usually mild, but fatalities have been reported in 5 percent of cases.5 As there are numerous reasons for neonatal neu-tropenia (i.e., congenital, antibody-mediated destruction, or infection that decreases hemopoietic cell production), detection of these maternal neutrophil antibodies is very important for determining the mechanism causing neutro-penia. This allows the physician to focus on the appropriate treatment. The presence of maternal antibodies can persist up to 6 months after birth although antibodies and clini-cal effects usually dissipate more quickly. The incidence of ANN has been estimated to be 0.1 to 0.2 percent.6,7 Cur-rently, neutrophil antibodies with the following specificities have been implicated in cases of ANN: HNA-1a,8–10 HNA-1b,11 HNA-1c,12 FcγRIIIb (CD16),13,14 HNA-2a,15 HNA-3a,16 and HNA-4a.17


Autoimmune Neutropenia Autoimmune neutropenia (AIN) occurs in both in-fants and adults. It is the most common form of chronic neutropenia in infants. Severe neutropenia is usually recog-nized 4 to 7 months after birth.4 In the vast majority of AIN cases infants exhibit relatively mild clinical issues such as stomatitis, otitis, and respiratory infections. The presence of clinical symptoms lessens as the child becomes older. Neutropenia in infants is self-limited with complete reso-lution commonly observed within 7 to 24 months.18 How-ever, this condition necessitates aggressive hygienic care of infants to prevent infection and vigorous therapy that may include severe antibiotic therapy along with treatment with recombinant G-CSF and IVIG when infection occurs. Cell destruction can occur in the peripheral circulation in addi-tion to interference with myelopoiesis in the bone marrow. Complete blood counts demonstrate absolute neutropenia, although random fluctuations in the neutrophil count can range from zero to near normal. AIN is also observed in adults. Monocytosis with or without lymphopenia may also be present.19 As in infants, the bone marrow commonly demonstrates an absence of mature cells with an increase in myeloid precursors. This apparent imbalance of precursors to mature segmented neutrophils is distinct in cause from the maturation arrest that occurs in congenital defects of neutrophils or hemato-logic malignancy. Unlike RBC autoantibodies, antibodies in AIN are often reported to have specificity that can include HNA-1a, HNA-1b, and HNA-2a.18–20

Drug-Induced Neutropenia Although neutrophil antibodies are believed to be in-volved in drug-induced neutropenia, neither the precise mechanisms nor the particular antigens on the cell surface have yet been clarified. Some cases of drug-induced granu-locytopenias probably result from direct marrow toxicity, although immune-mediated processes also occur. Quinine, quinidine, ibuprofen, and psychotropic medications such as clozapine are recognized as potential causes of neutrope-nia.21–23 Drug-dependent antibodies have been found to re-act with both granulocytes and granulocyte precursors. The FcγRIIIb and CD177 neutrophil glycoproteins have been reported to form neoantigens with drugs or their metabo-lites, which are recognized by drug-dependent neutrophil antibodies.

Transfusion Reactions Antibodies to neutrophil and HLA antigens can result in a variety of transfusion reactions. The spectrum of sever-ity can range from mild febrile reactions to death in the case of severe transfusion-related acute lung injury (TRALI). Re-cently TRALI has been shown to be the most common cause of transfusion-related fatalities in the United States.24

Febrile nonhemolytic transfusion reactions are com-mon and can occur when blood products containing WBCs are transfused into patients who have leukocyte antibodies.25

The occurrence of febrile reactions has been mitigated with the use of leukocyte-reduced RBCs and platelet products. As early as 1951 blood transfusions were implicated in noncardiogenic lung edema.26 The term TRALI was con-ceived in 1985 when Popovsky and Moore27 investigated a series of 36 patients with well-defined transfusion-related acute lung injury. They detected leukocyte (HLA and HNA) antibodies in 89 percent of the implicated donors. Seventy-two percent of these patients were treated with mechanical ventilation, and 6 percent died. The leading theories for the cause of TRALI involve the priming or activation of neu-trophils with antigen-antibody interactions, inducing an acute inflammatory response.28 Antibodies to HLA class I and class II as well as HNAs have all been implicated in the development of TRALI.28

Even though HLA class I alloantibodies are the most frequently encountered WBC antibodies in implicated blood donors, they may act as much weaker triggers of acute lung injury when compared with HLA class II and HNA antibod-ies. In a study designed to investigate leukocyte antibody specificities in severe TRALI reactions, Reil et al.29 reported that even though 73 percent of leukocyte antibodies identi-fied in their donor population were HLA class I, HLA class II and HNA antibodies were associated with 81 percent of the antibody-mediated TRALI cases.29 Specifically, in their 36 reported cases, 17 were elicited by blood products con-taining HLA class II antibodies, and HNA antibodies were implicated in 10 cases. Of the 10 fatal outcomes, 6 were linked to HNA-3a antibodies in the transfused blood prod-ucts, and HLA class II antibodies were associated with 3 fa-talities. Although leukocyte agglutinating (aggregating) anti-bodies have been frequently associated with TRALI,30–34 the ability of HNA-3a antibodies to also prime and activate neutrophils in the pulmonary vasculature is thought to be central to severe and fatal TRALI.35 Studies have now shown that in addition to HNA-3a antibodies, HNA-2a and HNA-4a antibodies also have the ability to prime neutrophils in vitro.36–38

Although the pathophysiology of TRALI appears com-plex and remains under investigation,39 substantial clinical and scientific information suggests that HLA (especially class II) and HNA antibodies do play a significant role in this disorder.28 Efforts are now being focused on detecting HLA and HNA antibodies in donors both to retrospectively investigate the cause of acute lung injury in blood recipients and to serve as a valuable strategy in preventing TRALI by identifying donors who may be responsible for the transfu-sion reaction.

Granulocyte Antigens Granulocyte-specific antigens are those with a tissue distribution restricted to granulocytes (neutrophils, eosino-phils, and basophils), whereas neutrophil-specific antigens are only present on neutrophils. Because of the difficulty in characterizing antigens on basophils and eosinophils, many

M.E. Clay et al.


Granuloycte serology: concepts and significance

antigens described as neutrophil-specific have not been tested to determine whether they are present only on neu-trophils or on all granulocytes. Although granulocyte (neu-trophil) antigen systems were initially identified through studies of ANN and AIN, both terms, granulocyte and neu-trophil, have been used to describe the alloantigens.

Nomenclature Lalezari and colleagues8 identified the first granulo-cyte antigen during their investigation of a neonate with transient ANN and subsequently proposed the first no-menclature system for these antigens. Because their stud-ies indicated that the antigen was neutrophil-specific, they designated it the N system and the antigens were named in chronologic order of discovery. The antigens were labeled alphabetically and the alleles were described numerically. The system eventually classified seven antigens: NA1,8 NA2,40 NB1,41 NB2,42,43 NC1,44 ND1,45 and NE1,46 which be-came the foundation for granulocyte serology. During this same period and subsequently, several dif-ferent granulocyte antigens were identified. Some were specific for granulocytes (or neutrophils): HGA-3, GA, GB, GC, Gr1, Gr247–49; some were shared with other cells or tissues: HGA-1, 5a, 5b, 9, MART47,50–52; and some had unclassified distribution: CN1, KEN, LAN, LEA, SL.53–56 All of these granulocyte antigens were detected with sera from patients with autoantibodies or alloantibodies di-rected against granulocytes, using traditional blood group serologic techniques such as agglutination, immunofluo-rescence, and cytotoxicity. Unfortunately, most of these antigens have not been further classified owing to the lack of sufficient biologic material for study. Although this led to a consolidation of individuals working in this field, advances were made and efforts were directed at im-munobiochemical, genetic, and structural or functional studies for a few of the well-classified granulocyte antigens. With the advent of this information came the need for a standardized granulocyte antigen no-menclature system based on the molecules carrying the antigens and the genes encod-ing each allele. In 1999, the Granulocyte Antigen Working Party of the International Society of Blood Transfusion (ISBT) estab-lished a new nomenclature system for the well-characterized granulocyte antigens, which was based on the glycoprotein loca-tion of the antigens.57 In this system the granulocyte antigens are called human neu-trophil alloantigens to indicate they are ex-pressed on neutrophils; however, this does not imply that they are neutrophil-specific. Each antigen group is assigned a number, and polymorphisms within the group are designated alphabetically in sequential order

of detection. The HNA system currently includes seven an-tigens, which are restricted to five antigen groups. The key features of the HNA system are summarized in Table 1. An extensive amount of information is now known about these antigens, and the reader is referred to Bux’s recent and comprehensive review about the antigens that constitute this system.58

HNA-1 is the best-characterized group, and it contains three antigens: HNA-1a, HNA-1b, and HNA-1c. The HNA-1 antigens are expressed only on neutrophils, and antibodies to these antigens are frequently implicated in cases of al-loimmune or autoimmune neutropenia.18,58 The antigens are epitopes that occur on the neutrophil low-affinity FcγRIIIb receptor (CD16). This molecule binds the Fc region of IgG antibodies complexed to other antigens or immunoglobu-lins. The gene frequencies of HNA-1a, HNA-1b, and HNA-1c vary among different ethnic populations.58 Furthermore, individuals lacking FcγRIIIb have been identified. These in-dividuals do not express the HNA-1 antigens on their neu-trophils, thus presenting as an HNA-1 null phenotype.54,62

The HNA-2 group has one characterized antigen—HNA-2a—which is located on a 58- to 64-kDa glycoprotein that is expressed only on neutrophils.63 Although HNA-2a is a high-frequency antigen phenotype (i.e., greater than 90% for most populations), it is unique in that it is expressed on subpopulations of neutrophils among antigen-positive people. The expression of HNA-2a is greater on neutrophils from women than men, and two or three subpopulations of expression are often detected: one population that lacks HNA-2a expression and one or two populations that express the antigen but with different intensities.64 The functional

Table 1. Human neutrophil antigens†

Antigen groups Antigens

Former name

Antigen location/ glycoprotein CD

numberCoding gene

alleles‡Clinical

significance

HNA-1 HNA-1aHNA-1bHNA-1c

NA1NA2SH

FcγRIIIb/CD16bFcγRIIIb/CD16bFcγRIIIb/CD16b

FCGR3B*01FCGR3B*02FCGR3B*03

ANN, AIN, TRALI,§

HNA-2 HNA-2a NB1 58–64 kDa/CD177 Unknown ANN, AIN, TRALI, febrile transfu-sion reactions,

drug-dependent neutropenia

HNA-3 HNA-3aHNA-3b

5b5a

CTL2/UnknownCTL2/Unknown

ANN, TRALI , febrile transfusion

reactions

HNA-4 HNA-4a MART CR3/CD11b (α M subunit)

ITGAM*01 (230G)

ANN

HNA-5 HNA-5a OND LFA-1/CD11a (α L subunit)

ITGAL*01 (2372G)

Unknown

†Information from references 20, 58, 59, 60.‡Alleles of the coding genes are named according to the ISGN Guidelines for Hu-man Gene Nomenclature.61

§Except HNA-1c.AIN = autoimmune neutropenia; ANN = alloimmune neonatal neutropenia; CD = clusters of differentiation; CR3 = complement receptor 3; CTL2 = choline trans-porter-like protein 2; FcγRIIIb = Fc gamma receptor IIIb; HNA = human neutrophil antigen; LFA-1 = leukocyte function antigen-1; TRALI = transfusion-related acute lung injury.


role of HNA-2a is not known, and antibodies to this antigen have been associated with AIN,20 ANN,65 febrile transfusion reactions,66 TRALI,67 and drug-dependent neutropenia.68

HNA-3a is expressed on granulocytes, lymphocytes, platelets, endothelial cells, kidney, spleen, and placental cells.50 It is a high-frequency antigen located on choline transporter-like protein 2 (CTL2), and its function is not known.59,60 Alloantibodies to HNA-3a have been associated with occasional cases of febrile transfusion reactions69 and one case of ANN.70 Although antibodies to HNA-3a are rare, their major clinical significance has been their association with serious and fatal TRALI events.32–34

The HNA-4 and HNA-5 antigens are located on subunits of the β2 integrin family (CD11a and CD11b molecules). In-tegrins are a large family of adhesive receptors that are es-sential for cell-cell interactions and cell trafficking. HNA-4a is an epitope on the α M (CD11b) chain of CD11b/CD18 (the CR3 molecule), and HNA-5a is an epitope on the α L chain (CD11a) of CD11a/CD18 (the leukocyte function antigen-1 [LFA-1] molecule).71 HNA-4a is expressed on granulocytes, monocytes, and natural killer cells, whereas HNA-5a is ex-pressed on all leukocytes.58 Alloantibodies to HNA-4a can cause ANN,72 and the CD11b/CD18 complex has been re-ported to be the target of autoantibodies.18,73 To date, HNA-5a antibodies have not been clinically associated with neutropenia. A general consensus exists that granulocytes do not ex-press ABO antigens74 or HLA class II antigens.75 Although the density of HLA class I antigens on granulocytes is fairly low, it can vary among individuals.76 Therefore, these anti-gens must be taken into consideration when patient samples are tested for granulocyte antibodies. HLA class I antigens may be of particular interest during granulocyte transfusion for neutropenic patients as these antigens, in granulocyte concentrates, may be introduced in high amounts to recipi-ents with preformed class I antibodies. Transfusion reac-tions including TRALI have been observed in this situation, and the antigen-antibody interaction has been proposed to limit effectiveness of the transfusion.77–79

Serologic Testing The detection of HNA antibodies is labor intensive and technically challenging. Unlike HLA testing, commercial test kits are not readily available for the detection of granu-locyte antibodies by solid-phase methodologies. Therefore, intact viable granulocytes are required for granulocyte an-tibody screening. Granulocytes are intrinsically designed to respond to physiologic priming signals, so they must be handled very carefully to prevent activation. Once activated it is impossible to interpret any serologic test results involv-ing these cells, as false-positive test results abound. Granu-locytes are also extremely labile and must be used within 24 hours of collection, necessitating ready access to panel

cell donors with the needed antigen phenotypes. Although many have tried, attempts to develop short-term or long-term granulocyte preservation procedures have been un-successful, thus requiring that fresh suspensions of granu-locytes be prepared daily for testing procedures. Finally, the presence of HLA antibodies in test sera makes identifying HNA antibodies difficult because granulocytes also express HLA class I antigens on their cell surface.

Preparation of Pure Granulocyte Suspensions The isolation of pure granulocyte suspensions from pe-ripheral blood was greatly facilitated and simplified with Bøyum’s introduction in 1968 of the Ficoll-Isopaque gradi-ent technique.80 Double-density gradient centrifugation is now the most widely used approach for the isolation and purification of granulocytes for serologic testing. The proce-dure makes use of discontinuous Ficoll gradients, and nu-merous publications exist describing the methodology and the recovery, purity, functional integrity, and activity of the isolated granulocytes.81–84

Granulocyte Antibody Detection and Antigen Typing Laboratory approaches for the detection of granulocyte antibodies require procedures that are accurate, reproduc-ible, and practical. Although numerous granulocyte sero-logic assays have been described, not all meet these criteria. In addition to the assay used, the ability to detect antibodies in test specimens or antigens on test cells may also depend on target cell antigen density and the concentration and immunoglobulin type of the antibody in the test reagent. Therefore, the successful detection of granulocyte antibod-ies often requires that a combination of methods be used. Although several methods for the detection of gran-ulocyte-specific antibodies are used by the few laborato-ries that perform this testing, the ISBT Working Party on Granulocyte Immunobiology, a worldwide consortium of 16 laboratories that participate in the annual International Granulocyte Immunology Workshop (IGIW), have recom-mended that granulocyte antibodies should be investigated using a minimum of two methods: the granulocyte immun-ofluorescence test (GIFT) and the granulocyte agglutination test (GAT).85 Multiple workshops have shown the sensitiv-ity of GIFT to far exceed that of GAT; however, agglutinat-ing antibodies such as HNA-1c, HNA-3a, and HNA-4a are much more readily detectable in GAT and in many cases it is the only reliable method for detecting these specificities. For example, the seventh IGIW included a test specimen that contained an HNA-3a antibody that could only be de-tected by GAT and the granulocyte chemiluminescence test (GCLT). Although GCLT has been useful in selective labora-tory settings, it is not routinely used for clinical granulocyte antibody detection and is not discussed.

M.E. Clay et al.



Antibody Detection Methods Granulocyte Agglutination Test Lalezari86 developed the basis of this assay in the early 1960s. Since that time, micro techniques and the use of pure granulocyte suspensions with optimized concentra-tions of EDTA have vastly improved the performance of this test. Typically, a panel of three to five donors is selected to include all known neutrophil antigens currently defined by the ISBT consortium. Purified granulocytes are isolated from EDTA-anticoagulated peripheral whole blood using a double-density gradient. These granulocytes are then washed in PBS before use. The test is biphasic and is based on the intrinsic response of granulocytes to aggregate when stimulated by antibodies reacting to corresponding cell surface antigens. The resulting agglutination is the conse-quence of the granulocyte activation that occurs during the sensitization phase, which induces the cells to form pseu-dopods and slowly migrate toward one another during the aggregation phase, until membrane contact is established. Typically, the serum or plasma from the specimen being investigated is incubated with the granulocyte suspension for 4.5 to 6 hours at 30ºC.87 The reactions are evaluated us-ing an inverted-phase microscope and graded from nega-tive to 4+ on the basis of the percentage of cells that are agglutinated. Our laboratory has determined cutoff scores for both adult and pediatric patients (<6 years old) on the basis of the reaction grades established during microscopic evaluation. Both IgG and IgM antibodies are detected by this method.

Granulocyte Immunofluorescence Test Developed by Verheugt et al.88 in the late 1970s, this fluorescent “antiglobulin” technique is used for the detec-tion of granulocyte alloantibodies and autoantibodies that are circulating and cell bound. As with the GAT, an anti-body screening panel of three to five donors is selected to include all currently defined HNA antigens. A purified granulocyte suspension is prepared and treated with 1% paraformaldehyde for a short time to prevent nonspecific immunoglobulin binding to the neutrophil Fc receptors and to stabilize the cell membrane. Serum or plasma from the patient is then incubated with an optimized concentration of granulocytes for 30 minutes at 37ºC. After a wash step that removes unbound antibodies, the granulocytes are then incubated with F(ab′)2 fragments of a fluorescent con-jugated anti-human antibody for approximately 30 min-utes at room temperature in a dark environment. The as-say’s performance is optimized with the use of a fluorescent secondary probe that can detect both IgM and IgG antibod-ies (to ensure the detection of both primary and secondary immune responses) and the use of F(ab′)2 Ig fragments to prevent the probe from binding to the high concentrations of Fc receptors on granulocyte surface membranes. The cells undergo another wash cycle, are resuspended, and then are analyzed. Detection of the immunofluorescence reactions

can be accomplished by using either a fluorescence-detect-ing microscope or a flow cytometer. Evaluation by a flow cytometer has in most cases replaced the fluorescence mi-croscope as more cells can be analyzed in a shorter time, resulting in improved assay sensitivity and reproducibility, and it does not require the expertise needed to evaluate the characteristic staining pattern seen with specific granulo-cyte antibodies by microscopic analysis.

Monoclonal Antibody Immobilization of Granulocyte Antigens The monoclonal antibody immobilization of granulo-cyte antigens assay (MAIGA) is based on the platelet ver-sion (MAIPA) developed by Kiefel et al. in 1987.89 MAIGA relies on the capture of neutrophil-specific antigen-antibody complexes by a murine monoclonal antibody onto a solid-phase surface.90 The benefits of this test are twofold: first, this is currently the most sensitive assay for the detection of granulocyte antibodies, and second, the assay is designed to detect only HNA antibodies even when HLA antibodies are present in the test specimen. The disadvantage of this pro-cedure is that it is very complex and requires highly skilled staff to perform the detailed techniques. Granulocytes are incubated with the test serum or plas-ma for 30 minutes at 37ºC. This granulocyte suspension is then washed to remove any unbound immunoglobulins and incubated with a murine monoclonal antibody to a specific neutrophil glycoprotein for an additional 30 minutes. Af-ter another wash step, the granulocyte membranes are dis-rupted in a mild detergent and centrifuged. The resulting lysate is then transferred to polystyrene microwells coated with anti-mouse immunoglobulins for incubation. The trimolecular neutrophil antigen–patient HNA antibody–murine monoclonal antibody complex present in the lysate is captured on the solid phase, whereas any HLA antibody-antigen complexes (if present) are removed by a subsequent wash step. Remaining complexes are then detected by the addition of anti-human IgG conjugated to horseradish peroxidase followed by a substrate (OPD dis-solved in 30% H2O2), and the reaction is analyzed with a spectrophotometer. MAIGA can be used to detect antibodies specific to HNA-1a, HNA-1b, and HNA-1c antigens located on FcγRIIIb (CD16); HNA-2a present on gp 58- to 64-kDa (CD177); HNA-4a on the complement component receptor C3bi (CD11b); and HNA-5a on the LFA-1 receptor (CD11a). HNA-4a and HNA-5a antibodies can also be detected by a common CD18 monoclonal antibody.

Antigen Detection and Typing Methods The same methodology that is used to detect antibodies in serum and plasma can be used to characterize the antigen phenotype of an individual’s granulocytes. Just as the short lifespan of granulocytes is a limitation to obtaining panel cells for antibody testing, it also restricts the ability to do antigen typing because granulocyte specimens must be tested


within 24 hours of collection to obtain reliable results. A sufficient volume of whole blood must be collected to obtain an adequate number of granulocytes for phenotyping. This can be a dilemma in pediatric patients or patients who are neutropenic. Because ample volume, freshness, and avail-ability of granulocytes is critical, this also acts as a practical barrier to doing donor-recipient crossmatch testing during investigations of suspected granulocyte-antibody–mediat-ed transfusion reactions such as TRALI. The highly charac-terized antisera used to phenotype granulocytes should be free of HLA class I antibodies as their presence can result in a false-positive HNA typing. At a minimum, the titer of the HLA antibody should be weaker than the HNA antibody as it can then be readily diluted to a nonreactive level. An-other constraint to phenotyping granulocytes is the limited availability of qualified antisera for the currently defined HNA antigens. Monoclonal antibodies specific to several HNA anti-gens are commercially available and have been used to type granulocytes by GIFT using the flow cytometer. An advan-tage to phenotyping by this method is that it can be done with whole blood instead of with isolated granulocytes. Because phenotyping granulocytes cannot often be per-formed or can be unreliable if samples are not handled in a careful and timely manner, genotyping by PCR, which has less stringent sample age and handling restraints, can serve either as an alternative to or as confirmation of serologic results.

Granulocyte Genotyping The discovery of the PCR in the 1980s facilitated the development of molecular methods that have now been ap-plied to delineate the genetics and allelic variations of the HNAs. Initially, the characterization of the genes encoding the HNA-1 antigen system (HNA-1a, HNA-1b, and HNA-1c) allowed for the development of PCR assays using sequence-specific primers (SSP) to differentiate the alleles. To com-plicate matters there is a high degree of homology between the FCGR3A gene that encodes the FcγRIIIa granulocyte receptor and the FCGR3B gene where the three different HNA-1 polymorphisms reside. HNA-1a, HNA-1b, and HNA-1c are encoded by FCGR3B*1, FCGR3B*2, and FCGR3B*3 genes, respectively. FCGR3B*1 differs from FCGR3B*2 by five nucleotide bases, and a single nucleotide polymor-phism (SNP) differentiates FCGR3B*2 from FCGR3B*3 (Table 2). FCGR3A differs from FCGR3B at five different nucleotide locations (Table 2). Methods are also available to genotype the HNA-4a and HNA-5a alleles.71 Both of these polymorphisms are the result of SNPs. Even though the molecular sequence of HNA-2a has been identified, genotyping methods to detect the gene that encodes this alloanti-gen are not available. The HNA-2a negative phenotype is attributable to CD177 mRNA

splicing defects, and no mutations have been detected in the CD177 introns or exons in these individuals.93 Because the gene encoding HNA-3a has only recently been identi-fied, there has not been a genotyping procedure for this antigen, but the clinical importance of this antigen is sup-porting efforts to get a molecular method developed. Genomic DNA (gDNA) can be isolated and purified from anticoagulated blood using any number of published methods. A unique set of sense and antisense oligonucle-otide primers that border the DNA fragment to be amplified are added to a master mix of reagents that includes deoxy-nucleoside triphosphates (dNTPs), Taq polymerase, a buf-fer solution containing Mg2+, and the individual’s gDNA. The master mix is subjected to a series of 30 to 40 tempera-ture changes that define the amplification cycles. Each cycle consists of three temperature steps that are critical in the amplification of the DNA target strands. The first step in the cycle typically involves heating the master mix to a tem-perature of 95ºC. This denaturation step results in double-stranded DNA separating into single strands by disrupting the hydrogen bonds between each nucleotide base pair. The annealing step follows in which the reaction temperature is lowered to 50ºC to 65ºC. This allows the primers to hybrid-ize to the opposing strands of the target DNA. The final ex-tension step heats the master mix from 71ºC to 72ºC, which results in DNA synthesis in the presence of Taq polymerase and dNTPs complementary to the oligonucleotide primers. A final extension step is added after all cycles have been completed to ensure that any remaining single-stranded DNA is fully elongated. The amplified double-stranded DNA (amplicon) is separated by size using agarose gel electrophoresis, stained with ethidium bromide, and visu-alized by fluorescence using ultraviolet light. The sizes of the amplicons are determined by comparison with a DNA ladder, which contains DNA fragments of known sizes that were run alongside the PCR products during the agarose gel electrophoresis step.

Future Directions Solid-Phase Technology The complement-dependent lymphocyte cytotoxicity assay was the first platform, developed nearly 50 years ago, for identifying HLA antibodies and remains the standard for the development of new methodologies in this field.

M.E. Clay et al.

Table 2. Nucleotide differences for the three HNA-1 alleles (1a, 1b, and 1c) on FcγRIIIb and among the FcγRIII genes†

HNA Gene Polymorphic nucleotides

141 147 227 266 277 349 473 505 559 641 733

1a FCGR3B*1 G C A C G G A C G C T

1b FCGR3B*2 C T G C A A A C G C T

1c FCGR3B*3 C T G A A A A C G C T

FCGR3A G C G C G A G T T T C

†Information from references 91 and 92.NOTE: Boldface indicates substituted nucleotides. FcγRIIIb = Fc gamma receptor IIIb; HNA = human neutrophil antigen.



Multiple new technologies are now being used by HLA laboratories, and in the past few years solid-phase assays have been developed in which purified HLA molecules are bound to a well in a microtiter plate or to a bead. HLA an-tibody is then detected either using an enzyme-linked im-munosorbent assay (ELISA) technique or by analyzing the beads with a flow cytometer, which uses a new technology developed by Luminex (Austin, TX).94 A critical advantage of this technology is its ability to measure multiple analytes simultaneously in a single reaction system. The commer-cially available test kits use purified HLA antigens bound to microbeads, which can be individually differentiated by varying ratios of internal fluorescent dye prepared during the manufacturing process. The test involves incubation of test sera with the panel of microbeads, and the bound HLA antibody is detected by a secondary R-phycoerythrin (PE) conjugated IgG (or IgM)–specific antibody. Analysis is done with the use of a Luminex flow analyz-er in which the beads pass through two lasers, similarly to classic flow cytometry. One laser identifies the specific bead being analyzed, thereby identifying the unique HLA anti-gen. The other laser detects the presence of bound Ig on the surface of the microbead. The color signals are then detect-ed and processed into data for each reaction. A significant fluorescence shift from the negative control range indicates the presence of specific antibody in the test sample. The widespread use of Luminex technology by HLA laboratories has fostered commercial activity directed at using this technology for the detection of HNA antibodies that have been related to febrile and pulmonary transfusion reactions. One Lambda, Inc. (Canoga Park, CA), has devel-oped recombinant cell lines for HNA-1a, -1b, -1c, -2a, and -4a, and these cell lines have allowed the production of pu-rified proteins that have been individually immobilized on Luminex microbeads for use in their LABScreen Multiflow bead assay. Stroncek and colleagues95 evaluated the specific-ity and sensitivity of this new procedure using 22 sera that had known HNA antibodies detected with the GA assay and 140 samples from nontransfused men. All HNA antibody specificities (except 4a) recognized by GAT were detected in the solid-phase flow bead system (8 HNA-1a, 6 HNA-1b, 1 HNA-1c, and 8 HNA-2a positive sera), and HNA-1c reac-tivity was detected in four specimens that were negative by GAT. In addition, all of the 140 negative control samples had negative reactions when tested by LABSreen Multi, and the system was able to detect both HNA and HLA antibod-ies (both class I and class II) in some individual specimens.95 Although preliminary, these results suggest that this solid-phase technology may emerge as a major achievement in the development of a new sensitive and specific platform for the detection of granulocyte antibodies in an automated manner with limited complexity and high cost efficiency.

Cell Lines Expressing HNA Antigens Another approach to eliminate the need for freshly isolated granulocytes for granulocyte antibody screening

has been the establishment of cell lines that express HNA antigens. In 1999 Bux’s group transfected Chinese ham-ster ovary (CHO) cells with HNA-1a, HNA-1b, and HNA-1c cDNA and established stable cell lines expressing these antigens.96 Although the cell lines were stable for 1 month at 4°C, they demonstrated high background fluorescence in the GIF assay and could only be used for the MAIGA, which limited their overall utility. More recently, Yasui et al.97,98 transduced HNA genes into the human erythroleukemia cell line K562 and established a panel of K562 cell lines that stably expressed HNA-1a, -1b, -1c, -2a, -4a, -4b, -5a, and -5b for at least 1 year while being maintained in culture. They were not able to establish a cell line expressing HNA-3a be-cause of the previous lack of biochemical and genetic infor-mation for this antigen. The group’s report demonstrated the utility of using the cultured cells in the GIF assay to de-tect HNA antibodies in serum samples from patients with HNA antibody–related disorders, but experience with this new cell line technology is very limited and it remains to be seen whether the cell lines can be maintained or preserved in a manner that would support the use of these cells for routine (nonresearch) granulocyte serology applications.

ISBT Working Party on Granulocyte Immunobiology In 1989, Professor Alan Waters organized the UK Plate-let and Granulocyte Serology Working Group, and 11 inter-national laboratories participated in the First International Workshop on Granulocyte Serology. The objectives of this first workshop were as follows: (1) to initiate an exchange of sera among laboratories to evaluate the techniques be-ing used for the detection of granulocyte antibodies, (2) to foster working relationships among laboratories involved in this field, and (3) to support the development of the sci-ence and technology for the detection of granulocyte anti-bodies.99

The Working Party was initially affiliated with the Brit-ish Blood Transfusion Society but is now affiliated with the ISBT. The goals of the Second Workshop, in 1996, were the (1) establishment of typed granulocyte panels by well-defined typing sera, (2) proficiency testing of granulocyte antibody detection, (3) investigation of uncharacterized sera, (4) proficiency testing of HNA genotyping, and (5) ex-change of information to establish standards in granulocyte serology.100 Fifteen international laboratories participated in the Fourth Workshop, in 2001, with the main objec-tive being the establishment of a formal quality assurance (QA) scheme for granulocyte serology and molecular typing methods.85

To date, the Working Party has conducted nine Granu-locyte Serology Workshops and comprises 16 international member laboratories, which are listed in Table 3. Also, the American Red Cross Mid-America Blood Services Neutro-phil & Platelet Immunology Laboratory has been a member of this consortium since its inception in 1989. The workshops continue to focus on the QA and standardization of the test systems, and the Working Party recently published its


serologic screening recommendations for the investigation and prevention of leukocyte antibody-mediated TRALI.101

Conclusions Granulocyte antibody and antigen testing has played and continues to play a critical role for diagnosing and investigating relatively rare but potentially lethal clini-cal conditions such as TRALI and immune neutropenias. Although mitigation strategies are already being imple-mented nationwide, TRALI still appears to be a leading and preventable cause of transfusion fatalities. As advancing technology allows granulocyte antibody testing to be done on a less manual and larger scale with more standardized and reproducible results, it now has the potential to become

even more valuable as a tool to increase blood safety by ac-tually preventing transfusion reactions without compro-mising an adequate supply of blood.

Acknowledgments The authors would like to thank Penny Milne and Bob-bie Gibson for their helpful assistance with the preparation of the manuscript.

References Lalezari P, Nussbaum M, Gelman, et al. Neonatal 1. neutropenia due to maternal isoimmunization. Blood 1960;15:236–43.Bux J, Jung KD, Kauth T, et al. Serological and clinical 2. aspects of granulocyte antibodies leading to alloimmune neonatal neutropenia. Transfus Med 1992;2:143–9.Rodwell RL, Gray PH, Taylor KM, et al. Granulocyte 3. colony stimulating factor treatment for alloimmune neonatal neutropenia. Arch Dis Child Fetal Neonatal Ed 1996;75:F57–8.Rodney GE, Lalezari P. HLA and neutrophil antigen 4. and antibody systems. In: Hoffman R, Benz E, Shattil SJ, eds. Hematology basic principles and practice. 3rd ed. New York: Churchill Livingstone, 2000:2220–41.Lalezari P. Alloimmune neonatal neutropenia. In: En-5. gelfriet CP,Van Logham JJ, von dem Borne AEGKr, eds. Immunohaematology. Amsterdam: Elsevier Sci-ence Publishers, 1984:179–86.Clay M, Kline W, McCullough J. Serological examina-6. tion of postpartum sera for the presence of neutrophil-specific antibodies (abstract). Transfusion 1981;21:616.Levine DH, Madyastha P, Wade R, Levkoff AH. Neo-7. natal isoimmune neutropenia (abstract). Pediatr Res 1981;15:296A.Lalezari P, Bernard GE. An isologous antigen-antibody 8. reaction with human neutrophils, related to neonatal neutropenia. J Clin Invest 1966;45:1741–50.Verheugt FWA, van Noord-Bokhorst JC, von dem 9. Borne AEGKr, et al. A family with alloimmune neonatal neutropenia: group specific pathogenicity of maternal antibodies. Vox Sang 1979;36:1–8.van der Weerdt CM, Lalezari P. Another example of 10. isoimmune neonatal neutropenia due to anti-NA1. Vox Sang 1972;22:438–46.Boxer LA, Yokoyama M, Lalezari P. Isoimmune neona-11. tal neutropenia. J Pediatr 1972;80:783–7.Bux J, Ernst-Ludwig Stein, Bierling P, et al. Character-12. ization of a new alloantigen (SH) on the human neutro-phil Fc gamma receptor IIIb. Blood 1997;89:1027–34.Schuller RM, Stroncek DF, Alpan O, et al. Different re-13. sponses observed between G-CSF and GM-CSF therapy in a case of neonatal alloimmune neutropenia (ab-stract). Transfusion 2008;48(Suppl):53A.Bachowski GJ, Mair DC, Schuller RM, et al. Alloimmune 14. neonatal neutropenia in monozygous twins caused by antibody to FcγRIIIb in a mother lacking CD16 antigen (abstract). Transfusion 2007;47(Suppl):21A.Stroncek DF, Sharpiro RS, Filipovich AH, et al. 15. Prolonged neutropenia resulting from antibodies to neutrophil-specific antigen NB1 following marrow transplantation. Transfusion 1993;33:158–63.

M.E. Clay et al.

Table 3. Members of the ISBT Working Party on Granulocyte Immunobiology

· Platelet and Leucocyte Immunology Laboratory, EFS Ile de France, Hopital Henri Mondor, Creteil, France

· Leucocyte and Platelet Immunology Laboratory, Blood Service West of the German Red Cross, Hagen, Germany

· The BloodCenter of Wisconsin, Platelet and Neutrophil Immunology Laboratory, Milwaukee, WI, USA

· Institute of Transfusion Medicine, University of Schleswig-Holstein, Kiel, Germany

· Australian Red Cross Blood Service-Queensland, Brisbane, Australia

· Platelet and Granulocyte Immunology Laboratory, National Blood Se vice, Bristol, UK

· Blood Transfusion Center of Slovenia, Ljubljana, Slovenia

· Centre de Transfusio I Banc de Teixitis, Hospital Vall d’Hebron, P. Vall d’Hebron, Barcelona, Spain

· Sanguin Diagnostics, Immunohaematology Diagnostic Department, Amsterdam, The Netherlands

· Institute for Clinical Immunology and Transfusion Medicine, Justus Liebig University, Giessen, Germany

· American Red Cross Mid-America Blood Services, Neutrophil & Platelet Immunology Laboratory, St. Paul, MN, USA

· Department of Transfusion Medicine, Graduate School of Medical Sc ences, University of Tokyo, Tokyo, Japan

· Department of Immunohaematology and Transfusion Medicine, Institute of Haematology and Blood Transfusion, Warsaw, Poland

· Aberdeen and North East Scotland Blood Centre, Scottish National Blood Transfusion Service, Aberdeen, UK

· Platelet and Leucocyte Immunology Laboratory, Institute of Biology, Nantes, France

· Department of Transfusion Medicine, Karolinska University Hospital, Stockholm, Sweden



de Haas M, Muniz-Diaz E, Alonso LG, et al. Neutrophil 16. antigen 5b is carried by a protein, migrating from 70 to 95 kDa, and may be involved in neonatal alloimmune neutropenia. Transfusion 2000;40:222–7.Fung L, Pitcher LA, Willett JE, et al. Alloimmune neo-17. natal neutropenia linked to anti-HNA-4a. Transfus Med 2003;13:49–52.Bux J, Behrens G, Jaeger G, et al. Diagnosis and clinical 18. course of autoimmune neutropenia in infancy: analysis of 240 cases. Blood 1998;91:181–6.Bux J, Kissel K, Nowak K, et al. Autoimmune neutrope-19. nia: clinical and laboratory studies in 143 patients. Ann Hematol 1991;63:249–52.Stroncek D. Granulocyte antigens and antibody detec-20. tion. Vox Sang 2004;87(Suppl 1):91–4.Sperner-Unterweger B, Czeipek I, Gaggl S, Geissler 21. D, Spiel G, Fleischhacker WW. Treatment of severe clozapine-induced neutropenia with granulocyte col-ony stimulating factor (G-CSF). Remission despite continuous treatment with clozapine. Br J Psychiatry 1998;172:82–4.Stroncek DF. Drug-induced immune neutropenia. 22. Transfus Med Rev 1993;7:268–74.Mamus SW, Burton JD, Groat JD, et al. Ibuprofen-23. associated pure white cell aplasia. N Engl J Med 1986;314:624–5.Holness L, Knippen MA, Simmons L, et al. Fatalities 24. caused by TRALI. Transfus Med Rev 2004;18:184–8.Brittingham TE. Immunologic studies on leukocytes. 25. Vox Sang 1952;2:242–8.Barnard, RD. Indiscriminate transfusion: a critique of 26. case reports illustrating hypersensitivity reactions. N Y State J Med 1951;51:2399–402.Popovsky MA, Moore SB. Diagnostic and pathogenic 27. considerations in transfusion-related acute lung injury. Transfusion 1985;25:573–7.Bux J, Sachs UJ. The pathogenesis of transfusion-28. related acute lung injury (TRALI). Br J Haematol 2007;136:788–99.Reil A, Keller-Stanislawski B, Gunay S, et al. Specifici-29. ties of leukocyte alloantibodies in transfusion-related acute lung injury and results of leukocyte antibody screening of blood donors. Vox Sang 2008;95:313–17.Ward HN. Pulmonary infiltrates associated with leu-30. koagglutinin transfusion reactions. Ann Intern Med 1970;73:689–94.Thompson JS, Severson CD, Parmely MJ, et al. Pul-31. monary ‘hypersensitivity’ reactions induced by trans-fusion of non-HLA lekoagglutinins. N Engl J Med 1971;284:1120–5.Nordhagen R, Conradi M, Drotorp SM. Pulmonary re-32. action associated with transfusion of plasma containing anti-5b. Vox Sang 1986;51:102–7.

Kopko PM, Marshall CS, MacKenzie MR, et al. Transfu-33. sion-related acute lung injury. Report of a clinical look-back investigation. JAMA 2002;287:1968–71.Davoren A, Curtis BR, Shulman IA, et al. TRALI due to 34. granulocyte-agglutinating human neutrophil antigen-3a (5b) alloantibodies in donor plasma: a report of 2 fatalities. Transfusion 2003;43:641–5.Silliman CC, Curtis BR, Kopko PM, et al. Donor anti-35. bodies to HNA-3a implicated in TRALI reactions prime neutrophils and cause PMN-mediated damage to hu-man pulmonary microvascular endothelial cells in a two-event in vitro model. Blood 2007;109:1752–5.Sachs UJ, Chavakis T, Fung L, et al. Human alloanti-36. body anti-MART interferes with Mac-1-dependent leu-kocyte adhesion. Blood 2004;104:727–34.Kopko PM, Curtis BR, Kelher M, et al. Merging the 37. pathogenesis of transfusion-related acute lung injury: the priming activity of the 5b (HNA-3) antibody (ab-stract). Transfusion 2004;44(Suppl 1):22A.Sachs UJ, Hattar K, Weissman N, et al. Antibody-38. induced neutrophil activation as a trigger for trans-fusion-related acute lung injury in an ex vivo rat lung model. Blood 2006;107:1217–9.Fung YL, Silliman CS. The role of neutrophils in the 39. pathogenesis of transfusion-related acute lung injury. Transfusion Med Rev 2009;23:266–83.Lalezari P, Radel E. Neutrophil-specific antigens: im-40. munology and clinical significance. Semin Hematol 1974;11:281–90.Lalezari P, Murphy GB, Allen FH. NB1, a new neutro-41. phil antigen involved in the pathogenesis of neonatal neutropenia. J Clin Invest 1971;50:1108–15.Huang ST, Lin J, McGowan El, et al. NB2, a new allele 42. of NB1 antigen involved in febrile transfusion reaction (abstract). Transfusion 1982;22:426.Lalezari P, Petrosova M, Jiang AF. NB2, an allele of 43. NB1 neutrophil specific antigen: relationship to 9a (ab-stract). Transfusion 1982;22:433.Lalezari P, Thelenfeld B, Weinstein WJ. The third neu-44. trophil antigen. In: Terasaki PI, ed. Histocompatibility testing. Baltimore: Williams & Wilkins, 1970:319–22.Verheugt FWA, von dem Borne AEGKr, van Noord-45. Bokhorst JC, et al. ND1, a new neutrophil granulocyte antigen. Vox Sang 1978;35:13–17.Claas FHJ, Langerak J, Sabbe LJM, et al. NE1, a 46. new neutrophil specific antigen. Tissue Antigens 1979;13:129–34.Thompson JS, Overlin VL, Herbick JM, et al. New 47. granulocyte antigens demonstrated by microgranulo-cytotoxicity assay. J Clin Invest 1980;65:1431–9.Hasegawa T, Bergh OJ, Mickay MR, et al. Prelimi-48. nary human granulocyte specificities. Transplant Pro 1975;7(Suppl 1):75–80.


Caplan SN, Berkman EM, Babior BM. Cytotoxins 49. against a granulocyte antigen system: detection by a new method employing cytochalasin-B-treated cells. Vox Sang 1977;33:206–11.Van Leeuwen A, Eerrise JG, van Room JJ. A new leu-50. kocyte group with two alleles: leukocyte group five. Vox Sang 1964;9:431–7.Van Rood JJ, van Leeuwen A, Schippers AMJ, et al. 51. Immunogenetics of the group four, five and nine sys-tems. In: Curton ES, Mattiuz PI, Tosi RM, eds. Histo-compatibility testing. Baltimore: Williams & Wilkins, 1967:203–19.Kline WE, Press C, Clay M, et al. Three sera defining a 52. new granulocyte-monocyte–T lymphocyte antigen. Vox Sang 1986;50:181–6.Madyastha PR, Glassman A, Levine D, et al. Identifi-53. cation of a new neutrophil antigen (CN1) more preva-lent among American blacks (abstract). Transfusion 1983;23:426.Schnell M, Halligan G, Herman J. A new granulocyte 54. antibody directed at a high frequency antigen causing neonatal alloimmune neutropenia (abstract). Transfu-sion 1989;29(Suppl):46S.Rodwell RL, Tudehope DI, O’Regan P, et al. Alloimmune 55. neonatal neutropenia in Australian aboriginals: an un-recognized disorder? Transfusion Med 1991;1:63–7.Stroncek DF, Ramsey G, Herr GP, et al. Identification of 56. a new white cell antigen. Transfusion 1994;34:706–11.Bux J, Bierling P, von dem Borne AE, et al. ISBT Granu-57. locyte Antigen Working Party. Nomenclature of granu-locyte alloantigens. (letter).Vox Sang 1999;77:251.Bux J. Human neutrophil alloantigens. Vox Sang 58. 2008;94:277–85.Greinacher A, Wesche J, Hammer E, et al. Character-59. ization of the human neutrophil alloantigen-3a. Nature Med 2010;16:45–8.Curtis BR, Cox NJ, Sullivan MJ, et al. The neutrophil 60. alloantigen HNA-3a (5b) is located on choline trans-porter-like protein 2 (CTL2) and appears to be en-coded by an R>Q 154 amino acid substitution. Blood 2010;115:2073-6. Shows TB, McAlpine PJ, Boucheix C, et al. Guidelines 61. for human gene nomenclature. An international system for human gene nomenclature (ISGN, 1987). Cytogenet Cell Genet 1987;46:11–28.Stroncek DF, Skubitz KM, Plachta LB, et al. Alloim-62. mune neonatal neutropenia due to an antibody to the neutrophil Fc-gamma receptor III with maternal defi-ciency of CD16 antigen. Blood 1991;77:1572–80.Stroncek DF, Skubitz K, McCullough JJ. Biochemical 63. nature of the neutrophil-specific antigen NB1. Blood 1990;75:744–55.Matsuo K, Lin A, Procter JL, et al. Variations in the 64. expression of granulocyte antigen NB1. Transfusion 2000;40:654–62.

Bux J, Jung KD, Kauth T, et al. Serological and clinical 65. aspects of granulocyte antibodies leading to alloimmune neonatal neutropenia. Transfusion Med 1992;2:143–9.Bux J. Granulocyte antibody mediated neutropenias 66. and transfusion reactions. Infusions Ther Transfusion Med 1999;26:152–7.Bux J, Becker F, Seeger W, et al. Transfusion-related 67. acute lung injury due to HLR-A2-specific antibodies in recipient and NB1-specific antibodies in donor blood. Br J Haematol 1996;93:707–13.Stroncek DF, Shankar RH, Herr GP. Quinine-68. dependent antibodies to neutrophils react with a 60 kD glycoprotein on which neutrophil-specific antigen NB1 is located and an 85 kD glycosylphosphatidylinos-itol–linked N-glycosylated plasma membrane protein. Blood 1993;81:2758–66.Lalezari P, Bernard GE. Identification of a specific 69. leukocyte antigen: another presumed example of 5b. Transfusion 1965;5:132–42.de Haas M, Muniz-Diaz E, Alonso LG, et al. Neutrophil 70. 5b is carried by a protein, migrating from 70 to 95 kDa, and may be involved in neonatal alloimmune neutrope-nia. Transfusion 2000;40:222–7.Simsek S, van der Schoot CE, Daams M, et al. Molecu-71. lar characterization of antigenic polymorphisms (ONDa

and MARTa) of the ß2 family recognized by human leu-kocyte alloantisera. Blood 1996;88:1350–8.Fung L, Pitcher LA, Willett JE, et al. Alloimmune neo-72. natal neutropenia linked to anti-HNA-4a. Transfusion Med 2003;13:49–52.Hartman KR, Wright DG. Identification of autoantibod-73. ies specific for the neutrophil adhesion glycoproteins CD11b/CD18 in patients with autoimmune neutro-penia. Blood 1991;78:1096–104.Kelton JG, Bebenek G. Granulocytes do not have sur-74. face ABO antigens. Transfusion 1985;25:567–9.Dunstan RA, Simpson MB, Sanfilippo FP. Absence of 75. specific HLA-DR antigens on human platelets and neu-trophils (abstract). Blood 1984;64:85a.Thorsby E. HLA antigens on human granulocytes stud-76. ied with cytotoxic iso-antisera obtained by skin graft-ing. Scan J Haematol 1969;6:119–27.Sachs UJ, Bux J. TRALI after transfusion of cross-match-77. positive granulocytes. Transfusion 2003;43:1683–6.Stroncek DF, Leonard K, Eiber G, et al. Alloimmuni-78. zation after granulocyte transfusions. Transfusion 1996;36:1009–15.McCullough J, Clay M, Hurd D, et al. Effect of leuko-79. cyte antibodies and HLA matching on the intravascular recovery, survival, and tissue localization of 111-indium granulocytes. Blood 1986;67:522–8.

M.E. Clay et al.



Bøyum A. Isolation of mononuclear cells and granu-80. locytes from blood. Isolation of mononuclear cells by one centrifugation and of granulocytes by combining centrifugation and sedimentation at 1g. Scan J Clin Lab Invest 1968;21(Suppl 97):77–89.English D, Anderson BR. Single-strep separation of red 81. blood cells, granulocytes, and mononuclear leukocytes on discontinuous density gradients of Ficoll-Hypaque. J Immunol Methods 1975;5:249–52.Ferrante A, Thong YH. A rapid one-step procedure for 82. purification of mononuclear and polymorphonuclear leukocytes from human blood using a modification of the Hypaque-Ficoll technique. J Immunol Methods 1978;24:389–93.Ferrante A, Thong HY. Optimal conditions for simultaneous 83. purification of mononuclear and polymorphonuclear leukocytes from human blood by the Hypaque-Ficoll method. J Immunol Methods 1980;36:109–17.Madyastha P, Madyastha KR, Wade T, et al. An im-84. proved method for rapid layering of Ficoll-Hypaque double density gradients suitable for granulocyte sepa-ration. J Immunol Methods 1982;42:281–6.Lucas G, Rogers S, de Haas M, et al. Report on the 85. Fourth International Granulocyte Immunology Work-shop: progress toward quality assessment. Transfusion 2002;42:462–8.Lalezari P, Pryce S. Detection of neutrophil and plate-86. let antibodies in immunologically induced neutrope-nia and thrombocytopenia. In: Rose NR, Friedman H, eds. Manual of clinical immunology. Washington, DC: American Society of Microbiology 1980;744–9.McCullough J, Clay ME, Press C, et al. Granulocyte se-87. rology: a clinical and laboratory guide. Chicago: ASCP Press, 1988:12–14, 168–71.Verheugt FWA, Von dern Borne AEGKr, Decary S, et 88. al. The detection of granulocyte alloantibodies with an indirect immunofluorescence test. Br J Haematol 1977;36:533–44.Kiefel V, Santoso S, Weisheit M, et al. Monoclonal anti-89. body-specific immobilization of platelet antigens (MAI-PA): a new tool for the identification of platelet reactive antibodies. Blood 1987;70:1722–6.Minchinton R, Noonan K, Johnson TJ. Examining 90. technical aspects of the monoclonal antibody im-mobilization of granulocyte antigen assay. Vox Sang 1997;73:87–92.Stroncek D. Neutrophil alloantigens. Transfusion Med 91. Rev 2002;16:67–75.Wang E, Marincola FM, Stroncek D. Human leukocyte 92. antigens and human neutrophil antigens systems. In: Hoffman R, Benz E, Shattil Sj, et al., eds. Hematology: basic principel and practices 4th ed. Philadelphia: El-sevier, 2005:2401–22.Kissel K, Scheffler S, Kerowgan M, et al. Molecu-93. lar basis of NB1 (HNA-2a, CD177) deficiency. Blood 2002;99:4231–3.

Vigrali DA. Multiplexed particle-based flow cytometric 94. assays. J Immunol Methods 2000;243:243–55.Stroncek DF, Adams S, Lee JH, et al. Neutrophil anti-95. body testing. ASHI Q 2008;32:68–72.Bux J, Kissel K, Hofmann C, et al. The use of allele-spe-96. cific recombinant Fcγ receptor IIIb antigens for the de-tection of granulocyte antibodies. Blood 1999;93:357–62.Yasui K, Miyazaki T, Matsuyama N, et al. Establish-97. ment of cell lines stably expressing HNA-1a, 1b, and -2a antigen with low background reactivity in flow cy-tometric analysis. Transfusion 2007;47:478–86.Yasui K, Hirayama F, Matsuyama N, et al. New cell lines 98. express HNA-1c, -4a, -4b, -5a, or -5b for identification of HNA antibodies. Transfusion 2008;48:1037–9.Lucas GF, Carrington PA. Results of the First Inter-99. national Granulocyte Serology Workshop. Vox Sang 1990;59:251–6.Bux J, Chapman J. Report on the Second Interna-tional Granulocyte Serology Workshop. Transfusion 1997;37:977–83.ISBT Working Party on Granulocyte Immunobiol-ogy. Recommendations of the ISBT Working Party on Granulocyte Immunobiology for leukocyte antibody screening in the investigation and prevention of antibody-mediated transfusion-related acute lung in-jury. Vox Sang 2009;96:266–7.

Mary E. Clay, MS, MT(ASCP), (corresponding author) Di-rector, Transfusion Medicine, Research and Development Program, University of Minnesota Medical School, De-partment of Laboratory Medicine and Pathology, MMC 198, D-251 Mayo Building, 420 Delaware Street, SE, Min-neapolis, MN 55455, and Consultant, Neutrophil & Plate-let Immunology Laboratory, American Red Cross, Mid-America Blood Services Region, 100 South Roberts Street, St. Paul, MN 55107, Randy M. Schuller, BS, MT(ASCP), Supervisor, and Gary J. Bachowski, MD, PhD, Assistant Medical Director, Neutrophil and Immunobiology Labo-ratory, American Red Cross, Mid-America Blood Services Region, St. Paul, MN.

100.

101.

For information concerning the National Reference Laboratory for Blood Group Serology, including the American Rare Donor Program, please contact Sandra Nance, by phone at (215) 451-4362, by fax at (215) 451-2538, or by e-mail at [email protected]


A review of the Colton blood group systemG.R. Halverson and T. Peyrard

History The idea that there must be openings in the cell mem-brane to permit the flow of water and salts to maintain cel-lular function and to eliminate the byproducts of cellular metabolism was recognized by the middle of the 19th cen-tury. Water is the major component of all living cells, in-cluding those of vertebrates, invertebrates, and unicellular organisms and plants. Salt water comprises approximately 70 percent of the human body. After debating for decades about the process of water transfer across cell membranes, most scientists finally decided that water must pass free-ly through biologic membranes by simple diffusion. Only a small number of scientists disagreed, and they had only indirect evidence that there were special channels for the transport of water. After all, there must be something to explain the high permeability of RBCs and renal tubules. Moreover, the water permeability of these tissues can be re-versibly inhibited by mercuric ions.1

By 1950 it had been shown that water moves quickly into and out of cells through pores that are selective for water molecules only. Every second, billions of water mol-ecules pass through a single channel. By the mid-1980s scientists had found an RBC membrane protein whose 28-kDa N-terminal amino acid sequence was related to the 26-kDa major intrinsic protein (MIP26) of bovine lens fi-ber cells.2,3 The 28-kDa protein was shown to be a unique molecule that is abundant on erythrocytes and on renal tu-bules. Preston and Agre4 succeeded in 1991 in isolating the same membrane protein from RBCs, which in 1997, was of-ficially named aquaporin-1 (AQP1) by the Human Genome Organization, for the water channel. AQP1 has been found in many tissues including several parts of the kidney, liver, gall bladder, eye, choroid plexus, hepatobiliary epithelium, and capillary endothelium. AQP1 turned out to be the first of the family of major intrinsic proteins (MIP), which are highly conserved membrane proteins that regulate small molecule transport. The MIP family of proteins is believed to date back 2.5 to 3 billion years in evolutionary time.5 It is believed that the MIP family evolved from two basal lineag-es: AqpZ-like water channels and GlpF-like glycerol facili-tators. These divergent lineages probably originated from an archaeal (AqpM-like) aquaporin.5 The MIP family is now divided into two groups: aquaporins (AqpZ, AQP0, AQP1, AQP2, AQP4, AQP5, AQP6, and AQP8) and aquaglycerol-porins (GlpF, AQP3, AQP7, AQP9, and AQP10).6–10 In 1994, AQP1 protein was characterized as carrying the Colton blood group antigens.11

Genetics and Inheritance In 1965 in Oslo, Norway, the discovery of an antibody that detected a “public” (or high prevalence) antigen was

linked to two other cases discovered earlier by workers in Minneapolis, Oxford, and London. The antigen was named Coa (Coltona) in 1967 for the first of the three producers of anti-Coa, who was from Minneapolis; it should actually have been named “Calton,” but the handwriting on the tube ap-parently was misread.12,13 Coa was shown to be an inherited characteristic.14 In 1970, the antithetical antigen, Cob, was reported by Giles et al (Table 1).15 In analysis of unrelated people of European extraction, the occurences of the three major Colton phenotypes were as follows: Co(a+b–) 0.914, Co(a+b+) 0.084, and Co(a–b+) 0.002 (Table 2).16 In 1971, the genetic independence of the Colton blood group system from Kell and Lutheran was reported, and it was confirmed that it is independent from MNSs, P, Rh, Duffy, Dombrock, and sex.17 Coa and Cob are codominant alleles that respec-tively encode the Coa and Cob antigens at the RBC surface. In 1974 the Co(a–b–) phenotype of three persons in a French Canadian family was reported.18 The serum from a patient in 1964 (Swarts) was eventually found to be negative only with these Co(a–b–) family members.16 Colton is the 15th human blood group system recognized by the ISBT (ISBT 015).

Molecular Basis The AQP1 locus was mapped to human chromosome 7p14 by fluorescent in situ hybridization.19 The Colton (Co) blood group antigens had been previously linked to the short arm of chromosome 7.20 Immunoprecipitation studies

Review

Table 1. Antigens of the Colton blood group system: their preva-lence, molecular basis, and sensitivity to enzyme and DTT treatment

NameISBT

symbolISBT

number PrevalenceMolecular

basisEffect of enzymes

Coa CO1 015001 99.8% Ala 45 PR TR CR NR DTTR

Cob CO2 015002 8.6% Val 45 PR TR CR NR DTTR

Co3 CO3 015003 >99.99% unknown PR TR CR NR DTTR

CR = α1-chymotrypsin resistant; DTTR = dithiothreitol resistant; NR = neuraminidase resistant; PR = papain resistant; TR = trypsin resistant.

Table 2. Phenotypes (% occurrence)

Phenotype % Occurrence (most populations)16

Co(a+b–) 91.4

Co(a–b+) 0.2

Co(a+b+) 8.4

Co(a–b–) <0.01


the Co protein have been reported to cause decreased or no expression of the Colton antigens. An amino acid change of Gln47Arg was observed to cause a Co(a–b+w) typing.29 The same amino acid substitution was recently fully inves-tigated in a Turkish female with the Co(a–b–) phenotype.30 Her RBCs were found by molecular studies to have normal water permeability by stopped-flow analysis and a normal amount of AQP1 protein in the RBC membrane. (See sec-tion on clinical significance). By PCR-RFLP the specimen was genotyped as Coa/Coa, but full sequencing revealed a 140A>G mutation leading to a Gln47Arg amino acid sub-stitution. This change in amino acids is close to the Coa/Cob polymorphism, and thus, the probable cause of the appar-ent discrepancy between phenotype and genotype. When using a specifically designed K-562 cell expression system for Colton antigens, the Gln47Arg substitution was shown to inhibit both Coa and Cob antigen expression.29 The antibody produced by these Co(a–b–) individuals with a Gln47Arg substitution is not a mixture of anti-Coa and -Cob, rather it is “anti-Co3–like,” reacting with both Co(a+b–) and Co(a–b+) RBCs, but more weakly than anti-Co3 produced by the true null phenotype. This suggests the existence of a new high-prevalence antigen in the Colton blood group system, with a corresponding antibody that Arnaud et al. proposed to name anti-Co4.30 Therefore, it is possible that some of the Co(a–b–) people known through-out the world, who would have not been fully investigated by molecular techniques, may actually not be “true” Conull but “CO:–1,–2,3,–4.” This may also be why some antibod-ies directed against a high-prevalence Colton antigen, mis-takenly considered as anti-Co3, were unexpectedly weakly reactive with Co:3 RBCs.31

Biochemical Physiology of the Colton Glycoprotein Protons are necessary for the bioenergetics of the living cell. The proton gradient drives many transport functions, membrane fusions, and the synthesis of ATP. Maintaining the proton gradient is essential, and indiscriminate pro-ton leakage across cellular membranes would be fatal to the living cell. At the same time, cells must maintain the balance of water’s ability to permeate the membrane bi-

layer. Aquaporins are strictly selective water chan-nels that have evolved for that purpose. AQP1 was found to have a configuration with intracellular N and C terminals, similar to that of other ion chan-nel proteins. Protein modeling from the cDNA sequences predicts a two-tandem repeat of three membrane-spanning helices that exist as tetram-ers within the cell membrane (Fig. 2). An electro-static field created by the highly conserved amino acid sequence NPA forms the water channel, which prevents proton loss while allowing water to perme-ate the membrane. The most prominent feature is the association of loop B with loop E driven by their positive N-terminal ends that brings together the NPA motifs at the center of the pore. This causes the formation of strong electrostatic fields that block

Colton blood group system review

and DNA sequencing showed that the Colton antigens are the result of a polymorphism in AQP1 at the extracellular site of loop A, which connects the first and second spanning domains. Figure 1 depicts the structure of the protein as it passes through the membrane, and indicated are the Coa/Cob polymorphic site at position 45 and the only N-glycan site at position 42. The Coa/Cob polymorphism arises from a single amino acid change of Ala45Val (alanine for Coa, va-line for Cob) caused by a nucleotide change of C>T at posi-tion 134 in exon 1 of AQP1 gene, named CO according to the ISBT nomenclature and AQP1 according to the Human Genome Organization (HUGO).11 The Colton gene consists of four exons distributed over 11.6 kbp of genomic DNA. The amino acid sequence of the wild-type AQP1 protein is shown in Table 3. Also known as the channel-forming inte-gral protein (CHIP), the Colton glycoprotein is a multipass protein consisting of three external loops and six mem-brane-spanning regions, as well as 2 cytoplasmic loops, with both the NH2 and COOH terminus located on the cyto-plasmic side of the membrane.22 Common to all MIP super-family proteins is the occurrence of the asparagine-proline-alanine (NPA) motifs, two being present in each half of the protein. An hourglass structural model has been proposed by Agre and colleagues.23,24

The molecular basis of the null Co(a–b–) phenotype has been attributed to several different molecular back-grounds; these are depicted in Table 4. Other changes in

Out

N-glycosylation site A = Coa

V = Cob

RBCMembrane

76 78

192 194

CE

B DIn

NH2 COOH

N P

PN

A

A1 2 3 4 5 6

A42 45N A/V

Fig. 1 Linear diagram depicting the Colton protein with the six membrane-spanning domains with both the N terminus and C terminus inside the cell membrane. The Coa/Cob polymorphism is indicated at position 45, as is the glycosylation site at N42. The signature NPA motifs are depicted on the internal loops B and D.21

Table 3. Amino acid sequence of wild-type AQP1 protein13

MASEFKKKLF WRAVVAEFLA TTLFVFISIG SALGFKYPVG NNQTAVQDNV 50

KVSLAFGLSI ATLAQSVGHI SGAHLNPAVT LGLLLSCQIS IFRALMYIIA 100

QCVGAIVATA ILSGITSSLT GNSLGRNDLA DGVNSGQGLG IEIIGTLQLV 150

LCVLATTDRR RRDLGGSAPL AIGLSVALGH LLAIDYTGCG INPARSFGSA 200

VITHNFSNHW IFWVGPFIGG ALAVLIYDFI LAPRSSDLTD RKVWTSGQV 250

EEYDLDADDI NSRVEMKPK 269

Amino acid sequence taken from GenBank, accession # M77829Also see the Blood Group Antigen Gene Mutation Database (dbRBC) at http://www.ncbi.nlm.nih.gov/gv/mhc/xslcgi.cgi?cmd=bgmut/home (accessed April 13, 2010).


the permeation of protons as well as H3O+ and other cat-ions. When Xenopus laevis oocytes were transfected with cRNA, they were found to have markedly increased water permeability, causing the oocytes to swell and burst.32 This permeability could be reversibly inhibited by Hg2+.33 When the same cRNA transcript was reconstituted into lipid vesi-cles, the same observations were made. This confirmed that membranes exhibit water permeability and showed that the water permeability of membranes with AQP1 was up to 100 times greater than that of those without it.

Colton Antigen Typing To date, anti-Coa and anti-Co3 reagents, either mono-clonal or polyclonal, are not commercialized. As a result, human polyclonal reagents are only available in immu-nohematology reference laboratories, but usually in small amounts. However, a human polyclonal anti-Cob is avail-able in Europe as a CE-marked in vitro diagnostic reagent (DiaMed, a division of Bio-Rad, Cressier sur Morat, Switzerland). The two major high-throughput molecu-lar genotyping platforms, a commercialized DNA glass array by Progenika (Barcelona, Spain) and a DNA bead array by BioArray/Immucor (Norcross, GA) allow Coa and Cob al-lele typing by searching for the 134C>T polymorphism in exon 1, with subsequent prediction of Colton phenotype. It is important, however, to be aware that prediction of the Co phenotype in Conull and Covariant people would be a source of misinterpretation with these DNA-based techniques.

Clinical Significance Antibodies to Colton blood group antigens are gener-ally IgG and react best by the antiglobulin test, especially when protease-treated RBCs are used.34,35 Some anti-Coa, -Cob and -Co3 may bind complement.36 Few reports of sig-nificant delayed or acute transfusion reactions or hemolytic disease of the fetus and newborn (HDFN) attributable to anti-Coa have been reported, although both are known to occur with severe morbidity.27,37–40 Anti-Cob is relatively rare and often found in patient’s sera containing other alloantibodies.34,35 Two reports of in

G.R. Halverson and T. Peyrard

Table 4. Molecular bases of the Colton-null [Co(a–b–)] phenotype: effect of the change at the DNA and protein level, and ancestry of the probands

Molecular alteration EffectObserved result on

AQP1 proteinPresence

of anti-Co3HDFN

reported Ancestry Reference

Conull phenotypes

Large deletion compris-ing most of exon 1

Absent or aberrant transcript

AQP1 not detected in RBC membrane extracts

Not reported No Northern European

Proband 1 in Preston et al.25

c.309insT Frameshift mutation AQP1 not detected in RBC membrane extracts

Yes No French Proband 2 in Preston et al.25

c.113C>T Missense mutation (p.Pro38Leu)

<1% AQP1 expression in RBC membrane extracts

Not reported No Northern European

Proband 3 in Preston et al.25

c.576C>A Missense mutation (p.Asn192Lys)

Not tested Yes No Portuguese Chrétien et al.26

c.232delG Frameshift mutation Not tested Yes Yes Indian Joshi et al.27

c.601delC Frameshift mutation Not tested Yes No Caucasian Nance et al.28; Reid and Lomas-Francis13

Altered Co phenotypes

c.140A>G Missense mutation (p.Gln47Arg)

Not tested No No Not reported, presumably Caucasian

Wagner and Flegel29

c.140A>G Missense mutation (p.Gln47Arg)

Normal AQP1 expression in RBC membrane

No No Turkish Arnaud et al.30

AQP1 = aquaporin 1; HDFN = hemolytic disease of the fetus and newborn.According to the recommendations of the Human Genome Variation Society and National Institutes of Health Single Nucleotide Poly-morphism database, “c.” means that the position of the polymorphism refers to the complementary DNA, “p” means that the position of the polymorphism refers to the protein.

Out

RBCMembrane

In

NH2 COOH

1 2 34 56H2O

N

N

A

A

P

P

Fig. 2 The AQP1 water channel is shown as an hourglass configu-ration created by the association of the six transmembrane domains and the crossing of loops B and E with the electrostatic field pro-duced by the NPA motifs forming the water channel.21


vivo survival studies of 51Cr-labeled Co(b+) RBCs in pa-tients with anti-Cob showed survival at 94 percent and 85 percent, respectively, at 1 hour, decreasing to 51 percent at 24 hours and 10 percent at 96 hours.41,42 Cob is fully devel-oped at birth, but no significant HDFN has been reported to date.34

Anti-Co3 was reported to cause severe HDFN requir-ing neonatal transfusion.43 Transfusion of incompatible Co(a+b–) RBCs in a patient with anti-Co3 was reported to be responsible for a mild hemolytic transfusion reaction.27 There are also reports of autoantibodies mimicking Colton specificities.44

AQP1-deficient individuals were studied for their renal function and capillary permeability before and after water deprivation.45 Baseline studies did not reveal any abnormali-ties; however, after water deprivation this study revealed an inability to concentrate urine. Thus, Colton-null individuals may be subject to serious hydroelectrolytic and metabolic disorders should they become severely dehydrated.

Acknowledgments The authors thank Robert Ratner for preparation of the manuscript and figures, and Hallie Lee-Stroka for her criti-cal review and helpful comments. This work was funded in part by a grant from the MetLife Foundation (GH).

ReferencesMacey RI. Transport of water and urea in red blood 1. cells. Am J Physiol 1984;246:C195–203.Agre P, Saboori AM, Asimos A, Smith BL. Purification 2. and partial characterization of the Mr 30,000 integral membrane protein associated with the erythrocyte Rh(D) antigen. J Biol Chem 1987;262:17497–503.Saboori AM, Smith BL, Agre P. Polymorphism in the 3. Mr 32,000 Rh protein purified from Rh(D)-positive and -negative erythrocytes. Proc Natl Acad Sci U S A 1988;85:4042–5.Preston GM, Agre P. Isolation of the cDNA for eryth-4. rocyte integral membrane protein of 28 kilodaltons: member of an ancient channel family. Proc Natl Acad Sci U S A 1991;88:11110–4.Kozono D, Ding X, Iwasaki I, et al. Functional expres-5. sion and characterization of an archaeal aquaporin. AqpM from Methanothermobacter marburgensis. J Biol Chem 2003;278:10649–56.Roudier N, Ripoche P, Gane P, et al. AQP3 deficiency in 6. humans and the molecular basis of a novel blood group system, GIL. J Biol Chem 2002;277:45854–9.Vajda Z, Pedersen M, Füchtbauer EM, et al. Delayed on-7. set of brain edema and mislocalization of aquaporin-4 in dystrophin-null transgenic mice. Proc Natl Acad Sci U S A 2002;99:13131–6.Ikeda M, Beitz E, Kozono D, Guggino WB, Agre P, Yasui 8. M. Characterization of aquaporin-6 as a nitrate channel in mammalian cells. Requirement of pore-lining resi-due threonine 63. J Biol Chem 2002;277:39873–9.

Carbrey JM, Gorelick-Feldman DA, Kozono D, Praeto-9. rius J, Nielsen S, Agre P. Aquaglyceroporin AQP9: sol-ute permeation and metabolic control of expression in liver. Proc Natl Acad Sci U S A 2003;100:2945–50.Endeward V, Musa-Aziz R, Cooper GJ, et al. Evidence 10. that aquaporin 1 is a major pathway for CO2 transport across the human erythrocyte membrane. FASEB J 2006;20:1974–81.Smith BL, Preston GM, Spring FA, Anstee DJ, Agre P. 11. Human red cell aquaporin CHIP. I. Molecular charac-terization of ABH and Colton blood group antigens. J Clin Invest 1994;94:1043–9.Heistö H, van der Hart M, Madsen G, et al. Three ex-12. amples of a new red cell antibody, anti-Coa. Vox Sang 1967;12:18–24.Reid ME, Lomas-Francis C. The blood group antigen 13. factsbook. 2nd ed. San Diego, CA: Academic Press, 2004.Wray E, Simpson S. A further example of anti-Co14. a and two informative families with Co(a–) members. Vox Sang 1968;14:130–2.Giles CM, Darnborough J, Aspinall P, Fletton MW. 15. Identification of the first example of anti-Cob. Br J Hae-matol 1970;19:267–9.Race RR, Sanger R. Blood groups in man. 6th ed. Ox-16. ford, England: Blackwell Scientific Publications, 1975.Lewis M, Kaita H, Anderson C, Chown B. Independence 17. of Colton blood group. Transfusion 1971;11:223–4.Rogers MJ, Stiles PA, Wright J. A new minus-minus 18. phenotype: three Co(a–b–) individuals in one family (abstract). Transfusion 1974;14:508.Moon C, Preston GM, Griffin CA, Jabs EW, Agre P. 19. The human aquaporin-CHIP gene. Structure, orga-nization, and chromosomal localization. J Biol Chem 1993;268:15772–8.Zelinski T, Kaita H, Gilson T, Coghlan G, Philipps S, 20. Lewis M. Linkage between the Colton blood group locus and ASSP11 on chromosome 7. Genomics 1990;6:623–5.Agre P, King L, Yasui M, et.al. Topical Review: Aqua-21. porin water channels—from atomic structure to clinical medicine. J Physiol. 2002;542:3–16.King LS, Agre P. Pathophysiology of the aquaporin wa-22. ter channels. Annu Rev Physiol 1996;58:619–48.Mathai JC, Agre P. Hourglass pore-forming domains 23. restrict aquaporin-1 tetramer assembly. Biochemistry 1999;38:923–8.Jung JS, Preston GM, Smith BL, Guggino WB, Agre 24. P. Molecular structure of the water channel through aquaporin CHIP. The hourglass model. J Biol Chem 1994;269:14648–54.Preston GM, Smith BL, Zeidel ML, Moulds JJ, Agre P. 25. Mutations in aquaporin-1 in phenotypically normal hu-mans without functional CHIP water channels. Science 1994;265:1585–7.

Colton blood group system review


Chrétien S, Catron JP, de Figueiredo M. A single mu-26. tation inside the NPA motif of aquaporin-1 found in a Colton-null phenotype (letter). Blood 1999;93:4021–3.Joshi SR, Wagner FF, Vasantha K, Panjwani SR, Fle-27. gel WA. An AQP1 null allele in an Indian woman with Co(a–b–) phenotype and high-titer anti-Co3 associated with mild HDN. Transfusion 2001;41:1273–8.Nance S, Kavitsky DL, Meny G, Vege, S, Westhoff CM. 28. An example of anti-Co3 not causing hemolytic disease of the newborn. Transfusion 2002;42(Suppl):105S.Wagner FF, Flegel WA. A clinically relevant Co(a)-like 29. allele encoded by AQP1 (Q47R) (abstract). Transfusion 2002;42(Suppl):24S–5S.Arnaud L, Helias V, Menanteau C, Peyrard T, et al. A 30. functional AQP1 allele producing a Co(a–b–) pheno-type revises and extends the Colton blood group sys-tem. Transfusion 2010. In press.Lacey PA, Robinson J, Collins ML, et al. Studies on the 31. blood of a Co (a–b–) proposita and her family. Transfu-sion 1987;27:268–71.Preston GM, Carroll TP, Guggino WB, Agre P. Appear-32. ance of water channels in Xenopus oocytes expressing red cell CHIP28 protein. Science 1992;256:385–7.Kozono D, Yasui M, King LS, Agre P. Aquaporin wa-33. ter channels: atomic structure and molecular dynamics meet clinical medicine. J Clin Invest 2002;109:1395–9.Daniels G. Human blood groups. 2nd ed. Malden, MA: 34. Blackwell Science, 2002.Issitt PD, Anstee DJ. Applied blood group serology. 4th 35. ed. Durham, NC: Montgomery Scientific Publications, 1998.Covin RB, Evans KS, Olshock R, Thompson HW. Acute 36. hemolytic transfusion reaction caused by anti-Coa. Im-munohematology 2001;17:45–9.Simpson WKH. Anti-Co37. a and severe haemolytic disease of the newborn. S Afr Med J 1973;47:1302–4.

Agre P, Smith BL, Baumgarten R, et al. Human red cell 38. aquaporin CHIP. II. Expression during normal fetal de-velopment and in a novel form of congenital dyseryth-ropoietic anemia. J Clin Invest 1994;94:1050–8.Kurtz SR, Kuszaj T, Ouellet R, Valeri CR. Survival of ho-39. mozygous Coa (Colton) red cells in a patient with anti-Coa. Vox Sang 1982;43:28–30.Michalewska B, Wielgos M, Zupanska B, Bartkowiak R. 40. Anti-Coa implicated in severe haemolytic disease of the foetus and newborn. Transfus Med 2008;18:71–3.Dzik WH, Blank J. Accelerated destruction of ra-41. diolabeled red cells due to anti-Coltonb. Transfusion 1986;26:246–8.Hoffmann JJML, Overbeeke MAM. Characteristics of 42. anti-Cob in vitro and in vivo: a case study. Immunohe-matology 1996;12:11–13.Savona-Ventura C, Grech ES, Zieba A. Anti-Co3 and se-43. vere hemolytic disease of the newborn. Obstet Gynecol 1989;73:870–2.Moulds M, Strohm P, McDowell MA, Moulds J. Autoan-44. tibody mimicking alloantibody in the Colton blood group system (abstract). Transfusion 1988;28(Suppl):36S.King LS, Choi M, Fernandez PC, Cartron JP, Agre P. De-45. fective urinary-concentrating ability due to a complete deficiency of aquaporin-1. N Engl J Med 2001;345:175–9.

Gregory R. Halverson, BS, MT(ASCP)SBB, DLM (cor-responding author), Laboratory of Immunochemistry, New York Blood Center, 310 East 67th Street, New York, NY 10065, and Thierry Peyrard, PharmD, MS, European Specialist in Clinical Chemistry and Laboratory Medicine, National Immunohematology Reference Laboratory, Na-tional Institute of Blood Transfusion, 20 rue Bouvier 75011 Paris, France.

G.R. Halverson and T. Peyrard

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Alloimmunization to the D antigen by a patient with weak D type 21

H. McGann and R.E. Wenk

Antibodies of apparent D specificity may be found in D+ pa-tients. We report a D+, multi-transfused Caucasian woman with myelodysplasia who exhibited several alloantibodies. One antibody was a moderately strong (2+) anti-D that per-sisted for 9 months, until the woman died. Molecular analy-sis of the patient’s RHD gene identified the rare weak D type 21 (938C>T) allele. D alloantibodies do not occur in patients with most weak D types, but some patients with a weak D phenotype, including those with type 21, can produce an-tibodies to nonself epitopes of the wild-type D antigen. Immunohematology 2010;26:27–29.

Key Words: alloimmunization, anti-D, RHD gene, weak D

A ntibodies with D specificity are unusual in D+ pa-tients. Alloantibodies to D may be (1) passively transfused in donor plasma products, (2) passively

transferred but actively produced by lymphocytes carried in transplanted organs, and (3) actively produced by an al-loimmunized patient. Autoantibodies to D may be warm- or cold-reactive antibodies with a preference for D+ RBCs or may be antibodies to LW that mimic anti-D specificity.1

In practice, the combination of clinical, serologic, and molecular investigations helps to determine why a D+ pa-tient has anti-D. Establishing the source of a D+ patient’s alloantibody or autoantibody with D specificity is important in deciding whether D+ or D– units should be transfused to optimize donor RBC survival (Table 1). In this case report, we describe a woman whose RBCs were strongly D+ at the phenotypic level, but had an RHD genotype consistent with weak D, and an alloanti-D.

Case Report A 72-year-old, gravida 3, Caucasian woman with myel-odysplasia and pancytopenia was transferred from another hospital, where in 6 months of treatment, she had received an unknown number of platelet transfusions, but no RBC transfusions. In our hospital and before any RBC transfusion, the patient’s RBCs serologically typed as D+ (4+) by tube ag-glutination at immediate spin phase, using a monoclonal-polyclonal blended anti-D (Ortho Clinical Diagnostics Co., Raritan, NJ). During a disease course of 13 months, the patient received 37 apheresis-processed platelet units and 108 leukocyte-reduced units of RBCs, which produced al-loimmunization to K, E, and Cw. After 9 months, her serum also demonstrated a 2+ anti-D in a gel-based, antibody de-tection system (Micro Typing Systems, Inc., Ortho Clinical Diagnostics, Pompano Beach, FL). The initial DAT (tube,

anti-IgG) and all but one subsequent DAT were negative as was the auto-control. On the one occasion, the DAT became weakly positive in a mixed-field reaction. Acid eluates pre-pared from the patient’s RBCs contained only anti-E. There was no history of organ transplantation or administration of a plasma product that might contain anti-D. The anti-D persisted throughout the patient’s course. Blood samples were collected and referred for RHD and RHCE gene analyses. Although continued transfusions with D– blood were prescribed, the patient refused all further medical treat-ment. Her myelodysplasia progressed to early acute leuke-mia before she expired at home.

Molecular Test Results Molecular tests were performed at the Molecular Red Cell and Platelet Testing Laboratory of the American Red Cross, Penn-Jersey Region, and the University of Penn-sylvania DNA Sequencing Facility, both in Philadelphia,

Case Report

Table 1. Evaluation and transfusion of patients with D+ RBCs and serum anti-D

Etiology/ pathogenesis Diagnostic evidence

Preferred RBC transfusion

Passive alloantibody History of transfusion with D– RBC, plasma, IVIG, RhIG, other product with anti-D

D– until anti-D wanes

Alloantibodies from passenger lymphocytes in donor organ

Organ transplant from D–donor to D+ recipient

D– until anti-D wanes

Autoantibody to D antigen

Positive direct/indirect antiglobulin test (anti-IgG)

D– over D+

Anti-D specificity ineluate or serum

History: pregnancy, warm autoimmune hemolytic anemia, malignancy

Autoantibodies or alloantibodies to LW

Antibody fails to react with RBC treated with dithiothreitol

D– over D+

Antibody is absorbed by D– RBC

Alloantibody to epitope absent from normal D protein

Anti-D fails to react with RBC of person who produced it

D– only

Anti-D fails to react with D– RBC


Pennsylvania. The patient was found to be heterozygous for RHD. No changes associated with any of the known partial D phenotypes were identified. A change (938C>T) was identified that is associated with weak D type 21 that is predicted to encode an intracellular amino acid change of Pro313Leu. The patient’s RH genotype could be interpreted to be RHD*weak D type 21, RHCE*Ce/RHCE*ce.

Discussion Initially, the finding of anti-D in a D+ patient was puz-zling and the several causes listed in Table 1 were consid-ered. Patients whose anti-D was passively transferred in plasma products (IVIG, RhIG, etc.) or whose serum anti-bodies were produced by a donor’s passenger lymphocytes may require D– RBCs until the acquired anti-D has weak-ened, usually in a matter of weeks to months. Passive anti-D, however, was ruled out by history. Be-fore hospital admission at our institution, the patient had been transfused only with apheresis platelets, but she had not been transfused with plasma or cellular products from D– donors, nor had she been infused with IVIG or injected with RhIG. Persistence of the anti-D was further evidence that the anti-D did not involve passive immunization. The transfer of passenger lymphocytes could be ex-cluded by history as well. The patient had never received a transplant. Passenger lymphocytes are alloantibody-secreting B cells that are carried in a transplanted donor organ to a recipient. An anti-D produced by passenger lymphocytes arises in the donor by alloimmunization to the D antigen before transplantation. Donor lymphocytes continue to manufacture detectable anti-D for about 3 months after transplantation. Many patients with autoimmune hemolytic anemia and mimicking antibodies to Rh antigens are treated medically and transfusion is avoided altogether. In other patients, transfusion can be delayed until the concentration of an-tibodies is reduced by medical treatment. Many mimicking warm autoantibodies have apparent Rh specificity, especially anti-e. Only rare D+ patients will possess warm autoan-tibodies that mimic anti-D. If a patient with autoantibodies requires transfusion, antigen-negative donor RBCs may be preferable because they may survive longer than antigen-positive RBCs.2

Antibodies to LW are almost always autoantibodies; those that are less potent appear to have anti-D specific-ity because the number of D antigen sites per RBC corre-lates with the number of LW antigen sites.3 Thus, D– RBCs, which have fewer LW antigen sites than D+ RBCs, may not be agglutinated by an anti-LW. However, anti-LW may be distinguished from anti-D by its failure to agglutinate RBCs treated with DTT. If transfusion is required for a patient with autoanti-LW, D– RBCs are preferable to D+ RBCs pro-viding such a patient is c+. Patient autoantibodies, including anti-LW, are often transient, but our patient’s anti-D persisted for 4 months at constant serologic strength (2+ by the IgG gel test).

Alloantibodies to Rh antigens are often durable (85%). Negative DATs and absence of RBC agglutination in auto-control tests also indicated the presence of D alloantibody. LW alloantibodies, which can mimic anti-D, arise only in very rare individuals whose RBCs are LW(a–b–) or LW(a–b+). Neither autoantibodies nor alloantibodies to LW were specifically evaluated in the patient described here. Alloantibodies to the D antigen occur in people who are known to lack one or more epitopes of D (partial D pheno-type). On the other hand, anti-D alloimmune responses in patients with other weak D types are unusual because their RBCs do not appear to lack D epitopes when tested with a standard battery of anti-D monoclonal reagents.4

Suspicion that the patient had been alloimmunized to D antigen was raised by the serologic finding of negative DATs and the antibody’s persistence. In addition, people who carry variants of the D antigen that are detectable only by molecular means are capable of producing antibodies to wild-type protein. In the present case, finding a variant RHD gene supported the clinical and serologic evidence of D alloimmunization. Historically, some D+ individuals who made anti-D were initially considered to express a weak D phenotype (first termed “DU”) when tested with human polyclonal reagents. Serologic evidence of alloimmunization to D in a D+ person included a negative DAT, a serum antibody, and, for some partial D phenotypes, the patient’s RBCs also expressed a low-prevalence Rh antigen.5 A D+ patient with alloanti-D was thought to lack part(s) of the D antigen, i.e., a “partial D” type. A person with a partial D phenotype would only produce antibody to those epitopes of the D antigen missing from his or her own RBCs. Investigative use of monoclonal antibody revealed many epitopes of the D antigen and sup-ported the concept that partial D types lacked one or more epitopes of their D antigen. The monoclonal antibodies also extended the identification and classification of the various partial D types initially defined by polyclonal anti-D. Many individuals, however, were found to have RBCs that agglutinated poorly with common anti-D reagents, but demonstrated the presence of all known epitopes. These D+ RBCs were called “weak D” in preference to DU. Weak D RBCs expressed fewer D molecules per RBC, a finding that accounted for their weak agglutination with reagent anti-bodies. Theoretically, individuals who carried these weak D types should not be able to develop alloantibodies to D. However, it is virtually impossible to distinguish a person with a weak D phenotype from a person with a weak par-tial D phenotype by serologic tests; only the production of alloanti-D will reveal someone to have a weak partial D phenotype. When molecular analysis became available, the RHD (and RHCE) gene(s) revealed far more extensive and com-plex polymorphisms than were evident by serologic meth-ods. Weakly agglutinating D serotypes could be related to point mutations, nonsense mutations, deletions, and tandem

H. McGann and R.E. Wenk


Anti-D in weak D type 21

rearrangements in the RHD and RHCE DNA. The cause of weak RBC agglutination was confirmed to be an absence of epitopes in partial D types, a decreased number of protein molecules in weak D types, or both. In addition, partial D phenotypes are expressions of genotypes that encode for changes in the amino acids on the outer surface of the RBC where the D protein loops outside the RBC membrane. Patients whose RBCs express partial D types are often im-munized to D+ RBCs by transfusion or pregnancy. On the other hand, genotypes that encode weak D phenotypes are predicted to result in intracellular or intramembranous changes in amino acids that, in theory, express all epitopes of D so that patients whose RBCs express weak D types are not expected to produce anti-D after immunization by transfusion or pregnancy. Theory is partly supported by evidence—the most common weak D variants (types 1–3), which constitute 70 to 90 percent of all weak D variants, do not produce alloanti-D.6 Molecular examination of the various partial and weak D types indicated that many serologic phenotypes had more than one basis, but did not suggest that new epitopes could be identified.7 However, serologic evidence suggested oth-erwise. Anti-D has been found in some weak D patients whose D protein is predicted by their DNA sequences to have altered intramembranous and intracellular amino ac-ids of the D protein. Thus, some weak D molecular alleles contain missense mutations that not only reduce the num-ber of antigens per RBC and cause weak RBC agglutination by reagent antibodies but also cause a loss of D epitopes and produce changes in the three-dimensional antigenic structure of the D protein. The case presented here indicates that the rare weak D type 21 is an allelic variant whose patient-carriers are susceptible to transfusion alloimmunization by wild-type D antigen. Weak D type 21 is the result of a point mutation that alters an amino acid thought to be in an intracellular region of the D protein.8 The rare allele was first discovered in one individual during investigation of 270 known weak D samples in people from northern Germany,9 and there has been one subsequent independent observation of it in Austria.10 In both cases, the allele has been found on the same chromosome (cis) as a RHCE*Ce allele in the haplo-type RHDweak D type 2-RHCE*Ce. The Pro313Leu change in the translated D protein is in its tenth intracellular helix, and all 37 tested epitopes of the D antigen appear to be present.9 As expected, the number of D antigen sites per RBC is reduced (5.2 × 103) when compared with the normal number (104 to 2.5 × 104) of sites.11 This antigen density is the highest among all known weak D alleles. The high density probably accounts for the patient’s 4+ serotype and explains why she was not identified as a weak D carrier.

Summary We evaluated a patient with a strongly expressed D an-tigen with a posttransfusion antibody to the D antigen. The antibody had clinical and serologic characteristics of an

alloantibody. After RHD and RHCE genotyping, the patient was found to have the RHDweakD type 21RHCE*Ce/RHCE*ce genotype (R1

weak D type 21r phenotype). Although most patients with a weak D phenotype do not produce alloanti-D, pa-tients who carry the rare weak D type 21 in the absence of a normal RHD allele appear susceptible to the production of D alloantibodies.

Acknowledgments The authors thank Connie Westhoff, PhD, and Sunitha Vege for the molecular analyses.

ReferencesLu RP, Clark P, Mintz PD. Apparent anti-D in a D+ in-1. dividual: a clinical and serologic approach. Transfusion 2007;47:1755.Leger, RM. Chapter 17: The positive direct antiglobu-2. lin test and immune-mediated hemolysis. In: Technical manual. 16th ed. Roback J, Combs MR, Grossman B, Hillyer, C, eds. Bethesda, MD: American Association of Blood Banks, 2008:499–524.Gibbs MB. The quantitative relationship of the Rh-like 3. (LW) and D antigens of human erythrocytes (letter). Nature 1966;210:642–3.Reid ME. The Rh antigen D: a review for clinicians. 4. Blood Bull 2008;10:1–2.Westhoff CM. Structure and function of the Rh blood 5. group antigens. Semin Hematol 2007;44:42–50.Flegel W. The genetics of the Rhesus blood group sys-6. tem. Dtsch Artzebl 2007;104(10):A651–7.Wagner FF, Frohmajer A, Ladewig B, et al. Weak D al-7. leles express distinct phenotypes. Blood 2000;95:2699–708.Wagner F. Weak D types. Available at: http://www.un-8. iulm.de/~fwagner/RH/RB/weakD.htm. Accessed Feb. 25, 2009. Le Van Kim C, Mouro I, Cherif-Zahar B, et al. Mo-9. lecular cloning and primary structure of the human blood group RhD polypeptide. Proc Nat Acad Sci U S A 1992;89:10925–9.Muller TH, Wagner FF, Trockenbacher A, et al. PCR 10. screening for common weak D types shows different distributions in three central European populations. Transfusion 2001;41:45–52.Polin H, Danzer M, Hofer K, Gassner W, Gabriel C. Ef-11. fective molecular RHD typing strategy for blood dona-tions. Transfusion 2007;47: 1350–5.Wagner FF, Gassner C, Muller TH, Schonitzer D, 12. Schunter F, Flegel WA. Molecular basis of weak D phe-notypes. Blood 1999;93:385–93.

Heather McGann, MT(ASCP) SBB (corresponding author), Supervisor, Blood Bank, Department of Pathology, and Robert E. Wenk, MD, Consulting Pathologist, Department of Pathology, Sinai Hospital, 2401 West Belvedere Avenue, Baltimore, MD 21215.


A review of the Chido/Rodgers blood groupR. Mougey

. . . and the blind, wise blood bankers said, “These an-tibodies are high-titered and have low avidity, they are red cell, white cell antibodies, they are clinically insignificant, are not reactive with enzyme-treated red cells, and can be absorbed with white cells!” Then the chorus of other blind, wise blood bankers said, “NO, THEY ARE NOT!” based on memories of discussions about these antibodies back in the 1970s and the fable of the elephant and blind wise men—r. mougey

It was a delight, an honor, and also a little intimidating to be asked to write a review of the Chido/Rodgers blood group system, especially considering that the last author to pen a review for Immunohematology on this subject was Carolyn M. Giles, the author of so many important publi-cations on Chido and Rodgers. Moreover, the recent dis-coveries about Chido and Rodgers have moved well beyond the field of blood group serology to an understanding of the functional protein and genetics at a molecular level. My delight was in the opportunity to revisit my early days in performing antibody identification and think again about the challenges that blood bankers faced then in work-ing with a group of antibodies that no one could agree about. Chido and Rodgers were usually lumped in with this group, which in light of current knowledge is quite understandable. As I recall even the pronunciation for Chido was a matter of some dispute, with adherents claiming that “Cheedo” with a short i vowel, not Chido with a long i sound, was correct. It occurred to me that this review should cover a historical perspective, which might be helpful to those blood bankers whose testing experience is derived from the newer meth-ods of gel column or solid-phase technology and who might be wondering what all the fuss was about. The review will then cover the current understanding of the serology, func-tion, and biochemistry, and then the genetics of C4 and the Chido/Rodgers blood group system. The nomenclature for the complement system is very confusing even for blood bankers who deal routinely with a nomenclature for a blood group that is just as confusing.

Historical Perspective In the beginning there was confusion and chaos. Dur-ing the 1960s and ’70s, the process for antibody identifica-tion was usually a straightforward and consistent exercise with good consensus between individual technical staff and among laboratories, except there was this group of anti-bodies that “would not cooperate.” In the reference labo-ratory where I worked, we called them “the grubbies,” but we were too frustrated by working with them to go much

further than that. The usual experience was to spend an inordinate amount of time performing every possible test, which led only to excluding what the antibody could not be. Then when additional samples were obtained to con-tinue the investigation, it was not uncommon to find that tests that had appeared to be nonreactive were now reactive with the new antibody sample or with a freshly collected sample of the test cell. It took me a while to recognize that with these antibodies, if you wanted to test for specificity by typing the patient’s RBCs or testing known antigen posi-tive or negative RBCs, it was best to use the freshest RBCs. If you wanted to find compatible blood, older donor units were more likely to be nonreactive. That is, it was gener-ally agreed that there were some patients whose antibod-ies could not be identified as any of the then known anti-bodies and whose reactivity was so weak there was a lot of disagreement as to which test RBCs were truly nonreactive. This made it very difficult to define and describe the true characteristics of the antigens and the antibodies and show that the antigens were expressed on the RBC as an inherited trait. This variability was partly a result of testing methods, which relied heavily on a multiphasic set of incubation tem-peratures and the use of a polyclonal antiglobulin reagent. In that period, blood bankers went to great lengths to de-tect and identify room temperature–reactive antibodies as well as antibodies active at 37°C. The use of fresh serum for testing was usually considered essential, with the end result that blood bankers were pretty good at detecting clinically significant antibodies but even better at finding clinically insignificant antibodies. Blood bankers got lots of prac-tice at identifying these clinically insignificant antibodies, and neutralizing them with human breast milk, avian egg albumin, hydatid cyst fluid, saliva, and human and guinea pig urine. The grubbies defied these efforts, and there was much discussion, some of it acrimonious, on how patients with these antibodies ought to be managed. The term high titer, low avidity was preferred by many if only for its ac-ronym HTLA, even though everyone knew that these anti-bodies were not always of high titer or of low avidity. When such weak antibodies were shown to be of high titer, it de-fied logic that the antibody should show such poor avidity. Obviously, the fault could lie with the antigen, either its ori-entation or its number of sites, or some other property not yet realized. Again, there was a contradiction in the patient making a high-titered antibody if there was so little antigen to stimulate the production of antibodies. The first glimmer of understanding in these contradictory findings was the re-ports on anti-Chido.

Review


Discovery and Serology of Chido/Rodgers The Chido antibody was first studied in a patient in 1962, and in a collaborative effort between laboratories in Minneapolis, Minnesota, and London, United Kingdom, it was found to be reactive with nearly all cells tested but was consistently nonreactive with the RBCs of a London panel donor. Six additional examples were found, and the anti-body was named Chido after one of the patients and the combined results were published by Harris et al. in 1967.1 Their title is as good a description of the grubbies as any, “A nebulous antibody responsible for crossmatching difficul-ties.” The authors reported that the antibodies could not be inhibited by saliva, urine, or hydatid cyst fluid. During this time, there was much speculation about these grubby antibodies being RBC-WBC antibodies, that came from some successful experiments using WBCs to ad-sorb these antibodies.2 This whole issue of mistaken identity was very understandable because of the not uncommon an-tibody problem known as anti-Bgs. Bg antibodies had been shown to be hemagglutinins with anti-HLA specificity, and the Bg antigens were thought to be remnants of those HLA antigens expressed on the RBC before the loss of its nucle-us. Because Bgs were antigens of lower frequency, it would have been so tidy to have them be the antithetical allele to Chido as the variable strength of reactivity seemed so simi-lar. Eventually it was realized that the “grubby” antibodies had nothing to do with WBC antigens. There may have been sufficient residual plasma in the WBC preparations used to adsorb the antibodies, now known to be capable of inhib-iting anti-Chido, to lead to this mistaken conclusion. The speculation that the Chido antigen was a WBC antigen did lead to studies for possible linkage with HLA, an important advance as linkage studies had shown no association with any other blood groups. Many additional examples of anti-Chido were found, and it was thought that these antibodies did not seem to cause obvious destruction of Chido-positive donor RBCs. The work of Middleton and Crookston3 published in 1972 provided the clue that led to recognizing the true nature of the blood group. In an attempt to enhance the reactivity of the anti-Chido they were studying, inert human plasma was added to the tests; a common practice of the time was to add a source of protein to enhance antibody binding for antihuman globulin (AHG) reactions when using polyspe-cific AHG. The authors recognized that when the antibody reactivity disappeared after the addition of plasma the Chido antigen must also be in plasma. They then found that performing Chido antigen typings using the plasma of the person being typed in a neutralization test was a much more reliable way to type individuals for the Chido antigen than testing their RBCs. They also noted that weak Chido antigen reactivity on an individual’s RBCs did not appear to correlate with the amount of antigen in that person’s plasma. The ability to neutralize anti-Chido was also a use-ful tool for antibody identification, especially in sera with multiple antibodies. This was the first way in which Chido

was distinguished from the other so-called HTLA specifici-ties Knops, Csa, and Yka, although some workers continued to think of anti-Yka as also showing some inhibition with plasma.4

At this point, the Chido antigen was thought to be a plasma antigen that was adsorbed onto the RBC much like the Lewis antigens. Once accurate, reproducible antigen typings were possible, family studies on the inheritance of the Chido gene led to the discovery that Chido was linked to HLA.5 This potential relationship to HLA prolonged the use of the term RBC-WBC antibodies, but the recognition of HLA linkage to Chido led to the studies to see whether other genes known to be linked to HLA would also show linkage to Chido. The description of anti-Rodgers, a second antibody re-active with an RBC antigen of high prevalence that could be neutralized by plasma, followed in 1976.6 The antibody was formed by a transfused patient whose RBCs were Chido-positive who also made an anti-E. The anti-Rga (as anti-Rg was referred to at that time) gave weak to 4+ reactions with E– RBCs (not your typical low-avidity antibody). Using plasma neutralization tests, Longster and Giles found that 3.1 percent of donors could not inhibit the antibody, i.e., were Rodgers-negative, although they found some donors who were only partial inhibitors. Nordhagen et al.7 were also able to demonstrate partial inhibition of anti-Chido by the plasma of Chido-positive subjects, and Giles8 showed that partial inhibition was an inherited trait. These findings would explain conflicting results in tests on Chido/Rodgers antibodies among laboratories if a single source of plasma was used in inhibition tests. The common practice now is to use a pool of inert plasmas when attempting to determine whether an unknown antibody is neutralizable by human plasma. Rodgers was also shown to be linked to HLA in two studies in 1976 that set the stage for the discovery of the relationship of Chido and Rodgers to the C4 protein.9,10 In 1978, O’Neill et al.11 showed that Chido and Rodgers are an-tigenic determinants expressed on the C4 complement pro-tein. Then Tilley et al.12 showed that the Chido and Rodgers determinants are on the C4d fragment of the C4 molecule. The small amount of C4d normally found on the RBC mem-brane is the limiting factor that explains the low avidity of anti-Chido or anti-Rodgers. In fact, if large amounts of C4d are bound to the RBC under low ionic conditions, anti-Ch or anti-Rg can be turned into a strongly reactive, direct ag-glutinin.13 The puzzle of incomplete or partial inhibition of the antibodies is caused by the extreme polymorphism of the C4 genes with multiple alleles that cause variations in the Ch or Rg determinants. All this progress made Chido and Rodgers antibodies easier to recognize with the exception of Chido-positive variant individuals who have made anti-Chido to the part of the Chido antigen that they lack.14 The further serologic complexities of Chido/Rodgers specificity will be discussed in the section on genetics.

Chido/Rodgers review


The inexorable unraveling of the puzzle of the true na-ture of the blood group Chido/Rodgers is an excellent ex-ample of the importance of recognizing anomalous findings in the study of antibodies that most blood bankers wished could just be ignored! The final refutation of the term HTLA came from the revelation that the Knops, McCoy, Swain-Langley, and York blood group sera recognize antigens that are carried on the RBC complement receptor CR1.15,16 How-ever, this finding seemed to have brought a level of kinship to these antibodies with Chido and Rodgers that must have been satisfying to those who wanted to believe that Chido, Knops, etc. must be related. Again the low avidity of most Knops antibodies is probably related to the low copy num-ber of the individual’s CR1 receptor. It also seems likely that these blood groups will be forever linked in the literature as those HTLAs when they are studied by aspiring blood bankers.

Clinical Significance of Chido/Rodgers It is well documented that many patients with Chido/Rodgers antibodies have been safely transfused with “ran-dom” RBC units, but there are also reports of significant re-actions to plasma or platelet transfusions in patients with anti-Chido or -Rodgers,17,18 a cautionary incident remind-ing blood bankers not to ignore the potential significance of an antibody based on specificity alone. Strupp et al.19 also pointed out the danger in assuming that antibodies that appear to be of low avidity may be safely ignored without further investigation. They reported 4 patients with sickle cell disease for whom the provision of compatible blood and benefit of transfusion were delayed because Dombrock an-tibodies were not initially recognized and identified. How-ever, there is a need to determine the best way to detect and identify these antibodies without incurring unnecessary delays and expense to the patient, but how much testing is enough? It sometimes seems that when we had the time, resources, and people to detect and identify these antibod-ies, we did not have a clue what they were. Now we know all about them and do not have the time, people, or resources to do much with them. In this area as with so many other blood group issues, perhaps molecular methods will identify good patient practices, and with the use of microarray tech-nology, definitive answers to some of these thorny issues of cost versus benefit will be found. Until that time, when patients with known anti-Chido or anti-Rodgers require RBC transfusions, antibody detection, identification, and crossmatching methods should include plasma-neutraliza-tion studies. After all, these patients are known antibody formers. If transfusion of plasma components is required, there should be some consideration given to the possible effects on the patient of receiving a large volume of plasma, such as the formation of precipitating immune complexes or anaphalaxis.17,18 Finally, the presence of antibodies to C4 proteins may indicate a potential disease association such as is seen with the patients who have anti-Ch and anti-Rg and are C4-deficient.

The story of Chido and Rodgers blood groups has moved well beyond the “crossmatching difficulties,” and there is now a wealth of new material that shows the C4 genes and proteins to be a fascinating model for the evolution of the genes for critical biologic processes that can both protect and attack their host organism.

Structure and Function of the C4 Protein The C4 protein expressing the Chido or Rodgers epitopes occurs in two isoforms, called C4A for acidic (for-merly C4F) and C4B for basic (formerly C4S).20 Awdeh and Alper20 proposed the change from F for fast and S for slow to conform with the then new International System for Hu-man Gene nomenclature and because in their experiments sialic acid was removed from plasma proteins before elec-trophoresis, resulting in distinct but different bands from those in the experiments of O’Neill et al.,11 which were the first to link the Chido/Rodgers antigen to C4 proteins. C4A proteins usually carry the Rodgers antigens and C4B, the Chido antigens, although a number of haplotypes have been discovered in which these associations were switched.21,22

C4A or C4B protein is expressed as a single-chain pre-cursor of 1744 amino acid residues, and the C4A or C4B amino acid sequences are nearly identical (99%) except near residues 1000 to 1200.23 The amino acid substitutions that occur at residues 1101 to 1106 distinguish C4A from C4B. Single-nucleotide polymorphisms (SNP) are also found in the region that distinguishes the alleles of C4A from each other and from C4B and its alleles. The Chido and Rodgers antigens are thought to differ by both amino acid substitu-tion as well as conformational binding sites.24

After translation, the pro-C4 protein is cleaved into al-pha, beta, and gamma polypeptide chains that are linked by disulfide bonds.25 However, incomplete processing ac-tually leads to multiple structural forms of the peptide be-ing secreted in the plasma.26 The activated complement component C1 initiates cleavage of 77 amino acids from the N-terminus of the alpha chain, releasing C4a, and the remainder of the molecule C4b changes shape, exposing a thioester bond between the sulfhydryl group of Cys-991 and the carbonyl group of Gln-994. This binding site that is exposed on activation will allow the formation of either a covalent ester or an amide linkage.27,28 Activated C4B is more efficient than C4A at pathogen cell lysis because of its histidine residue at position 1106 and its serine residue at position 1102.29

The liver is the primary site of C4 production, with smaller amounts produced by macrophages in other tis-sues.30 There is a wide variation in the amount of C4 pro-duced among individuals. The concentration of C4 in the plasma varies from 80 to 1000 µg/mL with variable con-centrations of C4 isotypes.31 Moulds et al.32 found variation in patterns of C4 protein levels in individuals that showed either a complete absence of C4A or C4B, more C4A than C4B, more C4B than C4A, or equal amounts of both. There

R. Mougey



is some correlation between C4 gene copy number varia-tions and certain polymorphisms with the level of C4 pro-tein produced, but the effect at the haplotype level can be lost in the diploid gene expression.33

C4 can be activated via the classical pathway or the mannan-binding lectin pathway, although this may be a simplistic view as a recent paper demonstrated the poten-tial for localized release of anaphylatoxins C3a and C4a by tryptase under certain conditions such as those found in the airways of asthmatics.34 Although it is simpler to consider organized pathways in which complement proteins perform their complex dance with the other parts of the innate and humoral immune response, it is likely that the protection or attack effect is far more chaotic and layered in multiple mechanisms that activate, block, or break down these pro-teins (see discussion on disease association). In the classical pathway, C4 is processed by activated C1, which removes C4a anaphylatoxin from the alpha chain. C4a is a mediator of local inflammation and can induce the contraction of smooth muscle, increase vascular permeabil-ity, and cause histamine release from mast and basophilic WBCs. C4a levels along with the anaphylatoxins C3a and C5a in plasma were shown to be elevated during normal pregnancy as an indication of the increased activation of the complement system, possibly as a protection against infec-tion.35

The remaining C4b fragment forms the enzymes C3 convertase (C4bC2a) and C5 convertase (C3bC4bC2a). These enzymes catalyze the membrane attack proteins, which open a channel into the cell membrane. C4 binding protein (C4bp) is another plasma protein that regulates C4 activity by blocking the formation of and promoting the decay of the classical pathway C3 convertase. Deficiency of C4bp would be expected to result in increased activation of C3 and complement activity in response to classical path-way or lectin pathway activation by immune complex for-mation, bacterial infections, apoptosis, and other triggering mechanisms.36

Activated C4B (C4b-B) binds to its target rapidly but has a short half-life of less than 1 second and has greater hemolytic activity than C4A.28 Activated C4A (C4b-A) is 10 times slower than C4B, but it forms amide bonds with amino group targets of immune complexes (IC) or protein antigens. C4B more efficiently forms ester bonds with car-bohydrate antigens, resulting in better clearance of patho-gens. Besides activation of the complement cascade via the classical or lectin pathways, the covalent binding of C4 to immunoglobulins and to IC enhances the solubilization of immune aggregates and the clearance of IC by binding to complement receptor on RBCs (CR-1). This IC binding on the RBC then acts as a shuttle and delivers the IC for phago-cytosis by macrophages. Thus C4 plays multiple roles for both the innate and adaptive immune systems in vertebrate animals.37

Part of this multiple role is the normal constant low level of activation of C3 via the classical pathway and the alternate pathway. In the classical pathway, it was thought that specific antigen binding by an antibody capable of ac-tivating C1 would initiate the pathway, but Manderson et al.38 have shown that C3 is also being activated by C1–C4 in the absence of antibody-antigen complexes. They propose that although circulating constant low levels of IC could account for their findings, the continual circulation of the normal levels of IgG antibodies may form “spontaneous un-stable Ig aggregates that would provide for a C3 tickover” as well. In their experiments, vascular stasis would encourage or initiate complement activation and would therefore be augmented at sites of inflammation. This would facilitate pathogen recognition by allowing the deposition of activated C3 fragments onto foreign surfaces (host cell surfaces with complement regulatory proteins would be unaffected). Thus the various polymorphisms of C4 affect the normal functioning of C4 and can be affected by the individual’s ability to produce suitable levels of C4 proteins. Deficiencies of C4 proteins whether acquired or inherited can increase the risk of severe viral and bacterial infections.33 How high levels of C4 may also contribute to the risk of diseases is just beginning to be understood (disease association will be discussed later).

Genetics of C4 and Chido/Rodgers The original notation of Cha and Rga implied the ex-pectation that there would be additional antithetical al-leles Chb and Rgb.1,6 However, it was soon realized that this blood group did not fit this pattern of inheritance. Chido, or Ch, and Rodgers, or Rg, are used to describe the cluster of determinants that are expressed on the C4A or C4B pro-teins that can neutralize the antibodies formed by Chido- or Rodgers-negative individuals. Also individuals can type as positive with anti-Chido, but form anti-Chido to the Chido epitopes they lack. Currently nine antigens have been de-scribed: six of high prevalence for Chido, two of high preva-lence for Rodgers, and one of low prevalence, WH.24 Eight phenotypes for the CH/RG system, with their respective oc-currences, are shown in Table 1 as the prevalence for the other antigen combinations has not been established (Table 1).39 The polymorphisms for the Ch and Rg epitopes as well

Table 1. Prevalence of Chido/Rodgers phenotypes in random samples

Chido phenotype

Prevalence (%)

Rodgers phenotype

Prevalence (%)

Ch:1,2,3 88.2 Rg:1,2 95.0

Ch:1,–2,3 4.9 Rg:1,–2 2.5

Ch:1,2,–3 3.1 Rg:–1,–2 2.5

Ch:–1,–2,–3 3.8

Ch:–1,2,–3 Rare

Ch:–1,2,3 (proposed but Rg:–1,2 (proposed but

Ch:–1,–2,3 may not exist) may not exist)


as the polymorphisms for the electrophoretic behavior of the C4 protein confounded the early investigations on the inheritance of the C4 genes. No attempt is going to be made by this author to explain the reported phenotypic frequen-cies of Ch and Rg antigens with the current understanding of the multiple copy number variation of the C4 gene. In early studies of the inheritance of Chido/Rodgers, it appeared that the genes must be encoded by two closely linked loci. The majority of persons were Chido-positive and Rodgers-positive. Approximately 1.7 percent of those stud-ied were Chido-negative, Rodgers-positive, and 3 percent were Chido-positive, Rodgers-negative.3,6 After the linkage of Chido/Rodgers with HLA was established and the dis-covery of C4 as the plasma protein carrier was made, family studies detecting both serologic allelic forms and electro-phoretic alleles seemed initially to be in agreement with this model. It was proposed that null genes or duplicated genes for C4A or C4B led to lack of Rodgers or Chido.40

As methods and antisera improved, numerous C4 al-leles were discovered and the results of a workshop to define and standardize nomenclature were published in a joint paper by Mauff et al.41 They described 14 determi-nants for the C4A gene and 17 for the C4B gene. Genes that express no C4A or C4B are designated with a *Q0 (mean-ing quantity naught). In patients with complete deficiency of C4 (C4A*Q0/B*Q0), null alleles were thought to be at-tributable to gene deletions or point mutations that cause a frameshift in the DNA sequence, leading to defective or no expression of the protein. One such example is a 2-bp insertion into the sequence for codon 1213 at exon 29 of the mutant C4A gene seen in both Caucasians and those of Af-rican descent, but very rarely in those of Asian descent.42

The origin of the complement system C3 and C4 pro-teins predates the ability to make lymphocytes or antibod-ies; the latter did not arise on the evolutionary scene until cartilaginous fish and sharks.43 Homology between C3 and C4 proteins indicates that the C4 gene may have evolved from C3 as a duplicated gene.44 The C4A gene is thought to have arisen by gene duplication and mutation from C4B because of its efficiency in facilitating the clearance of IC.28

The C4A or C4B gene consists of 41 exons that in ad-dition to encoding for an acidic or basic isotype can also exist as a long form of 20.6 kb or a short form of 14.2 kb.45 The long form has an insertion of a human endogenous ret-rovirus (HERV-K[C4]) into intron 9. The insertion of this retroviral material is thought to have occurred after dupli-cation of the ancestral mammalian C4A gene but before the divergence of humans from the ancestors of other primates. The reading frames of the retrovirus are all closed, but there is still promoter activity and the HERV-K(C4) insert is thought to be able to modulate the expression of the C4 gene.46 The long form was originally thought to be a C4A gene, but C4 genes can be C4A (long or short) or C4B (long or short). However, most C4A genes have the HERV-K(C4) insert and a greater proportion of C4B genes do not.

The genes for C4A and C4B were shown to be located between HLA-DR and HLA-B loci on the short arm of chro-mosome 6 along with C2 and factor B.40,47 C4A and C4B proteins are distinguished phenotypically by their migra-tion patterns in electrophoretic gels and genetically by four nucleotide substitutions that encoded changes at residues 1101 to 1106 where C4B encodes for LSPVIH and C4A en-codes for PCPVLD at the protein level. C4A and C4B are also distinguished phenotypically by their expression of ei-ther the Chido or the Rodgers antigen, which are the result of two amino acid substitutions at residues 1188 to 1191. The Ch1 epitope has ADLR and the Rg1 has VDLL. As data from family studies began to contradict a simple two-gene model, it was realized that a haplotype could have one, two, three, or four C4 genes and that the C4 genes were inher-ited as part of a complex of genes.48 The two-gene theory had to give way to the current hypothesis of the inheritance of a haplotype modular gene complex that can vary by C4 copy number and is part of a larger gene complex in the MHC locus. This model explains both the qualitative and quanti-tative nature of the C4 gene expression as regards epitopes, concentration of the C4 protein in the plasma, the frequency of certain haplotypes, and the association of C4 copy number variation with certain diseases. In fact, the C4 gene as part of the MHC complex can serve as an example of how the genes of an important biologic pathway evolve via the mechanics of meiosis and through the pressure of natural selection to con-serve important genes and to maximize the utility of poly-morphism potential. Gene duplication, translocation, single-nucleotide polymorphisms, insertions, and deletions all have played a role in the plastic nature of the C4 gene. Both forms of C4 are highly homologous but either by SNP or by insertions or deletions display great qualita-tive and quantitative diversity. The copy number variation comes from whether an individual has inherited haplotypes that have one, two, three, or four C4 genes.49 This gene clus-ter occurs as a complex in the MHC locus near the centromere with flanking genes in the following order: first an intact RP gene (a serine/threonine kinase gene), an intact C4 gene (either C4A or C4B), then an intact CYP21B gene (cy-tochrome P450 21-hydroxylase gene), and an intact TNXB (tenascin-X protein). Approximately 17 percent of persons of Northern European ancestry have this haplotype, termed monomodular.50 In 83 percent of those of Northern Euro-pean ancestry the C4 gene complex can contain one, two, or three duplicated gene clusters made of fragments or mu-tations of the above flanking genes and an intact C4 gene. The duplicated gene complex, called RCCX, occurs between the C4 gene and the intact CYP21B gene and consists of a CYP21 (usually a mutated form) fused to a 4.9-kb fragment of TNXA fused to an RP2 0.91-kb fragment and an intact C4 (A or B and long or short). Depending on the number of RCCX modules duplicated, the complete C4 gene may be a monomodular (no RCCX duplication), bimodular, trimod-ular, or quadrimodular form.

R. Mougey



This modular gene duplication maximizes opportuni-ties to conserve the C4 gene, whereas the opportunity for recombination allows greater diversity of polymorphisms. This great diversity should allow for a better response to pathogens but can create opportunities for unequal cross-ing over during meiosis. Thus low or high copy number variations could be as important a risk factor for disease as the actual C4 polymorphism. C4 gene copy number studies done on healthy subjects of different ages indicate that those individuals with a low copy number for C4B may be less healthy or have a shorter lifespan than those having more than two copies of the C4B gene.51 These studies found that in healthy individuals old-er than 50 years, there was a lower incidence of low copy number for the C4B gene when compared with the low copy number gene frequency in younger healthy individuals.

C4 and Disease Associations The association of C4 and a number of diseases has been a fruitful area of investigation, and at the genetic level, the frequency of C4 nulls or low copy variation shows a solid association with autoimmune disease, particularly systemic lupus erythematosus (SLE).52 Although the varying efficien-cies of complement function that result from partial or com-plete deficiency may have a profound effect on autoimmune susceptibility, above-average C4 protein levels may also in-crease risk. Once a loss of self-tolerance occurs, having high levels of C4 may exacerbate the disease. Moreover, it cannot be overstated that association is not the same as causation. SLE can be thought of as a prototype for the intricacies of the interaction of C4 proteins both in the development of autoimmunity and in disease progression. Inheritance of a C4A*Q0 null gene shows a greater risk in Caucasian and African populations, but C4B*Q0 nulls are more frequent in SLE patients of Asian descent.53 SLE is a heterogeneous disease that is characterized by the presence of multiple au-toantibody specificities that attack various tissues, result-ing in tissue damage, which then presents more antigen to the host’s immune system. It has become increasingly clear that multiple genetic pathways are likely involved and that C4 polymorphism, especially copy number variation, can be a negative or a positive risk factor in the development of autoimmune diseases.54 This postulates that the function of C4 in the initiation of autoimmunity may be related to its role in presenting antigen to the host immune system, which is separate from the role of complement in the in-flammatory process once autoantibodies have been formed. The current understanding of SLE genetics is that there are distinctions in severity, risk, and clinical manifestation that vary by ethnicity, geography, and sex with a greater preva-lence in women and some non-Caucasian ethnic groups. Although genetics undoubtedly plays a role, SLE may also be associated with poverty and exposure to infectious dis-eases.55,56

Progress both in human genetics, particularly by genome-wide association studies, and in the understand-ing of biologic pathways has led to the discovery of more than 20 different loci that show strong correlation to SLE.54 Most of the genes identified are involved in three kinds of biologic processes. Minor variations in the genes encoding the proteins of IC processing, production of interferon, or signal transduction of immune cells can greatly increase the susceptibility of an individual to SLE. It is to be expected that more than one kind of trigger, such as pathogens, al-lergens, or trauma, could then lead to disease. Inasmuch as a major function of the C4 protein is par-ticipation in innate immunity, there is a strong association between deficiencies of C4 and recurrent in-fections; although this is less frequent than other comple-ment deficiencies.57 Table 2 lists other reported diseases and their possible association with C4 or complement.

In their paper, Atkinson and Frank43 also warn that pa-tients with complement C2- or C4-deficient states (either acquired or inherited) have backup complement activation mechanisms that must be considered in defining the role of complement and a disease. For example, the immune re-sponse of specific antibody produced to a pathogen (antibody

Table 2. Disease associations with low levels of C4/complement Date Disease Citation

2009 Schizophrenia Shi58

2008 Role of complement in schizophrenia Mayilyan59

2009 Chronic fatigue syndrome Sorenson60

2008 Autism and missing or nonfunctional C4B gene

Sweetehn61

2008 Alzheimer’s and increased C4 deposition Zhou62

2008 Increased morbidity/mortality associated with low expression of C4B, smokers, and stroke

Szilagyi63

2007 Cardiovascular disease, smokers, and low expression of C4B

Arason64

2006 C4 nulls and adult rhinosinusitis Seppänen65

2002 Systemic sclerosis Arason66

1996 Insulin-dependent diabetes and C4 poly-morphisms

Lhotta67

1993 Graves’ disease Ratanachaiyavong68

1991 C4A deficiency and IgA nephropathy Wyatt69

1990 Felty’s syndrome Hillarby70

1989 Psoriasis Wyatt 71

1989 Rheumatoid arthritis Fielder72

1988 C4 allotypes in juvenile dermatomyositis Robb73

1987 C4B2 and primary biliary cirrhosis Briggs74

1984 Glomerulonephritis, Henoch-Schönlein purpura

McLean75


R. Mougey

excess) may be all that is needed to trigger more ancient pathways to perform cell lysis in the absence of sufficient complement proteins.

Summary The C4 protein plays an important role in maintaining health and, in some situations complicated by poor expres-sion of the C4 protein, may lead to or exacerbate certain diseases. The blood groups Chido and Rodgers are epitopes on the C4 protein, and polymorphisms associated with these epitopes may lead to the formation of antibodies to the Chido or Rodgers antigens in transfused patients. Iden-tification of anti-Ch or anti-Rg is still based on the antibody neutralization with plasma from Ch-positive or Rg-positive individuals and lack of reactivity with qualified Ch-negative or Rg-negative RBCs. These antibodies may be useful in genetic studies of C4 polymorphisms or, in the case of C4-deficient patients, a signal of the potential for serious ill-nesses. The recognition of the extreme polymorphism of the C4 gene and the gene complex RCCX should lead to more insights in the understanding of disease risk and potential treatment.

ReferencesHarris JP, Tegoli J, Swanson J, Fisher N, Gavin J, No-1. ades J. A nebulous antibody responsible for crossmatch-ing difficulties (Chido). Vox Sang 1967;12:140–2.Swanson J, Olson J, Azar M. Serological evidence that 2. antibodies of Chido-York-Csa specificity are white cell antibodies (abstract). Fed Proc 1971;30:248.Middleton J, Crookston MC. Chido substance in plas-3. ma. Vox Sang 1972;23:256–61.Swanson JL. Laboratory problems associated with leu-4. kocyte antibodies. In: Seminar on recent advances in immunohematology. Washington, DC: American Asso-ciation of Blood Banks.1973 121–55.Middleton J, Crookston MC, Falk JA, et al. Linkage of 5. Chido and HLA. Tissue Antigen 1974;4:366–73.Longster G, Giles CM. A new antibody specificity, anti-6. Rga, reacting with red cell and serum antigen. Vox Sang 1976;30:175–80.Nordhagen R, Olaisen B, Teisberg P, Gedde-Dahl T 7. Jr. Association between the electrophoretically deter-mined C4M haplotype product and partial inhibition of anti-Cha. J Immunogenet 1980;7:301–6.Giles CM. A new genetic variant for Chido. Vox Sang 8. 1984;46:149–56.Giles CM, Gedde-Dahl T Jr, Robson EB, et al. Rg9. a (Rod-gers) and the HLA region: linkage and association. Tis-sue Antigens 1976;8:143–49.James J, Stiles P, Boyce F, Wright J. The HL-A type of 10. Rg(a–) individuals. Vox Sang 1976;30:214–16.O’Neill GJ, Yang SY, Tegoli J, Berger R, Dupont B. 11. Chido and Rodgers blood groups are distinct anti-gen components of human complement C4. Nature 1978;273:668–70.

Tilley CA, Romans DG, Crookston MC. Localization of 12. Chido and Rodgers determinants to the C4d fragment of human C4. Nature 1978;273:713 -5.Judd WJ, Kraemer K, Moulds JJ. The rapid identifica-13. tion of Chido and Rodgers antibodies using C4d coated red blood cells. Transfusion 1981;21:189–92.Giles CM, Hoffman M, Moulds M, Harris M, Delmasso 14. A. Allo anti-Chido in a Ch-positive patient. Vox Sang 1987;52:129–33.Moulds JM, Nickells MW, Moulds JJ, et al. The C3b/15. C4b receptor is recognized by the Knops, McCoy, Swain-Langley, and York blood group sera. J Exp Med 1991;173:1159–63.Rao N, Ferguson DJ, Lee SF, Telen MJ. Identification 16. of human erythrocyte blood group antigens of C3b/C4b receptor. J Immunol 1991;146:3501–7.Lambin P, LePennec PY, Hauptmann G, et al. Adverse 17. transfusion reactions associated with a precipitating anti-C4 antibody of anti-Rodgers specificity. Vox Sang 1984;47:242–9.Westhoff CM, Sipherd BD, Wylie DE, Toalson LD. 18. Severe anaphylactic reactions following transfusions of platelets to a patient with anti-Ch. Transfusion 1992;32:576–9.Strupp A, Cash K, Uehlinger J. Difficulties in identi-19. fying antibodies in the Dombrock blood group sys-tem in multiply alloimmunized patients, Transfusion 1998;38:1022–5.Awdeh ZL, Alper CA. Inherited structural polymor-20. phism of the fourth component of human complement. Proc Natl Acad Sci U S A 1980;77:3576–80.Rittner C, Giles CM, Roos MH, Demant P, Mollenhauer 21. E. Genetics of human C4 polymorphism: detection and segregation of rare and duplicated haplotypes. Immu-nogenetics 1984;19:321–33.Roos MH, Giles CM, Demant P, Mollenhuuer E, Ritt-22. ner C. Rodgers (Rg) and Chido (Ch) determinants on human C4: characterization of two C4B5 subtypes, one of which contains Rg and Ch determinants. J Immunol 1984;133:2634–40.Yu CY. The complete exon-intron structure of a human 23. complement component C4A gene. DNA sequences, polymorphisms and linkage to 21-hydroxylase gene. J Immunol 1991;146:1057–66.Yu CY, Campbell RD, Porter RR. A structural model for 24. the location of the Rodgers and Chido antigenic deter-minants and their correlation with the human comple-ment components C4A/C4B isotypes. J Immunogenet 1988;27:399–405.Belt KT, Carroll MC, Porter RR. The structural basis of 25. the multiple forms of human complement component C4. Cell 1984;36:907–14.



Blanchong CA, Chung EK, Rupert KL, et al. Genetic, 26. structural and functional diversities of human comple-ment components C4A and C4B and their mouse homo-logues Slp and C4. Int Immunopharmacol 2001;1:365–92.Law SK, Lichtenberg NA, Levine RP. Covalent binding 27. and hemolytic activity of complement proteins. Proc Natl Acad Sci U S A 1980;77:7194–8.Dodds AW, Ren X-D, Willis AC, Law SKA. The reac-28. tion mechanism of the internal thioester in the human complement component C4. Nature 1996;379:177–9.Carroll MC, Fathallah DM, Bergamaschini L, Alicot EM, 29. Isenman DE. Substitution of a single amino acid (as-partic acid for histidine) converts the functional activity of human complement C4B to C4A. Proc Natl Acad Sci U S A 1990;87:6868–72.Colton HR. Genetics and synthesis of components of 30. the complement system. In: Ross GD, ed. Immunobi-ology of the complement system. An introduction for research and clinical medicine. New York: Academic Press, 1986:174–6.Uko G, Christiansen FT Dawkins RL, McCann VJ. 31. Reference ranges for serum C4 concentrations in sub-jects with and without C4 null alleles. J Clin Pathol 1986;39:573–6.Moulds JM, Arnett FC, Giles CM, Hamilton RG. A novel 32. immunoassay for the quantification of human C4 gene products. Complement Inflamm 1990;7:93–101.Chung EK, Yang Y, Rupert KL, et al. Determining the 33. one, two, three, or four long and short loci human com-plement C4 in a major histocompatibility complex hap-lotype encoding C4A or C4B proteins. Am J Hum Genet 2002;71:810–22.Fukuoka Y, Zia HZ, Sanchez-Munoz LB, Dellinger AL, 34. Escribano L, Schwartz B. Generation of anaphylatox-ins by human β-tryptase form C3, C4, C5. J Immunol 2008;180:6307–16.Richani K, Soto E, Romero R, et al. Normal pregnancy is 35. characterized by systemic activation of the complement system. J Matern Fetal Neonate Med 2005;17:239–45.Gigli I, Fugita T, Nussenzweig V. Modulation of the 36. classical pathway C3 convertase by plasma proteins C4 binding protein and C3b inactivator. Proc Natl Acad Sci U S A 1979;76:6596–600.Gatenby PA, Barbosa JE, Lachmann PJ. Differences 37. between C4A and C4B in the handling of immune com-plexes: the enhancement of CR1 binding is more impor-tant than the inhibition of immunoprecipitation. Clin Exp Immunol 1990;79:158–63.Manderson AP, Pickering MC, Botto M, Walport MJ, 38. Parrish CR. Continual low level activation of the classi-cal complement pathway. J Exp Med 2001;194:747–56.Giles CM. An update on Rodgers and Chido, the anti-39. genic determinants of human C4. Immunohematology 1989;5:1–6.

Carroll MC, Palsdottir A, Belt KT, Porter RR. Deletion 40. of complement C4 and 21-hydroxylase genes in the HLA class III region. EMBO J 1985;4:2547–52.Mauff G, Alper CA, Dawkins RL, et al. C4 nomenclature 41. statement. Complement Inflamm 1991;7:261–8.Barba G, Rittner C, Schneider PM. Genetic basis of 42. human complement C4A deficiency, detection of a point mutation leading to nonexpression. J Clin Invest 1993;91:1681–6.Atkinson JP, Frank MM. Bypassing complement: evo-43. lutionary lessons and future implications. J Clin Invest 2006;116:1215–8.Martinez OP, Longman-Jacobsen N, Davies R, et al. Ge-44. netics of human complement component C4 and evolu-tion of the central MHC. Front Biosci 2001;6:D904–13.Dangel AW, Mendoza AR, Baker BJ, et al. The dichoto-45. mous size variation of human complement C4 gene is mediated by a novel family of endogenous retroviruses which also establishes species-specific genomic pat-terns among Old World primates. Immunogenetics 1994;40:425–36.Mack M, Bender K, Schneider PM. Detection of retro-46. viral antisense transcripts and promoter activity of the HERV-K(C4) insertion in the MHC class III region. Im-munogenetics 2004;56:321–32.Yu CY, Campbell RD. Definitive RFLPS to distinguish 47. between the human complement C4/C4B isotypes and the major Rodgers/Chido determinants: applica-tion to the study of C4null alleles. Immunogenetics 1987;25:383–90.Yu CY, Yang Z, Blanchong CA, Miller W. The human 48. and mouse MHC class III region: a parade of the cen-tromeric segment with 21 genes. Immunol Today 2000;21:320–8.Chung EK, Yang Y, Rupert KL, et al. Determining the 49. one, two, three or four long and short loci of human complement C4 in a major histocompatibility complex haplotype encoding C4A or C4B proteins. Am J Hum Genet 2002;71:810–22.Wu YL, Yang Y, Chung EK, et al. Phenotypes, geno-50. types and disease susceptibility associated with gene copy number variations: complement C4 CNVs in Eu-ropean American healthy subjects and those with sys-temic lupus erythematosus. Cytogenet Genome Res 2008;123:131–41.Szilagyi A, Fust G. Diseases associated with low copy 51. number of the C4B gene encoding C4, the fourth component of complement. Cytogenet Genome Res 2008;123:118–30.


R. Mougey

Blanchong CA, Zhou B, Rupert KL, et al. Deficiencies 52. of human complement component C4A and C4B and heterozygosity in length variants of RP-C4-CYP21-TNX (RCCX) modules in Caucasians: the load of RCCX ge-netic diversity on major histocompatibility complex-associated disease. J Exp Med 2000;191:2183–96.Yang Y, Chung EK, Zhou B, et al. The intricate role of 53. complement component C4 in human systemic lupus erythematosus. Curr Dir Autoimmun. 2004;7:98–132.Harley IT, Kaufman KM, Langfeld CD, Harley JB, Kelly 54. JA. Genetic susceptibility to lupus: new insights from fine mapping and genome-wide association studies. Nat Rev Genet 2009;10:285–90.Crosslin KL, Wiginton KL. The impact of race and eth-55. nicity on disease severity in systemic lupus erythemato-sus. Ethn Dis 2009;19:301–7.Hiraki LT, Benseler SM, Tyrrell PN, Harvey E, Herbert D, 56. Silverman ED. Ethnic differences in pediatric systemic lupus erythematosus. J Rheumatol 2009;36:2539–46.Figueroa JE, Densen P. Infectious diseases associ-57. ated with complement deficiencies. Clin Microbiol Rev 1991;4:359–95.Shi J, Levinson DF, Duan J, et al. Common variants on 58. chromosome 6p22.1 are associated with schizophrenia. Nature 2009;460:753–7.Mayilyan LR, Weinberger DR, Sim RB. The comple-59. ment system in schizophrenia. Drug News Perspect 2008;21:200–10.Sorensen B, Jones JF, Vernon SD, Rajeevan MS. Tran-60. scription control of complement activation in an ex-ercise model of chronic fatigue syndrome. Mol Med 2009;15:34–42.Sweeten TL, Odell DW, Odell JD, Torres AR. C4B null 61. alleles are not associated with genetic polymorphisms in the adjacent gene CYP21A2 in autism. BMC Med Genet 2008;9:1–7.Zhou J, Fonseca MI, Pisalyaput K, Tenner AJ. Comple-62. ment C3 and C4 expression in C1q sufficient and de-ficient mouse models of Alzheimer’s disease. J Neuro-chem 2008;106:2080–92.Szilagyi A, Fust G. Diseases associated with the low 63. copy number of the C4B gene encoding C4, the fourth component of complement. Cytogenet Genome Res 2008;123:118–30.Arason GJ, Kramer J, Blasko B, et al. Smoking and a 64. complement gene polymorphism interact in promoting cardiovascular disease morbidity and mortality. Clin Exp Immunol 2007;149:132–8.

Seppänen M, Suvilehto J, Lokki M-L, et al. Immuno-65. globulins and complement factor C4 in adult rhinosi-nusitis. Clin Exp Immunol 2006;145:219–27.Arason GJ, Geirsson AJ, Kolka R, Vikingsdottir TH, 66. Valdimarrson H. Deficiency of complement dependent prevention of immune precipitation in systemic sclero-sis. Ann Rheum Dis 2002;61:257–60.Lhotta K, Auinger M, Kronenberg F, Irsigler K, Konig P. 67. Polymorphism of complement C4 and susceptibility to IDDM and microvascular complications. Diabetes Care 1996;19:53–5.Ratanachaiyavong S, Demaine AG, Campbell RD, 68. McGregore AM. Heat shock protein 70 (HS70) and complement C4 genotypes in patients with hyperthy-roid Graves’ disease. Clin Exp Immunol 1991;84:48–52.Wyatt RJ, Julian BA, Woodford SY. C4A deficiency and 69. poor prognosis in patients with IgA nephropathy. Clin Nephrol 1991;36:1–5.Hillarby MC, Strachan T, Grennan DM. Molecular 70. characterisation of C4 null alleles found in Felty’s syn-drome. Ann Rheum Dis 1990;49:763–7.Wyatt RJ, Wang C, Hudson EC, et al. Complement 71. phenotypes in patients with psoriasis. Hum Hered 1989;39:327–32.Fielder AH, Ollier W Lord DK, et al. HLA class III hap-72. lotypes in multicase rheumatoid arthritis families. Hum Immunol 1989;25:75–85.Robb SA, Fielder AHL, Saunders CEL. C4 complement 73. allotypes in juvenile dermatomyositis. Hum Immunol 1988;22:31–8.Briggs DC, Donaldson PT, Hayes P, et al. A major histo-74. compatibility complex class III allotype (C4B2) associ-ated with primary biliary cirrhosis (PBC). Tissue Anti-gens 1987;29:141–5.McLean RH, Wyatt RJ, Julian BA. Complement pheno-75. types in glomerulonephritis. Increased frequency of ho-mozygous null C4 phenotypes in IgA nephropathy and Henoch-Schönlein purpura. Kidney Int 1984;26:855–60.

Ruth Mougey MT(ASCP)SBB, President, Mougey, Incor-porated, 1705 Highland Avenue, Carrollton KY 41008.

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In Memoriam: Marie Cutbush Crookston 1920–2009

D. Mallory

Marie Cutbush Crookston, a dynamic person and ex-ceptional scientist, was born in 1920 in Victoria, Australia. She received a Bachelor of Science degree from the Univer-sity of Melbourne, and, in 1947, she set sail for England. There, she worked for 10 years with Dr. P. L. Mollison at the Medical Research Council Blood Transfusion Research Unit and contributed to the writing of the first edition of Dr. Mollison’s Blood Transfusion in Clinical Medicine. In 1957, she married Dr. John Crookston, a hematolo-gist from Toronto, who was in London working for Dr. J. V. Dacie. Giving up her studies for a PhD, she moved to To-ronto to raise two sons and to continue to work in the field of immunohematology. In 1964 she joined the University of Toronto, Department of Pathology, where she directed research, and in 1978 she became an associate professor. She also served as immunohematologist in the Department of Hematology at the Toronto General Hospital and acted as a consultant for the Blood Transfusion Laboratory until she retired in 1986. Marie’s contributions to immunohematology are out-standing and have made a permanent impact on the field. She and her late husband John helped transform the edu-cational level and direction of immunohematology in the 1960s when they returned to Toronto from London. There, they helped to organize the Ontario Antibody Club, which was active for 25 years. Marie’s studies on conversion of incomplete to complete antibodies by chemical modification were used by commer-cial companies to make chemically modified Rh antiserum. In addition, she was known for her discoveries of anti-Fya and anti-Lub and her early work in exchange transfusion of the newborn, long-term preservation of RBCs by freez-ing, and linkage of Chido and HLA. She and John Crook-ston also coined the term HEMPAS to describe an inherited chronic hemolytic anemia with multinucleated cells in the bone marrow associated with a positive acidified serum test (modified Ham’s solution). Everyone who knew Marie and read any of her 65 pub-lished papers remembers her wonderful ability to write in plain intelligible English, or as she would say, “deathless prose,” and how she kept strictly to proper blood group ter-minology. Her papers were always accepted as submitted. She was on the editorial boards of Immunohematology, Journal of Blood Group Serology and Education, Transfu-sion, and Vox Sanguinis.

These are some of her awards:The Karl Landsteiner Memorial Award in 1976 with Dr. • Eloise Giblett from the American Association of Blood Banks, awarded “for her discoveries of anti-Fya, anti-Lub, and anti-HEMPAS: for her studies of the relation-ship between blood-group antibody activity in vitro and in vivo: for her description of red cell uptake of Lea and Leb antigens from plasma; for her investigation of the Chido antigen in plasma; and for the detection of the linkage between Ch and HLA.” (quotation from citation on award)The Nuffield Foundation Award in 1952 with Dr. Her-• mann Lehmann for studying the blood groups of tribes in the Nilgiri Hills of South India.The Buchanan Award in 1980 from the Canadian Soci-• ety of Transfusion Medicine.The Canadian Blood Services Award in 2002 for “Life-• time Achievement.”

Marie always found the time to help students and scien-tists who needed directions with their studies or help with their manuscripts. She was a wonderful, caring person who was an enthusiastic, intelligent, and brilliant scientist and one who will be missed by her many friends and colleagues around the world.

Delores Mallory, MT(ASCP)SBBEmeritus Editorial Board


In Memoriam: Eloise Giblett 1921–2009D. Mallory

Eloise “Elo” Giblett was born in Tacoma, Washington, in 1921 and, in 1931, the family moved to Spokane. Elo de-veloped a keen interest in music at an early age, taking pi-ano, dance, and violin, the instrument on which she would focus, becoming concertmaster of her high school orches-tra. She put aside music, turned to science, and received a BS in bacteriology from the University of Washington (U of W) in 1942. World War II had started, so Elo joined the WAVES in 1944, became a medical laboratory technician, and served until 1946. She returned to the U of W and re-ceived her MS degree in microbiology in 1947 and then, in 1951, graduated first in her class in the second class to grad-uate from the new medical school at the U of W. During the medical internship and hematology fellow-ship at U of W, her interest in human genetics was encour-aged. In 1955, she became the first full-time physician at King County Blood Bank (now the Puget Sound Blood Cen-ter) as the Associate Director of the Typing and Crossmatch Laboratory and was given a 6-month sabbatical with Dr. Patrick Mollison at the Blood Transfusion Research Unit in London, England. There, she studied the Lewis blood group system, measured the rate of tagged RBC destruction, be-came acquainted with the clinical potential of the Coombs antiglobulin test, and mastered the tests used at that time in Dr. Mollison’s laboratory. When she returned to Seattle, she began to use the antiglobulin test to find RBC antibod-ies and discovered anti-V and anti-Jsa. Her numerous studies of the polymorphisms of hap-toglobin and transferrins and her interest in RBC, plasma, and protein polymorphisms resulted in the renowned 1969 publication of her book, Genetic Markers in Human Blood. In addition, she discovered that two forms of inherited im-munodeficiency disease were caused by deficiencies of the enzymes adenosine deaminase and purine nucleoside phos-phorylase. In 1979, Dr. Giblett became the Director of the Puget Sound Blood Center and remained so until she retired in 1987. Upon her retirement, she became Director Emeritus of the Blood Center and Professor Emeritus of the U of W Medical School. After 40 years, she returned to the violin and even helped found a new music school. Dr. Giblett was president of the American Society of Human Genetics (1973); a member of the Advisory Board of the American Society of Hematology (1980–1986); a mem-ber of the Editorial Board of Blood, Transfusion, and The American Journal of Human Genetics (among a few); and

chairperson of the Enzyme Nomenclature Committee of the International Workshop on Human Gene Mapping.She received many honors during her lengthy career, includ-ing two from the American Association of Blood Banks:

1975—Emily Cooley Lecture Award (American Associa-• tion of Blood Banks)1976—Karl Landsteiner Memorial Award (American • Association of Blood Banks)1978—The Philip S. Levine Award (American Society of • Clinical Pathology)1980—Election, National Academy of Science• 1987—Distinguished Alumna Award (University of • Washington)

Dr. Giblett has left a legacy of original scientific discov-eries, a book that was groundbreaking for the field of im-munohematology, and colleagues who were scientifically mentored and generously helped by her excellent editorial skills. Those who knew her will remember a very interesting and giving human being.

Delores Mallory, MT(ASCP)SBBEmeritus Editorial Board


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Ordering InformationThe book, which costs $25, can be ordered in two ways:

Order online from the publisher •at: www.sbbpocketbook.com

Order from the authors, who •will sign the book. Send a check, made payable to “New York Blood Center” and indicate “Pocketbook” on the memo line, to:

Marion Reid Laboratory of Immunochemistry New York Blood Center 310 East 67th Street New York, NY 10065

Please include the recipient’s complete mailing address.

Blood Group Antigens & AntibodiesA guide to clinical relevance & technical tipsby Marion E. Reid & Christine Lomas-Francis

Pocketbook Education FundThe authors are using royalties generated from the sale of this pocketbook for educational purposes to mentor people in the joys of immunohematology as a career. They will accomplish this in the following ways:

Sponsor workshops, seminars, and lectures•Sponsor students to attend a meeting•Provide copies of the pocketbook•

(See www.sbbpocketbook.com for details to apply for funds)

This compact “pocketbook” from the authors of the Blood Group Antigen FactsBook is a must for anyone who is involved in the laboratory or bedside care of patients with blood group alloantibodies.

The book contains clinical and technical information about the nearly 300 ISBT recognized blood group antigens and their corresponding antibodies. The information is listed in alphabetical order for ease of finding—even in the middle of the night. Included in the book is information relating to:

Clinical significance of antibodies in transfusion and HDN.•

Number of compatible donors that would be expected •to be found in testing 100 donors. Variations in different ethnic groups are given.

Characteristics of the antibodies and optimal technique(s) •for their detection.

Technical tips to aid their identification.•

Whether the antibody has been found as an autoantibody.•

IMMUNOHEMATOLOGY, Volume 26, Number 1, 2010 45Additional Information can be found by visiting the following Web sites: www.ascp.org, www.caahep.org and www.aabb.org Revised August 2007

Becoming a Specialist in Blood Banking (SBB)

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

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

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

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

How does one become an SBB?• Attend a CAAHEP-accredited Specialist in Blood Bank Technology Program OR • Sit for the examination based on criteria established by ASCP for education and experience

However: In recent years, a greater percentage of individuals who graduate from CAAHEP-accredited programs pass the SBB exam. Conclusion:The BEST route for obtaining an SBB certification is …to attend a CAAHEP-accredited Specialist in Blood Bank Technology Program

Contact the following programs for more information: Program Contact Name Phone Contact Email Contact Website On site

or On line Program

Walter Reed Army Medical Center William Turcan 202-782-6210 [email protected]

www.militaryblood.dod.mil On site

American Red Cross, Southern California Region Michael Coover 909-859-7496 [email protected] none On site

ARC-Central OH Region Joanne Kosanke 614-253-2740 x 2270 [email protected] none On site

Blood Center of Southeastern Wisconsin Lynne LeMense 414-937-6403 [email protected] www.bcw.edu On site

Community Blood Center/CTS Dayton, Ohio Nancy Lang 937-461-3293 [email protected] http://www.cbccts.org/education/sbb.htm

On line

Gulf Coast School of Blood Bank Technology Clare Wong 713-791-6201 [email protected] www.giveblood.org/education/distance/htm

On line

Hoxworth Blood Center, Univ. of Cincinnati Susan Wilkinson 513-558-1275 [email protected] www.hoxworth.org On site

Indiana Blood Center Jayanna Slayten 317-916-5186 [email protected] www.indianablood.org On line

Johns Hopkins Hospital Jan Light 410-955-6580 [email protected] http://pathology2.jhu/department/divisions/trnafusion/sbb.cfm

On site

Medical Center of Louisiana Karen Kirkley 504-903-3954 [email protected] none On site

NIH Clinical Center Dept.. of Transfusion Medicine Karen Byrne 301-496-8335 [email protected] www.cc.nih.gov/dtm On site

Rush University Veronica Lewis 312-942-2402 [email protected] www.rushu.rush.edu/health/dept.html

On line

Transfusion Medicine Center at Florida Blood Services

Marjorie Doty 727-568-5433 x 1514 [email protected] www.fbsblood.org On line

Univ. of Texas Health Science Center at San Antonio Linda Myers 210-731-5526 [email protected] www.uthscsa.edu On site

Univ. of Texas Medical Branch at Galveston Janet Vincent 409-772-3055 [email protected] www.utmb.edu/sbb On line

Univ. of Texas SW Medical Center Barbara Laird-Fryer

214-648-1785 [email protected] http://telecampus.utsystem.edu On line


IMMUNOHEMATOLOGY, Volume 26, Number 1, 201046I M M U N O H E M A T O L O G Y, V O L U M E 2 2 , N U M B E R 4 , 2 0 0 6 215

I. GENERAL INSTRUCTIONSBefore submitting a manuscript, consult current issues of

Immunohematology for style. Double-space throughout the manuscript.Number the pages consecutively in the upper right-hand corner, beginningwith the title page.

II. SCIENTIFIC ARTICLE, REVIEW, OR CASE REPORT WITHLITERATURE REVIEWA. Each component of the manuscript must start on a new page in thefollowing order:1. Title page2. Abstract3. Text4. Acknowledgments5. References6. Author information7. Tables8. Figures

B. Preparation of manuscript1. Title page

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

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

c. Running title of ≤40 characters, including spacesd. Three to ten key words

2. Abstracta. One paragraph, no longer than 300 wordsb. Purpose, methods, findings, and conclusion of study

3. Key wordsa. List under abstract

4. Text (serial pages): Most manuscripts can usually, but not necessarily,be divided into sections (as described below). Survey results andreview papers may need individualized sectionsa. Introduction

Purpose and rationale for study, including pertinent backgroundreferences

b. Case Report (if indicated by study)Clinical and/or hematologic data and background serology/molecular

c. Materials and MethodsSelection and number of subjects, samples, items, etc. studied anddescription of appropriate controls, procedures, methods, equipment,reagents, etc. Equipment and reagents should be identified inparentheses by model or lot and manufacturer’s name, city, and state.Do not use patient’s names or hospital numbers.

d. ResultsPresentation of concise and sequential results, referring to pertinenttables and/or figures, if applicable

e. DiscussionImplication and limitations of the study, links to other studies; ifappropriate, link conclusions to purpose of study as stated inintroduction

5. Acknowledgments: Acknowledge those who have made substantialcontributions to the study, including secretarial assistance; list any grants.

6. Referencesa. In text, use superscript, Arabic numbers.b. Number references consecutively in the order they occur in the text.

7. Tablesa. Head each with a brief title; capitalize the first letter of first word (e.g.,

Table 1. Results of . . .) use no punctuation at the end of the title.

b. Use short headings for each column needed and capitalize first letterof first word. Omit vertical lines.

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

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

b. Place caption for a figure on a separate page (e.g. Fig. 1 Results of. . . ),ending with a period. If figure is submitted as a glossy, place firstauthor’s name and figure number on back of each glossy submitted.

c. When plotting points on a figure, use the following symbols ifpossible: � � � � � �.

9. Author informationa. List first name, middle initial, last name, highest degree, position held,

institution and department, and complete address (including ZIPcode) for all authors. List country when applicable.

III. EDUCATIONAL FORUMA. All submitted manuscripts should be approximately 2000 to 2500words with pertinent references. Submissions may include:1. An immunohematologic case that illustrates a sound investigative

approach with clinical correlation, reflecting appropriate collaborationto sharpen problem solving skills

2. Annotated conference proceedingsB. Preparation of manuscript1. Title page

a. Capitalize first word of title.b. Initials and last name of each author (no degrees; all CAPs)

2. Texta. Case should be written as progressive disclosure and may include the

following headings, as appropriatei. Clinical Case Presentation: Clinical information and differential

diagnosisii. Immunohematologic Evaluation and Results: Serology and

molecular testingiii. Interpretation: Include interpretation of laboratory results,

correlating with clinical findingsiv. Recommended Therapy: Include both transfusion and

nontransfusion-based therapiesv. Discussion: Brief review of literature with unique features of this

casevi. Reference: Limited to those directly pertinentvii. Author information (see II.B.9.)viii. Tables (see II.B.7.)

IV. LETTER TO THE EDITORA. Preparation1. Heading (To the Editor)2. Title (first word capitalized)3. Text (written in letter [paragraph] format)4. Author(s) (type flush right; for first author: name, degree, institution,

address [including city, state, Zip code and country]; for other authors:name, degree, institution, city and state)

5. References (limited to ten)6. Table or figure (limited to one)

Send all manuscripts by e-mail to [email protected]

ImmunohematologyJOURNAL OF BLOOD GROUP SEROLOGY AND EDUCATION

Instructions to the Authors


Instructions for Authors

Musser Blood Center700 Spring Garden StreetPhiladelphia, PA 19123-3594

FIRST CLASSU.S. POSTAGE

PAIDSOUTHERN MD

PERMIT NO. 4144

vol. 26, no. 1, 2010

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