Journal of Blood Group Serology and Education

Immunohematology

Celebrating

2Yea5rs Volume 26, Number 1, 2010

Immunohematology Journal of Blood Group Serology and Education Volume 26, Number 1, 2010 Contents

Letter to the Readers 1 G.M. Meny

Review The Knops blood group system: a review 2 J.M. Moulds

Case Report Role for serial prenatal anti-Vel quantitative serologic monitoring 8 with 2-ME serum treatment W.J. Linz, J.T. Fueger, S. Allen, and S.T. Johnson

Review Granulocyte serology: current concepts and clinical significance 11 M.E. Clay, R.M. Schuller, and G.J. Bachowski

Review A review of the Colton blood group system 22 G.R. Halverson and T. Peyrard

Case Report 27 Alloimmunization to the D antigen by a patient with weak D type 21 H. McGann and R.E. Wenk

Review A review of the Chido/Rodgers blood group 30 R. Mougey

In Memoriam 39 Marie Cutbush Crookston (1920–2009) D. Mallory 40 In Memoriam Eloise Giblett (1921–2009) D. Mallory 41 Announcements

42 Advertisements

46 Instructions for Authors Editors-in-Chief Managing Editor Sandra Nance, MS, MT(ASCP)SBB Cynthia Flickinger, MT(ASCP)SBB Philadelphia, Pennsylvania Philadelphia, Pennsylvania

Connie M. Westhoff, PhD, MT(ASCP)SBB Technical Editors Philadelphia, Pennsylvania Christine Lomas-Francis, MSc New York City, New York Senior Medical Editor Geralyn M. Meny, MD Dawn M. Rumsey, ART (CSMLT) Philadelphia, Pennsylvania Glen Allen, Virginia

Associate Medical Editors David Moolten, MD Ralph R. Vassallo, MD Philadelphia, Pennsylvania Philadelphia, Pennsylvania

Editorial Board

Patricia Arndt, MT(ASCP)SBB Brenda J. Grossman, MD Mark Popovsky, MD Pomona, California St. Louis, Missouri Braintree, Massachusetts James P. AuBuchon, MD Christine Lomas-Francis, MSc Seattle, Washington New York City, New York Marion E. Reid, PhD, FIBMS New York City, New York Martha R. Combs, MT(ASCP)SBB Gary Moroff, PhD Durham, North Carolina Rockville, Maryland S. Gerald Sandler, MD Geoffrey Daniels, PhD John J. Moulds, MT(ASCP)SBB Washington, District of Columbia Bristol, United Kingdom Shreveport, Louisiana Jill R. Storry, PhD Anne F. Eder, MD Paul M. Ness, MD Lund, Sweden Washington, District of Columbia Baltimore, Maryland George Garratty, PhD, FRCPath Joyce Poole, FIBMS David F. Stroncek, MD Pomona, California Bristol, United Kingdom Bethesda, Maryland

Emeritus Editorial Board Delores Mallory, MT(ASCP)SBB Supply, North Carolina

Editorial Assistant Production Assistant Patti A. Brenner Marge Manigly Proofreader Copy Editor Lucy Oppenheim Electronic Publisher Mary L. Tod Wilson Tang

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

Immunohematology is indexed and included in Index Medicus and MEDLINE on the MEDLARS system. The contents are also cited in the EBASE/Excerpta Medica and Elsevier BIOBASE/Current Awareness in Biological Sciences (CABS) databases.

The subscription price is $40.00 (U.S.) and $50.00 (foreign) per year.

Subscriptions, Change of Address, and Extra Copies: Immunohematology, P.O. BOX 40325, Philadelphia, PA 19106 Or call (215) 451-4902 Web site: www.redcross.org/immunohematology

Letter to the Readers

G.M. Meny

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 description 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, MD Senior Medical Editor Immunohematology

On Our Cover El 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

IMMUNOHEMATOLOGY, Volume 26, Number 1, 2010 1 Review

The Knops blood group system: a review

J.M. Moulds

The Knops blood group system finds its roots in the The Knops blood group system began to expand when group of antibodies that were previously called high-titer, Molthan and Moulds6 described a new antigen, McCa, which low-avidity, or HTLA, antibodies. This term was used to de- seemed to be related to Kna. They reported that 53 percent scribe a group of alloantibodies that appeared to have many of McC(a–) samples were also Kn(a–). Interestingly, a ma- of the same characteristics, including very weak reactions jority of McCa antibody producers were Black whereas most at the anti-human globulin (AHG) phase of testing. Howev- of those making anti-Kna were Caucasian, thus suggesting er, as their specificities were more clearly defined, each was that ethnic differences might exist in their respective gene placed into a blood group system, and Knops became the frequencies. 22nd system recognized by the ISBT Committee on Termi- The next pair of alleles, Sla and Vil, was reported in nology for Red Cell Surface Antigens.1 Since that time, the separate abstracts with one author using the term McCc for protein bearing the Knops antigens has been identified, the Sla and McCd for Vil.7,8 The Sl terminology came from the gene has been cloned, and all of the known Knops antigens names of the first two antibody producers, i.e., Swain and have been characterized at the molecular level. This has al- Langley. It was preferred by these authors because they be- lowed investigators to more precisely study the role of the lieved that this antigen was independent from McCoy. After Knops blood group system in disease. identification of the protein bearing the Knops antigens, Sla was renamed S11 and Vil became S12.9 History The last high-incidence antigen identified in the Knops In 1965, the sera from three unrelated patients (Cope- system was KAM,10 later renamed KCAM by the ISBT Com- land, Stirling, and Wainright) were reported as having an mittee on Terminology for Red Cell Surface Antigens.11 The antibody with similar specificity.2 The antibody was named antibody producer was a Caucasian man who exhibited the after the first two antibody producers, Co and St (Cost), Helgeson phenotype, i.e., serologic Kn null. Like Sla, this and given the designation Csa. Ten years later a new anti- antigen showed a widely diverse frequency in Blacks vs. gen, York (Yka) was reported.3 The York serum was initially Caucasians (Table 1). Although KCAM is a high-frequency believed to be an example of anti-Csa until the RBCs of the antigen in Caucasians, only 20 percent of African Blacks donor were found to be Cs(a+). This same study found that were KCAM+. 12 of 1,246 Caucasian donors were both Cs(a–) and Yk(a–), suggesting that these antigens were at least phenotypically related. Neither, however, was given blood Table 1. Frequency of the Knops antigens in several ethnic groups group status, and they became part of the Population Kna Knb McCa McCb S11 S12 Yka KCAM Refs. HTLA group of antibodies. Even though Caucasians 98% 4% 98% 1% 99% <1% 90% 98% 3, 5, 8, Yka was thought to be associated with Csa 12, 13 at this time, it would later be assigned to West 100% NT 89–92% 49–54% 30–38% 95% NT 20% 10, 14 the Knops system. Africans Another similar antibody being inves- African 99% <1%* 90% 44% 51–61% 80%* 98% NT 8, 13, tigated in the 1960s was anti-Kna. Anti-Kna Americans 14 was described in a transfused Caucasian African 98% 2% 93% 42% 70% 86% NT 53% 12 woman who had a saline-reactive anti-K Brazilians plus an unidentified antiglobulin-reactive Asian 100% 0% 100% 0% 100% 0% NT 95% 12 antibody to a high-frequency antigen.4 Brazilians The antithetical Knb was later reported in *Marilyn Moulds, personal communication. Note: The publication by Covas et al.12 incorrectly lists 4870A the Hall serum, which contained a potent (isoleucine) as being KCAM negative. anti-Kpb and was being used to screen NT = not tested. Australian blood donors.5

2 IMMUNOHEMATOLOGY, Volume 26, Number 1, 2010 Knops blood group system

Knops Protein addition, when RBCs are stored, vesicles are budded from In 1991, two groups identified complement receptor one the membrane, and these vesicles contain CR1.21 Therefore, (CR1) as the protein carrying the Kna, McCa, and Sla blood the longer RBCs are stored, the weaker the Knops antigens group antigens.15,16 In addition, Moulds et al.15 also located become. This may explain why In(Lu) RBCs were initially Yka to CR1 and suggested that the Helgeson phenotype was reported to have weakened Knops antigens.22 attributable to low CR1 copy numbers on the erythrocytes (E-CR1). Several years later, Petty et al.17 confirmed these Table 2. Characteristics of the Knops antigens reports using monoclonal antibody-specific immobiliza- Inherited as Mendelian-dominant traits tion of erythrocyte antigens (MAIEA) analyses and also High-frequency RBC antigens (except Knb and McCb) confirmed that Csa was not on CR1. Thus, Csa and Csb have Developed on cord blood cells but may be weaker remained a collection as defined by ISBT. Weaker on stored RBCs Not destroyed by ficin or papain CR1 is a membrane-bound glycoprotein and, with the Destroyed by trypsin treatment exception of platelets, is found on most human periph- Not found on platelets eral blood cells. Depending on the methods used, erythro- Found in low levels in plasma but not in urine or saliva 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- Knops Antibodies ent in the peripheral circulation at concentrations 103-fold The Knops antibody characteristics are shown in Table higher than the peripheral blood mononuclear cells (PB- 3. Two characteristics that were often attributed to Knops MCs), they account for greater than 85 percent of CR1 in antibodies were (1) they were not neutralized with plasma, the blood. E-CR1 binds immune complexes (ICs) that are saliva, or urine, and (2) they were difficult to adsorb and shuttled to the liver or spleen for transfer to and ingestion elute. The latter most likely reflects the low density of the by macrophages, leading to their elimination. IC-free eryth- CR1 protein on the RBC membrane. However, Race and rocytes return to the circulation, where they may continue Sanger23 reported that adsorption performed with buffy participating in IC clearance. We have noted that individu- coats (WBCs) were able to remove anti-Kna from serum. als with high CR1 copy numbers may exhibit a weak false- This led to the speculation that anti-Kna and related speci- positive DAT in the gel technique, most likely as a result of ficities were WBC antibodies. The ability of antigen-positive increased binding of ICs. WBCs to adsorb Knops antibodies most likely relates to the fact that the CR1 copy number on WBCs is in the tens of Knops Antigen Characteristics thousands compared with a few hundred on RBCs. The Knops antigens can vary greatly in strength (Table 2), and weakly reactive cells can be falsely phenotyped as Table 3. Characteristics of Knops system antibodies negative if the cells are stored for some duration or have Titer is dependent on E-CR1 levels low E-CR1 numbers. Chemicals that can disrupt disulfide Can demonstrate variable reactions bonds, i.e., DTT and AET, can destroy Knops, McCoy, Not neutralized by pooled serum or other body fluids Swain-Langley, and York antigens because they destroy the Difficult to adsorb and elute from RBCs conformational structure of the short consensus repeats IgG reacting by AHG technique (SCRs). There are no examples of these antibodies that are Do not bind complement Usually not clinically significant 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- Although CR1 has not been found in the saliva, low rently identified are nonreactive with trypsin-treated RBCs. levels have been found in both urine24 and plasma.25 These It is known that a trypsin cleavage site exists in SCR 28 of are believed to be the result of proteolytic cleavage of CR1 the CR1 protein. Because the blood group antigens identi- from leukocytes.26 Serum CR1 is present only in nano- fied to date have been found in SCR 25, they are loston gram amounts,27 and therefore the levels are insufficient to trypsin treatment of the cells. This fact can be a useful tool neutralize Knops antibodies using routine serologic tech- not only in antibody identification but also for absorption to niques. Hence, Moulds and Rowe28 developed an inhibition remove other antibodies from a sample. technique using recombinant, soluble CR1 (sCR1). Because Although the variability in antigen strength can be in- their source of sCR1 was genetically positive for Kna, McCa, herited, it may also be acquired. Diseases that have high Sla, and Yka, it would only inhibit these antibodies and not levels of ICs being processed, e.g., systemic lupus erythe- inhibit anti-Knb or McCb. It must be remembered that the matosus or HIV infection, can cause an increased loss of Knops phenotype of the sCR1 will be dependent on the gene E-CR1, resulting in weakened Knops antigen strength.20 In chosen for its production. More recently, these investigators

IMMUNOHEMATOLOGY, Volume 26, Number 1, 2010 3 J.M. Moulds

have used mutated CR1 constructs to produce peptides ca- The third commonly recognized polymorphism is based pable of inhibiting anti-McCb and S12 (Vil).29 on quantitative differences in E-CR1. A HindIII RFLP is detected by two allelic fragments of 7.4 or 6.9 kb on South- Clinical Importance ern blots. Homozygotes for the 7.4-kb fragment are high Because the Knops antibodies are found in the serum CR1 expressors (H), and homozygotes for the 6.9-kb frag- of multiply transfused individuals, these sera often contain ment are low expressors (L) of CR1.32 Alternatively, a PCR- additional alloantibodies directed at (for example) K, E, RFLP can be used that results in a band of 1.8 kb for H or and Duffy antigens. Most of the Knops antibodies are not 1.3 and 0.5 kb for L alleles.33 Although this RFLP correlates considered clinically significant because they do not cause with RBC expression in Caucasian and Chinese individu- overt hemolytic transfusion reactions or hemolytic disease als,34 there is no relationship between this polymorphism of the fetus and newborn. Some examples of Knops sys- and CR1 expression in African Americans35 or West Afri- tem antibodies have been studied using in vitro tests such cans.36 The exact molecular mechanism regulating CR1 as the monocyte monolayer assay (MMA). Arndt and Gar- RBC expression has been elusive but does not appear to be ratty30 studied three anti-Kna, three anti-Yka, and five anti- part of the promoter or 3′ untranslated portions of the CR1 Kn/McC and found that only one had a macrophage index gene.37 of greater than 20, suggesting increased RBC destruction. Other MMA studies of two anti-Kna, two anti-Sla, one anti- Molecular Basis of Knops Antigens KCAM, and one anti-Yk/Cs all gave indexes of less than Using CR1 deletion constructs, Moulds et al.29 first lo- 0, indicating no enhanced RBC destruction (J.J. Moulds, calized the McCoy and S11 (Sla) antigens to LHR-D of CR1. unpublished data). By direct DNA sequencing they were then able to identify two separate mutations in SCR 25 that correlated with these The CR1 Gene two blood group antigens. The McCa/McCb polymorphism The CR1 gene resides on chromosome 1 (1q32) and is at base pair 4795, where an A encodes proline (McCa) comprises 39 exons spread out over approximately 133 and a G encodes aspartic acid (McCb). The S11/S12 muta- kilobase (kb) pairs of DNA.31 These exons encode SCRs tion is only 11 amino acids away; at base pair 4828, an A of approximately 60 amino acids in the functional CR1 encodes arginine and a G encodes glycine. Accordingly, the protein. Seven SCRs are organized into larger units called ISBT has now assigned these antigens to the Knops system long homologous repeats (LHRs). The most with the numbers shown in Table 4.38 common size protein product, CR1-1, is made Table 4. Molecular basis of the Knops antigens up of four LHRs (A, B, C, D), a transmembrane ISBT region (TM), and a cytoplasmic tail (CYT), as number Antigen Nucleotide Nucleotide† Amino acid shown in Figure 1. The binding sites for C3b KN1 Kna 4708G 4681G Va11561 and C4b have been localized to SCRs 8-9 and KN2 Knb 4708A 4681A Met1561 15-16 (LHRs B and C) and to SCRs 1-2 (LHR-A), respectively. KN3 McCa 4795A 4768A Lys1590 KN4 S11 4828A 4801A Arg1601 LHR-A LHR-B LHR-C LHR-D TM CYT KN5 Yka Unknown Unknown Unknown ●●●●●●●●●●●●●●●●●●●●●●●●●●●● ╫□ b SCR 1–7 SCR 8–14 SCR 15–21 SCR 22–27 KN6 McC 4795G 4768G Glu1590 KN7 S12 4828G 4801G Gly1601 Fig. 1. Schematic of the CR1-1 protein bearing the KN8 S13 (provisional) 4828A, 4855T* 4801A, 4828T* Arg1601, Ser1610 Knops antigens in SCR 25. KN9 KCAM 4870A 4843A Ile1615 The CR1 gene also exhibits two other poly- *Note: The publication by Moulds et al.39 incorrectly lists the 4855 mutation as morphisms besides the Knops blood group. A A>G. The correct substitution is T>A. structural, or size, polymorphism results from †Nucleotide number when nucleotides are counted beginning with the A of the AUG start codon four different genes encoding four different molecular weight proteins: 190 kDa (CR1*3), 220 kDa Two other mutations have been identified in SCR 25, (CR1*1), 250 kDa (CR1*2), and 280 kDa (CR1*4). Owing to one of which was found in a Caucasian and is related to the looping structure imparted by multiple disulfide bonds, S11. KMW was initially believed to have produced anti-Sla. the molecular weight shifts downward by approximately 30 However, after gene sequencing it was found that she was kDa when the proteins are separated on SDS-PAGE using not only homozygous for S11 but was homozygous for an- nonreducing conditions. It is now known that the molecu- other SNP at position 4855 that substituted threonine for lar weight polymorphism is independent from the blood serine at amino acid 1610. After extensive molecular stud- group polymorphism. ies, Moulds et al.39 proposed a model in which S13 was

4 IMMUNOHEMATOLOGY, Volume 26, Number 1, 2010 Knops blood group system

a conformational epitope needing S11 (1601R) and S14 Conclusions (1610S) to react. S14 and the antithetical antigen S15 are The molecular identification of the Knops blood group proposed epitopes awaiting the identification of the appro- polymorphisms holds the promise for a better means of typ- priate antibodies before they can be officially recognized. ing for these antigens, the only exception to this being the However, the related SNPs can be identified molecularly. yet unidentified Yka. Because of the inherited or acquired Limited population studies of Caucasians, Blacks, and changes in RBC expression of CR1, i.e., Knops antigens, Asians predict that S14 may be a high-frequency antigen in genotyping may become the method of choice for identify- all populations and that S15 predominantly occurs in Cau- ing these antigens, as it is for those of the Dombrock sys- casians.12,39 tem. It is interesting to note that all of the known Knops The final antigen assigned to the Knops system is KCAM polymorphisms are in SCR 25 of the CR1 protein. However, (initially reported as KAM). Interestingly, the SNP produc- additional SNPs have been found in the CR1 gene, which ing KCAM was already known but had not been associated suggests that the identification of new Knops blood group with a blood group specificity until a McCoy-like antibody antigens is not yet at an end and will surely provide a chal- was found in a Caucasian blood donor. On DNA sequenc- lenge to serologists in the future. ing, it was discovered that he was homozygous for an SNP at bp 4870 that substituted valine for isoleucine.10 Hence, References it was assigned the number KN9, and the antigen was re- 1. Daniels GL, Anstee DJ, Cartron JP, et al. Blood group named KCAM (KC for the city where the donor was found terminology 1995. Vox Sang 1995;69:265–79. and AM for the donor’s initials). 2. Giles CM, Huth MC, Wilson TE, Lewis HBM, Grove GEB. Three examples of a new antibody, anti-Csa, Disease Associations which reacts with 98% of red cell samples. Vox Sang In 1997, Rowe et al.40 identified CR1 as a ligand for 1965;10:405–15. the rosetting of Plasmodium falciparum–infected RBCs 3. Molthan L, Giles CM. A new antigen, Yka, and its rela- with uninfected cells. The ability of erythrocytes infected tionship to Csa (Cost). Vox Sang 1975;29:145–53. with P. falciparum to form rosettes is a property shown by 4. Helgeson M, Swanson J, Polesky HF. Knops-Helgeson only some parasite isolates, but is of importance because (Kna), a high frequency erythrocyte antigen. Transfu- it has been associated with severe malaria.41 These authors sion 1970;10:737–8. showed that CR1 on uninfected erythrocytes was required 5. Mallan MT, Grimm W, Hindley L, Knighton G, Moulds for the formation of rosettes by demonstrating that CR1- MK, Moulds JJ. The Hall serum: detecting Knb, the deficient erythrocytes (Helgeson phenotype) had reduced antithetical allele to Kna (abstract). Transfusion rosetting and soluble recombinant CR1 could inhibit ro- 1980;20:630. setting. RBCs having the Sl:–1 phenotype showed reduced 6. Molthan L, Moulds J. A new antigen, McCa (McCoy), binding to the parasite rosetting ligand PfEMP1.14 Thus, the and its relationship to Kna (Knops). Transfusion authors hypothesized that this polymorphism may have 1978;18:566–8. been selected for in malaria-endemic regions by provid- 7. Lacey P, Laird-Fryer B, Block U, Lair J, Guilbeau L. ing protection against severe malaria. The hypothesis was Moulds JJ. A new high incidence blood group factor, then tested by studies of Africans in Mali and Kenya, where Sla; and its hypothetical allele. (abstract).Transfusion it was found that the combined Knops haplotype Kn(a+)/ 1980;20:632. McC(a–)/Sl:–1 appeared to be protective. 8. Molthan L. Expansion of the York, Cost, McCoy, Knops CR1, as well as other complement receptors, has been blood group system: the new McCoy antigens McCc and identified as a receptor facilitating cell entry for a variety McCd. Med Lab Sci 1983;40:113–21. of pathogenic organisms. Pathogens using CR1 include 9. Daniels GL, Cartron JP, Fletcher A, et al. International Leishmania major (monocyte-macrophage),42 Legion- Society of Blood Transfusion Committee on terminol- ella pneumophila (monocyte-macrophage),43 Leishmania ogy for red cell surface antigens: Vancouver report. Vox panamensis,44 and Mycobacterium tuberculosis (mono- Sang 2003;84:244–7. cyte-macrophage).45 In an AABB abstract, Noumsi et al.46 10. Moulds JM, Pierce S, Peck KB, Tulley ML, Doumbo O, reported that individuals heterozygous for McCa and McCb Moulds JJ. KAM: a new allele in the Knops Blood Group were less likely to be infected by M. tuberculosis (odds ra- System (abstract). Transfusion 2005;45(Suppl):27A. tio, 0.42; 95% confidence interval, 0.22 to 0.81; P = 0.007). 11. Daniels G, Castilho L, Flegel WA. International Society These results suggested that McCb may have evolved among of Blood Transfusion Committee on terminology for African populations in the context of M. tuberculosis pres- red cell surface antigens: Cape Town report. Vox Sang sure, and conferred a survival advantage in its heterozygous 2007;92:250–3. form.

IMMUNOHEMATOLOGY, Volume 26, Number 1, 2010 5 J.M. Moulds

12. Covas DT, de Oliveira FS, Rodrigues ES, Abe-Sandes 28. Moulds JM, Rowe KE. Neutralization of Knops system K, Silva WA Jr, Fontes AM. Knops blood group hap- antibodies using soluble complement receptor 1. Trans- lotypes among distinct Brazilian populations. Transfu- fusion 1996;36:517–20. sion 2007;47:147–53. 29. Moulds JM, Zimmerman PA, Doumbo OK, et al. Molecu- 13. Molthan L. The serology of the York-Cost-McCoy-Knops lar identification of Knops blood group polymorphisms red blood cell system. Am J Med Technol 1983;49:49– found in long homologous region D of complement re- 56. ceptor 1. Blood 2001;97:2879–85. 14. Moulds JM, Kassambara L, Middleton JJ. Identifica- 30. Arndt PA, Garratty G. A retrospective analysis of the tion of complement receptor one (CR1) polymorphisms value of monocyte monolayer assay results for predict- in west Africa. Genes Immun 2000;1:325–9. ing the clinical significance of blood group alloantibod- 15. Moulds JM, Nickells MW, Moulds JJ, Brown M, At- ies. Transfusion 2004;44:1273–81. kinson JP. The C3b/C4b receptor is recognized by the 31. Vik DP, Wong WW. Structure of the gene for the F al- Knops, McCoy, Swain-Langley, and York blood group lele of complement receptor type 1 and sequence of sera. J Exp Med 1991;173:1159–63. the coding region unique to the S allele. J Immunol 16. Rao N, Ferguson DJ, Lee SF, Telen MJ. Identification 1993;151:6214–24. of human erythrocyte blood group antigens on the C3b/ 32. Wilson JG, Murphy EE, Wong WW, Klickstein LB, C4b receptor. J Immunol 1991;146:3501–7. Weis JH, Fearon DT. Identification of a restriction 17. Petty AC, Green CA, Poole J, Daniels GL. Analysis of fragment polymorphism by a CR1 cDNA that correlates Knops blood group antigens on CR1 (CD35) by the with the number of CR1 on erythrocytes. J Exp Med MAIEA test and by immunoblotting. Transfus Med 1986;164:50–9. 1997;7:55–62. 33. Cornillet P, Philbert F, Kazatchkine MD, Cohen JHM. 18. Moulds MK. Serological investigation and clinical sig- Genomic determination of the CR1 (CD35) density nificance of high-titer, low-avidity (HTLA) antibodies. polymorphism on erythrocytes using polymerase chain Am J Med Technol 1981;10:789–95. reaction amplification and HindIII restriction enzyme 19. Reid ME, Lomas-Francis C. Knops blood group system. digestion. J Immunol Methods 1991;136:193–7. In: The blood group antigen factsbook. 2nd ed. San Di- 34. Moulds JM, Brai M, Cohen JHM, et al. Reference typ- ego, CA: Academic Press, 2004:439–54. ing report for complement receptor 1 (CR1). Exp Clin 20. Holme E, Fyfe A, Zoma A, Veitch J, Hunter J, Whaley Immunogenet 1998;15:291–4. K. Decreased C3b receptors (CR1) on erythrocytes from 35. Herrera AH, Xiang L, Martin SG, Lewis J, Wilson JG. patients with systemic lupus erythematosus. Clin Exp Analysis of complement receptor type 1 (CR1) Immunol 1986;63:41–8. expression on erythrocytes and of CR1 allelic markers 21. Pascual M, Lutz HU, Steiger G, Stammler P, Schifferli in Caucasian and African American populations. Clin JA. Release of vesicles enriched in complement receptor Immunol Immunopathol 1998;87:176–83. 1 from human erythrocytes. J Immunol 1993;151:397– 36. Rowe JA, Plowe CV, Diallo DA, et al. Erythrocyte com- 404. plement receptor 1 expression level does not correlate 22. Daniels GL, Shaw MA, Lomas CG, Leak MR, Tippett P. with HindIII restriction fragment length polymorphism The effect of In(Lu) on some high-frequency antigens. in ; implications for studies on malaria susceptibility. Transfusion 1986;26:171–2. Genes Immun 2002;8:497–500. 23. Race RR, Sanger R. Some very frequent antigens. In: 37. Cockburn IA, Rowe JA. Erythrocyte complement recep- Blood groups in man. 6th ed. Oxford: Blackwell Scien- tor 1 (CR1) expression level is not associated with poly- tific Publications, 1975:410–30. morphisms in the promoter or 3′ untranslated regions 24. Pascual M, Steiger G, Sadallah S, et al. Identification of the CR1 gene. Int J Immunogenet 2006;33:17–20. of membrane-bound CR1 (CD35) in human urine: ev- 38. Daniels GL, Anstee DJ, Cartron JP, et al. Interna- idence for its release by glomerular podocytes. J Exp tional Society of Blood Transfusion working party on Med 1994;179:889–99. terminology for red cell surface antigens. Vox Sang 25. Yoon SH, Fearon DT. Characterization of a soluble form 2001;80:193–6. of the C3b/C4b receptor (CR1) in human plasma. J Im- 39. Moulds JM, Zimmerman PA, Doumbo OK, et al. Ex- munol 1985;134:3332–8. pansion of the Knops blood group system and subdivi- 26. Danielsson C, Pascual M, Larsen B, Steiger G, Schifferli sion of Sla. Transfusion 2002;42:251–6. JA. Soluble complement receptor type 1 (CD35) is re- 40. Rowe JA, Moulds JM, Newbold CI, Miller LH. P. falci- leased from leukocytes by surface cleavage. Eur J Im- parum rosetting mediated by a parasite-variant eryth- munol 1994;24:2725–31. rocyte protein and complement-receptor 1. Nature 27. Pascual M, Duchosal MA, Steiger G, et al. Circulating 1997;388:292–5. soluble CR1 (CD35). J Immunol 1993;151:1702–11.

6 IMMUNOHEMATOLOGY, Volume 26, Number 1, 2010 Knops blood group system

41. Rowe JA, Obeiro J, Newbold CI, Marsh K. Plasmodium 45. Schlesinger LS, Bellinger-Kawahara CG, Payne NR, falciparum rosetting is associated with malaria severity Horwitz MA. Phagocytosis of Mycobacterium tuber- in Kenya. Infect Immun 1995;63:2323–6. culosis is mediated by human monocyte complement 42. Da Silva RP, Hall BF, Joiner KA, Sacks DL. CR1, the receptors and complement component C3. J Immunol C3b receptor, mediates binding of infective Leishmania 1990;144:2771–80. major metacyclic promastigotes to human macrophag- 46. Noumsi GT, Tounkara A, Diallo D, Moulds JM. Knops es. J Immunol 1989;143:617–22. blood group polymorphism and protection from My- 43. Payne NR, Horwitz MA. Phagocytosis of legionella cobacterium tuberculosis (abstract). Transfusion pneumophila is mediated by human monocyte comple- 2006;46(Suppl):19A. ment receptors. J Exp Med 1987;166:1377–89. 44. Robledo S, Wozencraft A, Valencia AZ, Saravia N. Hu- Joann M. Moulds, PhD, MT(ASCP)SBB, Director, Clinical man monocyte infection by Leishmania (viannia) pan- Immunogenetics, LifeShare Blood Centers, 8910 Linwood amensis. J Immunol 1994;152:1265–75. Avenue, Shreveport, LA 71106.

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

Attention: State Blood Bank Meeting Organizers For information concerning Immunohematology, Journal of Blood Group Serology and Education, or If you are planning a state meeting and would like cop- the Immunohematology Methods and Procedures ies of Immunohematology for distribution, please send manual, contact us by e-mail at immuno@usa. request, 4 months in advance, to immuno@usa. redcross.org redcross.org.

IMMUNOHEMATOLOGY, Volume 26, Number 1, 2010 7 Case Report

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. reticulocytosis. As a corollary to the van Gammeren case re- Its clinical significance and management in the prenatal setting are port, we present a case of an anti-Vel for which the IgG titer not well characterized. We present a case that demonstrates the as determined by 2-ME treatment was stable through preg- utility of serial prenatal anti-Vel quantitative serologic monitoring nancy despite a significant rise in the titer of the anti-Vel in with 2-ME serum treatment during pregnancy. The patient is a the untreated serum. At birth, the child showed no evidence 23-year-old Hispanic woman with history of prior pregnancy and prior transfusion who was discovered to have an antibody to the of HDFN. This case report supports the importance of serial high-prevalence Vel antigen in the first trimester (week 7) of her determination of the prenatal IgG titer to help assess risk second pregnancy. Interval measurements of the serologic antibody of HDFN. titers were performed during the next 26 weeks. The untreated serum (IgM and IgG) titer increased from a baseline of 4 to 16 during Case Report that interval, while the 2-ME (presumed IgG component) titer re- A 23-year-old Hispanic woman, with a history of prior mained stable at 4. Responding to ultrasound findings suspicious blood transfusion and one successful pregnancy, presented for fetal anemia, the child was delivered without complications at initially early in the first trimester of the index pregnancy. 34 weeks’ gestation. At birth, the DAT was negative and there was Her RBCs were typed as group O, D+. Routine serologic no evidence of HDN. Placed in the context of other similar reports, testing demonstrated variable reactivity using gel technolo- this case demonstrates the importance of separately reporting the IgG fraction (after either DTT treatment or 2-ME treatment) from gy. The autocontrol was negative. Further testing using tube the untreated (IgM and IgG) fraction and the importance of cor- technology with PEG as enhancement also demonstrated relating the treated serum titer with potential clinical significance. variable weak reactivity, again with no specificity. No reac- Immunohematology 2010;26:08–10. tivity was demonstrated when using tube technology with LISS. A repeat serologic evaluation was performed 4 weeks Key Words: Vel antigen collection, anti-Vel, prenatal serologic later to help clarify the nature of this reactivity. The subse- monitoring, 2-ME-treated serum, pregnancy quent serologic evaluation demonstrated reactivity with all RBCs tested whether using gel or test tube technology with el is a high-prevalence antigen first described by LISS or PEG methods. The autocontrol was consistently Sussman et al. in 1952 in association with a trans- negative. As an antibody to a high-prevalence antigen was 1 fusion reaction. Although several additional cases suspected, the sample was referred to a large nationally rec- V 2 quickly confirmed these authors’ findings, our knowledge ognized immunohematology reference laboratory. With use of the Vel antigen (collection) remains incomplete. The of rare RBCs lacking various high-prevalence antigens, re- presence of the Vel-negative phenotype in the general pop- activity was determined to be that of anti-Vel. The patient’s 3 ulation is estimated to be 1 in 4000 individuals. Vel alloim- RBCs were shown to lack the Vel antigen. The initial quan- munization can be mediated through IgG or IgM. Anti-Vel titative serologic titer was 4 when tested against Vel+ RBCs is thought to be associated rarely with hemolytic disease of using both 2-ME-treated and untreated serum. The Marsh the fetus and newborn (HDFN) because many examples of score ranged from 14 with untreated serum to 4 with 2-ME- anti-Vel are mostly IgM and Vel is weakly expressed on the treated serum. The developing baby’s RBCs were presumed surface of fetal RBCs. Although the risk may be low, it is rea- to be Vel+ as the father’s RBCs were demonstrated to ex- sonable to monitor prenatal serologic titers serially during press the Vel antigen. Subsequent quantitative serologic 4 pregnancy. The findings published in a recent case report monitoring was performed in approximately 4- to 6-week 5 by van Gammeren et al., of severe HDFN from anti-Vel, intervals for the next 20 weeks of pregnancy (Table 1). support this approach. The van Gammeren case report de- At 33 2/7 weeks’ gestation, Doppler ultrasonography scribes discovery in a prenatal patient of an anti-Vel, which showed an increase in middle cerebral artery velocity (>1.5 at initial presentation was determined to be composed ex- multiples of the median [MoM] for gestational age) suspi- clusively of IgM. At the end of the pregnancy, an anti-Vel cious for fetal anemia. This finding was a significant change titer of 64 was demonstrated in untreated serum, whereas from previous Doppler ultrasound studies and correspond- the DTT-treated serum (presumed IgG content) titer rose ed with the change in quantitative serologic titer of the to 16. In their case, the infant exhibited severe jaundice and

8 IMMUNOHEMATOLOGY, Volume 26, Number 1, 2010 Role for anti-Vel prenatal monitoring

untreated serum from 4 at presentation to 16 by week 26 performed at the immunohematology reference laboratory (Table 1). A multidisciplinary risk-benefit analysis, includ- used tube method. The patient’s serum was diluted in 6% ing consideration of potential maternal and fetal need for albumin. A 30-minute 37°C incubation was performed, fol- transfusion with an extremely limited blood supply (two lowed by a saline IAT (Anti-IgG, ImmucorGamma Inc.).6 available units), the unpredictable timing issues encoun- Reaction scoring was performed using the system originally tered with induction, and the limited time-availability for described by Marsh.7 The patient’s serum was treated with transfusion after thawing the available units, prompted 2-ME (Fisher Scientific Co., Hanover Park, IL) and dialyzed the decision to move toward delivery in a manner that op- overnight using dialysis membranes (Spectra/Por2, 12,000 timized the availability of blood for the newborn, if it was to 14,000 dalton molecular weight cut-off [MWCO], Spec- indeed anemic. The patient underwent cesarean delivery at trum Laboratories, Inc., Rancho Domingtuez, CA) in PBS, 34 3/7 weeks’ gestational age, 2 days after the initial admin- pH 7.3. Dialyzed serum was tested using the methods previ- istration of Celestone to promote fetal pulmonary maturity. ously described. A 3088-g male newborn with Apgar scores of 7 at 1 minute and 9 at 5 minutes was born without complication. The Discussion child’s RBCs typed as group A, D+, and the antibody screen This case adds to the limited published information was negative. Initial laboratory data were also remarkable regarding the natural history of anti-Vel in the prenatal for a Hb level of 16.6 g/dL (normal range), reticulocyte count setting, and it supports the value of sorting immunoglobu- of 3.5% (normal range), and negative DAT. Approximately lin classes as an aid to proper prenatal management. It is 1 week after delivery, a mild rise in bilirubin was observed, worth noting that at initial presentation, weak, variable se- reaching a peak of 10 mg/dL. Given that the DAT and the rologic reactivity was demonstrated. Despite use of several initial screen were negative, the mild hyperbilirubinemia standard methods, specificity could not be assigned to the was thought not likely to have resulted from transplacen- serologic reactivity. In the initial serologic evaluation, reac- tal anti-Vel or a possible anti-A from a group O mother. tivity was present using gel technology and tube technology Moreover, although laboratory data are a bit limited, there with PEG as enhancement. Tube testing with LISS showed was no evidence of anemia or hemolysis. Importantly, the no reactivity. Although a LISS tube method is considered mild hyperbilirubinemia quickly resolved, and the newborn an appropriate antibody detection method in the prenatal was released to home and is doing well approximately 10 setting,8 this method would not have detected the antibody. months after birth. A repeat study approximately 7 weeks after the initial study did demonstrate the presence of strong serologic reactivity Table 1. Quantitative serologic monitoring using tube testing with LISS and PEG as well as with gel. Approxi- 2-ME- 2-ME- The titer of the untreated serum and 2-ME–treated serum mate ges- Sero- Untreated Untreated treated treated was 4. Presumably, late in the first trimester of pregnancy tational logic serum serum serum serum age (wk) findings titer score titer score the mother was exposed to fetal RBCs expressing the Vel antigen because by week 14 she demonstrated a specific se- 7 Weak, N/A N/A N/A N/A variable rologic immune response. Interestingly, for the remainder reactivity of the pregnancy, the titer of the anti-Vel in the 2-ME (pre- 14 Anti-Vel 4 24 4 24 sumed IgG component)–treated serum remained stable al- 18 Anti-Vel 4 27 4 14 though the titer in the untreated serum (presumed IgM and IgG) increased by two dilutions. This observation may be 22 Anti-Vel 8 N/T N/T N/T useful to laboratories without the capacity to treat patient 26 Anti-Vel 16 25 4 18 serum with 2-ME or DTT. Namely, in this case anti-Vel titer 34 Anti-Vel 16 30 4 14 in the untreated serum appeared to rise before the anti-Vel N/A = not applicable; N/T = not tested. IgG titer in the 2-ME–treated serum. The rise in antibody titer in the untreated serum com- Methods bined with the abnormal Doppler velocimetry prompted ABO and D testing and DAT were performed using the preterm delivery. At birth, the DAT and antibody screen commercially available reagents according to manufactur- were negative, and the hemoglobin and reticulocyte count ers’ protocols using tube method (Immucor Gamma Inc., were in the normal range. As the anti-Vel present in the ma- Norcross, GA). Antibody detection and identification were ternal serum contained an IgG component, the negative DAT performed using either gel technology according to the man- suggests that Vel antigen on the fetal RBCs may either be ufacturer’s protocol using reagent cells (RBCs 0.8% Surgis- weakly or partially expressed9 or the antibody avidity to the creen and reagent RBCs 0.8% Resolve Antigram, ID-MTS antigen was poor. Regardless, in this case, an anti-Vel titer of Gel Test; Ortho-Clinical Diagnostics, Raritan, NJ) or tube 4 does not appear to be independently sufficient for HDFN, method with commercially available cells and reagents whereas a titer of 16 in the case reported by van Gammeren (PEG and LISS, Immucor Gamma Inc.). Titration studies et al.5 was associated with HDFN. The cause of the rise in

IMMUNOHEMATOLOGY, Volume 26, Number 1, 2010 9 W.J. Linz et al. the bilirubin level after birth remains uncertain, although 7. Marsh WL. Scoring of hemagglutination reactions. liver immaturity is the most likely explanation. Finally, the Transfusion 1972;12:352–3. abnormal Doppler velocimetry in the absence of true fetal 8. Judd WJ; Scientific Section Coordinating Commit- anemia reflects the inherent limitations of a screening test tee of the AABB. Practice guidelines for prenatal and that is known to have a 12 percent rate of false-positive re- perinatal immunohematology, revisited. Transfusion sults.10 2001;41:1445–52. The Vel antigen (collection) and anti-Vel remain enig- 9. Reid ME, Lomas-Francis C. The blood group antigen matic and are in need of further study. To date, it is known factsbook. 2nd ed. San Diego, CA: Academia Press, that the Vel-negative phenotype is universally uncommon. 2004:503-4. The incidence of the Vel-negative phenotype in the Hispanic 10. Giancarlo M. Noninvasive diagnosis by Doppler ultra- population is not known with certainty. Anti-Vel is famous- sonography of fetal anemia due to maternal red cell ally associated with in vitro hemolysis, severe transfusion re- loimmunization. N Engl J Med 2000;42:9–14. actions, and shortened RBC survival,11 although there is at 11. Tilley CA, Crookston MC, Haddad SA, Shumak KN. Red least one case report of Vel+ blood being successfully trans- blood cell survival studies in patients with anti-Cha, fused to a recipient possessing anti-Vel.12 An auto-anti-Vel13 anti-Yka, anti-Ge and anti-Vel. Transfusion 1977;17:169– has been described, although it is uncertain to what degree 72. the autoantibody contributed to the patient’s RBC destruc- 12. Issitt PD, Anstee DJ. Applied blood group serology. 4th tion. Anti-Vel can be difficult to detect, and it can simulate ed. Durham, NC: Montgomery Scientific Publications, the serologic profile of a cold-reactive autoantibody, with 1998: 802. disastrous consequences if improper analytic methods are 13. Becton DL, Kinney TR. An infant girl with severe auto- used.14 The Vel antigen appears to have a variable antigenic immune hemolytic anemia: apparent anti-Vel specific- expression and appears to be best expressed on adult cells. ity. Vox Sang 1986;51:108–11. When viewed in concert with the recent case report by 14. Storry JR, Mallory D. Misidentification of anti-Vel due van Gammeren et al.,5 our case report further supports the to inappropriate use of prewarming and adsorption need not only to properly identify anti-Vel in the prenatal techniques. Immunohematology 1994;10:83–6. setting but also to perform serial quantitative serologic monitoring using methods to clearly separate the IgG frac- Walter J. Linz, MD, MBA, Medical Director, Scott and tion (DTT treatment or 2-ME treatment) from the untreat- White Transfusion Service and Donor Center, Assistant ed (IgM and IgG) fraction. Although both the untreated Professor of Pathology, Texas A&M UHSC, 2401 South 31st and treated serum titer and score results may be reported, Street, Temple, TX 76508; Judith T. Fueger, MT(ASCP)SBB, changes in the treated serum fraction, representing the IgG Immunohematology Reference Laboratory, Diagnostic component, may be a better trigger to initiate additional Laboratories, BloodCenter of Wisconsin, Milwaukee, WI; clinical investigation. Additional cases may help identify Steven Allen, MD, Associate Professor, Texas A&M UHSC/ the most appropriate critical 2-ME or DTT titer for anti- Scott and White Memorial Hospital, Chair, Department Vel. of Ob/Gyn, Director, Division of Obstetrics and Gynecol- ogy, Scott & White Healthcare and Texas A&M University References Health Science Center College of Medicine, Temple, TX; 1. Sussman LN, Miller EB. New blood factor: Vel. Rev He- and Susan T. Johnson, MSTM, MT(ASCP)SBB, Immuno- matol 1952;7:368–71. hematology Reference Laboratory, Diagnostic Laborato- 2. Levine P, Robinson EA, Herrington LB, Sussman LN. ries, BloodCenter of Wisconsin, Milwaukee, WI. Second example of the antibody for the high-incidence blood factor Vel. Am J Clin Pathol 1955;25:751–4. 3. Issitt PD, Oyen R, Reihart JK, Adebahr ME, Allen FH, Kuhns WJ. Anti-Vel 2, a new antibody showing heterogeneity of Vel system antibodies. Vox Sang 1968;15:125–32. 4. Tarsa M, Kelly TF. Managing a pregnancy with antibodies: a clinician’s perspective. Immunohematology 2008;24:52–7. Notice to Readers 5. van Gammeren AJ, Overbeeke MA, Idema RN, van Beek RH, Ten Kate-Booij MJ, Ermens AA. Haemolytic Immunohematology, Journal of Blood Group disease of the newborn because of rare anti-Vel. Trans- Serology and Education, is printed on acid-free fus Med 2008;18:197–8. paper. 6. Roback JD. Technical manual. 16th ed. Bethesda, MD: AABB 2008: 900.

10 IMMUNOHEMATOLOGY, Volume 26, Number 1, 2010 Review

Granulocyte serology: current concepts and clinical signifcance

M.E. Clay, R.M. Schuller, and G.J. Bachowski

Applying serologic procedures to the detection of RBC and lym- Clinical Significance phocyte antigens has facilitated the identification of granulo- Alloimmune Neonatal Neutropenia cyte antigens with established clinical significance, which are Neutropenia observed in the neonate is primarily the now classified in the human neutrophil antigen system. Granu- result of neutrophil-specific alloantibodies transplacentally locyte alloantibodies and autoantibodies have been implicated transferred to the fetus from the maternal circulation. The in a variety of clinical conditions including alloimmune neutropenia, autoimmune neutropenia, febrile and severe pulmonary presence of these human neutrophil antigen (HNA) anti- transfusion reactions, drug-induced neutropenia, refractori- bodies most likely is the result of prenatal maternal sensi- ness to granulocyte transfusions, and immune neutropenia tization by fetal neutrophils crossing the placental barrier, after hematopoietic stem cell transplantation. Although the although the antibodies may have also arisen in association intrinsically fragile nature of granulocytes contributes to the in- with autoimmune diseases such as systemic lupus herent challenges of granulocyte serology, several advances in erythematosus or rheumatoid arthritis.2,3 Mater- laboratory procedures have improved detection of granulocyte nal alloimmunization can occur anytime after the first antibodies. This review will provide a current perspective about trimester of pregnancy. Once present in the fetal circula- the importance and use of granulocyte serology for detection tion, maternal HNA antibodies will bind to the mature fetal of granulocyte antibodies that have significant medical effects. Immunohematology 2010;26:11–21. cells expressing the corresponding neutrophil alloantigens, as the neutrophil precursor cells are spared.4 Neonates af- Key Words: granulocyte, neutrophil, antigens, antibodies, HNA, fected by alloimmune neonatal neutropenia (ANN) are al- alloimmune neonatal neutropenia, autoimmune neutropenia, most always neutropenic at birth, although cases have been transfusion-related acute lung injury, TRALI reported in which the neutropenia has been delayed by 1 to 3 days.4 ANN is often asymptomatic and goes undiscovered t has now been half a century since Lalezari1 described unless the deficit of the neutrophils is profound enough the first case of neonatal neutropenia attributable to (absolute neutrophil count < 1.5 × 109/L) that the newborn Itransplacental transfer of granulocyte-specific antibod- becomes susceptible and succumbs to infection caused by ies, thus initiating the field of granulocyte serology. This bacterial or fungal pathogens. These children commonly event fostered several decades of work directed at the iden- present with fever, malaise, lethargy, skin infections such tification of granulocyte-specific antigens (and antibodies), as cellulitis and omphalitis, mucosal and respiratory infec- methods for their detection, and an appreciation for their tions (stomatitis, otitis media, and pneumonia), urinary clinical significance. In many ways, granulocyte serology infections, and very rarely septicemia. These infections are developed along the laboratory footprints of RBC and hu- usually mild, but fatalities have been reported in 5 percent man lymphocyte antigen (HLA) serology with one major of cases.5 As there are numerous reasons for neonatal neu- exception—granulocytes could not be preserved for testing tropenia (i.e., congenital, antibody-mediated destruction, purposes. The requirement for fresh cells was and contin- or infection that decreases hemopoietic cell production), ues to be a major impediment for wide-scale implementa- detection of these maternal neutrophil antibodies is very tion of granulocyte immunobiology studies. Although the important for determining the mechanism causing neutro- number of laboratories worldwide that specialize in this penia. This allows the physician to focus on the appropriate technology is limited, the clinical significance of granulo- treatment. The presence of maternal antibodies can persist cyte antibodies has not diminished and is now supporting up to 6 months after birth although antibodies and clini- the development of new methodologies that have the poten- cal effects usually dissipate more quickly. The incidence of tial to enhance the utility of testing and growth of this field. ANN has been estimated to be 0.1 to 0.2 percent.6,7 Cur- Therefore, as we move ahead, it is important to (1) focus rently, neutrophil antibodies with the following specificities on the clinical events that support the need for granulocyte have been implicated in cases of ANN: HNA-1a,8–10 HNA- serology, (2) understand the benefits and limitations of the 1b,11 HNA-1c,12 FcγRIIIb (CD16),13,14 HNA-2a,15 HNA-3a,16 current serologic assays, and (3) evaluate the application of and HNA-4a.17 new technologies for the detection of granulocyte antigens and antibodies.

IMMUNOHEMATOLOGY, Volume 26, Number 1, 2010 11 M.E. Clay et al.

Autoimmune Neutropenia The occurrence of febrile reactions has been mitigated with Autoimmune neutropenia (AIN) occurs in both in- the use of leukocyte-reduced RBCs and platelet products. fants and adults. It is the most common form of chronic As early as 1951 blood transfusions were implicated in neutropenia in infants. Severe neutropenia is usually recog- noncardiogenic lung edema.26 The term TRALI was con- nized 4 to 7 months after birth.4 In the vast majority of AIN ceived in 1985 when Popovsky and Moore27 investigated a cases infants exhibit relatively mild clinical issues such as series of 36 patients with well-defined transfusion-related stomatitis, otitis, and respiratory infections. The presence acute lung injury. They detected leukocyte (HLA and HNA) of clinical symptoms lessens as the child becomes older. antibodies in 89 percent of the implicated donors. Seventy- Neutropenia in infants is self-limited with complete reso- two percent of these patients were treated with mechanical lution commonly observed within 7 to 24 months.18 How- ventilation, and 6 percent died. The leading theories for the ever, this condition necessitates aggressive hygienic care of cause of TRALI involve the priming or activation of neu- infants to prevent infection and vigorous therapy that may trophils with antigen-antibody interactions, inducing an include severe antibiotic therapy along with treatment with acute inflammatory response.28 Antibodies to HLA class I recombinant G-CSF and IVIG when infection occurs. Cell and class II as well as HNAs have all been implicated in the destruction can occur in the peripheral circulation in addi- development of TRALI.28 tion to interference with myelopoiesis in the bone marrow. Even though HLA class I alloantibodies are the most Complete blood counts demonstrate absolute neutropenia, frequently encountered WBC antibodies in implicated blood although random fluctuations in the neutrophil count can donors, they may act as much weaker triggers of acute lung range from zero to near normal. injury when compared with HLA class II and HNA antibod- AIN is also observed in adults. Monocytosis with or ies. In a study designed to investigate leukocyte antibody without lymphopenia may also be present.19 As in infants, specificities in severe TRALI reactions, Reil et al.29 reported the bone marrow commonly demonstrates an absence of that even though 73 percent of leukocyte antibodies identi- mature cells with an increase in myeloid precursors. This fied in their donor population were HLA class I, HLA class apparent imbalance of precursors to mature segmented II and HNA antibodies were associated with 81 percent of neutrophils is distinct in cause from the maturation arrest the antibody-mediated TRALI cases.29 Specifically, in their that occurs in congenital defects of neutrophils or hemato- 36 reported cases, 17 were elicited by blood products con- logic malignancy. Unlike RBC autoantibodies, antibodies in taining HLA class II antibodies, and HNA antibodies were AIN are often reported to have specificity that can include implicated in 10 cases. Of the 10 fatal outcomes, 6 were HNA-1a, HNA-1b, and HNA-2a.18–20 linked to HNA-3a antibodies in the transfused blood products, and HLA class II antibodies were associated with 3 fa- Drug-Induced Neutropenia talities. Although neutrophil antibodies are believed to be in- Although leukocyte agglutinating (aggregating) anti- volved in drug-induced neutropenia, neither the precise bodies have been frequently associated with TRALI,30–34 mechanisms nor the particular antigens on the cell surface the ability of HNA-3a antibodies to also prime and activate have yet been clarified. Some cases of drug-induced granu- neutrophils in the pulmonary vasculature is thought to be locytopenias probably result from direct marrow toxicity, central to severe and fatal TRALI.35 Studies have now shown although immune-mediated processes also occur. Quinine, that in addition to HNA-3a antibodies, HNA-2a and HNA- quinidine, ibuprofen, and psychotropic medications such 4a antibodies also have the ability to prime neutrophils in as clozapine are recognized as potential causes of neutrope- vitro.36–38 nia.21–23 Drug-dependent antibodies have been found to re- Although the pathophysiology of TRALI appears com- act with both granulocytes and granulocyte precursors. The plex and remains under investigation,39 substantial clinical FcγRIIIb and CD177 neutrophil glycoproteins have been and scientific information suggests that HLA (especially reported to form neoantigens with drugs or their metabo- class II) and HNA antibodies do play a significant role in lites, which are recognized by drug-dependent neutrophil this disorder.28 Efforts are now being focused on detecting antibodies. HLA and HNA antibodies in donors both to retrospectively investigate the cause of acute lung injury in blood recipients Transfusion Reactions and to serve as a valuable strategy in preventing TRALI by Antibodies to neutrophil and HLA antigens can result identifying donors who may be responsible for the transfu- in a variety of transfusion reactions. The spectrum of sever- sion reaction. ity can range from mild febrile reactions to death in the case of severe transfusion-related acute lung injury (TRALI). Re- Granulocyte Antigens cently TRALI has been shown to be the most common cause Granulocyte-specific antigens are those with a tissue of transfusion-related fatalities in the United States.24 distribution restricted to granulocytes (neutrophils, eosino- Febrile nonhemolytic transfusion reactions are com- phils, and basophils), whereas neutrophil-specific antigens mon and can occur when blood products containing WBCs are only present on neutrophils. Because of the difficulty in are transfused into patients who have leukocyte antibodies.25 characterizing antigens on basophils and eosinophils, many

12 IMMUNOHEMATOLOGY, Volume 26, Number 1, 2010 Granuloycte serology: concepts and significance antigens described as neutrophil-specific have not been of detection. The HNA system currently includes seven an- tested to determine whether they are present only on neu- tigens, which are restricted to five antigen groups. The key trophils or on all granulocytes. Although granulocyte (neu- features of the HNA system are summarized in Table 1. An trophil) antigen systems were initially identified through extensive amount of information is now known about these studies of ANN and AIN, both terms, granulocyte and neu- antigens, and the reader is referred to Bux’s recent and trophil, have been used to describe the alloantigens. comprehensive review about the antigens that constitute this system.58 Nomenclature HNA-1 is the best-characterized group, and it contains Lalezari and colleagues8 identified the first granulo- three antigens: HNA-1a, HNA-1b, and HNA-1c. The HNA-1 cyte antigen during their investigation of a neonate with antigens are expressed only on neutrophils, and antibodies transient ANN and subsequently proposed the first no- to these antigens are frequently implicated in cases of al- menclature system for these antigens. Because their stud- loimmune or autoimmune neutropenia.18,58 The antigens are ies indicated that the antigen was neutrophil-specific, they epitopes that occur on the neutrophil low-affinity FcγRIIIb designated it the N system and the antigens were named in receptor (CD16). This molecule binds the Fc region of IgG chronologic order of discovery. The antigens were labeled antibodies complexed to other antigens or immunoglobu- alphabetically and the alleles were described numerically. lins. The gene frequencies of HNA-1a, HNA-1b, and HNA- The system eventually classified seven antigens: 8NA1, 1c vary among different ethnic populations.58 Furthermore, NA2,40 NB1,41 NB2,42,43 NC1,44 ND1,45 and NE1,46 which be- individuals lacking FcγRIIIb have been identified. These in- came the foundation for granulocyte serology. dividuals do not express the HNA-1 antigens on their neu- During this same period and subsequently, several dif- trophils, thus presenting as an HNA-1 null phenotype.54,62 ferent granulocyte antigens were identified. Some were The HNA-2 group has one characterized antigen— specific for granulocytes (or neutrophils): HGA-3, GA, HNA-2a—which is located on a 58- to 64-kDa glycoprotein GB, GC, Gr1, Gr247–49; some were shared with other cells that is expressed only on neutrophils.63 Although HNA-2a is or tissues: HGA-1, 5a, 5b, 9, MART47,50–52; and some had a high-frequency antigen phenotype (i.e., greater than 90% unclassified distribution: CN1, KEN, LAN, LEA,53–56 SL. for most populations), it is unique in that it is expressed All of these granulocyte antigens were detected with sera on subpopulations of neutrophils among antigen-positive from patients with autoantibodies or alloantibodies di- people. The expression of HNA-2a is greater on neutrophils rected against granulocytes, using traditional blood group from women than men, and two or three subpopulations serologic techniques such as agglutination, immunofluo- of expression are often detected: one population that lacks rescence, and cytotoxicity. Unfortunately, most of these HNA-2a expression and one or two populations that express antigens have not been further classified owing to the lack the antigen but with different intensities.64 The functional of sufficient biologic material for study. Table 1. Human neutrophil antigens† Although this led to a consolidation of individuals working in this field, advances Antigen location/ Antigen Former glycoprotein CD Coding gene Clinical were made and efforts were directed at im- groups Antigens name number alleles‡ significance munobiochemical, genetic, and structural HNA-1 HNA-1a NA1 FcγRIIIb/CD16b FCGR3B*01 ANN, AIN, or functional studies for a few of the well- HNA-1b NA2 FcγRIIIb/CD16b FCGR3B*02 TRALI,§ classified granulocyte antigens. With the HNA-1c SH FcγRIIIb/CD16b FCGR3B*03 advent of this information came the need HNA-2 HNA-2a NB1 58–64 kDa/CD177 Unknown ANN, AIN, TRALI, for a standardized granulocyte antigen no- febrile transfu- menclature system based on the molecules sion reactions, drug-dependent carrying the antigens and the genes encod- neutropenia ing each allele. In 1999, the Granulocyte HNA-3 HNA-3a 5b CTL2/Unknown ANN, TRALI , Antigen Working Party of the International HNA-3b 5a CTL2/Unknown febrile transfusion Society of Blood Transfusion (ISBT) estab- reactions lished a new nomenclature system for the HNA-4 HNA-4a MART CR3/CD11b ITGAM*01 ANN well-characterized granulocyte antigens, (α M subunit) (230G) which was based on the glycoprotein loca- HNA-5 HNA-5a OND LFA-1/CD11a ITGAL*01 Unknown 57 tion of the antigens. In this system the (α L subunit) (2372G) granulocyte antigens are called human neu- †Information from references 20, 58, 59, 60. trophil alloantigens to indicate they are ex- ‡Alleles of the coding genes are named according to the ISGN Guidelines for Hu- man Gene Nomenclature.61 pressed on neutrophils; however, this does §Except HNA-1c. not imply that they are neutrophil-specific. AIN = autoimmune neutropenia; ANN = alloimmune neonatal neutropenia; CD = Each antigen group is assigned a number, clusters of differentiation; CR3 = complement receptor 3; CTL2 = choline transporter-like protein 2; FcγRIIIb = Fc gamma receptor IIIb; HNA = human neutrophil and polymorphisms within the group are antigen; LFA-1 = leukocyte function antigen-1; TRALI = transfusion-related acute designated alphabetically in sequential order lung injury.

IMMUNOHEMATOLOGY, Volume 26, Number 1, 2010 13 M.E. Clay et al. role of HNA-2a is not known, and antibodies to this antigen cell donors with the needed antigen phenotypes. Although have been associated with AIN,20 ANN,65 febrile transfusion many have tried, attempts to develop short-term or long- reactions,66 TRALI,67 and drug-dependent neutropenia.68 term granulocyte preservation procedures have been un- HNA-3a is expressed on granulocytes, lymphocytes, successful, thus requiring that fresh suspensions of granu- platelets, endothelial cells, kidney, spleen, and placental locytes be prepared daily for testing procedures. Finally, the cells.50 It is a high-frequency antigen located on choline presence of HLA antibodies in test sera makes identifying transporter-like protein 2 (CTL2), and its function is not HNA antibodies difficult because granulocytes also express known.59,60 Alloantibodies to HNA-3a have been associated HLA class I antigens on their cell surface. with occasional cases of febrile transfusion reactions69 and one case of ANN.70 Although antibodies to HNA-3a are rare, Preparation of Pure Granulocyte Suspensions their major clinical significance has been their association The isolation of pure granulocyte suspensions from pe- with serious and fatal TRALI events.32–34 ripheral blood was greatly facilitated and simplified with The HNA-4 and HNA-5 antigens are located on subunits Bøyum’s introduction in 1968 of the Ficoll-Isopaque gradi- of the β2 integrin family (CD11a and CD11b molecules). In- ent technique.80 Double-density gradient centrifugation is tegrins are a large family of adhesive receptors that are es- now the most widely used approach for the isolation and sential for cell-cell interactions and cell trafficking. HNA-4a purification of granulocytes for serologic testing. The proce- is an epitope on the α M (CD11b) chain of CD11b/CD18 (the dure makes use of discontinuous Ficoll gradients, and nu- CR3 molecule), and HNA-5a is an epitope on the α L chain merous publications exist describing the methodology and (CD11a) of CD11a/CD18 (the leukocyte function antigen-1 the recovery, purity, functional integrity, and activity of the [LFA-1] molecule).71 HNA-4a is expressed on granulocytes, isolated granulocytes.81–84 monocytes, and natural killer cells, whereas HNA-5a is expressed on all leukocytes.58 Alloantibodies to HNA-4a can Granulocyte Antibody Detection and Antigen Typing cause ANN,72 and the CD11b/CD18 complex has been re- Laboratory approaches for the detection of granulocyte ported to be the target of autoantibodies.18,73 To date, HNA- antibodies require procedures that are accurate, reproduc- 5a antibodies have not been clinically associated with ible, and practical. Although numerous granulocyte sero- neutropenia. logic assays have been described, not all meet these criteria. A general consensus exists that granulocytes do not ex- In addition to the assay used, the ability to detect antibodies press ABO antigens74 or HLA class II antigens.75 Although in test specimens or antigens on test cells may also depend the density of HLA class I antigens on granulocytes is fairly on target cell antigen density and the concentration and low, it can vary among individuals.76 Therefore, these anti- immunoglobulin type of the antibody in the test reagent. gens must be taken into consideration when patient samples Therefore, the successful detection of granulocyte antibod- are tested for granulocyte antibodies. HLA class I antigens ies often requires that a combination of methods be used. may be of particular interest during granulocyte transfusion Although several methods for the detection of gran- for neutropenic patients as these antigens, in granulocyte ulocyte-specific antibodies are used by the few laborato- concentrates, may be introduced in high amounts to recipi- ries that perform this testing, the ISBT Working Party on ents with preformed class I antibodies. Transfusion reac- Granulocyte Immunobiology, a worldwide consortium of tions including TRALI have been observed in this situation, 16 laboratories that participate in the annual International and the antigen-antibody interaction has been proposed to Granulocyte Immunology Workshop (IGIW), have recom- limit effectiveness of the transfusion.77–79 mended that granulocyte antibodies should be investigated using a minimum of two methods: the granulocyte immun- Serologic Testing ofluorescence test (GIFT) and the granulocyte agglutination The detection of HNA antibodies is labor intensive and test (GAT).85 Multiple workshops have shown the sensitiv- technically challenging. Unlike HLA testing, commercial ity of GIFT to far exceed that of GAT; however, agglutinat- test kits are not readily available for the detection of granu- ing antibodies such as HNA-1c, HNA-3a, and HNA-4a are locyte antibodies by solid-phase methodologies. Therefore, much more readily detectable in GAT and in many cases it intact viable granulocytes are required for granulocyte an- is the only reliable method for detecting these specificities. tibody screening. Granulocytes are intrinsically designed For example, the seventh IGIW included a test specimen to respond to physiologic priming signals, so they must be that contained an HNA-3a antibody that could only be de- handled very carefully to prevent activation. Once activated tected by GAT and the granulocyte chemiluminescence test it is impossible to interpret any serologic test results involv- (GCLT). Although GCLT has been useful in selective labora- ing these cells, as false-positive test results abound. Granu- tory settings, it is not routinely used for clinical granulocyte locytes are also extremely labile and must be used within antibody detection and is not discussed. 24 hours of collection, necessitating ready access to panel

14 IMMUNOHEMATOLOGY, Volume 26, Number 1, 2010 Granuloycte serology: concepts and significance

Antibody Detection Methods can be accomplished by using either a fluorescence-detect- Granulocyte Agglutination Test ing microscope or a flow cytometer. Evaluation by a flow Lalezari86 developed the basis of this assay in the early cytometer has in most cases replaced the fluorescence mi- 1960s. Since that time, micro techniques and the use of croscope as more cells can be analyzed in a shorter time, pure granulocyte suspensions with optimized concentra- resulting in improved assay sensitivity and reproducibility, tions of EDTA have vastly improved the performance of and it does not require the expertise needed to evaluate the this test. Typically, a panel of three to five donors is selected characteristic staining pattern seen with specific granulo- to include all known neutrophil antigens currently defined cyte antibodies by microscopic analysis. by the ISBT consortium. Purified granulocytes are isolated from EDTA-anticoagulated peripheral whole blood using Monoclonal Antibody Immobilization of Granulocyte Antigens a double-density gradient. These granulocytes are then The monoclonal antibody immobilization of granulo- washed in PBS before use. The test is biphasic and is based cyte antigens assay (MAIGA) is based on the platelet ver- on the intrinsic response of granulocytes to aggregate when sion (MAIPA) developed by Kiefel et al. in 1987.89 MAIGA stimulated by antibodies reacting to corresponding cell relies on the capture of neutrophil-specific antigen-antibody surface antigens. The resulting agglutination is the conse- complexes by a murine monoclonal antibody onto a solid- quence of the granulocyte activation that occurs during the phase surface.90 The benefits of this test are twofold: first, sensitization phase, which induces the cells to form pseu- this is currently the most sensitive assay for the detection of dopods and slowly migrate toward one another during the granulocyte antibodies, and second, the assay is designed to aggregation phase, until membrane contact is established. detect only HNA antibodies even when HLA antibodies are Typically, the serum or plasma from the specimen being present in the test specimen. The disadvantage of this pro- investigated is incubated with the granulocyte suspension cedure is that it is very complex and requires highly skilled for 4.5 to 6 hours at 30ºC.87 The reactions are evaluated us- staff to perform the detailed techniques. ing an inverted-phase microscope and graded from nega- Granulocytes are incubated with the test serum or plas- tive to 4+ on the basis of the percentage of cells that are ma for 30 minutes at 37ºC. This granulocyte suspension is agglutinated. Our laboratory has determined cutoff scores then washed to remove any unbound immunoglobulins and for both adult and pediatric patients (<6 years old) on the incubated with a murine monoclonal antibody to a specific basis of the reaction grades established during microscopic neutrophil glycoprotein for an additional 30 minutes. Af- evaluation. Both IgG and IgM antibodies are detected by ter another wash step, the granulocyte membranes are dis- this method. rupted in a mild detergent and centrifuged. The resulting lysate is then transferred to polystyrene microwells coated Granulocyte Immunofluorescence Test with anti-mouse immunoglobulins for incubation. The Developed by Verheugt et al.88 in the late 1970s, this trimolecular neutrophil antigen–patient HNA antibody– fluorescent “antiglobulin” technique is used for the detec- murine monoclonal antibody complex present in the lysate tion of granulocyte alloantibodies and autoantibodies that is captured on the solid phase, whereas any HLA are circulating and cell bound. As with the GAT, an anti- antibody-antigen complexes (if present) are removed by body screening panel of three to five donors is selected a subsequent wash step. Remaining complexes are then to include all currently defined HNA antigens. A purified detected by the addition of anti-human IgG conjugated to granulocyte suspension is prepared and treated with 1% horseradish peroxidase followed by a substrate (OPD dis- paraformaldehyde for a short time to prevent nonspecific solved in 30% H2O2), and the reaction is analyzed with a immunoglobulin binding to the neutrophil Fc receptors and spectrophotometer. to stabilize the cell membrane. Serum or plasma from the MAIGA can be used to detect antibodies specific patient is then incubated with an optimized concentration to HNA-1a, HNA-1b, and HNA-1c antigens located on of granulocytes for 30 minutes at 37ºC. After a wash step FcγRIIIb (CD16); HNA-2a present on gp 58- to 64-kDa that removes unbound antibodies, the granulocytes are (CD177); HNA-4a on the complement component receptor then incubated with F(ab′)2 fragments of a fluorescent con- C3bi (CD11b); and HNA-5a on the LFA-1 receptor (CD11a). jugated anti-human antibody for approximately 30 min- HNA-4a and HNA-5a antibodies can also be detected by a utes at room temperature in a dark environment. The as- common CD18 monoclonal antibody. say’s performance is optimized with the use of a fluorescent secondary probe that can detect both IgM and IgG antibod- Antigen Detection and Typing Methods ies (to ensure the detection of both primary and secondary The same methodology that is used to detect antibodies immune responses) and the use of F(ab′)2 Ig fragments to in serum and plasma can be used to characterize the antigen prevent the probe from binding to the high concentrations phenotype of an individual’s granulocytes. Just as the short of Fc receptors on granulocyte surface membranes. The cells lifespan of granulocytes is a limitation to obtaining panel undergo another wash cycle, are resuspended, and then are cells for antibody testing, it also restricts the ability to do analyzed. Detection of the immunofluorescence reactions antigen typing because granulocyte specimens must be tested

IMMUNOHEMATOLOGY, Volume 26, Number 1, 2010 15 M.E. Clay et al. within 24 hours of collection to obtain reliable results. A splicing defects, and no mutations have been detected in sufficient volume of whole blood must be collected to obtain the CD177 introns or exons in these individuals.93 Because an adequate number of granulocytes for phenotyping. This the gene encoding HNA-3a has only recently been identi- can be a dilemma in pediatric patients or patients who are fied, there has not been a genotyping procedure for this neutropenic. Because ample volume, freshness, and avail- antigen, but the clinical importance of this antigen is sup- ability of granulocytes is critical, this also acts as a practical porting efforts to get a molecular method developed. barrier to doing donor-recipient crossmatch testing during Genomic DNA (gDNA) can be isolated and purified investigations of suspected granulocyte-antibody–mediat- from anticoagulated blood using any number of published ed transfusion reactions such as TRALI. The highly charac- methods. A unique set of sense and antisense oligonucle- terized antisera used to phenotype granulocytes should be otide primers that border the DNA fragment to be amplified free of HLA class I antibodies as their presence can result in are added to a master mix of reagents that includes deoxy- a false-positive HNA typing. At a minimum, the titer of the nucleoside triphosphates (dNTPs), Taq polymerase, a buf- HLA antibody should be weaker than the HNA antibody fer solution containing Mg2+, and the individual’s gDNA. as it can then be readily diluted to a nonreactive level. An- The master mix is subjected to a series of 30 to 40 tempera- other constraint to phenotyping granulocytes is the limited ture changes that define the amplification cycles. Each cycle availability of qualified antisera for the currently defined consists of three temperature steps that are critical in the HNA antigens. amplification of the DNA target strands. The first stepin Monoclonal antibodies specific to several HNA anti- the cycle typically involves heating the master mix to a tem- gens are commercially available and have been used to type perature of 95ºC. This denaturation step results in double- granulocytes by GIFT using the flow cytometer. An advan- stranded DNA separating into single strands by disrupting tage to phenotyping by this method is that it can be done the hydrogen bonds between each nucleotide base pair. The with whole blood instead of with isolated granulocytes. annealing step follows in which the reaction temperature is Because phenotyping granulocytes cannot often be per- lowered to 50ºC to 65ºC. This allows the primers to hybrid- formed or can be unreliable if samples are not handled in a ize to the opposing strands of the target DNA. The final ex- careful and timely manner, genotyping by PCR, which has tension step heats the master mix from 71ºC to 72ºC, which less stringent sample age and handling restraints, can serve results in DNA synthesis in the presence of Taq polymerase either as an alternative to or as confirmation of serologic and dNTPs complementary to the oligonucleotide primers. results. A final extension step is added after all cycles have been completed to ensure that any remaining single-stranded Granulocyte Genotyping DNA is fully elongated. The amplified double-stranded The discovery of the PCR in the 1980s facilitated the DNA (amplicon) is separated by size using agarose gel development of molecular methods that have now been ap- electrophoresis, stained with ethidium bromide, and visu- plied to delineate the genetics and allelic variations of the alized by fluorescence using ultraviolet light. The sizes of HNAs. Initially, the characterization of the genes encoding the amplicons are determined by comparison with a DNA the HNA-1 antigen system (HNA-1a, HNA-1b, and HNA-1c) ladder, which contains DNA fragments of known sizes that allowed for the development of PCR assays using sequence- were run alongside the PCR products during the agarose gel specific primers (SSP) to differentiate the alleles. To com- electrophoresis step. plicate matters there is a high degree of homology between the FCGR3A gene that encodes the FcγRIIIa granulocyte Future Directions receptor and the FCGR3B gene where the three different Solid-Phase Technology HNA-1 polymorphisms reside. HNA-1a, HNA-1b, and HNA- The complement-dependent lymphocyte cytotoxicity 1c are encoded by FCGR3B*1, FCGR3B*2, and FCGR3B*3 assay was the first platform, developed nearly 50 years ago, genes, respectively. FCGR3B*1 differs from FCGR3B*2 for identifying HLA antibodies and remains the standard by five nucleotide bases, and a single nucleotide polymor- for the development of new methodologies in this field. phism (SNP) differentiates FCGR3B*2 from Table 2. Nucleotide differences for the three HNA-1 alleles (1a, 1b, and 1c) on FCGR3B*3 (Table 2). FCGR3A differs from FcγRIIIb and among the FcγRIII genes† FCGR3B at five different nucleotide locations HNA Gene Polymorphic nucleotides (Table 2). Methods are also available to genotype 141 147 227 266 277 349 473 505 559 641 733 the HNA-4a and HNA-5a alleles.71 Both of 1a FCGR3B*1 G C A C G G A C G C T these polymorphisms are the result of SNPs. 1b FCGR3B*2 C T G C A A A C G C T Even though the molecular sequence of HNA- 1c FCGR3B*3 C T G A A A A C G C T 2a has been identified, genotyping methods FCGR3A G C G C G A G T T T C to detect the gene that encodes this alloanti- †Information from references 91 and 92. gen are not available. The HNA-2a negative NOTE: Boldface indicates substituted nucleotides. phenotype is attributable to CD177 mRNA FcγRIIIb = Fc gamma receptor IIIb; HNA = human neutrophil antigen.

16 IMMUNOHEMATOLOGY, Volume 26, Number 1, 2010 Granuloycte serology: concepts and significance

Multiple new technologies are now being used by HLA has been the establishment of cell lines that express HNA laboratories, and in the past few years solid-phase assays antigens. In 1999 Bux’s group transfected Chinese ham- have been developed in which purified HLA molecules are ster ovary (CHO) cells with HNA-1a, HNA-1b, and HNA- bound to a well in a microtiter plate or to a bead. HLA an- 1c cDNA and established stable cell lines expressing these tibody is then detected either using an enzyme-linked im- antigens.96 Although the cell lines were stable for 1 month munosorbent assay (ELISA) technique or by analyzing the at 4°C, they demonstrated high background fluorescence in beads with a flow cytometer, which uses a new technology the GIF assay and could only be used for the MAIGA, which developed by Luminex (Austin, TX).94 A critical advantage limited their overall utility. More recently, Yasui et al.97,98 of this technology is its ability to measure multiple analytes transduced HNA genes into the human erythroleukemia simultaneously in a single reaction system. The commer- cell line K562 and established a panel of K562 cell lines that cially available test kits use purified HLA antigens bound stably expressed HNA-1a, -1b, -1c, -2a, -4a, -4b, -5a, and -5b to microbeads, which can be individually differentiated by for at least 1 year while being maintained in culture. They varying ratios of internal fluorescent dye prepared during were not able to establish a cell line expressing HNA-3a be- the manufacturing process. The test involves incubation of cause of the previous lack of biochemical and genetic infor- test sera with the panel of microbeads, and the bound HLA mation for this antigen. The group’s report demonstrated antibody is detected by a secondary R-phycoerythrin (PE) the utility of using the cultured cells in the GIF assay to de- conjugated IgG (or IgM)–specific antibody. tect HNA antibodies in serum samples from patients with Analysis is done with the use of a Luminex flow analyz- HNA antibody–related disorders, but experience with this er in which the beads pass through two lasers, similarly to new cell line technology is very limited and it remains to be classic flow cytometry. One laser identifies the specific bead seen whether the cell lines can be maintained or preserved being analyzed, thereby identifying the unique HLA anti- in a manner that would support the use of these cells for gen. The other laser detects the presence of bound Ig on the routine (nonresearch) granulocyte serology applications. surface of the microbead. The color signals are then detected and processed into data for each reaction. A significant ISBT Working Party on Granulocyte Immunobiology fluorescence shift from the negative control range indicates In 1989, Professor Alan Waters organized the UK Plate- the presence of specific antibody in the test sample. let and Granulocyte Serology Working Group, and 11 inter- The widespread use of Luminex technology by HLA national laboratories participated in the First International laboratories has fostered commercial activity directed at Workshop on Granulocyte Serology. The objectives of this using this technology for the detection of HNA antibodies first workshop were as follows: (1) to initiate an exchange that have been related to febrile and pulmonary transfusion of sera among laboratories to evaluate the techniques be- reactions. One Lambda, Inc. (Canoga Park, CA), has devel- ing used for the detection of granulocyte antibodies, (2) to oped recombinant cell lines for HNA-1a, -1b, -1c, -2a, and foster working relationships among laboratories involved -4a, and these cell lines have allowed the production of pu- in this field, and (3) to support the development of the sci- rified proteins that have been individually immobilized on ence and technology for the detection of granulocyte anti- Luminex microbeads for use in their LABScreen Multiflow bodies.99 bead assay. Stroncek and colleagues95 evaluated the specific- The Working Party was initially affiliated with the Brit- ity and sensitivity of this new procedure using 22 sera that ish Blood Transfusion Society but is now affiliated with the had known HNA antibodies detected with the GA assay and ISBT. The goals of the Second Workshop, in 1996, were 140 samples from nontransfused men. All HNA antibody the (1) establishment of typed granulocyte panels by well- specificities (except 4a) recognized by GAT were detected defined typing sera, (2) proficiency testing of granulocyte in the solid-phase flow bead system (8 HNA-1a, 6 HNA-1b, antibody detection, (3) investigation of uncharacterized 1 HNA-1c, and 8 HNA-2a positive sera), and HNA-1c reac- sera, (4) proficiency testing of HNA genotyping, and (5) ex- tivity was detected in four specimens that were negative by change of information to establish standards in granulocyte GAT. In addition, all of the 140 negative control samples serology.100 Fifteen international laboratories participated had negative reactions when tested by LABSreen Multi, and in the Fourth Workshop, in 2001, with the main objec- the system was able to detect both HNA and HLA antibod- tive being the establishment of a formal quality assurance ies (both class I and class II) in some individual specimens.95 (QA) scheme for granulocyte serology and molecular typing Although preliminary, these results suggest that this solid- methods.85 phase technology may emerge as a major achievement in To date, the Working Party has conducted nine Granu- the development of a new sensitive and specific platform locyte Serology Workshops and comprises 16 international for the detection of granulocyte antibodies in an automated member laboratories, which are listed in Table 3. Also, the manner with limited complexity and high cost efficiency. American Red Cross Mid-America Blood Services Neutro- phil & Platelet Immunology Laboratory has been a member Cell Lines Expressing HNA Antigens of this consortium since its inception in 1989. The workshops Another approach to eliminate the need for freshly continue to focus on the QA and standardization of the test isolated granulocytes for granulocyte antibody screening systems, and the Working Party recently published its

IMMUNOHEMATOLOGY, Volume 26, Number 1, 2010 17 M.E. Clay et al. serologic screening recommendations for the investigation References and prevention of leukocyte antibody-mediated TRALI.101 1. Lalezari P, Nussbaum M, Gelman, et al. Neonatal neutropenia due to maternal isoimmunization. Blood Conclusions 1960;15:236–43. Granulocyte antibody and antigen testing has played 2. Bux J, Jung KD, Kauth T, et al. Serological and clinical and continues to play a critical role for diagnosing and aspects of granulocyte antibodies leading to alloimmune investigating relatively rare but potentially lethal clini- neonatal neutropenia. Transfus Med 1992;2:143–9. cal conditions such as TRALI and immune neutropenias. 3. Rodwell RL, Gray PH, Taylor KM, et al. Granulocyte Although mitigation strategies are already being imple- colony stimulating factor treatment for alloimmune mented nationwide, TRALI still appears to be a leading and neonatal neutropenia. Arch Dis Child Fetal Neonatal preventable cause of transfusion fatalities. As advancing Ed 1996;75:F57–8. technology allows granulocyte antibody testing to be done 4. Rodney GE, Lalezari P. HLA and neutrophil antigen on a less manual and larger scale with more standardized and antibody systems. In: Hoffman R, Benz E, Shattil and reproducible results, it now has the potential to become SJ, eds. Hematology basic principles and practice. 3rd ed. New York: Churchill Livingstone, 2000:2220–41. Table 3. Members of the ISBT Working Party on Granulocyte 5. Lalezari P. Alloimmune neonatal neutropenia. In: En- Immunobiology gelfriet CP,Van Logham JJ, von dem Borne AEGKr, · Platelet and Leucocyte Immunology Laboratory, EFS Ile de France, eds. Immunohaematology. Amsterdam: Elsevier Sci- Hopital Henri Mondor, Creteil, France ence Publishers, 1984:179–86. · Leucocyte and Platelet Immunology Laboratory, Blood Service West of 6. Clay M, Kline W, McCullough J. Serological examina- the German Red Cross, Hagen, Germany tion of postpartum sera for the presence of neutrophil- · The BloodCenter of Wisconsin, Platelet and Neutrophil Immunology specific antibodies (abstract). Transfusion 1981;21:616. Laboratory, Milwaukee, WI, USA 7. Levine DH, Madyastha P, Wade R, Levkoff AH. Neo- · Institute of Transfusion Medicine, University of Schleswig-Holstein, Kiel, Germany natal isoimmune neutropenia (abstract). Pediatr Res · Australian Red Cross Blood Service-Queensland, Brisbane, Australia 1981;15:296A. · Platelet and Granulocyte Immunology Laboratory, National Blood Se 8. Lalezari P, Bernard GE. An isologous antigen-antibody vice, Bristol, UK reaction with human neutrophils, related to neonatal · Blood Transfusion Center of Slovenia, Ljubljana, Slovenia neutropenia. J Clin Invest 1966;45:1741–50. · Centre de Transfusio I Banc de Teixitis, Hospital Vall d’Hebron, P. Vall 9. Verheugt FWA, van Noord-Bokhorst JC, von dem d’Hebron, Barcelona, Spain Borne AEGKr, et al. A family with alloimmune neonatal · Sanguin Diagnostics, Immunohaematology Diagnostic Department, neutropenia: group specific pathogenicity of maternal Amsterdam, The Netherlands antibodies. Vox Sang 1979;36:1–8. · Institute for Clinical Immunology and Transfusion Medicine, Justus 10. van der Weerdt CM, Lalezari P. Another example of Liebig University, Giessen, Germany isoimmune neonatal neutropenia due to anti-NA1. Vox · American Red Cross Mid-America Blood Services, Neutrophil & Platelet Immunology Laboratory, St. Paul, MN, USA Sang 1972;22:438–46. · Department of Transfusion Medicine, Graduate School of Medical Sc 11. Boxer LA, Yokoyama M, Lalezari P. Isoimmune neona- ences, University of Tokyo, Tokyo, Japan tal neutropenia. J Pediatr 1972;80:783–7. · Department of Immunohaematology and Transfusion Medicine, Institute 12. Bux J, Ernst-Ludwig Stein, Bierling P, et al. Character- of Haematology and Blood Transfusion, Warsaw, Poland ization of a new alloantigen (SH) on the human neutro- · Aberdeen and North East Scotland Blood Centre, Scottish National phil Fc gamma receptor IIIb. Blood 1997;89:1027–34. Blood Transfusion Service, Aberdeen, UK 13. Schuller RM, Stroncek DF, Alpan O, et al. Different re- · Platelet and Leucocyte Immunology Laboratory, Institute of Biology, sponses observed between G-CSF and GM-CSF therapy Nantes, France in a case of neonatal alloimmune neutropenia (ab- · Department of Transfusion Medicine, Karolinska University Hospital, Stockholm, Sweden stract). Transfusion 2008;48(Suppl):53A. 14. Bachowski GJ, Mair DC, Schuller RM, et al. Alloimmune even more valuable as a tool to increase blood safety by ac- neonatal neutropenia in monozygous twins caused by tually preventing transfusion reactions without compro- antibody to FcγRIIIb in a mother lacking CD16 antigen mising an adequate supply of blood. (abstract). Transfusion 2007;47(Suppl):21A. 15. Stroncek DF, Sharpiro RS, Filipovich AH, et al. Acknowledgments Prolonged neutropenia resulting from antibodies to The authors would like to thank Penny Milne and Bob- neutrophil-specific antigen NB1 following marrow bie Gibson for their helpful assistance with the preparation transplantation. Transfusion 1993;33:158–63. of the manuscript.

18 IMMUNOHEMATOLOGY, Volume 26, Number 1, 2010 Granuloycte serology: concepts and significance

16. de Haas M, Muniz-Diaz E, Alonso LG, et al. Neutrophil 33. Kopko PM, Marshall CS, MacKenzie MR, et al. Transfu- antigen 5b is carried by a protein, migrating from 70 to sion-related acute lung injury. Report of a clinical look- 95 kDa, and may be involved in neonatal alloimmune back investigation. JAMA 2002;287:1968–71. neutropenia. Transfusion 2000;40:222–7. 34. Davoren A, Curtis BR, Shulman IA, et al. TRALI due to 17. Fung L, Pitcher LA, Willett JE, et al. Alloimmune neo- granulocyte-agglutinating human neutrophil antigen- natal neutropenia linked to anti-HNA-4a. Transfus 3a (5b) alloantibodies in donor plasma: a report of 2 Med 2003;13:49–52. fatalities. Transfusion 2003;43:641–5. 18. Bux J, Behrens G, Jaeger G, et al. Diagnosis and clinical 35. Silliman CC, Curtis BR, Kopko PM, et al. Donor anti- course of autoimmune neutropenia in infancy: analysis bodies to HNA-3a implicated in TRALI reactions prime of 240 cases. Blood 1998;91:181–6. neutrophils and cause PMN-mediated damage to hu- 19. Bux J, Kissel K, Nowak K, et al. Autoimmune neutrope- man pulmonary microvascular endothelial cells in a nia: clinical and laboratory studies in 143 patients. Ann two-event in vitro model. Blood 2007;109:1752–5. Hematol 1991;63:249–52. 36. Sachs UJ, Chavakis T, Fung L, et al. Human alloanti- 20. Stroncek D. Granulocyte antigens and antibody detec- body anti-MART interferes with Mac-1-dependent leu- tion. Vox Sang 2004;87(Suppl 1):91–4. kocyte adhesion. Blood 2004;104:727–34. 21. Sperner-Unterweger B, Czeipek I, Gaggl S, Geissler 37. Kopko PM, Curtis BR, Kelher M, et al. Merging the D, Spiel G, Fleischhacker WW. Treatment of severe pathogenesis of transfusion-related acute lung injury: clozapine-induced neutropenia with granulocyte col- the priming activity of the 5b (HNA-3) antibody (ab- ony stimulating factor (G-CSF). Remission despite stract). Transfusion 2004;44(Suppl 1):22A. continuous treatment with clozapine. Br J Psychiatry 38. Sachs UJ, Hattar K, Weissman N, et al. Antibody- 1998;172:82–4. induced neutrophil activation as a trigger for trans- 22. Stroncek DF. Drug-induced immune neutropenia. fusion-related acute lung injury in an ex vivo rat lung Transfus Med Rev 1993;7:268–74. model. Blood 2006;107:1217–9. 23. Mamus SW, Burton JD, Groat JD, et al. Ibuprofen- 39. Fung YL, Silliman CS. The role of neutrophils in the associated pure white cell aplasia. N Engl J Med pathogenesis of transfusion-related acute lung injury. 1986;314:624–5. Transfusion Med Rev 2009;23:266–83. 24. Holness L, Knippen MA, Simmons L, et al. Fatalities 40. Lalezari P, Radel E. Neutrophil-specific antigens: im- caused by TRALI. Transfus Med Rev 2004;18:184–8. munology and clinical significance. Semin Hematol 25. Brittingham TE. Immunologic studies on leukocytes. 1974;11:281–90. Vox Sang 1952;2:242–8. 41. Lalezari P, Murphy GB, Allen FH. NB1, a new neutro- 26. Barnard, RD. Indiscriminate transfusion: a critique of phil antigen involved in the pathogenesis of neonatal case reports illustrating hypersensitivity reactions. N Y neutropenia. J Clin Invest 1971;50:1108–15. State J Med 1951;51:2399–402. 42. Huang ST, Lin J, McGowan El, et al. NB2, a new allele 27. Popovsky MA, Moore SB. Diagnostic and pathogenic of NB1 antigen involved in febrile transfusion reaction considerations in transfusion-related acute lung injury. (abstract). Transfusion 1982;22:426. Transfusion 1985;25:573–7. 43. Lalezari P, Petrosova M, Jiang AF. NB2, an allele of 28. Bux J, Sachs UJ. The pathogenesis of transfusion- NB1 neutrophil specific antigen: relationship to 9a (ab- related acute lung injury (TRALI). Br J Haematol stract). Transfusion 1982;22:433. 2007;136:788–99. 44. Lalezari P, Thelenfeld B, Weinstein WJ. The third neu- 29. Reil A, Keller-Stanislawski B, Gunay S, et al. Specifici- trophil antigen. In: Terasaki PI, ed. Histocompatibility ties of leukocyte alloantibodies in transfusion-related testing. Baltimore: Williams & Wilkins, 1970:319–22. acute lung injury and results of leukocyte antibody 45. Verheugt FWA, von dem Borne AEGKr, van Noord- screening of blood donors. Vox Sang 2008;95:313–17. Bokhorst JC, et al. ND1, a new neutrophil granulocyte 30. Ward HN. Pulmonary infiltrates associated with leu- antigen. Vox Sang 1978;35:13–17. koagglutinin transfusion reactions. Ann Intern Med 46. Claas FHJ, Langerak J, Sabbe LJM, et al. NE1, a 1970;73:689–94. new neutrophil specific antigen. Tissue Antigens 31. Thompson JS, Severson CD, Parmely MJ, et al. Pul- 1979;13:129–34. monary ‘hypersensitivity’ reactions induced by trans- 47. Thompson JS, Overlin VL, Herbick JM, et al. New fusion of non-HLA lekoagglutinins. N Engl J Med granulocyte antigens demonstrated by microgranulo- 1971;284:1120–5. cytotoxicity assay. J Clin Invest 1980;65:1431–9. 32. Nordhagen R, Conradi M, Drotorp SM. Pulmonary re- 48. Hasegawa T, Bergh OJ, Mickay MR, et al. Prelimi- action associated with transfusion of plasma containing nary human granulocyte specificities. Transplant Pro anti-5b. Vox Sang 1986;51:102–7. 1975;7(Suppl 1):75–80.

IMMUNOHEMATOLOGY, Volume 26, Number 1, 2010 19 M.E. Clay et al.

49. Caplan SN, Berkman EM, Babior BM. Cytotoxins 65. Bux J, Jung KD, Kauth T, et al. Serological and clinical against a granulocyte antigen system: detection by a aspects of granulocyte antibodies leading to alloimmune new method employing cytochalasin-B-treated cells. neonatal neutropenia. Transfusion Med 1992;2:143–9. Vox Sang 1977;33:206–11. 66. Bux J. Granulocyte antibody mediated neutropenias 50. Van Leeuwen A, Eerrise JG, van Room JJ. A new leu- and transfusion reactions. Infusions Ther Transfusion kocyte group with two alleles: leukocyte group five. Vox Med 1999;26:152–7. Sang 1964;9:431–7. 67. Bux J, Becker F, Seeger W, et al. Transfusion-related 51. Van Rood JJ, van Leeuwen A, Schippers AMJ, et al. acute lung injury due to HLR-A2-specific antibodies in Immunogenetics of the group four, five and nine sys- recipient and NB1-specific antibodies in donor blood. tems. In: Curton ES, Mattiuz PI, Tosi RM, eds. Histo- Br J Haematol 1996;93:707–13. compatibility testing. Baltimore: Williams & Wilkins, 68. Stroncek DF, Shankar RH, Herr GP. Quinine- 1967:203–19. dependent antibodies to neutrophils react with a 60 52. Kline WE, Press C, Clay M, et al. Three sera defining a kD glycoprotein on which neutrophil-specific antigen new granulocyte-monocyte–T lymphocyte antigen. Vox NB1 is located and an 85 kD glycosylphosphatidylinos- Sang 1986;50:181–6. itol–linked N-glycosylated plasma membrane protein. 53. Madyastha PR, Glassman A, Levine D, et al. Identifi- Blood 1993;81:2758–66. cation of a new neutrophil antigen (CN1) more preva- 69. Lalezari P, Bernard GE. Identification of a specific lent among American blacks (abstract). Transfusion leukocyte antigen: another presumed example of 5b. 1983;23:426. Transfusion 1965;5:132–42. 54. Schnell M, Halligan G, Herman J. A new granulocyte 70. de Haas M, Muniz-Diaz E, Alonso LG, et al. Neutrophil antibody directed at a high frequency antigen causing 5b is carried by a protein, migrating from 70 to 95 kDa, neonatal alloimmune neutropenia (abstract). Transfu- and may be involved in neonatal alloimmune neutrope- sion 1989;29(Suppl):46S. nia. Transfusion 2000;40:222–7. 55. Rodwell RL, Tudehope DI, O’Regan P, et al. Alloimmune 71. Simsek S, van der Schoot CE, Daams M, et al. Molecu- neonatal neutropenia in Australian aboriginals: an un- lar characterization of antigenic polymorphisms (ONDa a recognized disorder? Transfusion Med 1991;1:63–7. and MART ) of the ß2 family recognized by human leu- 56. Stroncek DF, Ramsey G, Herr GP, et al. Identification of kocyte alloantisera. Blood 1996;88:1350–8. a new white cell antigen. Transfusion 1994;34:706–11. 72. Fung L, Pitcher LA, Willett JE, et al. Alloimmune neo- 57. Bux J, Bierling P, von dem Borne AE, et al. ISBT Granu- natal neutropenia linked to anti-HNA-4a. Transfusion locyte Antigen Working Party. Nomenclature of granu- Med 2003;13:49–52. locyte alloantigens. (letter).Vox Sang 1999;77:251. 73. Hartman KR, Wright DG. Identification of autoantibod- 58. Bux J. Human neutrophil alloantigens. Vox Sang ies specific for the neutrophil adhesion glycoproteins 2008;94:277–85. CD11b/CD18 in patients with autoimmune neutro- 59. Greinacher A, Wesche J, Hammer E, et al. Character- penia. Blood 1991;78:1096–104. ization of the human neutrophil alloantigen-3a. Nature 74. Kelton JG, Bebenek G. Granulocytes do not have sur- Med 2010;16:45–8. face ABO antigens. Transfusion 1985;25:567–9. 60. Curtis BR, Cox NJ, Sullivan MJ, et al. The neutrophil 75. Dunstan RA, Simpson MB, Sanfilippo FP. Absence of alloantigen HNA-3a (5b) is located on choline trans- specific HLA-DR antigens on human platelets and neu- porter-like protein 2 (CTL2) and appears to be en- trophils (abstract). Blood 1984;64:85a. coded by an R>Q 154 amino acid substitution. Blood 76. Thorsby E. HLA antigens on human granulocytes stud- 2010;115:2073-6. ied with cytotoxic iso-antisera obtained by skin graft- 61. Shows TB, McAlpine PJ, Boucheix C, et al. Guidelines ing. Scan J Haematol 1969;6:119–27. for human gene nomenclature. An international system 77. Sachs UJ, Bux J. TRALI after transfusion of cross-match- for human gene nomenclature (ISGN, 1987). Cytogenet positive granulocytes. Transfusion 2003;43:1683–6. Cell Genet 1987;46:11–28. 78. Stroncek DF, Leonard K, Eiber G, et al. Alloimmuni- 62. Stroncek DF, Skubitz KM, Plachta LB, et al. Alloim- zation after granulocyte transfusions. Transfusion mune neonatal neutropenia due to an antibody to the 1996;36:1009–15. neutrophil Fc-gamma receptor III with maternal defi- 79. McCullough J, Clay M, Hurd D, et al. Effect of leuko- ciency of CD16 antigen. Blood 1991;77:1572–80. cyte antibodies and HLA matching on the intravascular 63. Stroncek DF, Skubitz K, McCullough JJ. Biochemical recovery, survival, and tissue localization of 111-indium nature of the neutrophil-specific antigen NB1. Blood granulocytes. Blood 1986;67:522–8. 1990;75:744–55. 64. Matsuo K, Lin A, Procter JL, et al. Variations in the expression of granulocyte antigen NB1. Transfusion 2000;40:654–62.

20 IMMUNOHEMATOLOGY, Volume 26, Number 1, 2010 Granuloycte serology: concepts and significance

80. Bøyum A. Isolation of mononuclear cells and granu- 94. Vigrali DA. Multiplexed particle-based flow cytometric locytes from blood. Isolation of mononuclear cells by assays. J Immunol Methods 2000;243:243–55. one centrifugation and of granulocytes by combining 95. Stroncek DF, Adams S, Lee JH, et al. Neutrophil anti- centrifugation and sedimentation at 1g. Scan J Clin Lab body testing. ASHI Q 2008;32:68–72. Invest 1968;21(Suppl 97):77–89. 96. Bux J, Kissel K, Hofmann C, et al. The use of allele-spe- 81. English D, Anderson BR. Single-strep separation of red cific recombinant Fcγ receptor IIIb antigens for the de- blood cells, granulocytes, and mononuclear leukocytes tection of granulocyte antibodies. Blood 1999;93:357– on discontinuous density gradients of Ficoll-Hypaque. 62. J Immunol Methods 1975;5:249–52. 97. Yasui K, Miyazaki T, Matsuyama N, et al. Establish- 82. Ferrante A, Thong YH. A rapid one-step procedure for ment of cell lines stably expressing HNA-1a, 1b, and purification of mononuclear and polymorphonuclear -2a antigen with low background reactivity in flow cy- leukocytes from human blood using a modification of tometric analysis. Transfusion 2007;47:478–86. the Hypaque-Ficoll technique. J Immunol Methods 98. Yasui K, Hirayama F, Matsuyama N, et al. New cell lines 1978;24:389–93. express HNA-1c, -4a, -4b, -5a, or -5b for identification 83. Ferrante A, Thong HY. Optimal conditions for simultaneous of HNA antibodies. Transfusion 2008;48:1037–9. purification of mononuclear and polymorphonuclear 99. Lucas GF, Carrington PA. Results of the First Inter- leukocytes from human blood by the Hypaque-Ficoll national Granulocyte Serology Workshop. Vox Sang method. J Immunol Methods 1980;36:109–17. 1990;59:251–6. 84. Madyastha P, Madyastha KR, Wade T, et al. An im- 100. Bux J, Chapman J. Report on the Second Interna- proved method for rapid layering of Ficoll-Hypaque tional Granulocyte Serology Workshop. Transfusion double density gradients suitable for granulocyte sepa- 1997;37:977–83. ration. J Immunol Methods 1982;42:281–6. 101. ISBT Working Party on Granulocyte Immunobiol- 85. Lucas G, Rogers S, de Haas M, et al. Report on the ogy. Recommendations of the ISBT Working Party on Fourth International Granulocyte Immunology Work- Granulocyte Immunobiology for leukocyte antibody shop: progress toward quality assessment. Transfusion screening in the investigation and prevention of 2002;42:462–8. antibody-mediated transfusion-related acute lung in- 86. Lalezari P, Pryce S. Detection of neutrophil and plate- jury. Vox Sang 2009;96:266–7. let antibodies in immunologically induced neutropenia and thrombocytopenia. In: Rose NR, Friedman H, Mary E. Clay, MS, MT(ASCP), (corresponding author) Di- eds. Manual of clinical immunology. Washington, DC: rector, Transfusion Medicine, Research and Development American Society of Microbiology 1980;744–9. Program, University of Minnesota Medical School, De- 87. McCullough J, Clay ME, Press C, et al. Granulocyte se- partment of Laboratory Medicine and Pathology, MMC rology: a clinical and laboratory guide. Chicago: ASCP 198, D-251 Mayo Building, 420 Delaware Street, SE, Min- Press, 1988:12–14, 168–71. neapolis, MN 55455, and Consultant, Neutrophil & Plate- 88. Verheugt FWA, Von dern Borne AEGKr, Decary S, et let Immunology Laboratory, American Red Cross, Mid- al. The detection of granulocyte alloantibodies with America Blood Services Region, 100 South Roberts Street, an indirect immunofluorescence test. Br J Haematol St. Paul, MN 55107, Randy M. Schuller, BS, MT(ASCP), 1977;36:533–44. Supervisor, and Gary J. Bachowski, MD, PhD, Assistant 89. Kiefel V, Santoso S, Weisheit M, et al. Monoclonal anti- Medical Director, Neutrophil and Immunobiology Labo- body-specific immobilization of platelet antigens (MAI- ratory, American Red Cross, Mid-America Blood Services PA): a new tool for the identification of platelet reactive Region, St. Paul, MN. antibodies. Blood 1987;70:1722–6. 90. Minchinton R, Noonan K, Johnson TJ. Examining technical aspects of the monoclonal antibody immobilization of granulocyte antigen assay. Vox Sang 1997;73:87–92. 91. Stroncek D. Neutrophil alloantigens. Transfusion Med Rev 2002;16:67–75. For information concerning the National 92. Wang E, Marincola FM, Stroncek D. Human leukocyte Reference Laboratory for Blood Group antigens and human neutrophil antigens systems. In: Serology, including the American Rare Hoffman R, Benz E, Shattil Sj, et al., eds. Hematology: Donor Program, please contact Sandra basic principel and practices 4th ed. Philadelphia: El- Nance, by phone at (215) 451-4362, by fax at sevier, 2005:2401–22. (215) 451-2538, or by e-mail at snance@usa. 93. Kissel K, Scheffler S, Kerowgan M, et al. Molecu- redcross.org lar basis of NB1 (HNA-2a, CD177) deficiency. Blood 2002;99:4231–3.

IMMUNOHEMATOLOGY, Volume 26, Number 1, 2010 21 Review

A review of the Colton blood group system

G.R. Halverson and T. Peyrard

History linked to two other cases discovered earlier by workers in The idea that there must be openings in the cell mem- Minneapolis, Oxford, and London. The antigen was named brane to permit the flow of water and salts to maintain cel- Coa (Coltona) in 1967 for the first of the three producers of lular function and to eliminate the byproducts of cellular anti-Coa, who was from Minneapolis; it should actually have metabolism was recognized by the middle of the 19th cen- been named “Calton,” but the handwriting on the tube ap- tury. Water is the major component of all living cells, in- parently was misread.12,13 Coa was shown to be an inherited cluding those of vertebrates, invertebrates, and unicellular characteristic.14 In 1970, the antithetical antigen, Cob, was organisms and plants. Salt water comprises approximately reported by Giles et al (Table 1).15 In analysis of unrelated 70 percent of the human body. After debating for decades people of European extraction, the occurences of the three about the process of water transfer across cell membranes, major Colton phenotypes were as follows: Co(a+b–) 0.914, most scientists finally decided that water must pass free- Co(a+b+) 0.084, and Co(a–b+) 0.002 (Table 2).16 In 1971, ly through biologic membranes by simple diffusion. Only the genetic independence of the Colton blood group system a small number of scientists disagreed, and they had only from Kell and Lutheran was reported, and it was confirmed indirect evidence that there were special channels for the that it is independent from MNSs, P, Rh, Duffy, Dombrock, transport of water. After all, there must be something to and sex.17 Coa and Cob are codominant alleles that respec- explain the high permeability of RBCs and renal tubules. tively encode the Coa and Cob antigens at the RBC surface. In Moreover, the water permeability of these tissues can be re- 1974 the Co(a–b–) phenotype of three persons in a French versibly inhibited by mercuric ions.1 Canadian family was reported.18 The serum from a patient By 1950 it had been shown that water moves quickly in 1964 (Swarts) was eventually found to be negative only into and out of cells through pores that are selective for with these Co(a–b–) family members.16 Colton is the 15th water molecules only. Every second, billions of water mol- human blood group system recognized by the ISBT (ISBT ecules pass through a single channel. By the mid-1980s 015). scientists had found an RBC membrane protein whose 28- kDa N-terminal amino acid sequence was related to the Molecular Basis 26-kDa major intrinsic protein (MIP26) of bovine lens fi- The AQP1 locus was mapped to human chromosome ber cells.2,3 The 28-kDa protein was shown to be a unique 7p14 by fluorescent in situ hybridization.19 The Colton (Co) molecule that is abundant on erythrocytes and on renal tu- blood group antigens had been previously linked to the bules. Preston and Agre4 succeeded in 1991 in isolating the short arm of chromosome 7.20 Immunoprecipitation studies same membrane protein from RBCs, which in 1997, was officially named aquaporin-1 (AQP1) by the Human Genome Table 1. Antigens of the Colton blood group system: their preva- Organization, for the water channel. AQP1 has been found lence, molecular basis, and sensitivity to enzyme and DTT treatment in many tissues including several parts of the kidney, liver, ISBT ISBT Molecular Effect of gall bladder, eye, choroid plexus, hepatobiliary epithelium, Name symbol number Prevalence basis enzymes and capillary endothelium. AQP1 turned out to be the first Coa CO1 015001 99.8% Ala 45 PR TR CR of the family of major intrinsic proteins (MIP), which are NR DTTR highly conserved membrane proteins that regulate small Cob CO2 015002 8.6% Val 45 PR TR CR molecule transport. The MIP family of proteins is believed NR DTTR to date back 2.5 to 3 billion years in evolutionary time.5 It is Co3 CO3 015003 >99.99% unknown PR TR CR believed that the MIP family evolved from two basal lineag- NR DTTR CR = -chymotrypsin resistant; DTTR = dithiothreitol resistant; es: AqpZ-like water channels and GlpF-like glycerol facili- α1 NR = neuraminidase resistant; PR = papain resistant; TR = trypsin tators. These divergent lineages probably originated from resistant. 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- Table 2. Phenotypes (% occurrence) porins (GlpF, AQP3, AQP7, AQP9, and AQP10).6–10 In 1994, Phenotype % Occurrence (most AQP1 protein was characterized as carrying the Colton populations)16 blood group antigens.11 Co(a+b–) 91.4 Co(a–b+) 0.2 Genetics and Inheritance Co(a+b+) 8.4 In 1965 in Oslo, Norway, the discovery of an antibody Co(a–b–) <0.01 that detected a “public” (or high prevalence) antigen was

22 IMMUNOHEMATOLOGY, Volume 26, Number 1, 2010 Colton blood group system review and DNA sequencing showed that the Colton antigens are the Co protein have been reported to cause decreased or no the result of a polymorphism in AQP1 at the extracellular expression of the Colton antigens. An amino acid change site of loop A, which connects the first and second spanning of Gln47Arg was observed to cause a Co(a–b+w) typing.29 domains. Figure 1 depicts the structure of the protein as it The same amino acid substitution was recently fully inves- passes through the membrane, and indicated are the Coa/ tigated in a Turkish female with the Co(a–b–) phenotype.30 Cob polymorphic site at position 45 and the only N-glycan Her RBCs were found by molecular studies to have normal site at position 42. The Coa/Cob polymorphism arises from water permeability by stopped-flow analysis and a normal a single amino acid change of Ala45Val (alanine for Coa, va- amount of AQP1 protein in the RBC membrane. (See sec- line for Cob) caused by a nucleotide change of C>T at position on clinical significance). By PCR-RFLP the specimen tion 134 in exon 1 of AQP1 gene, named CO according to was genotyped as Coa/Coa, but full sequencing revealed a the ISBT nomenclature and AQP1 according to the Human 140A>G mutation leading to a Gln47Arg amino acid sub- Genome Organization (HUGO).11 The Colton gene consists stitution. This change in amino acids is close to the Coa/Cob of four exons distributed over 11.6 kbp of genomic DNA. polymorphism, and thus, the probable cause of the appar- The amino acid sequence of the wild-type AQP1 protein is ent discrepancy between phenotype and genotype. When shown in Table 3. Also known as the channel-forming inte- using a specifically designed K-562 cell expression system gral protein (CHIP), the Colton glycoprotein is a multipass for Colton antigens, the Gln47Arg substitution was protein consisting of three external loops and six mem- shown to inhibit both Coa and Cob antigen expression.29 brane-spanning regions, as well as 2 cytoplasmic loops, The antibody produced by these Co(a–b–) individuals a with both the NH2 and COOH terminus located on the cyto- with a Gln47Arg substitution is not a mixture of anti-Co plasmic side of the membrane.22 Common to all MIP super- and -Cob, rather it is “anti-Co3–like,” reacting with both family proteins is the occurrence of the asparagine-proline- Co(a+b–) and Co(a–b+) RBCs, but more weakly than anti- alanine (NPA) motifs, two being present in each half of the Co3 produced by the true null phenotype. This suggests the protein. An hourglass structural model has been proposed existence of a new high-prevalence antigen in the Colton by Agre and colleagues.23,24 blood group system, with a corresponding antibody that Arnaud et al. proposed to name anti-Co4.30 Therefore, it is

a N-glycosylation site A = Co possible that some of the Co(a–b–) people known through- V = Cob E N A/V 45 42 A C out the world, who would have not been fully investigated Out 192 194 by molecular techniques, may actually not be “true” Conull N A 1 2 3 4 5 P 6 RBC but “CO:–1,–2,3,–4.” This may also be why some antibod- Membrane P ies directed against a high-prevalence Colton antigen, mis- N A 76 78 takenly considered as anti-Co3, were unexpectedly weakly In B D reactive with Co:3 RBCs.31

NH2 COOH Biochemical Physiology of the Colton Glycoprotein Protons are necessary for the bioenergetics of the living Fig. 1 Linear diagram depicting the Colton protein with the six membrane-spanning domains with both the N terminus and C cell. The proton gradient drives many transport functions, terminus inside the cell membrane. The Coa/Cob polymorphism is membrane fusions, and the synthesis of ATP. Maintaining indicated at position 45, as is the glycosylation site at N42. The the proton gradient is essential, and indiscriminate pro- signature NPA motifs are depicted on the internal loops B and D.21 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- 13 Table 3. Amino acid sequence of wild-type AQP1 protein layer. Aquaporins are strictly selective water chan- MASEFKKKLF WRAVVAEFLA TTLFVFISIG SALGFKYPVG NNQTAVQDNV 50 nels that have evolved for that purpose. AQP1 was KVSLAFGLSI ATLAQSVGHI SGAHLNPAVT LGLLLSCQIS IFRALMYIIA 100 found to have a configuration with intracellular N QCVGAIVATA ILSGITSSLT GNSLGRNDLA DGVNSGQGLG IEIIGTLQLV 150 and C terminals, similar to that of other ion chan- LCVLATTDRR RRDLGGSAPL AIGLSVALGH LLAIDYTGCG INPARSFGSA 200 nel proteins. Protein modeling from the cDNA sequences predicts a two-tandem repeat of three VITHNFSNHW IFWVGPFIGG ALAVLIYDFI LAPRSSDLTD RKVWTSGQV 250 membrane-spanning helices that exist as tetram- EEYDLDADDI NSRVEMKPK 269 ers within the cell membrane (Fig. 2). An electro- Amino acid sequence taken from GenBank, accession # M77829 static field created by the highly conserved amino Also see the Blood Group Antigen Gene Mutation Database (dbRBC) at http://www.ncbi.nlm.nih.gov/gv/mhc/xslcgi.cgi?cmd=bgmut/home acid sequence NPA forms the water channel, which (accessed April 13, 2010). prevents proton loss while allowing water to permeate the membrane. The most prominent feature is the association of loop B with loop E driven by their The molecular basis of the null Co(a–b–) phenotype positive N-terminal ends that brings together the has been attributed to several different molecular back- NPA motifs at the center of the pore. This causes grounds; these are depicted in Table 4. Other changes in the formation of strong electrostatic fields that block

IMMUNOHEMATOLOGY, Volume 26, Number 1, 2010 23 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 Observed result on Presence HDFN Molecular alteration Effect AQP1 protein of anti-Co3 reported Ancestry Reference

Conull phenotypes Large deletion compris- Absent or aberrant AQP1 not detected in Not reported No Northern Proband 1 in Preston ing most of exon 1 transcript RBC membrane extracts European et al.25 c.309insT Frameshift mutation AQP1 not detected in Yes No French Proband 2 in Preston RBC membrane extracts et al.25 c.113C>T Missense mutation <1% AQP1 expression in Not reported No Northern Proband 3 in Preston (p.Pro38Leu) RBC membrane extracts European et al.25 c.576C>A Missense mutation Not tested Yes No Portuguese Chrétien et al.26 (p.Asn192Lys) 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 Not tested No No Not reported, Wagner and Flegel29 (p.Gln47Arg) presumably Caucasian c.140A>G Missense mutation Normal AQP1 expression No No Turkish Arnaud et al.30 (p.Gln47Arg) in RBC membrane 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.

Colton Antigen Typing the permeation of protons as well as H3O+ and other cations. When Xenopus laevis oocytes were transfected with To date, anti-Coa and anti-Co3 reagents, either mono- cRNA, they were found to have markedly increased water clonal or polyclonal, are not commercialized. As a result, permeability, causing the oocytes to swell and burst.32 This human polyclonal reagents are only available in immu- permeability could be reversibly inhibited by Hg2+.33 When nohematology reference laboratories, but usually in small the same cRNA transcript was reconstituted into lipid vesi- amounts. However, a human polyclonal anti-Cob is avail- cles, the same observations were made. This confirmed that able in Europe as a CE-marked in vitro diagnostic reagent membranes exhibit water permeability and showed that the (DiaMed, a division of Bio-Rad, Cressier sur Morat, water permeability of membranes with AQP1 was up to 100 Switzerland). The two major high-throughput molecu- times greater than that of those without it. 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 allele typing by searching for the 134C>T polymorphism in Out exon 1, with subsequent prediction of Colton phenotype. It P is important, however, to be aware that prediction of the Co N A 1 6 2 4 3 5 phenotype in Conull and Covariant people would be a source of RBC H O misinterpretation with these DNA-based techniques. Membrane 2 A P N Clinical Significance Antibodies to Colton blood group antigens are gener- In ally IgG and react best by the antiglobulin test, especially when protease-treated RBCs are used.34,35 Some anti-Coa, b 36 NH -Co and -Co3 may bind complement. Few reports of sig- 2 COOH nificant delayed or acute transfusion reactions or hemolytic disease of the fetus and newborn (HDFN) attributable to Fig. 2 The AQP1 water channel is shown as an hourglass configu- anti-Coa have been reported, although both are known to ration created by the association of the six transmembrane domains occur with severe morbidity.27,37–40 and the crossing of loops B and E with the electrostatic field pro- Anti-Cob is relatively rare and often found in patient’s duced by the NPA motifs forming the water channel.21 sera containing other alloantibodies.34,35 Two reports of in

24 IMMUNOHEMATOLOGY, Volume 26, Number 1, 2010 Colton blood group system review vivo survival studies of 51Cr-labeled Co(b+) RBCs in pa- 9. Carbrey JM, Gorelick-Feldman DA, Kozono D, Praeto- tients with anti-Cob showed survival at 94 percent and 85 rius J, Nielsen S, Agre P. Aquaglyceroporin AQP9: sol- percent, respectively, at 1 hour, decreasing to 51 percent at ute permeation and metabolic control of expression in 24 hours and 10 percent at 96 hours.41,42 Cob is fully devel- liver. Proc Natl Acad Sci U S A 2003;100:2945–50. oped at birth, but no significant HDFN has been reported to 10. Endeward V, Musa-Aziz R, Cooper GJ, et al. Evidence 34 date. that aquaporin 1 is a major pathway for CO2 transport Anti-Co3 was reported to cause severe HDFN requir- across the human erythrocyte membrane. FASEB J ing neonatal transfusion.43 Transfusion of incompatible 2006;20:1974–81. Co(a+b–) RBCs in a patient with anti-Co3 was reported to 11. Smith BL, Preston GM, Spring FA, Anstee DJ, Agre P. be responsible for a mild hemolytic transfusion reaction.27 Human red cell aquaporin CHIP. I. Molecular charac- There are also reports of autoantibodies mimicking Colton terization of ABH and Colton blood group antigens. J specificities.44 Clin Invest 1994;94:1043–9. AQP1-deficient individuals were studied for their renal 12. Heistö H, van der Hart M, Madsen G, et al. Three ex- function and capillary permeability before and after water amples of a new red cell antibody, anti-Coa. Vox Sang deprivation.45 Baseline studies did not reveal any abnormali- 1967;12:18–24. ties; however, after water deprivation this study revealed an 13. Reid ME, Lomas-Francis C. The blood group antigen inability to concentrate urine. Thus, Colton-null individuals factsbook. 2nd ed. San Diego, CA: Academic Press, may be subject to serious hydroelectrolytic and metabolic 2004. disorders should they become severely dehydrated. 14. Wray E, Simpson S. A further example of anti-Coa and two informative families with Co(a–) members. Vox Acknowledgments Sang 1968;14:130–2. The authors thank Robert Ratner for preparation of the 15. Giles CM, Darnborough J, Aspinall P, Fletton MW. manuscript and figures, and Hallie Lee-Stroka for her criti- Identification of the first example of anti-Cob. Br J Hae- cal review and helpful comments. This work was funded in matol 1970;19:267–9. part by a grant from the MetLife Foundation (GH). 16. Race RR, Sanger R. Blood groups in man. 6th ed. Ox- ford, England: Blackwell Scientific Publications, 1975. References 17. Lewis M, Kaita H, Anderson C, Chown B. Independence 1. Macey RI. Transport of water and urea in red blood of Colton blood group. Transfusion 1971;11:223–4. cells. Am J Physiol 1984;246:C195–203. 18. Rogers MJ, Stiles PA, Wright J. A new minus-minus 2. Agre P, Saboori AM, Asimos A, Smith BL. Purification phenotype: three Co(a–b–) individuals in one family and partial characterization of the Mr 30,000 integral (abstract). Transfusion 1974;14:508. membrane protein associated with the erythrocyte 19. Moon C, Preston GM, Griffin CA, Jabs EW, Agre P. Rh(D) antigen. J Biol Chem 1987;262:17497–503. The human aquaporin-CHIP gene. Structure, orga- 3. Saboori AM, Smith BL, Agre P. Polymorphism in the nization, and chromosomal localization. J Biol Chem Mr 32,000 Rh protein purified from Rh(D)-positive 1993;268:15772–8. and -negative erythrocytes. Proc Natl Acad Sci U S A 20. Zelinski T, Kaita H, Gilson T, Coghlan G, Philipps S, 1988;85:4042–5. Lewis M. Linkage between the Colton blood group locus 4. Preston GM, Agre P. Isolation of the cDNA for eryth- and ASSP11 on chromosome 7. Genomics 1990;6:623–5. rocyte integral membrane protein of 28 kilodaltons: 21. Agre P, King L, Yasui M, et.al. Topical Review: Aqua- member of an ancient channel family. Proc Natl Acad porin water channels—from atomic structure to clinical Sci U S A 1991;88:11110–4. medicine. J Physiol. 2002;542:3–16. 5. Kozono D, Ding X, Iwasaki I, et al. Functional expres- 22. King LS, Agre P. Pathophysiology of the aquaporin wa- sion and characterization of an archaeal aquaporin. ter channels. Annu Rev Physiol 1996;58:619–48. AqpM from Methanothermobacter marburgensis. J 23. Mathai JC, Agre P. Hourglass pore-forming domains Biol Chem 2003;278:10649–56. restrict aquaporin-1 tetramer assembly. Biochemistry 6. Roudier N, Ripoche P, Gane P, et al. AQP3 deficiency in 1999;38:923–8. humans and the molecular basis of a novel blood group 24. Jung JS, Preston GM, Smith BL, Guggino WB, Agre system, GIL. J Biol Chem 2002;277:45854–9. P. Molecular structure of the water channel through 7. Vajda Z, Pedersen M, Füchtbauer EM, et al. Delayed on- aquaporin CHIP. The hourglass model. J Biol Chem set of brain edema and mislocalization of aquaporin-4 1994;269:14648–54. in dystrophin-null transgenic mice. Proc Natl Acad Sci 25. Preston GM, Smith BL, Zeidel ML, Moulds JJ, Agre P. U S A 2002;99:13131–6. Mutations in aquaporin-1 in phenotypically normal hu- 8. Ikeda M, Beitz E, Kozono D, Guggino WB, Agre P, Yasui mans without functional CHIP water channels. Science M. Characterization of aquaporin-6 as a nitrate channel 1994;265:1585–7. in mammalian cells. Requirement of pore-lining residue threonine 63. J Biol Chem 2002;277:39873–9.

IMMUNOHEMATOLOGY, Volume 26, Number 1, 2010 25 G.R. Halverson and T. Peyrard

26. Chrétien S, Catron JP, de Figueiredo M. A single mu- 38. Agre P, Smith BL, Baumgarten R, et al. Human red cell tation inside the NPA motif of aquaporin-1 found in a aquaporin CHIP. II. Expression during normal fetal de- Colton-null phenotype (letter). Blood 1999;93:4021–3. velopment and in a novel form of congenital dyseryth- 27. Joshi SR, Wagner FF, Vasantha K, Panjwani SR, Fle- ropoietic anemia. J Clin Invest 1994;94:1050–8. gel WA. An AQP1 null allele in an Indian woman with 39. Kurtz SR, Kuszaj T, Ouellet R, Valeri CR. Survival of ho- a Co(a–b–) phenotype and high-titer anti-Co3 associated mozygous Co (Colton) red cells in a patient with anti- a with mild HDN. Transfusion 2001;41:1273–8. Co . Vox Sang 1982;43:28–30. 28. Nance S, Kavitsky DL, Meny G, Vege, S, Westhoff CM. 40. Michalewska B, Wielgos M, Zupanska B, Bartkowiak R. a An example of anti-Co3 not causing hemolytic disease Anti-Co implicated in severe haemolytic disease of the of the newborn. Transfusion 2002;42(Suppl):105S. foetus and newborn. Transfus Med 2008;18:71–3. 29. Wagner FF, Flegel WA. A clinically relevant Co(a)-like 41. Dzik WH, Blank J. Accelerated destruction of ra- b allele encoded by AQP1 (Q47R) (abstract). Transfusion diolabeled red cells due to anti-Colton . Transfusion 2002;42(Suppl):24S–5S. 1986;26:246–8. 30. Arnaud L, Helias V, Menanteau C, Peyrard T, et al. A 42. Hoffmann JJML, Overbeeke MAM. Characteristics of b functional AQP1 allele producing a Co(a–b–) pheno- anti-Co in vitro and in vivo: a case study. Immunohe- type revises and extends the Colton blood group sys- matology 1996;12:11–13. tem. Transfusion 2010. In press. 43. Savona-Ventura C, Grech ES, Zieba A. Anti-Co3 and se- 31. Lacey PA, Robinson J, Collins ML, et al. Studies on the vere hemolytic disease of the newborn. Obstet Gynecol blood of a Co (a–b–) proposita and her family. Transfu- 1989;73:870–2. sion 1987;27:268–71. 44. Moulds M, Strohm P, McDowell MA, Moulds J. Autoan- 32. Preston GM, Carroll TP, Guggino WB, Agre P. Appear- tibody mimicking alloantibody in the Colton blood group ance of water channels in Xenopus oocytes expressing system (abstract). Transfusion 1988;28(Suppl):36S. red cell CHIP28 protein. Science 1992;256:385–7. 45. King LS, Choi M, Fernandez PC, Cartron JP, Agre P. De- 33. Kozono D, Yasui M, King LS, Agre P. Aquaporin wa- fective urinary-concentrating ability due to a complete ter channels: atomic structure and molecular dynamics deficiency of aquaporin-1. N Engl J Med 2001;345:175–9. meet clinical medicine. J Clin Invest 2002;109:1395–9. 34. Daniels G. Human blood groups. 2nd ed. Malden, MA: Gregory R. Halverson, BS, MT(ASCP)SBB, DLM (cor- Blackwell Science, 2002. responding author), Laboratory of Immunochemistry, 35. Issitt PD, Anstee DJ. Applied blood group serology. 4th New York Blood Center, 310 East 67th Street, New York, ed. Durham, NC: Montgomery Scientific Publications, NY 10065, and Thierry Peyrard, PharmD, MS, European 1998. Specialist in Clinical Chemistry and Laboratory Medicine, 36. Covin RB, Evans KS, Olshock R, Thompson HW. Acute National Immunohematology Reference Laboratory, Na- hemolytic transfusion reaction caused by anti-Coa. Im- tional Institute of Blood Transfusion, 20 rue Bouvier 75011 munohematology 2001;17:45–9. Paris, France. 37. Simpson WKH. Anti-Coa and severe haemolytic disease of the newborn. S Afr Med J 1973;47:1302–4.

Free Classified Ads and Announcements

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

Deadlines 1st week in January for the March issue 1st week in April for the June issue 1st week in July for the September issue 1st week in October for the December issue

E-mail or fax these items to [email protected] or (215) 451-2538.

26 IMMUNOHEMATOLOGY, Volume 26, Number 1, 2010 Case Report

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- Table 1. Evaluation and transfusion of patients with D+ RBCs and tients. We report a D+, multi-transfused Caucasian woman serum anti-D with myelodysplasia who exhibited several alloantibodies. Etiology/ Preferred RBC One antibody was a moderately strong (2+) anti-D that per- pathogenesis Diagnostic evidence transfusion sisted for 9 months, until the woman died. Molecular analy- Passive alloantibody History of transfusion D– until anti-D sis of the patient’s RHD gene identified the rare weak D type with D– RBC, plasma, wanes 21 (938C>T) allele. D alloantibodies do not occur in patients IVIG, RhIG, other product with most weak D types, but some patients with a weak D with anti-D phenotype, including those with type 21, can produce antibodies to nonself epitopes of the wild-type D antigen. Alloantibodies from Organ transplant from D– D– until anti-D Immunohematology 2010;26:27–29. passenger lymphocytes donor to D+ recipient wanes in donor organ Key Words: alloimmunization, anti-D, RHD gene, weak D Autoantibody to D Positive direct/indirect D– over D+ antigen antiglobulin test (anti-IgG) ntibodies with D specificity are unusual in D+ pa- Anti-D specificity in tients. Alloantibodies to D may be (1) passively eluate or serum A transfused in donor plasma products, (2) passively History: pregnancy, warm transferred but actively produced by lymphocytes carried autoimmune hemolytic in transplanted organs, and (3) actively produced by an al- anemia, malignancy loimmunized patient. Autoantibodies to D may be warm- or cold-reactive antibodies with a preference for D+ RBCs or Autoantibodies or Antibody fails to react D– over D+ 1 alloantibodies to LW with RBC treated with may be antibodies to LW that mimic anti-D specificity. dithiothreitol In practice, the combination of clinical, serologic, and Antibody is absorbed by molecular investigations helps to determine why a D+ pa- D– RBC tient has anti-D. Establishing the source of a D+ patient’s alloantibody or autoantibody with D specificity is important Alloantibody to epitope Anti-D fails to react with D– only in deciding whether D+ or D– units should be transfused to absent from normal D RBC of person who optimize donor RBC survival (Table 1). protein produced it In this case report, we describe a woman whose RBCs Anti-D fails to react with D– RBC were strongly D+ at the phenotypic level, but had an RHD genotype consistent with weak D, and an alloanti-D. anti-IgG) and all but one subsequent DAT were negative as Case Report was the auto-control. On the one occasion, the DAT became A 72-year-old, gravida 3, Caucasian woman with myel- weakly positive in a mixed-field reaction. Acid eluates pre- odysplasia and pancytopenia was transferred from another pared from the patient’s RBCs contained only anti-E. There hospital, where in 6 months of treatment, she had received was no history of organ transplantation or administration an unknown number of platelet transfusions, but no RBC of a plasma product that might contain anti-D. The anti-D transfusions. persisted throughout the patient’s course. Blood samples In our hospital and before any RBC transfusion, the were collected and referred for RHD and RHCE gene analyses. patient’s RBCs serologically typed as D+ (4+) by tube ag- Although continued transfusions with D– blood were glutination at immediate spin phase, using a monoclonal- prescribed, the patient refused all further medical treat- polyclonal blended anti-D (Ortho Clinical Diagnostics Co., ment. Her myelodysplasia progressed to early acute leuke- Raritan, NJ). During a disease course of 13 months, the mia before she expired at home. patient received 37 apheresis-processed platelet units and 108 leukocyte-reduced units of RBCs, which produced al- Molecular Test Results loimmunization to K, E, and Cw. After 9 months, her serum Molecular tests were performed at the Molecular Red also demonstrated a 2+ anti-D in a gel-based, antibody de- Cell and Platelet Testing Laboratory of the American Red tection system (Micro Typing Systems, Inc., Ortho Clinical Cross, Penn-Jersey Region, and the University of Penn- Diagnostics, Pompano Beach, FL). The initial DAT (tube, sylvania DNA Sequencing Facility, both in Philadelphia,

IMMUNOHEMATOLOGY, Volume 26, Number 1, 2010 27 H. McGann and R.E. Wenk

Pennsylvania. The patient was found to be heterozygous Alloantibodies to Rh antigens are often durable (85%). for RHD. No changes associated with any of the known Negative DATs and absence of RBC agglutination in auto- partial D phenotypes were identified. A change (938C>T) control tests also indicated the presence of D alloantibody. was identified that is associated with weak D type 21 that is LW alloantibodies, which can mimic anti-D, arise predicted to encode an intracellular amino acid change of only in very rare individuals whose RBCs are LW(a–b–) Pro313Leu. The patient’s RH genotype could be interpreted or LW(a–b+). Neither autoantibodies nor alloantibodies to be RHD*weak D type 21, RHCE*Ce/RHCE*ce. to LW were specifically evaluated in the patient described here. Discussion Alloantibodies to the D antigen occur in people who are Initially, the finding of anti-D in a D+ patient was puz- known to lack one or more epitopes of D (partial D pheno- zling and the several causes listed in Table 1 were consid- type). On the other hand, anti-D alloimmune responses in ered. Patients whose anti-D was passively transferred in patients with other weak D types are unusual because their plasma products (IVIG, RhIG, etc.) or whose serum anti- RBCs do not appear to lack D epitopes when tested with a bodies were produced by a donor’s passenger lymphocytes standard battery of anti-D monoclonal reagents.4 may require D– RBCs until the acquired anti-D has weak- Suspicion that the patient had been alloimmunized to ened, usually in a matter of weeks to months. D antigen was raised by the serologic finding of negative Passive anti-D, however, was ruled out by history. Be- DATs and the antibody’s persistence. In addition, people fore hospital admission at our institution, the patient had who carry variants of the D antigen that are detectable only been transfused only with apheresis platelets, but she had by molecular means are capable of producing antibodies not been transfused with plasma or cellular products from to wild-type protein. In the present case, finding a variant D– donors, nor had she been infused with IVIG or injected RHD gene supported the clinical and serologic evidence of with RhIG. Persistence of the anti-D was further evidence D alloimmunization. that the anti-D did not involve passive immunization. Historically, some D+ individuals who made anti-D were The transfer of passenger lymphocytes could be ex- initially considered to express a weak D phenotype (first cluded by history as well. The patient had never received a termed “DU”) when tested with human polyclonal reagents. transplant. Passenger lymphocytes are alloantibody-secreting Serologic evidence of alloimmunization to D in a D+ person B cells that are carried in a transplanted donor organ to a included a negative DAT, a serum antibody, and, for some recipient. An anti-D produced by passenger lymphocytes partial D phenotypes, the patient’s RBCs also expressed a arises in the donor by alloimmunization to the D antigen low-prevalence Rh antigen.5 A D+ patient with alloanti-D before transplantation. Donor lymphocytes continue to was thought to lack part(s) of the D antigen, i.e., a “partial manufacture detectable anti-D for about 3 months after D” type. A person with a partial D phenotype would only transplantation. produce antibody to those epitopes of the D antigen missing Many patients with autoimmune hemolytic anemia and from his or her own RBCs. Investigative use of monoclonal mimicking antibodies to Rh antigens are treated medically antibody revealed many epitopes of the D antigen and sup- and transfusion is avoided altogether. In other patients, ported the concept that partial D types lacked one or more transfusion can be delayed until the concentration of an- epitopes of their D antigen. The monoclonal antibodies also tibodies is reduced by medical treatment. Many mimicking extended the identification and classification of the various warm autoantibodies have apparent Rh specificity, especially partial D types initially defined by polyclonal anti-D. anti-e. Only rare D+ patients will possess warm autoan- Many individuals, however, were found to have RBCs tibodies that mimic anti-D. If a patient with autoantibodies that agglutinated poorly with common anti-D reagents, but requires transfusion, antigen-negative donor RBCs may be demonstrated the presence of all known epitopes. These D+ preferable because they may survive longer than antigen- RBCs were called “weak D” in preference to DU. Weak D positive RBCs.2 RBCs expressed fewer D molecules per RBC, a finding that Antibodies to LW are almost always autoantibodies; accounted for their weak agglutination with reagent anti- those that are less potent appear to have anti-D specific- bodies. Theoretically, individuals who carried these weak ity because the number of D antigen sites per RBC corre- D types should not be able to develop alloantibodies to D. lates with the number of LW antigen sites.3 Thus, D– RBCs, However, it is virtually impossible to distinguish a person which have fewer LW antigen sites than D+ RBCs, may not with a weak D phenotype from a person with a weak par- be agglutinated by an anti-LW. However, anti-LW may be tial D phenotype by serologic tests; only the production distinguished from anti-D by its failure to agglutinate RBCs of alloanti-D will reveal someone to have a weak partial D treated with DTT. If transfusion is required for a patient phenotype. with autoanti-LW, D– RBCs are preferable to D+ RBCs pro- When molecular analysis became available, the RHD viding such a patient is c+. (and RHCE) gene(s) revealed far more extensive and com- Patient autoantibodies, including anti-LW, are often plex polymorphisms than were evident by serologic meth- transient, but our patient’s anti-D persisted for 4 months ods. Weakly agglutinating D serotypes could be related to at constant serologic strength (2+ by the IgG gel test). point mutations, nonsense mutations, deletions, and tandem

28 IMMUNOHEMATOLOGY, Volume 26, Number 1, 2010 Anti-D in weak D type 21 rearrangements in the RHD and RHCE DNA. The cause of alloantibody. After RHD and RHCE genotyping, the patient weak RBC agglutination was confirmed to be an absence of was found to have the RHDweakD type 21RHCE*Ce/RHCE*ce weak D type 21 epitopes in partial D types, a decreased number of protein genotype (R1 r phenotype). Although most patients molecules in weak D types, or both. In addition, partial D with a weak D phenotype do not produce alloanti-D, pa- phenotypes are expressions of genotypes that encode for tients who carry the rare weak D type 21 in the absence of a changes in the amino acids on the outer surface of the RBC normal RHD allele appear susceptible to the production of where the D protein loops outside the RBC membrane. D alloantibodies. Patients whose RBCs express partial D types are often im- munized to D+ RBCs by transfusion or pregnancy. On the Acknowledgments other hand, genotypes that encode weak D phenotypes are The authors thank Connie Westhoff, PhD, and Sunitha predicted to result in intracellular or intramembranous Vege for the molecular analyses. changes in amino acids that, in theory, express all epitopes of D so that patients whose RBCs express weak D types References are not expected to produce anti-D after immunization by 1. Lu RP, Clark P, Mintz PD. Apparent anti-D in a D+ in- transfusion or pregnancy. Theory is partly supported by dividual: a clinical and serologic approach. Transfusion evidence—the most common weak D variants (types 1–3), 2007;47:1755. which constitute 70 to 90 percent of all weak D variants, do 2. Leger, RM. Chapter 17: The positive direct antiglobu- not produce alloanti-D.6 lin test and immune-mediated hemolysis. In: Technical Molecular examination of the various partial and weak manual. 16th ed. Roback J, Combs MR, Grossman B, D types indicated that many serologic phenotypes had more Hillyer, C, eds. Bethesda, MD: American Association of than one basis, but did not suggest that new epitopes could Blood Banks, 2008:499–524. be identified.7 However, serologic evidence suggested oth- 3. Gibbs MB. The quantitative relationship of the Rh-like erwise. Anti-D has been found in some weak D patients (LW) and D antigens of human erythrocytes (letter). whose D protein is predicted by their DNA sequences to Nature 1966;210:642–3. have altered intramembranous and intracellular amino ac- 4. Reid ME. The Rh antigen D: a review for clinicians. ids of the D protein. Thus, some weak D molecular alleles Blood Bull 2008;10:1–2. contain missense mutations that not only reduce the num- 5. Westhoff CM. Structure and function of the Rh blood ber of antigens per RBC and cause weak RBC agglutination group antigens. Semin Hematol 2007;44:42–50. by reagent antibodies but also cause a loss of D epitopes 6. Flegel W. The genetics of the Rhesus blood group sys- and produce changes in the three-dimensional antigenic tem. Dtsch Artzebl 2007;104(10):A651–7. structure of the D protein. 7. Wagner FF, Frohmajer A, Ladewig B, et al. Weak D al- The case presented here indicates that the rare weak leles express distinct phenotypes. Blood 2000;95:2699– D type 21 is an allelic variant whose patient-carriers are 708. susceptible to transfusion alloimmunization by wild-type D 8. Wagner F. Weak D types. Available at: http://www.un- antigen. Weak D type 21 is the result of a point mutation iulm.de/~fwagner/RH/RB/weakD.htm. Accessed Feb. that alters an amino acid thought to be in an intracellular 25, 2009. region of the D protein.8 The rare allele was first discovered 9. Le Van Kim C, Mouro I, Cherif-Zahar B, et al. Mo- in one individual during investigation of 270 known weak lecular cloning and primary structure of the human D samples in people from northern Germany,9 and there blood group RhD polypeptide. Proc Nat Acad Sci U S A has been one subsequent independent observation of it in 1992;89:10925–9. Austria.10 In both cases, the allele has been found on the 10. Muller TH, Wagner FF, Trockenbacher A, et al. PCR same chromosome (cis) as a RHCE*Ce allele in the haplo- screening for common weak D types shows different type RHDweak D type 2-RHCE*Ce. The Pro313Leu change in the distributions in three central European populations. translated D protein is in its tenth intracellular helix, and all Transfusion 2001;41:45–52. 37 tested epitopes of the D antigen appear to be present.9 As 11. Polin H, Danzer M, Hofer K, Gassner W, Gabriel C. Ef- expected, the number of D antigen sites per RBC is reduced fective molecular RHD typing strategy for blood dona- (5.2 × 103) when compared with the normal number (104 tions. Transfusion 2007;47: 1350–5. to 2.5 × 104) of sites.11 This antigen density is the highest 12. Wagner FF, Gassner C, Muller TH, Schonitzer D, among all known weak D alleles. The high density probably Schunter F, Flegel WA. Molecular basis of weak D phe- accounts for the patient’s 4+ serotype and explains why she notypes. Blood 1999;93:385–93. was not identified as a weak D carrier. Heather McGann, MT(ASCP) SBB (corresponding author), Summary Supervisor, Blood Bank, Department of Pathology, and We evaluated a patient with a strongly expressed D an- Robert E. Wenk, MD, Consulting Pathologist, Department tigen with a posttransfusion antibody to the D antigen. The of Pathology, Sinai Hospital, 2401 West Belvedere Avenue, antibody had clinical and serologic characteristics of an Baltimore, MD 21215.

IMMUNOHEMATOLOGY, Volume 26, Number 1, 2010 29 Review

A review of the Chido/Rodgers blood group

R. Mougey

. . . and the blind, wise blood bankers said, “These an- further than that. The usual experience was to spend an tibodies are high-titered and have low avidity, they are red inordinate amount of time performing every possible test, cell, white cell antibodies, they are clinically insignificant, which led only to excluding what the antibody could not are not reactive with enzyme-treated red cells, and can be be. Then when additional samples were obtained to con- absorbed with white cells!” Then the chorus of other blind, tinue the investigation, it was not uncommon to find that wise blood bankers said, “NO, THEY ARE NOT!” tests that had appeared to be nonreactive were now reactive b a s e d o n m e m o r i e s o f discussions a b o u t t h e s e a n t i b o d i e s with the new antibody sample or with a freshly collected b a c k i n t h e 1970s a n d t h e f a b l e o f t h e e l e p h a n t a n d b l i n d w i s e sample of the test cell. It took me a while to recognize that m e n —r. m o u g e y with these antibodies, if you wanted to test for specificity It was a delight, an honor, and also a little intimidating by typing the patient’s RBCs or testing known antigen posi- to be asked to write a review of the Chido/Rodgers blood tive or negative RBCs, it was best to use the freshest RBCs. group system, especially considering that the last author to If you wanted to find compatible blood, older donor units pen a review for Immunohematology on this subject was were more likely to be nonreactive. That is, it was gener- Carolyn M. Giles, the author of so many important publi- ally agreed that there were some patients whose antibod- cations on Chido and Rodgers. Moreover, the recent dis- ies could not be identified as any of the then known anti- coveries about Chido and Rodgers have moved well beyond bodies and whose reactivity was so weak there was a lot of the field of blood group serology to an understanding of the disagreement as to which test RBCs were truly nonreactive. functional protein and genetics at a molecular level. This made it very difficult to define and describe the true My delight was in the opportunity to revisit my early characteristics of the antigens and the antibodies and show days in performing antibody identification and think again that the antigens were expressed on the RBC as an inherited about the challenges that blood bankers faced then in work- trait. This variability was partly a result of testing methods, ing with a group of antibodies that no one could agree about. which relied heavily on a multiphasic set of incubation tem- Chido and Rodgers were usually lumped in with this group, peratures and the use of a polyclonal antiglobulin reagent. which in light of current knowledge is quite understandable. In that period, blood bankers went to great lengths to de- As I recall even the pronunciation for Chido was a matter of tect and identify room temperature–reactive antibodies as some dispute, with adherents claiming that “Cheedo” with well as antibodies active at 37°C. The use of fresh serum for a short i vowel, not Chido with a long i sound, was correct. testing was usually considered essential, with the end result It occurred to me that this review should cover a historical that blood bankers were pretty good at detecting clinically perspective, which might be helpful to those blood bankers significant antibodies but even better at finding clinically whose testing experience is derived from the newer meth- insignificant antibodies. Blood bankers got lots of prac- ods of gel column or solid-phase technology and who might tice at identifying these clinically insignificant antibodies, be wondering what all the fuss was about. The review will and neutralizing them with human breast milk, avian egg then cover the current understanding of the serology, func- albumin, hydatid cyst fluid, saliva, and human and guinea tion, and biochemistry, and then the genetics of C4 and the pig urine. The grubbies defied these efforts, and there was Chido/Rodgers blood group system. The nomenclature for much discussion, some of it acrimonious, on how patients the complement system is very confusing even for blood with these antibodies ought to be managed. The term high bankers who deal routinely with a nomenclature for a blood titer, low avidity was preferred by many if only for its ac- group that is just as confusing. ronym HTLA, even though everyone knew that these antibodies were not always of high titer or of low avidity. When Historical Perspective such weak antibodies were shown to be of high titer, it de- In the beginning there was confusion and chaos. Dur- fied logic that the antibody should show such poor avidity. ing the 1960s and ’70s, the process for antibody identifica- Obviously, the fault could lie with the antigen, either its ori- tion was usually a straightforward and consistent exercise entation or its number of sites, or some other property not with good consensus between individual technical staff and yet realized. Again, there was a contradiction in the patient among laboratories, except there was this group of anti- making a high-titered antibody if there was so little antigen bodies that “would not cooperate.” In the reference labo- to stimulate the production of antibodies. The first glimmer ratory where I worked, we called them “the grubbies,” but of understanding in these contradictory findings was the re- we were too frustrated by working with them to go much ports on anti-Chido.

30 IMMUNOHEMATOLOGY, Volume 26, Number 1, 2010 Chido/Rodgers review

Discovery and Serology of Chido/Rodgers was distinguished from the other so-called HTLA specifici- The Chido antibody was first studied in a patient in ties Knops, Csa, and Yka, although some workers continued 1962, and in a collaborative effort between laboratories in to think of anti-Yka as also showing some inhibition with Minneapolis, Minnesota, and London, United Kingdom, it plasma.4 was found to be reactive with nearly all cells tested but was At this point, the Chido antigen was thought to be a consistently nonreactive with the RBCs of a London panel plasma antigen that was adsorbed onto the RBC much like donor. Six additional examples were found, and the anti- the Lewis antigens. Once accurate, reproducible antigen body was named Chido after one of the patients and the typings were possible, family studies on the inheritance of combined results were published by Harris et al. in 1967.1 the Chido gene led to the discovery that Chido was linked Their title is as good a description of the grubbies as any, “A to HLA.5 This potential relationship to HLA prolonged the nebulous antibody responsible for crossmatching difficul- use of the term RBC-WBC antibodies, but the recognition of ties.” The authors reported that the antibodies could not be HLA linkage to Chido led to the studies to see whether other inhibited by saliva, urine, or hydatid cyst fluid. genes known to be linked to HLA would also show linkage During this time, there was much speculation about to Chido. these grubby antibodies being RBC-WBC antibodies, that The description of anti-Rodgers, a second antibody re- came from some successful experiments using WBCs to ad- active with an RBC antigen of high prevalence that could be sorb these antibodies.2 This whole issue of mistaken identity neutralized by plasma, followed in 1976.6 The antibody was was very understandable because of the not uncommon an- formed by a transfused patient whose RBCs were Chido- tibody problem known as anti-Bgs. Bg antibodies had been positive who also made an anti-E. The anti-Rga (as anti-Rg shown to be hemagglutinins with anti-HLA specificity, and was referred to at that time) gave weak to 4+ reactions with the Bg antigens were thought to be remnants of those HLA E– RBCs (not your typical low-avidity antibody). Using antigens expressed on the RBC before the loss of its nucle- plasma neutralization tests, Longster and Giles found that us. Because Bgs were antigens of lower frequency, it would 3.1 percent of donors could not inhibit the antibody, i.e., have been so tidy to have them be the antithetical allele to were Rodgers-negative, although they found some donors Chido as the variable strength of reactivity seemed so simi- who were only partial inhibitors. Nordhagen et al.7 were lar. Eventually it was realized that the “grubby” antibodies also able to demonstrate partial inhibition of anti-Chido by had nothing to do with WBC antigens. There may have been the plasma of Chido-positive subjects, and Giles8 showed sufficient residual plasma in the WBC preparations used to that partial inhibition was an inherited trait. These findings adsorb the antibodies, now known to be capable of inhib- would explain conflicting results in tests on Chido/Rodgers iting anti-Chido, to lead to this mistaken conclusion. The antibodies among laboratories if a single source of plasma speculation that the Chido antigen was a WBC antigen did was used in inhibition tests. The common practice now is to lead to studies for possible linkage with HLA, an important use a pool of inert plasmas when attempting to determine advance as linkage studies had shown no association with whether an unknown antibody is neutralizable by human any other blood groups. plasma. Many additional examples of anti-Chido were found, Rodgers was also shown to be linked to HLA in two and it was thought that these antibodies did not seem to studies in 1976 that set the stage for the discovery of the cause obvious destruction of Chido-positive donor RBCs. relationship of Chido and Rodgers to the C4 protein.9,10 In The work of Middleton and Crookston3 published in 1972 1978, O’Neill et al.11 showed that Chido and Rodgers are an- provided the clue that led to recognizing the true nature of tigenic determinants expressed on the C4 complement pro- the blood group. In an attempt to enhance the reactivity tein. Then Tilley et al.12 showed that the Chido and Rodgers of the anti-Chido they were studying, inert human plasma determinants are on the C4d fragment of the C4 molecule. was added to the tests; a common practice of the time was The small amount of C4d normally found on the RBC mem- to add a source of protein to enhance antibody binding for brane is the limiting factor that explains the low avidity of antihuman globulin (AHG) reactions when using polyspe- anti-Chido or anti-Rodgers. In fact, if large amounts of C4d cific AHG. The authors recognized that when the antibody are bound to the RBC under low ionic conditions, anti-Ch reactivity disappeared after the addition of plasma the or anti-Rg can be turned into a strongly reactive, direct ag- Chido antigen must also be in plasma. They then found glutinin.13 The puzzle of incomplete or partial inhibition of that performing Chido antigen typings using the plasma of the antibodies is caused by the extreme polymorphism of the person being typed in a neutralization test was a much the C4 genes with multiple alleles that cause variations in more reliable way to type individuals for the Chido antigen the Ch or Rg determinants. than testing their RBCs. They also noted that weak Chido All this progress made Chido and Rodgers antibodies antigen reactivity on an individual’s RBCs did not appear easier to recognize with the exception of Chido-positive to correlate with the amount of antigen in that person’s variant individuals who have made anti-Chido to the part plasma. The ability to neutralize anti-Chido was also a use- of the Chido antigen that they lack.14 The further serologic ful tool for antibody identification, especially in sera with complexities of Chido/Rodgers specificity will be discussed multiple antibodies. This was the first way in which Chido in the section on genetics.

IMMUNOHEMATOLOGY, Volume 26, Number 1, 2010 31 R. Mougey

The inexorable unraveling of the puzzle of the true na- The story of Chido and Rodgers blood groups has moved ture of the blood group Chido/Rodgers is an excellent ex- well beyond the “crossmatching difficulties,” and there is ample of the importance of recognizing anomalous findings now a wealth of new material that shows the C4 genes and in the study of antibodies that most blood bankers wished proteins to be a fascinating model for the evolution of the could just be ignored! The final refutation of the term HTLA genes for critical biologic processes that can both protect came from the revelation that the Knops, McCoy, Swain- and attack their host organism. Langley, and York blood group sera recognize antigens that are carried on the RBC complement receptor CR1.15,16 How- Structure and Function of the C4 Protein ever, this finding seemed to have brought a level of kinship The C4 protein expressing the Chido or Rodgers to these antibodies with Chido and Rodgers that must have epitopes occurs in two isoforms, called C4A for acidic (for- been satisfying to those who wanted to believe that Chido, merly C4F) and C4B for basic (formerly C4S).20 Awdeh and Knops, etc. must be related. Again the low avidity of most Alper20 proposed the change from F for fast and S for slow Knops antibodies is probably related to the low copy num- to conform with the then new International System for Hu- ber of the individual’s CR1 receptor. It also seems likely that man Gene nomenclature and because in their experiments these blood groups will be forever linked in the literature as sialic acid was removed from plasma proteins before elec- those HTLAs when they are studied by aspiring blood bankers. trophoresis, resulting in distinct but different bands from those in the experiments of O’Neill et al.,11 which were the Clinical Significance of Chido/Rodgers first to link the Chido/Rodgers antigen to C4 proteins. C4A It is well documented that many patients with Chido/ proteins usually carry the Rodgers antigens and C4B, the Rodgers antibodies have been safely transfused with “ran- Chido antigens, although a number of haplotypes have been dom” RBC units, but there are also reports of significant re- discovered in which these associations were switched.21,22 actions to plasma or platelet transfusions in patients with C4A or C4B protein is expressed as a single-chain pre- anti-Chido or -Rodgers,17,18 a cautionary incident remind- cursor of 1744 amino acid residues, and the C4A or C4B ing blood bankers not to ignore the potential significance of amino acid sequences are nearly identical (99%) except near an antibody based on specificity alone. Strupp et al.19 also residues 1000 to 1200.23 The amino acid substitutions that pointed out the danger in assuming that antibodies that occur at residues 1101 to 1106 distinguish C4A from C4B. appear to be of low avidity may be safely ignored without Single-nucleotide polymorphisms (SNP) are also found in further investigation. They reported 4 patients with sickle the region that distinguishes the alleles of C4A from each cell disease for whom the provision of compatible blood and other and from C4B and its alleles. The Chido and Rodgers benefit of transfusion were delayed because Dombrock an- antigens are thought to differ by both amino acid substitu- tibodies were not initially recognized and identified. How- tion as well as conformational binding sites.24 ever, there is a need to determine the best way to detect and After translation, the pro-C4 protein is cleaved into al- identify these antibodies without incurring unnecessary pha, beta, and gamma polypeptide chains that are linked delays and expense to the patient, but how much testing is by disulfide bonds.25 However, incomplete processing ac- enough? It sometimes seems that when we had the time, tually leads to multiple structural forms of the peptide be- resources, and people to detect and identify these antibod- ing secreted in the plasma.26 The activated complement ies, we did not have a clue what they were. Now we know all component C1 initiates cleavage of 77 amino acids from about them and do not have the time, people, or resources the N-terminus of the alpha chain, releasing C4a, and the to do much with them. In this area as with so many other remainder of the molecule C4b changes shape, exposing blood group issues, perhaps molecular methods will identify a thioester bond between the sulfhydryl group of Cys-991 good patient practices, and with the use of microarray tech- and the carbonyl group of Gln-994. This binding site that nology, definitive answers to some of these thorny issues is exposed on activation will allow the formation of either of cost versus benefit will be found. Until that time, when a covalent ester or an amide linkage.27,28 Activated C4B is patients with known anti-Chido or anti-Rodgers require more efficient than C4A at pathogen cell lysis because of its RBC transfusions, antibody detection, identification, and histidine residue at position 1106 and its serine residue at crossmatching methods should include plasma-neutraliza- position 1102.29 tion studies. After all, these patients are known antibody The liver is the primary site of C4 production, with formers. If transfusion of plasma components is required, smaller amounts produced by macrophages in other tis- there should be some consideration given to the possible sues.30 There is a wide variation in the amount of C4 pro- effects on the patient of receiving a large volume of plasma, duced among individuals. The concentration of C4 in the such as the formation of precipitating immune complexes plasma varies from 80 to 1000 µg/mL with variable con- or anaphalaxis.17,18 Finally, the presence of antibodies to C4 centrations of C4 isotypes.31 Moulds et al.32 found variation proteins may indicate a potential disease association such in patterns of C4 protein levels in individuals that showed as is seen with the patients who have anti-Ch and anti-Rg either a complete absence of C4A or C4B, more C4A than and are C4-deficient. C4B, more C4B than C4A, or equal amounts of both. There

32 IMMUNOHEMATOLOGY, Volume 26, Number 1, 2010 Chido/Rodgers review is some correlation between C4 gene copy number varia- Part of this multiple role is the normal constant low tions and certain polymorphisms with the level of C4 pro- level of activation of C3 via the classical pathway and the tein produced, but the effect at the haplotype level can be alternate pathway. In the classical pathway, it was thought lost in the diploid gene expression.33 that specific antigen binding by an antibody capable of ac- C4 can be activated via the classical pathway or the tivating C1 would initiate the pathway, but Manderson et mannan-binding lectin pathway, although this may be a al.38 have shown that C3 is also being activated by C1–C4 in simplistic view as a recent paper demonstrated the poten- the absence of antibody-antigen complexes. They propose tial for localized release of anaphylatoxins C3a and C4a by that although circulating constant low levels of IC could tryptase under certain conditions such as those found in the account for their findings, the continual circulation of the airways of asthmatics.34 Although it is simpler to consider normal levels of IgG antibodies may form “spontaneous un- organized pathways in which complement proteins perform stable Ig aggregates that would provide for a C3 tickover” as their complex dance with the other parts of the innate and well. In their experiments, vascular stasis would encourage humoral immune response, it is likely that the protection or initiate complement activation and would therefore be or attack effect is far more chaotic and layered in multiple augmented at sites of inflammation. This would facilitate mechanisms that activate, block, or break down these pro- pathogen recognition by allowing the deposition of activated teins (see discussion on disease association). C3 fragments onto foreign surfaces (host cell surfaces with In the classical pathway, C4 is processed by activated complement regulatory proteins would be unaffected). C1, which removes C4a anaphylatoxin from the alpha chain. Thus the various polymorphisms of C4 affect the normal C4a is a mediator of local inflammation and can induce the functioning of C4 and can be affected by the individual’s contraction of smooth muscle, increase vascular permeabil- ability to produce suitable levels of C4 proteins. Deficiencies ity, and cause histamine release from mast and basophilic of C4 proteins whether acquired or inherited can increase WBCs. C4a levels along with the anaphylatoxins C3a and the risk of severe viral and bacterial infections.33 How high C5a in plasma were shown to be elevated during normal levels of C4 may also contribute to the risk of diseases is pregnancy as an indication of the increased activation of the just beginning to be understood (disease association will be complement system, possibly as a protection against infec- discussed later). tion.35 The remaining C4b fragment forms the enzymes C3 Genetics of C4 and Chido/Rodgers convertase (C4bC2a) and C5 convertase (C3bC4bC2a). The original notation of Cha and Rga implied the ex- These enzymes catalyze the membrane attack proteins, pectation that there would be additional antithetical al- which open a channel into the cell membrane. C4 binding leles Chb and Rgb.1,6 However, it was soon realized that this protein (C4bp) is another plasma protein that regulates C4 blood group did not fit this pattern of inheritance. Chido, activity by blocking the formation of and promoting the or Ch, and Rodgers, or Rg, are used to describe the cluster decay of the classical pathway C3 convertase. Deficiency of of determinants that are expressed on the C4A or C4B pro- C4bp would be expected to result in increased activation of teins that can neutralize the antibodies formed by Chido- or C3 and complement activity in response to classical path- Rodgers-negative individuals. Also individuals can type as way or lectin pathway activation by immune complex for- positive with anti-Chido, but form anti-Chido to the Chido mation, bacterial infections, apoptosis, and other triggering epitopes they lack. Currently nine antigens have been de- mechanisms.36 scribed: six of high prevalence for Chido, two of high preva- Activated C4B (C4b-B) binds to its target rapidly but lence for Rodgers, and one of low prevalence, WH.24 Eight has a short half-life of less than 1 second and has greater phenotypes for the CH/RG system, with their respective oc- hemolytic activity than C4A.28 Activated C4A (C4b-A) is currences, are shown in Table 1 as the prevalence for the 10 times slower than C4B, but it forms amide bonds with other antigen combinations has not been established (Table amino group targets of immune complexes (IC) or protein 1).39 The polymorphisms for the Ch and Rg epitopes as well antigens. C4B more efficiently forms ester bonds with car- Table 1. Prevalence of Chido/Rodgers phenotypes in random samples bohydrate antigens, resulting in better clearance of patho- Chido Rodgers gens. phenotype Prevalence (%) phenotype Prevalence (%) Besides activation of the complement cascade via the Ch:1,2,3 88.2 Rg:1,2 95.0 classical or lectin pathways, the covalent binding of C4 to immunoglobulins and to IC enhances the solubilization of Ch:1,–2,3 4.9 Rg:1,–2 2.5 immune aggregates and the clearance of IC by binding to Ch:1,2,–3 3.1 Rg:–1,–2 2.5 complement receptor on RBCs (CR-1). This IC binding on Ch:–1,–2,–3 3.8 the RBC then acts as a shuttle and delivers the IC for phago- Ch:–1,2,–3 Rare cytosis by macrophages. Thus C4 plays multiple roles for Ch:–1,2,3 (proposed but Rg:–1,2 (proposed but both the innate and adaptive immune systems in vertebrate Ch:–1,–2,3 may not exist) may not exist) animals.37

IMMUNOHEMATOLOGY, Volume 26, Number 1, 2010 33 R. Mougey as the polymorphisms for the electrophoretic behavior of The genes for C4A and C4B were shown to be located the C4 protein confounded the early investigations on the between HLA-DR and HLA-B loci on the short arm of chro- inheritance of the C4 genes. No attempt is going to be made mosome 6 along with C2 and factor B.40,47 C4A and C4B by this author to explain the reported phenotypic frequen- proteins are distinguished phenotypically by their migra- cies of Ch and Rg antigens with the current understanding tion patterns in electrophoretic gels and genetically by four of the multiple copy number variation of the C4 gene. nucleotide substitutions that encoded changes at residues In early studies of the inheritance of Chido/Rodgers, 1101 to 1106 where C4B encodes for LSPVIH and C4A en- it appeared that the genes must be encoded by two closely codes for PCPVLD at the protein level. C4A and C4B are linked loci. The majority of persons were Chido-positive and also distinguished phenotypically by their expression of ei- Rodgers-positive. Approximately 1.7 percent of those stud- ther the Chido or the Rodgers antigen, which are the result ied were Chido-negative, Rodgers-positive, and 3 percent of two amino acid substitutions at residues 1188 to 1191. were Chido-positive, Rodgers-negative.3,6 After the linkage The Ch1 epitope has ADLR and the Rg1 has VDLL. As data of Chido/Rodgers with HLA was established and the dis- from family studies began to contradict a simple two-gene covery of C4 as the plasma protein carrier was made, family model, it was realized that a haplotype could have one, two, studies detecting both serologic allelic forms and electro- three, or four C4 genes and that the C4 genes were inher- phoretic alleles seemed initially to be in agreement with ited as part of a complex of genes.48 The two-gene theory had this model. It was proposed that null genes or duplicated to give way to the current hypothesis of the inheritance of a genes for C4A or C4B led to lack of Rodgers or Chido.40 haplotype modular gene complex that can vary by C4 copy As methods and antisera improved, numerous C4 al- number and is part of a larger gene complex in the MHC leles were discovered and the results of a workshop to locus. This model explains both the qualitative and quanti- define and standardize nomenclature were published in tative nature of the C4 gene expression as regards epitopes, a joint paper by Mauff et al.41 They described 14 determi- concentration of the C4 protein in the plasma, the frequency nants for the C4A gene and 17 for the C4B gene. Genes that of certain haplotypes, and the association of C4 copy number express no C4A or C4B are designated with a *Q0 (mean- variation with certain diseases. In fact, the C4 gene as part of ing quantity naught). In patients with complete deficiency the MHC complex can serve as an example of how the genes of C4 (C4A*Q0/B*Q0), null alleles were thought to be at- of an important biologic pathway evolve via the mechanics of tributable to gene deletions or point mutations that cause meiosis and through the pressure of natural selection to con- a frameshift in the DNA sequence, leading to defective or serve important genes and to maximize the utility of poly- no expression of the protein. One such example is a 2-bp morphism potential. Gene duplication, translocation, single- insertion into the sequence for codon 1213 at exon 29 of the nucleotide polymorphisms, insertions, and deletions all have mutant C4A gene seen in both Caucasians and those of Af- played a role in the plastic nature of the C4 gene. rican descent, but very rarely in those of Asian descent.42 Both forms of C4 are highly homologous but either The origin of the complement system C3 and C4 pro- by SNP or by insertions or deletions display great qualita- teins predates the ability to make lymphocytes or antibod- tive and quantitative diversity. The copy number variation ies; the latter did not arise on the evolutionary scene until comes from whether an individual has inherited haplotypes cartilaginous fish and sharks.43 Homology between C3 and that have one, two, three, or four C4 genes.49 This gene clus- C4 proteins indicates that the C4 gene may have evolved ter occurs as a complex in the MHC locus near the centromere from C3 as a duplicated gene.44 The C4A gene is thought with flanking genes in the following order: first an intact to have arisen by gene duplication and mutation from C4B RP gene (a serine/threonine kinase gene), an intact C4 because of its efficiency in facilitating the clearance of IC.28 gene (either C4A or C4B), then an intact CYP21B gene (cy- The C4A or C4B gene consists of 41 exons that in ad- tochrome P450 21-hydroxylase gene), and an intact TNXB dition to encoding for an acidic or basic isotype can also (tenascin-X protein). Approximately 17 percent of persons exist as a long form of 20.6 kb or a short form of 14.2 kb.45 of Northern European ancestry have this haplotype, termed The long form has an insertion of a human endogenous ret- monomodular.50 In 83 percent of those of Northern Euro- rovirus (HERV-K[C4]) into intron 9. The insertion of this pean ancestry the C4 gene complex can contain one, two, retroviral material is thought to have occurred after dupli- or three duplicated gene clusters made of fragments or mu- cation of the ancestral mammalian C4A gene but before the tations of the above flanking genes and an intact C4 gene. divergence of humans from the ancestors of other primates. The duplicated gene complex, called RCCX, occurs between The reading frames of the retrovirus are all closed, but the C4 gene and the intact CYP21B gene and consists of a there is still promoter activity and the HERV-K(C4) insert CYP21 (usually a mutated form) fused to a 4.9-kb fragment is thought to be able to modulate the expression of the C4 of TNXA fused to an RP2 0.91-kb fragment and an intact gene.46 The long form was originally thought to be a C4A C4 (A or B and long or short). Depending on the number of gene, but C4 genes can be C4A (long or short) or C4B (long RCCX modules duplicated, the complete C4 gene may be a or short). However, most C4A genes have the HERV-K(C4) monomodular (no RCCX duplication), bimodular, trimod- insert and a greater proportion of C4B genes do not. ular, or quadrimodular form.

34 IMMUNOHEMATOLOGY, Volume 26, Number 1, 2010 Chido/Rodgers review

This modular gene duplication maximizes opportuni- Progress both in human genetics, particularly by ties to conserve the C4 gene, whereas the opportunity for genome-wide association studies, and in the understand- recombination allows greater diversity of polymorphisms. ing of biologic pathways has led to the discovery of more This great diversity should allow for a better response to than 20 different loci that show strong correlation to SLE.54 pathogens but can create opportunities for unequal cross- Most of the genes identified are involved in three kinds of ing over during meiosis. Thus low or high copy number biologic processes. Minor variations in the genes encoding variations could be as important a risk factor for disease as the proteins of IC processing, production of interferon, or the actual C4 polymorphism. signal transduction of immune cells can greatly increase the C4 gene copy number studies done on healthy subjects susceptibility of an individual to SLE. It is to be expected of different ages indicate that those individuals with a low that more than one kind of trigger, such as pathogens, al- copy number for C4B may be less healthy or have a shorter lergens, or trauma, could then lead to disease. lifespan than those having more than two copies of the C4B Inasmuch as a major function of the C4 protein is par- gene.51 These studies found that in healthy individuals old- ticipation in innate immunity, there is a strong er than 50 years, there was a lower incidence of low copy association between deficiencies of C4 and recurrent in- number for the C4B gene when compared with the low copy fections; although this is less frequent than other comple- number gene frequency in younger healthy individuals. ment deficiencies.57 Table 2 lists other reported diseases and their possible association with C4 or complement. C4 and Disease Associations The association of C4 and a number of diseases has been Table 2. Disease associations with low levels of C4/complement a fruitful area of investigation, and at the genetic level, the Date Disease Citation frequency of C4 nulls or low copy variation shows a solid 2009 Schizophrenia Shi58 association with autoimmune disease, particularly systemic 2008 Role of complement in schizophrenia Mayilyan59 lupus erythematosus (SLE).52 Although the varying efficien- cies of complement function that result from partial or com- 2009 Chronic fatigue syndrome Sorenson60 plete deficiency may have a profound effect on autoimmune 2008 Autism and missing or nonfunctional C4B Sweetehn61 susceptibility, above-average C4 protein levels may also in- gene crease risk. Once a loss of self-tolerance occurs, having high 2008 Alzheimer’s and increased C4 deposition Zhou62 levels of C4 may exacerbate the disease. Moreover, it cannot 2008 Increased morbidity/mortality associated Szilagyi63 be overstated that association is not the same as causation. with low expression of C4B, smokers, and SLE can be thought of as a prototype for the intricacies of stroke the interaction of C4 proteins both in the development of 2007 Cardiovascular disease, smokers, and low Arason64 autoimmunity and in disease progression. Inheritance of a expression of C4B C4A*Q0 null gene shows a greater risk in Caucasian and 2006 C4 nulls and adult rhinosinusitis Seppänen65 African populations, but C4B*Q0 nulls are more frequent 2002 Systemic sclerosis Arason66 in SLE patients of Asian descent.53 SLE is a heterogeneous 1996 Insulin-dependent diabetes and C4 poly- Lhotta67 disease that is characterized by the presence of multiple au- morphisms toantibody specificities that attack various tissues, result- 1993 Graves’ disease Ratanachaiyavong68 ing in tissue damage, which then presents more antigen to 1991 C4A deficiency and IgA nephropathy Wyatt69 the host’s immune system. It has become increasingly clear 1990 Felty’s syndrome Hillarby70 that multiple genetic pathways are likely involved and that 1989 Psoriasis Wyatt 71 C4 polymorphism, especially copy number variation, can be a negative or a positive risk factor in the development 1989 Rheumatoid arthritis Fielder72 of autoimmune diseases.54 This postulates that the function 1988 C4 allotypes in juvenile dermatomyositis Robb73 of C4 in the initiation of autoimmunity may be related to 1987 C4B2 and primary biliary cirrhosis Briggs74 its role in presenting antigen to the host immune system, 1984 Glomerulonephritis, Henoch-Schönlein McLean75 which is separate from the role of complement in the in- purpura flammatory process once autoantibodies have been formed. The current understanding of SLE genetics is that there are In their paper, Atkinson and Frank43 also warn that pa- distinctions in severity, risk, and clinical manifestation that tients with complement C2- or C4-deficient states (either vary by ethnicity, geography, and sex with a greater preva- acquired or inherited) have backup complement activation lence in women and some non-Caucasian ethnic groups. mechanisms that must be considered in defining the role of Although genetics undoubtedly plays a role, SLE may also complement and a disease. For example, the immune re- be associated with poverty and exposure to infectious dis- sponse of specific antibody produced to a pathogen (antibody eases.55,56

IMMUNOHEMATOLOGY, Volume 26, Number 1, 2010 35 R. Mougey excess) may be all that is needed to trigger more ancient 12. Tilley CA, Romans DG, Crookston MC. Localization of pathways to perform cell lysis in the absence of sufficient Chido and Rodgers determinants to the C4d fragment complement proteins. of human C4. Nature 1978;273:713 -5. 13. Judd WJ, Kraemer K, Moulds JJ. The rapid identifica- Summary tion of Chido and Rodgers antibodies using C4d coated The C4 protein plays an important role in maintaining red blood cells. Transfusion 1981;21:189–92. health and, in some situations complicated by poor expres- 14. Giles CM, Hoffman M, Moulds M, Harris M, Delmasso sion of the C4 protein, may lead to or exacerbate certain A. Allo anti-Chido in a Ch-positive patient. Vox Sang diseases. The blood groups Chido and Rodgers are epitopes 1987;52:129–33. on the C4 protein, and polymorphisms associated with 15. Moulds JM, Nickells MW, Moulds JJ, et al. The C3b/ these epitopes may lead to the formation of antibodies to C4b receptor is recognized by the Knops, McCoy, the Chido or Rodgers antigens in transfused patients. Iden- Swain-Langley, and York blood group sera. J Exp Med tification of anti-Ch or anti-Rg is still based on the antibody 1991;173:1159–63. neutralization with plasma from Ch-positive or Rg-positive 16. Rao N, Ferguson DJ, Lee SF, Telen MJ. Identification individuals and lack of reactivity with qualified Ch-negative of human erythrocyte blood group antigens of C3b/C4b or Rg-negative RBCs. These antibodies may be useful in receptor. J Immunol 1991;146:3501–7. genetic studies of C4 polymorphisms or, in the case of C4- 17. Lambin P, LePennec PY, Hauptmann G, et al. Adverse deficient patients, a signal of the potential for serious ill- transfusion reactions associated with a precipitating nesses. The recognition of the extreme polymorphism of the anti-C4 antibody of anti-Rodgers specificity. Vox Sang C4 gene and the gene complex RCCX should lead to more 1984;47:242–9. insights in the understanding of disease risk and potential 18. Westhoff CM, Sipherd BD, Wylie DE, Toalson LD. treatment. Severe anaphylactic reactions following transfusions of platelets to a patient with anti-Ch. Transfusion References 1992;32:576–9. 1. Harris JP, Tegoli J, Swanson J, Fisher N, Gavin J, No- 19. Strupp A, Cash K, Uehlinger J. Difficulties in identi- ades J. A nebulous antibody responsible for crossmatch- fying antibodies in the Dombrock blood group sys- ing difficulties (Chido). Vox Sang 1967;12:140–2. tem in multiply alloimmunized patients, Transfusion 2. Swanson J, Olson J, Azar M. Serological evidence that 1998;38:1022–5. antibodies of Chido-York-Csa specificity are white cell 20. Awdeh ZL, Alper CA. Inherited structural polymor- antibodies (abstract). Fed Proc 1971;30:248. phism of the fourth component of human complement. 3. Middleton J, Crookston MC. Chido substance in plas- Proc Natl Acad Sci U S A 1980;77:3576–80. ma. Vox Sang 1972;23:256–61. 21. Rittner C, Giles CM, Roos MH, Demant P, Mollenhauer 4. Swanson JL. Laboratory problems associated with leu- E. Genetics of human C4 polymorphism: detection and kocyte antibodies. In: Seminar on recent advances in segregation of rare and duplicated haplotypes. Immu- immunohematology. Washington, DC: American Asso- nogenetics 1984;19:321–33. ciation of Blood Banks.1973 121–55. 22. Roos MH, Giles CM, Demant P, Mollenhuuer E, Ritt- 5. Middleton J, Crookston MC, Falk JA, et al. Linkage of ner C. Rodgers (Rg) and Chido (Ch) determinants on Chido and HLA. Tissue Antigen 1974;4:366–73. human C4: characterization of two C4B5 subtypes, one 6. Longster G, Giles CM. A new antibody specificity, anti- of which contains Rg and Ch determinants. J Immunol Rga, reacting with red cell and serum antigen. Vox Sang 1984;133:2634–40. 1976;30:175–80. 23. Yu CY. The complete exon-intron structure of a human 7. Nordhagen R, Olaisen B, Teisberg P, Gedde-Dahl T complement component C4A gene. DNA sequences, Jr. Association between the electrophoretically deter- polymorphisms and linkage to 21-hydroxylase gene. J mined C4M haplotype product and partial inhibition of Immunol 1991;146:1057–66. anti-Cha. J Immunogenet 1980;7:301–6. 24. Yu CY, Campbell RD, Porter RR. A structural model for 8. Giles CM. A new genetic variant for Chido. Vox Sang the location of the Rodgers and Chido antigenic deter- 1984;46:149–56. minants and their correlation with the human comple- 9. Giles CM, Gedde-Dahl T Jr, Robson EB, et al. Rga (Rod- ment components C4A/C4B isotypes. J Immunogenet gers) and the HLA region: linkage and association. Tis- 1988;27:399–405. sue Antigens 1976;8:143–49. 25. Belt KT, Carroll MC, Porter RR. The structural basis of 10. James J, Stiles P, Boyce F, Wright J. The HL-A type of the multiple forms of human complement component Rg(a–) individuals. Vox Sang 1976;30:214–16. C4. Cell 1984;36:907–14. 11. O’Neill GJ, Yang SY, Tegoli J, Berger R, Dupont B. Chido and Rodgers blood groups are distinct antigen components of human complement C4. Nature 1978;273:668–70.

36 IMMUNOHEMATOLOGY, Volume 26, Number 1, 2010 Chido/Rodgers review

26. Blanchong CA, Chung EK, Rupert KL, et al. Genetic, 40. Carroll MC, Palsdottir A, Belt KT, Porter RR. Deletion structural and functional diversities of human comple- of complement C4 and 21-hydroxylase genes in the ment components C4A and C4B and their mouse homo- HLA class III region. EMBO J 1985;4:2547–52. logues Slp and C4. Int Immunopharmacol 2001;1:365– 41. Mauff G, Alper CA, Dawkins RL, et al. C4 nomenclature 92. statement. Complement Inflamm 1991;7:261–8. 27. Law SK, Lichtenberg NA, Levine RP. Covalent binding 42. Barba G, Rittner C, Schneider PM. Genetic basis of and hemolytic activity of complement proteins. Proc human complement C4A deficiency, detection of a Natl Acad Sci U S A 1980;77:7194–8. point mutation leading to nonexpression. J Clin Invest 28. Dodds AW, Ren X-D, Willis AC, Law SKA. The reac- 1993;91:1681–6. tion mechanism of the internal thioester in the human 43. Atkinson JP, Frank MM. Bypassing complement: evo- complement component C4. Nature 1996;379:177–9. lutionary lessons and future implications. J Clin Invest 29. Carroll MC, Fathallah DM, Bergamaschini L, Alicot EM, 2006;116:1215–8. Isenman DE. Substitution of a single amino acid (as- 44. Martinez OP, Longman-Jacobsen N, Davies R, et al. Ge- partic acid for histidine) converts the functional activity netics of human complement component C4 and evolu- of human complement C4B to C4A. Proc Natl Acad Sci tion of the central MHC. Front Biosci 2001;6:D904– U S A 1990;87:6868–72. 13. 30. Colton HR. Genetics and synthesis of components of 45. Dangel AW, Mendoza AR, Baker BJ, et al. The dichoto- the complement system. In: Ross GD, ed. Immunobi- mous size variation of human complement C4 gene is ology of the complement system. An introduction for mediated by a novel family of endogenous retroviruses research and clinical medicine. New York: Academic which also establishes species-specific genomic pat- Press, 1986:174–6. terns among Old World primates. Immunogenetics 31. Uko G, Christiansen FT Dawkins RL, McCann VJ. 1994;40:425–36. Reference ranges for serum C4 concentrations in sub- 46. Mack M, Bender K, Schneider PM. Detection of retro- jects with and without C4 null alleles. J Clin Pathol viral antisense transcripts and promoter activity of the 1986;39:573–6. HERV-K(C4) insertion in the MHC class III region. Im- 32. Moulds JM, Arnett FC, Giles CM, Hamilton RG. A novel munogenetics 2004;56:321–32. immunoassay for the quantification of human C4 gene 47. Yu CY, Campbell RD. Definitive RFLPS to distinguish products. Complement Inflamm 1990;7:93–101. between the human complement C4/C4B isotypes 33. Chung EK, Yang Y, Rupert KL, et al. Determining the and the major Rodgers/Chido determinants: applica- one, two, three, or four long and short loci human com- tion to the study of C4null alleles. Immunogenetics plement C4 in a major histocompatibility complex hap- 1987;25:383–90. lotype encoding C4A or C4B proteins. Am J Hum Genet 48. Yu CY, Yang Z, Blanchong CA, Miller W. The human 2002;71:810–22. and mouse MHC class III region: a parade of the cen- 34. Fukuoka Y, Zia HZ, Sanchez-Munoz LB, Dellinger AL, tromeric segment with 21 genes. Immunol Today Escribano L, Schwartz B. Generation of anaphylatox- 2000;21:320–8. ins by human β-tryptase form C3, C4, C5. J Immunol 49. Chung EK, Yang Y, Rupert KL, et al. Determining the 2008;180:6307–16. one, two, three or four long and short loci of human 35. Richani K, Soto E, Romero R, et al. Normal pregnancy is complement C4 in a major histocompatibility complex characterized by systemic activation of the complement haplotype encoding C4A or C4B proteins. Am J Hum system. J Matern Fetal Neonate Med 2005;17:239–45. Genet 2002;71:810–22. 36. Gigli I, Fugita T, Nussenzweig V. Modulation of the 50. Wu YL, Yang Y, Chung EK, et al. Phenotypes, geno- classical pathway C3 convertase by plasma proteins C4 types and disease susceptibility associated with gene binding protein and C3b inactivator. Proc Natl Acad Sci copy number variations: complement C4 CNVs in Eu- U S A 1979;76:6596–600. ropean American healthy subjects and those with sys- 37. Gatenby PA, Barbosa JE, Lachmann PJ. Differences temic lupus erythematosus. Cytogenet Genome Res between C4A and C4B in the handling of immune com- 2008;123:131–41. plexes: the enhancement of CR1 binding is more impor- 51. Szilagyi A, Fust G. Diseases associated with low copy tant than the inhibition of immunoprecipitation. Clin number of the C4B gene encoding C4, the fourth Exp Immunol 1990;79:158–63. component of complement. Cytogenet Genome Res 38. Manderson AP, Pickering MC, Botto M, Walport MJ, 2008;123:118–30. Parrish CR. Continual low level activation of the classical complement pathway. J Exp Med 2001;194:747–56. 39. Giles CM. An update on Rodgers and Chido, the antigenic determinants of human C4. Immunohematology 1989;5:1–6.

IMMUNOHEMATOLOGY, Volume 26, Number 1, 2010 37 R. Mougey

52. Blanchong CA, Zhou B, Rupert KL, et al. Deficiencies 65. Seppänen M, Suvilehto J, Lokki M-L, et al. Immuno- of human complement component C4A and C4B and globulins and complement factor C4 in adult rhinosi- heterozygosity in length variants of RP-C4-CYP21-TNX nusitis. Clin Exp Immunol 2006;145:219–27. (RCCX) modules in Caucasians: the load of RCCX ge- 66. Arason GJ, Geirsson AJ, Kolka R, Vikingsdottir TH, netic diversity on major histocompatibility complex- Valdimarrson H. Deficiency of complement dependent associated disease. J Exp Med 2000;191:2183–96. prevention of immune precipitation in systemic sclero- 53. Yang Y, Chung EK, Zhou B, et al. The intricate role of sis. Ann Rheum Dis 2002;61:257–60. complement component C4 in human systemic lupus 67. Lhotta K, Auinger M, Kronenberg F, Irsigler K, Konig P. erythematosus. Curr Dir Autoimmun. 2004;7:98–132. Polymorphism of complement C4 and susceptibility to 54. Harley IT, Kaufman KM, Langfeld CD, Harley JB, Kelly IDDM and microvascular complications. Diabetes Care JA. Genetic susceptibility to lupus: new insights from 1996;19:53–5. fine mapping and genome-wide association studies. 68. Ratanachaiyavong S, Demaine AG, Campbell RD, Nat Rev Genet 2009;10:285–90. McGregore AM. Heat shock protein 70 (HS70) and 55. Crosslin KL, Wiginton KL. The impact of race and eth- complement C4 genotypes in patients with hyperthy- nicity on disease severity in systemic lupus erythemato- roid Graves’ disease. Clin Exp Immunol 1991;84:48– sus. Ethn Dis 2009;19:301–7. 52. 56. Hiraki LT, Benseler SM, Tyrrell PN, Harvey E, Herbert D, 69. Wyatt RJ, Julian BA, Woodford SY. C4A deficiency and Silverman ED. Ethnic differences in pediatric systemic poor prognosis in patients with IgA nephropathy. Clin lupus erythematosus. J Rheumatol 2009;36:2539–46. Nephrol 1991;36:1–5. 57. Figueroa JE, Densen P. Infectious diseases associ- 70. Hillarby MC, Strachan T, Grennan DM. Molecular ated with complement deficiencies. Clin Microbiol Rev characterisation of C4 null alleles found in Felty’s syn- 1991;4:359–95. drome. Ann Rheum Dis 1990;49:763–7. 58. Shi J, Levinson DF, Duan J, et al. Common variants on 71. Wyatt RJ, Wang C, Hudson EC, et al. Complement chromosome 6p22.1 are associated with schizophrenia. phenotypes in patients with psoriasis. Hum Hered Nature 2009;460:753–7. 1989;39:327–32. 59. Mayilyan LR, Weinberger DR, Sim RB. The comple- 72. Fielder AH, Ollier W Lord DK, et al. HLA class III hap- ment system in schizophrenia. Drug News Perspect lotypes in multicase rheumatoid arthritis families. Hum 2008;21:200–10. Immunol 1989;25:75–85. 60. Sorensen B, Jones JF, Vernon SD, Rajeevan MS. Tran- 73. Robb SA, Fielder AHL, Saunders CEL. C4 complement scription control of complement activation in an ex- allotypes in juvenile dermatomyositis. Hum Immunol ercise model of chronic fatigue syndrome. Mol Med 1988;22:31–8. 2009;15:34–42. 74. Briggs DC, Donaldson PT, Hayes P, et al. A major histo- 61. Sweeten TL, Odell DW, Odell JD, Torres AR. C4B null compatibility complex class III allotype (C4B2) associ- alleles are not associated with genetic polymorphisms ated with primary biliary cirrhosis (PBC). Tissue Anti- in the adjacent gene CYP21A2 in autism. BMC Med gens 1987;29:141–5. Genet 2008;9:1–7. 75. McLean RH, Wyatt RJ, Julian BA. Complement pheno- 62. Zhou J, Fonseca MI, Pisalyaput K, Tenner AJ. Comple- types in glomerulonephritis. Increased frequency of ho- ment C3 and C4 expression in C1q sufficient and de- mozygous null C4 phenotypes in IgA nephropathy and ficient mouse models of Alzheimer’s disease. J Neuro- Henoch-Schönlein purpura. Kidney Int 1984;26:855– chem 2008;106:2080–92. 60. 63. Szilagyi A, Fust G. Diseases associated with the low copy number of the C4B gene encoding C4, the fourth Ruth Mougey MT(ASCP)SBB, President, Mougey, Incor- component of complement. Cytogenet Genome Res porated, 1705 Highland Avenue, Carrollton KY 41008. 2008;123:118–30. 64. Arason GJ, Kramer J, Blasko B, et al. Smoking and a complement gene polymorphism interact in promoting cardiovascular disease morbidity and mortality. Clin Exp Immunol 2007;149:132–8.

Important Notice About Manuscripts Immunohematology is on the Web! for Immunohematology www.redcross.org/immunohematology

Please e-mail all manuscripts for consideration to For more information, send an e-mail message [email protected] to [email protected]

38 IMMUNOHEMATOLOGY, Volume 26, Number 1, 2010 In Memoriam: Marie Cutbush Crookston 1920–2009

D. Mallory

Marie Cutbush Crookston, a dynamic person and ex- These are some of her awards: ceptional scientist, was born in 1920 in Victoria, Australia. • The Karl Landsteiner Memorial Award in 1976 with Dr. She received a Bachelor of Science degree from the Univer- Eloise Giblett from the American Association of Blood sity of Melbourne, and, in 1947, she set sail for England. Banks, awarded “for her discoveries of anti-Fya, anti- There, she worked for 10 years with Dr. P. L. Mollison at Lub, and anti-HEMPAS: for her studies of the relation- the Medical Research Council Blood Transfusion Research ship between blood-group antibody activity in vitro and Unit and contributed to the writing of the first edition of Dr. in vivo: for her description of red cell uptake of Lea and Mollison’s Blood Transfusion in Clinical Medicine. Leb antigens from plasma; for her investigation of the In 1957, she married Dr. John Crookston, a hematolo- Chido antigen in plasma; and for the detection of the gist from Toronto, who was in London working for Dr. J. V. linkage between Ch and HLA.” (quotation from citation Dacie. Giving up her studies for a PhD, she moved to To- on award) ronto to raise two sons and to continue to work in the field • The Nuffield Foundation Award in 1952 with Dr. Her- of immunohematology. In 1964 she joined the University mann Lehmann for studying the blood groups of tribes of Toronto, Department of Pathology, where she directed in the Nilgiri Hills of South India. research, and in 1978 she became an associate professor. • The Buchanan Award in 1980 from the Canadian Soci- She also served as immunohematologist in the Department ety of Transfusion Medicine. of Hematology at the Toronto General Hospital and acted • The Canadian Blood Services Award in 2002 for “Life- as a consultant for the Blood Transfusion Laboratory until time Achievement.” she retired in 1986. Marie always found the time to help students and scien- Marie’s contributions to immunohematology are out- tists who needed directions with their studies or help with standing and have made a permanent impact on the field. their manuscripts. She was a wonderful, caring person who She and her late husband John helped transform the edu- was an enthusiastic, intelligent, and brilliant scientist and cational level and direction of immunohematology in the one who will be missed by her many friends and colleagues 1960s when they returned to Toronto from London. There, around the world. they helped to organize the Ontario Antibody Club, which was active for 25 years. Delores Mallory, MT(ASCP)SBB Marie’s studies on conversion of incomplete to complete Emeritus Editorial Board antibodies by chemical modification were used by commercial 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 published 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 terminology. 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.

IMMUNOHEMATOLOGY, Volume 26, Number 1, 2010 39 In Memoriam: Eloise Giblett 1921–2009

D. Mallory

Eloise “Elo” Giblett was born in Tacoma, Washington, chairperson of the Enzyme Nomenclature Committee of the in 1921 and, in 1931, the family moved to Spokane. Elo de- International Workshop on Human Gene Mapping. veloped a keen interest in music at an early age, taking pi- She received many honors during her lengthy career, includ- ano, dance, and violin, the instrument on which she would ing two from the American Association of Blood Banks: focus, becoming concertmaster of her high school orches- • 1975—Emily Cooley Lecture Award (American Associa- tra. She put aside music, turned to science, and received a tion of Blood Banks) BS in bacteriology from the University of Washington (U • 1976—Karl Landsteiner Memorial Award (American of W) in 1942. World War II had started, so Elo joined the Association of Blood Banks) WAVES in 1944, became a medical laboratory technician, • 1978—The Philip S. Levine Award (American Society of and served until 1946. She returned to the U of W and re- Clinical Pathology) ceived her MS degree in microbiology in 1947 and then, in • 1980—Election, National Academy of Science 1951, graduated first in her class in the secondclass to grad- • 1987—Distinguished Alumna Award (University of uate from the new medical school at the U of W. Washington) During the medical internship and hematology fellow- Dr. Giblett has left a legacy of original scientific discov- ship at U of W, her interest in human genetics was encour- eries, a book that was groundbreaking for the field of im- aged. In 1955, she became the first full-time physician at munohematology, and colleagues who were scientifically King County Blood Bank (now the Puget Sound Blood Cen- mentored and generously helped by her excellent editorial ter) as the Associate Director of the Typing and Crossmatch skills. Those who knew her will remember a very interesting Laboratory and was given a 6-month sabbatical with Dr. and giving human being. Patrick Mollison at the Blood Transfusion Research Unit in London, England. There, she studied the Lewis blood group Delores Mallory, MT(ASCP)SBB system, measured the rate of tagged RBC destruction, be- Emeritus Editorial Board 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 antibodies 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 member of the Editorial Board of Blood, Transfusion, and The American Journal of Human Genetics (among a few); and

40 IMMUNOHEMATOLOGY, Volume 26, Number 1, 2010 Announcements

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

Applications are invited from medical or science graduates for the Master of Science (MSc) degree in Transfusion and Transplantation Sciences at the University of Bristol. The course starts in October 2010 and will last for 1 year. A part-time option lasting 2 or 3 years is also available. There may also be opportunities to continue studies for PhD or MD following the MSc. The syllabus is organized jointly by The Bristol Institute for Transfusion Sciences and the University of Bristol, Department of Pathology and Microbiology. It includes:

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

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

The course is accredited by the Institute of Biomedical Sciences.

Further information can be obtained from the Web site: http://www.blood.co.uk/ibgrl/MscHome.htm

For further details and application forms please contact:

Dr Patricia Denning-Kendall University of Bristol, Paul O’Gorman Lifeline Centre, Department of Pathology and Microbiology, Southmead Hospital, Westbury-on-Trym, Bristol BS10 5NB, England Fax +44 1179 595 342, Telephone +44 1779 595 455, e-mail: p.a.denning-kendall@bristol. ac.uk.

IMMUNOHEMATOLOGY, Volume 26, Number 1, 2010 41 Announcements, cont., and advertisements

Specialist in Blood Bank (SBB) Program The Department of Transfusion Medicine, National Institutes of Health, is accepting applications for its 1-year Specialist in Blood Bank Technology Program. Students are federal employees who work 32 hours/week. This program introduces students to all areas of transfusion medicine, including reference serology, cell processing, HLA, and infectious disease testing. Students also design and conduct a research project. NIH is an Equal Opportunity Organization. Application deadline is December 31, 2010, for the July 2011 class. See www.cc.nih.gov/dtm > education for brochure and application. For further information contact Karen M. Byrne at (301) 451-8645 or [email protected].

Monoclonal antibodies available at no charge The New York Blood Center has developed a wide range of monoclonal antibodies (both murine and humanized) that are useful for donor screening and for typing RBCs with a positive DAT. These include anti-A1, -M, -s, -U, -D, -Rh17, -K, -k, -Kpa, -Jsb, -Fya, -Fy3, -Fy6, Wrb, -Xga, -CD99, -Dob, -H, -Ge2, -Ge3, -CD55 (both SCR2/3 and SCR4), -Oka, -I, and anti- CD59. Most of the antibodies are murine IgG and require the use of anti-mouse IgG for detection (anti-K, k, and -Kpa). Some are directly agglutinating (anti-A1, -M, -Wrb and -Rh17) and a few have been humanized into the IgM isoform (anti- Jsb). The antibodies are available at no charge to anyone who requests them. Please visit our Web site for a complete list of available monoclonal antibodies and the procedure for obtaining them.

For additional information, contact: Gregory Halverson, New York Blood Center, 310 East 67th Street, New York, NY 10021; e-mail: [email protected]; phone: (212) 570-3026; fax: (212) 737-4935; or visit the web site at http:// www.nybloodcenter.org >research >immunochemistry >current list of monoclonal antibodies available.

National Platelet Serology Reference Laboratory National Neutrophil Serology Reference Laboratory Diagnostic testing for: Our laboratory specializes in granulocyte antibody detection • Neonatal alloimmune thrombocytopenia (NAIT) and granulocyte antigen typing. • Posttransfusion purpura (PTP) Indications for granulocyte serology testing include: • Refractoriness to platelet transfusion • Heparin-induced thrombocytopenia (HIT) • Alloimmune neonatal neutropenia (ANN) • Alloimmune idiopathic thrombocytopenia purpura (AITP) • Autoimmune neutropenia (AIN) • Transfusion related acute lung injury (TRALI) Medical consultation available Methodologies employed: Test methods: • Granulocyte agglutination (GA) • GTI systems tests • Granulocyte immunofluorescence by flow cytometry (GIF) — detection of glycoprotein-specific platelet antibodies • Monoclonal antibody immobilization of neutrophil antigens — detection of heparin-induced antibodies (PF4 ELISA) (MAINA) • Platelet suspension immunofluorescence test (PSIFT) TRALI investigations also include: • Solid phase red cell adherence (SPRCA) assay • Monoclonal immobilization of platelet antigens (MAIPA) • HLA (PRA) Class I and Class II antibody detection • Molecular analysis for HPA-1a/1b For further information contact: For further information, contact Neutrophil Serology Laboratory (651) 291-6797 Platelet Serology Laboratory (215) 451-4205 Randy Schuller(651) 291-6758 [email protected] Maryann Keashen-Schnell (215) 451-4041 office [email protected] Sandra Nance (215) 451-4362 American Red Cross Blood Services [email protected] Neutrophil Serology Laboratory American Red Cross Blood Services 100 South Robert Street Musser Blood Center St. Paul, MN 55107 700 Spring Garden Street Philadelphia, PA 19123-3594

CLIA licensed CLIA licensed

42 IMMUNOHEMATOLOGY, Volume 26, Number 1, 2010 Advertisements, cont.

Reference and Consultation Services IgA/Anti-IgA Testing

Antibody identification and problem resolution IgA and anti-IgA testing is available to do the HLA-A, B, C, and DR typing following: HLA-disease association typing • Identify IgA-deficient patients Paternity testing/DNA • Investigate anaphylactic reactions • Confirm IgA-deficient donors

For information, contact Our ELISA for IgA detects protein to 0.05 mg/dL. Mehdizadeh Kashi at (503) 280-0210, or write to: For additional information contact Cindy Flickinger at Pacific Northwest Regional Blood Services (215) 451-4909, or e-mail: [email protected], ATTENTION: Tissue Typing Laboratory or write to: American Red Cross 3131 North Vancouver American Red Cross Blood Services Musser Blood Center Portland, OR 97227 700 Spring Garden Street Philadelphia, PA 19123-3594 CLIA licensed, ASHI accredited ATTN: Cindy Flickinger

National Reference Laboratory for Blood Group Serology

Immunohematology Reference Laboratory CLIA licensed

AABB, ARC, New York State, and CLIA licensed Donor IgA Screening (215) 451-4901— 24-hr. phone number (215) 451-2538— Fax • Effective tool for screening large volumes of American Rare Donor Program donors • Gel diffusion test that has a 15-year proven track (215) 451-4900— 24-hr. phone number record: (215) 451-2538— Fax Approximately 90 percent of all donors [email protected] identified as IgA deficient by are confirmed by the more sensitive testing methods Immunohematology Journal of Blood Group Serology and Education For additional information, call Kathy Kaherl at: (860)678-2764, e-mail: [email protected] (215) 451-4902— Phone, business hours (215) 451-2538— Fax or write to: [email protected] Reference Laboratory Quality Control of Cryoprecipitated-AHF American Red CrossBiomedical Services Connecticut Region (215) 451-4903— Phone, business hours 209 Farmington Ave. (215) 451-2538— Fax Farmington, CT 06032

CLIA licensed

IMMUNOHEMATOLOGY, Volume 26, Number 1, 2010 43 Advertisements, cont.

Blood Group Antigens & Antibodies A guide to clinical relevance & technical tips

by Marion E. Reid & Christine Lomas-Francis 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 Ordering Information to be found in testing 100 donors. Variations in different The book, which costs $25, can be ethnic groups are given. ordered in two ways: • Characteristics of the antibodies and optimal technique(s) • Order online from the publisher for their detection. at: www.sbbpocketbook.com • Technical tips to aid their identification. • Order from the authors, who • Whether the antibody has been found as an autoantibody. will sign the book. Send a check, made payable to “New York Blood Center” and indicate “Pocket Pocketbook Education Fund book” on the memo line, to: The authors are using royalties generated from the sale of this pocketbook for educational purposes to mentor people Marion Reid in the joys of immunohematology as a career. They will Laboratory of Immunochemistry accomplish this in the following ways: New York Blood Center • Sponsor workshops, seminars, and lectures 310 East 67th Street • Sponsor students to attend a meeting New York, NY 10065 • Provide copies of the pocketbook Please include the recipient’s (See www.sbbpocketbook.com for details to apply for funds) complete mailing address.

44 IMMUNOHEMATOLOGY, Volume 26, Number 1, 2010 Advertisements, cont. Becoming a Specialist in Blood Banking (SBB)

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

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

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

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

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

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 MIL 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/ On line sbb.htm Gulf Coast School of Blood Bank Technology Clare Wong 713-791-6201 [email protected] www.giveblood.org/education/ On line distance/htm 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/ On site department/divisions/trnafusion/ sbb.cfm 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. On line html Transfusion Medicine Center at Florida Blood Marjorie Doty 727-568-5433 x 1514 [email protected] www.fbsblood.org On line Services 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- 214-648-1785 [email protected] http://telecampus.utsystem.edu On line Fryer Additional Information can be found by visiting the following Web sites: www.ascp.org, www.caahep.org and www.aabb.org Revised August 2007 IMMUNOHEMATOLOGY, Volume 26, Number 1, 2010 45 ImmunohematologyImmunohematology JournalJOURNAL ofOF BloodBLOOD GroupGROUP SEROLOGY Serology AND and EDUCATION Education Instructions to for the Authors Authors

I. GENERAL INSTRUCTIONS b. Use short headings for each column needed and capitalize first letter Before submitting a manuscript, consult current issues of of first word. Omit vertical lines. Immunohematology for style. Double-space throughout the manuscript. c. Place explanation in footnotes (sequence: *, †, ‡, §, ¶, **, ††). Number the pages consecutively in the upper right-hand corner, beginning 8. Figures with the title page. a. Figures can be submitted either by e-mail or as photographs (5″×7″ II. SCIENTIFIC ARTICLE, REVIEW, OR CASE REPORT WITH glossy). LITERATURE REVIEW b. Place caption for a figure on a separate page (e.g. Fig. 1 Results of...), A. Each component of the manuscript must start on a new page in the ending with a period. If figure is submitted as a glossy, place first following order: author’s name and figure number on back of each glossy submitted. 1. Title page c. When plotting points on a figure, use the following symbols if 2. Abstract possible: � � � � � �. 3. Text 9. Author information 4. Acknowledgments a. List first name, middle initial, last name, highest degree, position held, 5. References institution and department, and complete address (including ZIP 6. Author information code) for all authors. List country when applicable. 7. Tables 8. Figures III. EDUCATIONAL FORUM B. Preparation of manuscript A. All submitted manuscripts should be approximately 2000 to 2500 1. Title page words with pertinent references. Submissions may include: a. Full title of manuscript with only first letter of first word capitalized 1. An immunohematologic case that illustrates a sound investigative (bold title) approach with clinical correlation, reflecting appropriate collaboration b. Initials and last name of each author (no degrees; all CAPS), e.g., M.T. JONES, J.H. BROWN, AND S.R. SMITH to sharpen problem solving skills c. Running title of ≤40 characters, including spaces 2. Annotated conference proceedings d. Three to ten key words B. Preparation of manuscript 2. Abstract 1. Title page a. One paragraph, no longer than 300 words a. Capitalize first word of title. b. Purpose, methods, findings, and conclusion of study b. Initials and last name of each author (no degrees; all CAPs) 3. Key words 2. Text a. List under abstract a. Case should be written as progressive disclosure and may include the 4. Text (serial pages): Most manuscripts can usually, but not necessarily, following headings, as appropriate be divided into sections (as described below). Survey results and i. Clinical Case Presentation: Clinical information and differential review papers may need individualized sections diagnosis a. Introduction ii. Immunohematologic Evaluation and Results: Serology and Purpose and rationale for study, including pertinent background references molecular testing b. Case Report (if indicated by study) iii. Interpretation: Include interpretation of laboratory results, Clinical and/or hematologic data and background serology/molecular correlating with clinical findings c. Materials and Methods iv. Recommended Therapy: Include both transfusion and Selection and number of subjects, samples, items, etc. studied and nontransfusion-based therapies description of appropriate controls, procedures, methods, equipment, v. Discussion: Brief review of literature with unique features of this reagents, etc. Equipment and reagents should be identified in case parentheses by model or lot and manufacturer’s name, city, and state. vi. Reference: Limited to those directly pertinent Do not use patient’s names or hospital numbers. vii. Author information (see II.B.9.) d. Results viii. Tables (see II.B.7.) Presentation of concise and sequential results, referring to pertinent tables and/or figures, if applicable IV. LETTER TO THE EDITOR e. Discussion A. Preparation Implication and limitations of the study, links to other studies; if 1. Heading (To the Editor) appropriate, link conclusions to purpose of study as stated in 2. Title (first word capitalized) introduction 3. Text (written in letter [paragraph] format) 5. Acknowledgments: Acknowledge those who have made substantial contributions to the study, including secretarial assistance; list any grants. 4. Author(s) (type flush right; for first author: name, degree, institution, 6. References address [including city, state, Zip code and country]; for other authors: a. In text, use superscript, Arabic numbers. name, degree, institution, city and state) b. Number references consecutively in the order they occur in the text. 5. References (limited to ten) 7. Tables 6. Table or figure (limited to one) a. 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. Send all manuscripts by e-mail to [email protected]

IMMUNOHEMATOLOGY, VOLUME 22, NUMBER 4, 2006 215 46 IMMUNOHEMATOLOGY, Volume 26, Number 1, 2010

FIRST CLASS U.S. POSTAGE PAID SOUTHERN MD Musser Blood Center PERMIT NO. 4144 700 Spring Garden Street Philadelphia, PA 19123-3594