Immunohematology

Journal of Blood Group Serology and Education Celebrating

2 Yea5rs Volume 25, Number 2, 2009

Immunohematology Journal of Blood Group Serology and Education Volume 25, Number 2, 2009 Contents

Invited Review: Milestones Series 39 Milestones in laboratory procedures and techniques M.E. Reid

Case Report 44 Anti-Kpa–induced severe delayed hemolytic transfusion reaction R. Koshy, B. Patel, and J.S. Harrison

Review 48 The ABO blood group system revisited: a review and update J.R. Storry and M.L. Olsson

Case Report 60 Clinical evaluation for lymphoproliferative disease prompted by finding of IgM warm autoanti-IT in two cases R.M. Leger, F. Lowder, M.C. Dungo, W. Chen, H.M. Mason, and G. Garratty

Original Report 63 Southeast Asian ovalocytosis is associated with increased expression of Duffy antigen receptor for chemokines (DARC) I.J. Woolley, P. Hutchinson, J.C. Reeder, J.W. Kazura, and A. Cortés

Review 67 Sickle cell disease: a review S.D. Roseff

Report 75 Consortium for Blood Group Genes (CBGG): 2008 report G.A. Denomme, C.M. Westhoff, L. Castilho, and M.E. Reid for the CBGG

Special Dedication 81 W. John Judd, FIBMS, MIBiol D. Mallory

82 Letter from the Editors

83 Announcements

84 Advertisements

88 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 Editor Philadelphia, Pennsylvania Christine Lomas-Francis, MSc New York City, New York Senior Medical Editor Geralyn M. Meny, MD Philadelphia, Pennsylvania

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 Joyce Poole, FIBMS Pomona, California St. Louis, Missouri Bristol, United Kingdom

James P. AuBuchon, MD W. John Judd, FIBMS, MIBiol Mark Popovsky, MD Lebanon, New Hampshire Ann Arbor, Michigan Braintree, Massachusetts

Martha R. Combs, MT(ASCP)SBB Christine Lomas-Francis, MSc Marion E. Reid, PhD, FIBMS Durham, North Carolina New York City, New York New York City, New York

Geoffrey Daniels, PhD Gary Moroff, PhD S. Gerald Sandler, MD Bristol, United Kingdom Rockville, Maryland Washington, District of Columbia

Anne F. Eder, MD John J. Moulds, MT(ASCP)SBB Jill R. Storry, PhD Washington, District of Columbia Shreveport, Louisiana Lund, Sweden

George Garratty, PhD, FRCPath Paul M. Ness, MD David F. Stroncek, MD Pomona, California Baltimore, Maryland Bethesda, Maryland

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

Editorial Assistant Production Assistant Judith Abrams Marge Manigly Proofreader Copy Editor Lucy Oppenheim Electronic Publisher Mary L. Tod Wilson Tang

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Milestones in laboratory procedures and techniques

M.E. Reid

hen I left England in 1969, hemagglutination was tory used plasma-suspended RBCs, which neutralized most done by sedimentation. In New York, I was intro- antibodies to Lewis and Ch/Rg antigens and thus they did Wduced to centrifugation as a method to speed the not spend time identifying these clinically insignificant an- process. Both approaches accomplished similar goals, but tibodies. Abandonment of unnecessary testing dramatically one was more time efficient. Use of other techniques, such improved the efficiency of testing by manual hemagglutina- as Western immunoblotting, cloning genes, and polymerase tion and did not have a noticeable negative impact on the chain reaction, made it possible to gain much knowledge efficacy of transfusion. Other changes, which occurred with about blood group antigens. the same goal, include using anti-IgG instead of the broad- spectrum reagent in routine antiglobulin tests, eliminating Immunohematology Beginnings the routine direct antiglobulin test (DAT), and not prepar- In 1975, Sandy Ellisor and I from Central California ing eluates from all samples whose RBCs were positive in Region of the American Red Cross (ARC) and Helen Glid- the DAT. den from ARC Headquarters volunteered to co-edit the first Also in the late 1970s, an extraordinary number of American Red Cross Reference Laboratory Newsletter. crossmatches were performed. Indeed, the number was ex- The purpose of this newsletter, which was for the technolo- cessive. The concepts of a “type and screen”2 and a “maxi- gists and by the technologists, was to share techniques and mum surgical blood order schedule”3 were introduced. technical tips, educate, and mentor in technical writing. The These approaches were quickly adopted, and they reduced first year (1976), the newsletter was low-tech; it was simply not only the number of crossmatches but also the number photocopied. That year, the ARC Reference Laboratory of RBC components reserved for a particular patient. Soon Committee ran a competition and by popular vote the news- after, the suggestion to drop the antiglobulin crossmatch letter was renamed the Red Cell Free Press. And to improve for patients who had a negative antibody screen was units appearance it was printed. This informal newsletter was leashed.4–6 Despite concerns that antibodies to antigens published for 8 years, until in 1984 it evolved into Immuno- not expressed on screening RBCs (e.g., anti-Wra) would hematology under the able editorship of Delores Mallory.1 be missed, this practice is now commonplace. Indeed, the During the 25-year life of Immunohematology, techniques physical immediate-spin crossmatch is now frequently re- evolved that allowed the gathering of an amazing amount placed by a computer crossmatch.7 of knowledge about blood groups and the membrane components on which they are carried. Despite this evolution, Use of Enhancement Reagents and Nonserologic to this day we still mainly rely on hemagglutination (direct Techniques and indirect) for detection and identification of antibodies Old standby techniques such as saline and albumin fell to blood group antigens and for testing for incompatibility. from favor and were largely replaced by low-ionic-strength This amazing technique is hard to beat in terms of sensitivi- solution (LISS) methods in the mid-1970s8 and by LISS-ad- ty and specificity that are appropriate for safe transfusions. ditive solutions shortly thereafter. LISS rapidly gained popu- larity because it simultaneously reduced incubation time and Early Immunohematology Testing Practices increased sensitivity. In 1984, the same year that the first vol- In the era before the ARC Newsletter, it was common- ume of Immunohematology appeared, a solid-phase adher- place to perform excessive testing (e.g., testing at room ence assay for detection of antibody-antigen reactions was temperature, use of enzyme-treated RBCs at temperatures introduced. For a review of this technology, see Beck et al.9 below 37ºC, absorbing eluates from autoimmune hemo- Subsequently, other techniques were reported and quickly lytic anemia cases, and performing minor crossmatches) became popular; two examples are the use of polyethylene on a single blood sample. Fortunately, Eloise Giblett in the glycol (PEG) as a potentiator of antibody-antigen reactions early 1960s advocated stopping unnecessary testing; she in 198710 and the column agglutination technology, using believed testing should be done as close to physiologic con- a gel as the support medium, in 1990.11 Automated assays ditions as possible, i.e., testing at 37ºC using plasma-sus- based on hemagglutination and manual or automated solid- pended RBCs. Her reasoning was scholarly and logical and phase adherence assays are now in use, but they have not changed the way testing was (and is) done. Giblett’s labora- replaced hemagglutination tests in tubes in many settings.

IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009 39 M.E. Reid

Many reagents became commercially available, and as biology techniques have made it possible to convert murine compliance requirements became more stringent, labora- IgG monoclonal antibodies to human IgG or directly agglu- tories reduced the number of reagents they prepared in- tinating IgM.20 house. Safety concerns prompted many changes. Favorite methods fell from grace, such as the use of ether—as a re- Techniques to Predict Clinical Significance of Anti- sult of concerns about its low flash point. Also, teratogenic, bodies carcinogenic, and neurotoxic chemicals became less read- Hemagglutination detects antibodies, but in itself does ily available. Awareness of the potential hazards of work- not predict their clinical significance. Criteria such as titer, ing with certain chemicals and with blood samples that may phase of reactivity, Ig class, IgG subclass, and specificity contain various pathogens led to the strict requirement to (and the historic knowledge of the clinical significance of an wear personal protective equipment such as gloves, labora- antibody in other patients) are useful indicators but are not tory coats, eye protection, and closed-toed shoes. The long- unequivocally good predictors of clinical significance. The standing practice of drinking, eating, and smoking in the monocyte monolayer assay (MMA) is as close to conditions laboratory was abolished. in vivo as is possible to establish in vitro and provides in- The value of proteolytic enzymes, notably ficin, for en- sight into the potential clinical significance of an antibody. hancing the reactivity of some antibody-antigen reactions It has been applied to predicting the clinical relevance of while weakening or ablating others has been appreciated antibodies in transfusion21 and hemolytic disease of the for many years.12 Other red cell modifications such as di- newborn,22–24 but is a highly specialized and technically dif- thiothreitol (DTT), trypsin, and α-chymotrypsin13 were in- ficult test that belongs in a small number of laboratories. troduced. However, it took some time before the full power Another technique, flow cytometry, has been used to detect of using the combination (i.e., testing RBCs treated with minor RBC populations in a fetal maternal hemorrhage and ficin or papain, trypsin, α-chymotrypsin, or DTT) of these to follow the survival of transfused RBCs,25,26 to determine methods to aid in antibody identification was appreciated. the dosage of a blood group antigen on RBCs,27 and quanti- Although classic antibody identification approaches (and tation of RBC-bound IgG.28 appropriate controls) are still necessary to name an anti- Numerous techniques have been used to identify al- body specificity positively, a system of parallel testing of the loantibodies underlying autoantibodies.29–32 All are time- same RBCs untreated and treated with different reagents consuming, as are methods to separate patient RBCs from provides a valuable tool in helping to guide us in the right di- those of transfused donor RBCs so that the patient’s RBCs rection, without requiring a vast library of rare reagents.14,15 can be phenotyped.33 Washing peripheral blood RBCs from When using enzymes for this purpose, it is important transfused patients with sickle cell disease with a hypoton- to include a negative (or auto) control, as a proportion of ic solution is a simple and clever technique to obtain the human sera contain antibodies that attach to RBCs treated patient’s RBCs for typing by hemagglutination.34 Although with a proteolytic enzyme. these techniques have value, they also have limitations.

Use of Monoclonal Reagents Molecular Testing For many years, we were dependent on polyclonal anti- Although hemagglutination remains the predominant bodies from human or rabbit sera or from plant lectins such test for detecting antibody-antigen interactions, unrelated as Vicia graminea, Ulex europeaus, and Dolichos biflorus techniques have provided insight into RBC components for blood grouping reagents. In 1975, Köhler and Milstein that carry blood group antigens. In the decade before described a technique to fuse murine myeloma cells with Immunohematology first appeared, the Western immuno- antibody-secreting B cells to produce monoclonal antibod- blotting method was used extensively to study characteris- ies.16 This technique was embraced, and monoclonal an- tics of components carrying blood groups. This approach, tibodies were produced to highly immunogenic antigens together with cloning and sequencing of the genes encoding such as to carbohydrate human blood group antigens (A blood group antigens,35 has revealed sequence homology to and B, Lewis, and P1) and high copy number glycoproteins known proteins in other cell types and provided insights (M, N).17 It took several years before human B cells were into the structure and function of the RBC membrane com- used in place of mouse cells. With this advancement came ponents carrying blood groups (Table 1).36 the production of monoclonal anti-D in 1983.19 Monoclo- The cloning and sequencing of genes laid the ground- nal antibodies with other specificities have been made in work for analysis of many alleles encoding variant blood the last 25 years.19 They are now the predominant source group antigens and phenotypes and, thus, our ability to test of typing reagents, and three international workshops have DNA by the polymerase chain reaction (PCR) to predict a been held to study reaction characteristics of numerous blood group. Subsequent manufacturing of machines to monoclonal antibodies. Monoclonal antibodies have had automatically perform PCR amplification made it possible a major impact on our ability to define variant RBCs, espe- to perform this technique in a clinical laboratory setting. cially partial D antigens, and to optimize techniques such PCR-based tests on DNA to predict a blood type have several as flow cytometry and Western immunoblotting. Molecular applications that provide a substitute for phenotyping.37 The

40 IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009 Milestones in laboratory procedures and techniques

presence of donor RBCs in a patient’s blood sample or IgG Table 2. Potential uses of DNA-based assays on RBCs (positive DAT) does not interfere with the tests. To type patients who have been recently transfused RBCs are not required to type a fetus at risk for hemolytic To identify a fetus at risk for hemolytic disease of the newborn disease of the newborn (Table 2). Thus, during the life of To type patients whose RBCs are coated with immunoglobulin (positive Immunohematology, we have had a reversal of phenotype DAT) and genotype. We used to phenotype RBCs to predict the To type patients with AIHA* to select antigen-negative RBCs for absorp- genotype; now we test DNA to predict the phenotype, both tion of autoantibodies when searching for underlying alloantibodies with the understanding that variant and null alleles exist. To type donors, including mass screening for antigen-negative donors, If high-throughput platforms were inexpensive enough, when appropriate antisera are not readily available DNA testing and prediction of antigen types could be used To type donors for use on antibody identification panels when antisera are as a screening method to increase antigen-negative inven- not available tories. Where antisera are available, negative DNA screening To type patients who have an antigen that is expressed weakly on RBCs results could be confirmed by hemagglutination methods, To resolve blood group A, B, and D discrepancies thereby conserving reagents. When antisera are not available, (e.g., anti-Dob, anti-Hy) the predicted type is often To study unusual and novel serologic reactions more reliable than the crossmatch. The availability of *AIHA = autoimmune hemolytic anemia increased inventories of RBC components with various combinations of antigen-negativity would make it possible Modifications of classic hemagglutination, which in- to more precisely match a patient’s phenotype, before the creased its sensitivity and specificity and reduced the time patient was immunized. The technique could be used to es- it takes to perform the test, have contributed to the safe sentially eliminate repetitive time-consuming techniques transfusion practice as we know it today. Although West- needed to identify underlying alloantibodies in cases of ern immunoblotting and PCR-based assays have led to an warm autoimmune hemolytic anemia and antibodies to understanding of the function and structure of RBC membrane high-prevalence antigens. components carrying blood groups, hemagglutination remains the workhorse of immunohematology testing. However, despite all our advances, errors in identification and clerical errors are still the big problem, Table 1. Structure and function of RBC membrance components and ABO incompatibility remains a primary carrying blood group antigens cause of preventable transfusion-related fatali- Function Carbohydrate Single-pass Multi-pass GPI-linked Adsorbed ties.38–40 Approximately half of the errors occur Glycocalyx ABO MNS LE when blood is transfused to the wrong patient. P Sample collection error, i.e., drawing blood H I from the wrong patient or mislabeling the GLOB sample, is also responsible for the blood being 41 Structural GE DI transfused to the wrong patient. Hemaggluti- RH nation in tubes does not require sophisticated XK equipment. It is simple, inexpensive (although Transport CO antibodies are becoming expensive and tech- DI nologist costs constantly rise), sensitive, spe- GIL cific, and reproducible. It remains the basic JK RH method for detecting reactions between RBCs RHAG and antibodies. XK Adhesion/ IN FY JMH Acknowledgments receptor LU RAPH I thank Steve Pierce for helpful sugges- LW tions and Robert Ratner for help in preparing MNS OK the manuscript. SC XG Enzyme KEL DO YT Complement KN CR CH/RG regulator MSN

IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009 41 M.E. Reid

References 18. Crawford DH, Barlow MJ, Harrison JF, Winger L, 1. Mallory D. Milestones in Immunohematology 1984- Huehns ER. Production of human monoclonal anti- 2009, Immunohematology 2009;25:6–8. body to rhesus D antigen. Lancet 1983;1:386–8. 2. Boral LI, Henry JB. The type and screen: a safe alter- 19. Anstee DJ, Parsons SF, Mallinson G, Spring FA, Judson native and supplement in selected surgical procedures. PA, Smythe J. Characterization of monoclonal antibod- Transfusion 1977;17:163–8. ies against erythrocyte sialoglycoproteins by serological 3. Friedman BA, Oberman HA, Chadwick AR, Kingdon analysis, immunoblotting and flow cytometry. Rev Fr KI. The maximum surgical blood order schedule and Transfus Immunohematol 1988;31:317–32. surgical blood use in the United States. Transfusion 20. Huang TJ, Reid ME, Halverson GR, Yazdanbakhsh K. 1976;16:380–7. Production of recombinant murine-human chimeric 4. Oberman HA, Barnes BA, Friedman BA. The risk of ab- IgM and IgG anti-Jsb for use in the clinical laboratory. breviating the major crossmatch in urgent or massive Transfusion 2003;43:758–64. transfusion. Transfusion 1978;18:137–41. 21. Nance SJ, Arndt P, Garratty G. Predicting the clinical 5. Garratty G. The role of compatibility tests. (Report of significance of red cell alloantibodies using a monocyte a meeting sponsored by the Bureau of Biologics for monolayer assay. Transfusion 1987;27:449–52. the Blood Products Advisory Committee). Transfusion 22. Nance SJ, Nelson JM, Horenstein J, Arndt PA, Platt 1982;22:169–72. LD, Garratty G. Monocyte monolayer assay: an effi- 6. Garratty G. Abbreviated pretransfusion testing (edito- cient noninvasive technique for predicting the severity rial). Transfusion 1986;26:217–19. of hemolytic disease of the newborn. Am J Clin Pathol 7. Butch SH, Judd WJ, Steiner EA, Stoe M, Oberman HA. 1989;92:89–92. Electronic verification of donor-recipient compatibility: 23. Lucas GF, Hadley AG, Nance SJ, Garratty G. Predicting the computer crossmatch. Transfusion 1994;34:105–9. hemolytic disease of the newborn: a comparison of the 8. Moore HC, Mollison PL. Use of a low-ionic-strength monocyte monolayer assay and the chemiluminescence medium in manual tests for antibody detection. Trans- test. Transfusion 1993;33:484–7. fusion 1976;16:291–6. 24. Hunt JS, Beck ML, Wood GW. Monocyte-mediated 9. Beck ML, Plapp FV, Sinor LT, Rachel JM. Solid-phase erythrocyte destruction: a comparative study of current techniques in blood transfusion serology (review). Crit methods. Transfusion 1981;21:735–8. Rev Clin Lab Sci 1986;22:317–42. 25. Nance SJ, Nelson JM, Arndt PA, Lam HC, Garratty 10. Nance SJ, Garratty G. A new potentiator of red blood G. Quantitation of fetal-maternal hemorrhage by flow cell antigen-antibody reactions. Am J Clin Pathol cytometry: a simple and accurate method. Am J Clin 1987;87:633–5. Pathol 1989;91:288–92. 11. Lapierre Y, Rigal D, Adam J, et al. The gel test: a new 26. Nance SJ, Garratty G. Application of flow cytometry to way to detect red cell antigen-antibody reactions. immunohematology. J Immunol Meth 1987;101:127– Transfusion 1990;30:109–13. 31. 12. Morton JA, Pickles MM. The proteolytic enzyme test 27. Oien L, Nance S, Arndt P, Garratty G. Determination of for detecting incomplete antibodies. J Clin Pathol zygosity using flow cytometric analysis of red cell anti- 1951;4:189–99. gen strength. Transfusion 1988;28:541–4. 13. Judson PA, Anstee DJ. Comparative effect of trypsin 28. Garratty G, Nance SJ. Correlation between in vivo and chymotrypsin on blood group antigens. Med Lab hemolysis and the amount of red cell-bound IgG mea- Sci 1977;34:1–6. sured by flow cytometry. Transfusion 1990;30:617–21. 14. Daniels G. Effect of enzymes on and chemical modifica- 29. Reid ME, Ellisor SS. Absorption of warm autoantibod- tions of high-frequency red cell antigens. Immunohe- ies using glutaraldehyde-treated human red cells. Am J matology 1992;8:53–7. Med Technol 1982;48:679–84. 15. Reid ME, Lomas-Francis C. Blood group antigens and 30. Branch DR, Petz LD. A new reagent (ZZAP) having antibodies: a guide to clinical relevance and technical multiple applications in immunohematology. Am J tips. New York: Star Bright Books, 2007. Clin Pathol 1982;78:161–7. 16. Kohler G, Milstein C. Continuous cultures of fused cells 31. Reid ME, Toy PTCY. Use of preserved autologous red secreting antibody of predefined specificity. Nature blood cells to absorb warm autoantibodies from the 1975;256:495–7. serum of patients receiving blood transfusion therapy. 17. Barnstable CJ, Bodmer WF, Brown G, et al. Production Vox Sang 1984;46:355–9. of monoclonal antibodies to group A erythrocytes, HLA 32. Leger RM, Garratty G. Evaluation of methods for de- and other human cell surface antigens: new tools for tecting alloantibodies underlying warm autoantibod- genetic analysis. Cell 1978;14:9–20. ies. Transfusion 1999;39:11–16.

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33. Reid ME, Toy PTCY. Simplified method for recovery of autologous red blood cells from transfused patients. Am J Clin Pathol 1983;79:364–6. 34. Brown DJ. A rapid method for harvesting autologous red cells from patients with hemoglobin S disease. Transfusion 1988;28:21–3. 35. Lögdberg L, Reid ME, Lamont RE, Zelinski T. Human blood group genes 2004: chromosomal locations and cloning strategies. Transf Med Rev 2005;19:45–57. 36. Reid ME, Mohandas N. Red blood cell blood group antigens: structure and function. Semin Hematol 2004;41:93–117. 37. Reid ME. Applications of DNA-based assays in blood group antigen and antibody identification. Transfusion Marion E. Reid, PhD, FIBMS 2003;43:1748–57. Editorial Board 38. Linden JV, Paul B, Dressler KP. A report of 104 Immunohematology transfusion errors in New York State. Transfusion 1992;32:601–6. 39. Shulman IA, Kent D. Unit placement errors: a potential risk factor for ABO and Rh incompatible blood transfusions. Lab Med 1991;22:194–6. 40. Williamson LM, Lowe S, Love EM, et al. Serious hazards of transfusion (SHOT) initiative: analysis of the first two annual reports. BMJ 1999;319:16–19. 41. Sazama K. Reports of 355 transfusion-associated deaths: 1976 through 1985. Transfusion 1990;30:583–90.

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IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009 43 Case Report

Anti-Kpa–induced severe delayed hemolytic transfusion reaction

R. Koshy, B. Patel, and J.S. Harrison

Kpa is a low-frequency antigen occurring in less than 2 percent rectal bleeding, nausea, weakness, and dizziness. Rectal ex- of the Caucasian population. Mild to moderate delayed hemolytic amination confirmed the rectal fissures and bleeding. There transfusion reactions (DHTR) and hemolytic disease of the fetus were no other remarkable findings noted on physical exam- a and newborn attributable to anti-Kp have been reported. Severe ination. ECG showed sinus tachycardia at 106 beats/min. overt DHTR has not been reported with anti-Kpa. A case of a se- She was admitted for further workup and for RBC transfu- vere DHTR attributed to anti-Kpa after multiple RBC transfusions is being reported. A 52-year-old Caucasian woman received mul- sions. tiple units of RBCs for a lower gastrointestinal bleed. She was re- Past history was negative for prior transfusions. Patient ferred to our institution for hepatic and renal failure, which was has an adult son who was listed as a contact person, and no supported by laboratory findings of peak LDH, bilirubin, BUN, other pregnancy history was available. She had left renal and creatinine elevations. Hemoglobin had dropped on Day 10 angioplasty several years ago for left renal artery stenosis. after transfusion. The DAT and antibody screen (ABS) were nega- She had a history of rectal fissures. She is a smoker (2 packs tive. Initial workup and subsequent ABS were negative. Anti-Kpa per day) and drinks occasionally. She has a history of al- was identified when an additional RBC panel was tested. One of lergy to amitriptyline. the RBC units transfused was incompatible by antihuman globulin (AHG) crossmatch with the patient’s plasma and typed positive for Kpa. DHTR was confirmed after extensive workup. The patient responded to supportive therapy and experienced an uneventful recovery. DHTR may not be considered when DAT and ABS are negative. However, correlation of recent transfusion with signs and symptoms should alert the clinician to entertain and investigate a DHTR that should include the AHG crossmatch of all implicated RBC units. The severity of the reaction also raises concerns as to when and what antigen specificity should be considered for inclusion in the antibody screening cells. Immunohematology 2009;25:44–47.

pa (KEL 3, Penney) is one of 31 antigens in the Kell (ISBT symbol, KEL) blood group system. The Kell Kantigens, encoded by KEL, located on chromosome 7q33, appear to be erythroid specific and are found in the fetal liver and in bone marrow cells.1 Kpa is a low-incidence Fig. 1 Hemoglobin (Hb; g/dL) and hematocrit (Hct; %) values for day 0 to day 13 are shown. antigen found in less than 2 percent of Caucasians.1,2 The antigen is resistant to the effects of enzyme treatment but is sensitive to treatment with dithiothreitol and acid. Mild to moderate delayed hemolytic transfusion reaction (DHTR) and mild to moderate hemolytic disease of the fetus or newborn (HDFN) have been reported as well as a case of hydrops fetalis attributed to anti-Kpa.3 The antibody is an IgG.4–6 Suppression of erythropoiesis by anti-Kpa has been reported as the cause of decreased hemoglobin in HDFN.7 Reports of an overt DHTR attributable to several antibodies missed in the antibody screen and immediate-spin crossmatch mention anti-Kpa as one of the antibodies.8 However, there have been no reported cases of severe overt DHTR as a result of anti-Kpa as per MEDLINE search.

Case Report Fig. 2 Alkaline phosphatase (Alk Phos), lactate dehydrogenase (LDH), A 52-year-old Caucasian woman presented to the emer- and creatine phosphokinase (CPK) values for day 0 to day 14 are gency room complaining of being light-headed and having shown.

44 IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009 Anti-Kpa–induced DHTR

Table 1. Laboratory data from the referring hospital transfused. Urinalysis on Day 11 Tests Normal range Patient results showed evidence of increased free Admission Day 2 Day 7 Day 10 Day 11 Day 12 hemoglobin, bilirubin, and uro- Hemoglobin 4.5–11 g/dL 6.1 12 — 8.7 10.2 9.9 bilinogen. Computed tomographic scan and magnetic resonance im- Hematocrit 36%–48% 19.6 — — 26.9 31.2 29.3 aging to rule out acute cholecystitis WBC 4.5–11.0 x 9.9 — — — — — revealed an enlarged gallbladder 10 9/dL with thickened wall and inflamma- Platelets 120,000– 628,000 — — — — — tion of adjacent areas, reported as 450,000/µL suspicious for cholecystitis. Liver, Reticulocytes 0.9%–1.9% — — — — 4.1 — spleen, and pancreas were within PT 11.5–14.7 s 13.8 — — 13.5 15.2 18.1 normal limits. There was also evi- Glucose 65–100 mg/dL 125 — — — dence of atrophic left kidney and Alkaline phosphatase 30–133 U/L 138 142 203 211 422 393 hypertrophic right kidney with se- BUN 7–21 mg/dL 18 — 18 36 46 51 vere stenosis of a segment of the right renal artery close to its origin. Creatinine 0.5–1.4 mg/dL 0.8 — 1.0 4.2 5.6 6.2 The patient received 3 additional AST 5–39 U/L 31 — — 161 529 303 units of RBCs between Days 10 and ALT 7–56 U/L 19 — — 113 168 120 11 for the drop in hemoglobin. LDH 333–699 U/L — 521 3983 — 7905 2215 On the 13th day of her hospital 7637 course, she was admitted to the re- CPK 35–230 U/L 87 425 — — — — ferral hospital, our institution, for Total bilirubin 0.2–1.3 mg/dL 0.3 — — 1.8 23.5 18.3 investigation of acute liver and re- Direct bilirubin 0.0–0.4 mg/dL — — — — 24.7 16.1 nal failure. DHTR was suspected, and a workup was ordered by the ABO, D — O — — O O — D positive D positive D positive hematologist. Pigment nephropa- thy attributable to intravascular Antibody screen — Negative — — Negative Negative — hemolysis was considered as the DAT — — — — — Negative — cause for the acute renal failure. HBsAg, HBc antibody, HBs antibody, and hepatitis C antibody—all nonreactive The cause of the severe hepatotox- Urinalysis — — — — — icity could not be ascertained. Bilirubin Negative Moderate Urobilinogen <1 mg/dL 4 mg/dL Free hemoglobin Negative Large Materials and Methods At the referral hospital, ABO and ALT = alanine aminotransferase; AST = aspartate aminotransferase; BUN = blood urea nitrogen; D typings were performed using the CPK = creatine phosphokinase; HBc antibody = hepatitis B core antibody; HBs antibody = Ortho A/B/D Monoclonal and Re- hepatitis B surface antibody; HBsAg = hepatitis B surface antigen; PT = prothrombin time; WBC verse Grouping Card (Ortho-Clinical = white blood cell count. Diagnostics, Inc., Raritan, NJ). Affir- magen reagent RBCs (pooled cells), Admission Hb was 6.1 g/dL. She was transfused with 0.8%, were used for reverse typing, and antibody screen was 5 units of type-specific, leukoreduced or washed (as per done by IgG gel card, 0.8% Surgiscreen (Johnson & Johnson, hospital policy), immediate-spin, crossmatch-compatible New Brunswick, NJ). Antibody panel was performed using RBC during the course of 24 hours of admission, which 0.8% Resolve Panel A (Ortho-Clinical Diagnostics, Inc.) and raised her Hb to 12 g/dL. She remained hospitalized for Panocell 16, by Immucor, Inc. (Norcross, GA). The DAT was further evaluation for gastrointestinal bleed. On the 10th performed using anti-IgG-C3d with check cells, anti-IgG and posttransfusion day, her Hb dropped to 8.7 g/dL and her check cells, and anti-C3b/C3d with complement check cells Hct was 26.9%. Her reticulocyte count was 4.1% (reference (Immucor, Inc.). Reagents used for phenotyping were from range, 0.9 to 1.9%) on Day 11. There was no evidence of con- Immucor, Inc. Elution was performed with Gamma ELU-KIT tinued rectal bleed. There was a gradual increase of several II (Gamma Biologicals, Inc., Houston, TX) following initial sa- chemistry results, which peaked on the 10th and 11th post- line wash (Fisher Diagnostics, Middleton, VA) of patient cells. transfusion days (Table 1; Figs. 1–4). The patient appeared ABO and D typings and a three-cell antibody screen were severely jaundiced with signs of acute liver and renal fail- performed by gel technique and read through the MTS reader ure, which was supported by her laboratory values. Investi- SA (Ortho-Clinical Diagnostics, Inc.). DAT was performed gation for a suspected DHTR revealed a negative DAT and with polyclonal AHG, anti-IgG, and anti-C3d by tube method a negative antibody screen. Investigation did not proceed and eluate panel by gel method using Ortho and Immucor to antihuman globulin (AHG) crossmatch for all RBC units panel cells.

IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009 45 R. Koshy et al.

Results The DHTR investigation on the day of admission at the referral hospital revealed a negative antibody screen with the three-cell antibody screen. The DAT was negative (including a 15-minute incubation) with polyspecific, IgG, and C3b/C3d AHG. At the referral hospital, tests with a 16-cell reagent RBC panel (Immucor, Inc.) revealed the presence of anti-Kpa. All other clinically significant antibodies were ruled out. Eluate prepared from DAT-negative RBCs was negative against Kp(a+) RBC on Immucor Panocell 16. The patient typed negative for Kpa. AHG crossmatch was done from samples obtained from donor segments of the trans- Fig. 3 Total and direct bilirubin values for day 0 to day 20 are shown. fused RBC units received from the blood supplier. One of the five RBC units transfused on Day 1 of admission was incompatible with the patient’s plasma and typed positive for Kpa. Haptoglobin was 1 mg/dL (reference range, 34–200 mg/dL). A diagnosis of severe DHTR with intravascular hemolysis attributable to anti-Kpa was confirmed. As the patient’s renal and liver functions improved, she did not undergo dialysis or consideration for further liver or renal evaluation. The patient was monitored and maintained on supportive therapy. Her hospital course was uneventful. All laboratory results returned to normal values (Figs. 1–4). She was discharged 10 days after her admission to the referral hos- Fig. 4 Alanine aminotransferase (ALT) and aspartate aminotrans- pital. ferase (AST) values for day 0 to day 20 are shown. Table 2 presents the patient’s laboratory data on admission to the referral hospital on Day 13. Table 2. Laboratory data on admission at the referral hospital, Day reside on the C-terminal domain of Kell in the structural 13 sequence N-terminal to the zinc-binding catalytic motif, Test Result which is the major site for endothelin-3–converting enzyme activity. Haptoglobin (reference range 1 mg/dL 34–200 mg/dL) Kell antigens are important in transfusion medicine owing to their strong immunogenicity, and the correspond- ABO, D (by Ortho gel) O, D positive ing antibodies are clinically significant.9 Anti-Kpa has been Antibody screen (0.8% Surgiscreen) Negative implicated in mild to moderate HDFN and DHTR. Severe Panel cells, A Negative DHTR as presented in this case has not been reported. Panocell 16 Positive (anti-Kpa identified) Clinical signs and the laboratory values presenting on Day 7 Kpa typing of patient’s RBCs Negative with peak laboratory values at Days 10 and 11 after transfu- a DAT—using polyclonal, IgG, and Negative sion of a unit of RBCs positive for Kp support a diagnosis C3b/C3d AHG in this case of a severe DHTR with intravascular hemoly- a a Panel with eluate Negative sis attributable to anti-Kp . The cause of the primary Kp exposure could not be determined from the patient’s his- Crossmatch with 1 of 5 units trans- Incompatible, Kp(a+) fused at referring hospital tory. AHG crossmatch of all implicated RBC units despite the negative DAT or antibody screen would have positively Kpa typing of incompatible unit Kp(a+): 1+ by tube and 2+ by gel techniques identified the incompatible Kp(a+) RBC as the implicated unit for the DHTR. Discussion The patient under discussion experienced unusually Kpa was first described by Allen and Lewis in 1957.2 The severe hepatotoxicity, and the reasons for this remain un- Kpa antigen is a member of the Kell blood group system and clear: there was no prior history of liver disease. Severe is carried on the Kell glycoprotein. The prototypical gene hemolysis with resultant high levels of free heme can cause for the Kell protein family was cloned and characterized in undesirable toxicity, leading to organ, tissue, and cellu- the early 1990s.9 As discussed by Lee and colleagues,9 the lar injury. The mechanism of action is through free heme antigens of the Kell blood group system result from single that catalyzes the oxidation, covalent crosslinking, and ag- nucleotide changes in the Kell protein. Most Kell antigens gregate formation of protein and its degradation to small

46 IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009 Anti-Kpa–induced DHTR peptides. It also catalyzes the formation of cytotoxic lipid 3. Smoleniec J, Anderson N, Poole G. Hydrops fetalis peroxide via lipid peroxidation and damages DNA through caused by a blood group antibody usually undetected oxidative stress. Heme, being a lipophilic molecule, inter- in routine screening. Arch Dis Child Fetal Neonatal Ed calates in the membrane and impairs lipid bilayers and or- 1994;71:F216–7. ganelles, such as mitochondria and nuclei, and destabilizes 4. Pinkerton PH, Coovadia AS, Goldstein J. Frequency of the cytoskeleton. It also causes endothelial cell injury, lead- delayed hemolytic transfusion reactions following an- ing to vascular inflammation, and stimulates the expression tibody screening and immediate-spin crossmatching. of intracellular adhesion molecules. As a proinflammatory Transfusion 1992;32:814–17. molecule, heme induces inflammation that results in toxic 5. Garratty G. How concerned should we be about miss- effects on the kidney, liver, central nervous system, and car- ing antibodies to low incidence antigens? Transfusion diac tissue.10 The severe heme toxicity may also be attribut- 2003;43:844–47. able to the markedly compromised renal condition owing to 6. Reid ME, Lomas-Francis C. The blood group antigen the severe stenosis of the right renal artery. factsbook. San Diego, CA: Academic Press, 1997:182– The severity of the DHTR in this case may warrant con- 4. sideration for inclusion of clinically significant low-frequency 7. Tuson M, Koyal K, Desai R, et al. Probable suppression antigens that may be missed by current screening cells for of fetal erythropoiesis by anti-Kp (abstract). Transfu- detection of clinically significant RBC antibodies. - How sion 2007;47(3 Suppl):166A. ever, the risk of overt DHTRs to low-incidence antigens is 8. Shulman IA. The risk of an overt hemolytic transfusion estimated at 1 per 650,000 crossmatches.5 The calculation reaction following the use of an immediate spin cross- was based on report of probability of acute overt hemolytic match. Arch Pathol Lab Med 1990;114:412–14. transfusion reaction at 1 per 260,000 with immediate-spin 9. Lee S, Zambas ED, Marsh WL, Redman CM. Molecular crossmatches of 1.3 million negative antibody screens.8 As cloning and primary structure of Kell blood group pro- the risk of overt DHTR is extremely small, the cost benefit tein. Proc Natl Acad Sci U S A 1991;88:6353–7. should be considered for inclusion of low-frequency an- 10. Kumar S, Bandyopadhyay U. Free heme toxicity and tigens such as Wra, Cw, and Kpa in the antibody screening its detoxification systems in human. Toxicol Lett cells.5 We concur with the previous observation. AHG phase 2005;157:175–88. crossmatch must be included in the investigation of any suspected DHTR irrespective of a negative DAT or nega- Ranie Koshy, MD (corresponding author), Director, Blood tive antibody screen. Appropriate and timely patient care to Bank and Transfusion Services, and Bhishma Patel, SBB, avert further patient harm depends on a thorough investi- Blood Bank and Transfusion Safety Officer, Department gation. of Pathology and Laboratory Medicine, NJMS/UMDNJ, C101, University Hospital, Newark, NJ 07103; and Jona- References than S. Harrison, MD, Department of Medicine, Hema- 1. Roback, JD, ed. Technical Manual. 16th ed. Bethesda, tology, Robert Wood Johnson Medical School—Pisc/New MD: American Association of Blood Banks, 2008. Brunswick, New Brunswick, NJ. 2. Allen FH Jr, Lewis SJ. Kpa (Penney), a new antigen in the Kell blood group system. Vox Sang 1957;2:81–7.

IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009 47 Review The ABO blood group system revisited: a review and update

J.R. Storry and M.L. Olsson

The antigens of the ABO system were the first to be recognized as Variation in A antigen expression was also recognized blood groups and actually the first human genetic markers known. very early in the twentieth century (reviewed in Race and Their presence and the realization of naturally occurring antibod- 10 Sanger ), and the A blood group was divided into A1 and ies to those antigens lacking from the cells made sense of the A .1,11 Other descriptions of weakened A antigen expression erratic failure of blood transfusion hitherto and opened up the 2 followed, and the A blood group was subdivided further possibility of a safe treatment practice in life-threatening blood loss. Although initially apparently simple, the ABO system has based on characteristic reactivity with human polyclonal come to grow in complexity over the years. The mass of knowl- antisera, i.e., strength of reactivity and presence of mixed edge relating to carbohydrate chemistry, enzymology, molecular field agglutination; presence of anti-A1; and whether A or H genetics, and structural and evolutionary biology is now enormous blood group substance was present in the saliva of secretor thanks to more than a century of research using ABO as a principal subjects (Table 1). Weak forms of B antigen were also found model. This has provided us with data to form a solid platform but are typically more difficult to define serologically into of evidence-based transfusion and transplantation medicine used specific categories although some subgroups (e.g., elB ) are every day in laboratories and clinics around the globe. This review analogous to their A counterparts (A ). aims to summarize key findings and recent progress made toward el The frequency of the common ABO phenotypes (A , A , further understanding of this surprisingly polymorphic system. 1 2 B, A B, A B, and O) varies greatly among different popula- Immunohematology 2009;25:48–59. 1 2 tions.12,13 Populations with a high frequency of A phenotype Key Words: ABO, blood group, antigen, allele, genotype are found mainly in Northern and Central Europe, and the

here have been many reviews of the ABO blood group Table 1. Subgroups of A—agglutination reaction patterns adapted from textbooks system written throughout the years, covering different aspects of this fascinating topic. A limited se- Sub- Reactions* with Sub- Anti- T group stances A1 in Anti-A Anti-A,B Anti-A1 Anti-H lection of such reviews can be found in the reference list, of A in saliva† serum particularly for readers who want to focus more on the dis- A ++++ ++++ ++++ 0 A, H No covery,1 biochemistry,2,3 enzyme structure,4 and molecular 1 A ++++ ++++ 0 ++++ A, H Some- genetics.5,6 Our intention is not to reproduce them but to 2 times follow the guidelines of this new series of blood group sys- A ++++ ++++ ++(+) +++ A, H No tematic reviews in Immunohematology to provide a brief int A ++(+) mf ++(+)mf 0 ++++ A, H Some- introduction to this amazingly complex blood group system. 3 times A 0/+ ++(+) 0 ++++ H Often History x

The discovery of the ABO blood group system ranges Aend + + 0 ++++ H Some- from myth and folk legend all the way to the Nobel Prize. Karl times

Landsteiner, a Viennese pathologist, made the observation Am 0/+ 0/+ 0 ++++ A, H No that when his serum and that of five colleagues were mixed Afinn + + 0 ++++ H Yes individually with their saline-suspended RBCs, agglutina- Abantu +(+) +(+) 0 ++++ H Yes tion was observed with some mixtures but not with others. A 0‡ 0 +++** ++++ H Yes*** He reported this as a footnote to a paper published in 19007 1ae A 0‡ 0 0 ++++ A, H No followed by a more comprehensive paper in 1901.8 Transla- y A 0‡ 0 0 ++++ H Some- tions of both papers can be found in the review by Camp el times and Ellis.1 In these early studies, he showed that two each of the six sera discriminated among three blood groups: A, *A negative reaction is denoted by 0. Positive reactions are denoted as from + (weak agglutination) to ++++ (maximal agglutination). B, and C (later renamed O from the German ohne, meaning **Dolichos biflorus only. without). Thus, Landsteiner demonstrated that a person’s ***Serum reactivity against both A1 and A2 RBC. serum contained antibodies to the antigen(s) lacking from †Blood group ABO substances in saliva and other body fluids of their RBCs. The fourth blood group, AB, was described a secretors. ‡ year later by Decastello and Sturli9 in four individuals in a Despite lack of agglutination, anti-A can be adsorbed to and eluted from cells in this subgroup. larger study of 34 healthy subjects and 121 patients. mf = mixed field agglutination.

48 IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009 ABO blood group system

A2 phenotype reaches its peak among the Lapps in North- dbRBC Web site (http://www.ncbi.nlm.nih.gov/projects/ ern Scandinavia but is very rare in Asia. The B phenotype gv/rbc20). In this review, alleles will be referred to in the is most frequent in Central Asia and almost absent in Am- traditional way but with dbRBC terminology given in brack- erindians. Blood group O is the most frequent phenotype ets, e.g., A1 [A101] and O1 [O01]. in a global perspective, with Native American Indians be- It can be specifically noted that it is important for clar- ing almost exclusively blood group O. Parts of Africa and ity and consistency to use subscripts, superscripts, and 1 Australia also show high frequencies of blood group O. The italics appropriately. For example, A1, A1, and A mean the reason for the differences observed among populations is antigen, the phenotype, and the allele, respectively. not fully understood although several theories have arisen. The concept of evolutionary selection based on pathogen- Inheritance and Molecular Genetics driven blood group changes will be discussed later (see The A and B antigens are inherited as Mendelian char- section on pathogen interactions). The early importance of acteristics in a codominant autosomal fashion. In 1908, Ep- ABO diversity is supported by reports in which the group stein and Ottenberg21 were the first to suggest in a short case O-defining single base pair deletion at nucleotide position report that ABO blood groups might be inherited. This was 261 (see section on molecular genetics) has been found in later proved by von Dungern and Hirszfeld in 1910 (trans- both Neandertal people14 and ancient Egyptian mummies,15 lated by Pohlmann22). In fact, ABO inheritance was one of suggesting that selection pressure and survival of the fittest the first genetic markers to be used in paternity testing and were indeed early features during our co-evolution with in forensic medicine.23,24 pathogens like malaria parasites and many others. Unlike the majority of blood groups, the antigens of the six currently known carbohydrate systems are not coded Nomenclature by genes directly. Instead, these blood group genes encode The ABO system was the first to be discovered and has glycosyltransferases that in turn synthesize the oligosac- therefore been given the number 001 in the official Inter- charide epitopes (Fig. 1). Thus, the A and B antigens are national Society of Blood Transfusion (ISBT) terminology. made by A and B glycosyltransferase, respectively, encoded Nomenclature for the ABO antigens is actually straightfor- by the ABO gene carried on the long arm of chromosome 9 ward since the antigen status is determined by the pres- (9q34). As with many of the blood group genes, the position ence or absence of specific carbohydrate molecules. This is of the ABO locus was known for many years before the gene particularly true for the A and B antigens but may be less was cloned.25 The genes encoding the A-synthesizing 3-α-N- clear for the two other antigens (A,B and A1) given official acetylgalactosaminyltransferase and the B-synthesizing antigen status by the ISBT. The A,B antigen is thought to 3-α-N-galactosaminyltransferase were cloned by Yamamo- be an epitope not involving the A versus B–differentiating to and colleagues in 199026,27 after purification and partial surfaces, but consists of a common recognition motif16 amino acid sequencing of A transferase from lung tissue.28 found when either the A or B antigens are present. The de- Probing cDNA libraries obtained from human adenocar- bate surrounding the real identity of the A1 antigen is still cinoma cell lines of different ABO types, the main alleles 17,18 26 ongoing, but the A1 phenotype reveals many differences were defined. They determined that the B-specific mRNA compared with A2, both quantitative and qualitative, so the differed from the A-specific gene by only 7 of 1062 coding question is quite complex (see section on biochemistry). nucleotides, of which 4 result in amino acid differences in Table 2 shows both the numerical and traditional nomen- the enzyme product. The difference between the A1 [A101] clature according to the ISBT Working Party on Terminol- and O1 [O01] genes was shown to be a single guanosine (G) ogy for Red Cell Surface Antigens.19 deletion, which alters and severely truncates the open reading

frame (ORF) as shown in Figure 2. The A2 phenotype was Table 2. ABO nomenclature shown to depend on a cytosine deletion in the 5′ end of the System ABO antigen notation gene, resulting in elongation of the ORF.29 The organization ISBT number 001 ABO1 ABO2 ABO3 ABO4 of the ABO locus30,31 is shown in Figure 3. The gene consists (001001) (001002) (001003) (001004)

ISBT name ABO A B A,B A1 O H O H C H O H C H 2 O H 2 Fig. 1 Principal O O O H O H H O C H O H H O C H 2 O H 2 structure of the A, B, O O Blood group allele nomenclature including ABO is un- A c N H O H and H oligosaccha- O O O O H O O H der consideration by a subcommittee of the aforementioned rides. The difference C H C H 3 O 3 O ISBT Working Party. In the absence of an officially agreed- between the A and O H O H H O H O on terminology, several different variants have evolved (see B structures is high- O H O H O H A t r i s a c c h a r i d e B t r i s a c c h a r i d e 6 C H 2O H Table 7 in Chester and Olsson ). Typically, alleles are re- lighted by arrows. O ferred to by their serologic activity and a number. An un- H O O O H official but often used terminology is found at the Blood C H 3 O O H Group Antigen Gene Mutation Database, also known as the H O O H

H d i s a c c h a r i d e

IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009 49 J.R. Storry and M.L. Olsson of seven exons (plus an alternative exon 1a located upstream have been described that encode glycosyltransferases ca- of exon 132), with the majority of the catalytic domain of the pable of synthesizing easily detectable levels of both A and enzyme encoded by exon 7. B antigens. This group includes alleles conveying the two Since the groundbreaking cloning paper, 215 ABO se- unusual phenotypes cisAB and B(A). Fifty-eight alleles are quence entries have been submitted to the dbRBC Web predicted to give rise to proteins without enzymatic activity site20 (accessed on April 15, 2009) and curated by the dbR- of which 45 contain 261delG, the mutation that alters the BC staff and experts, but new alleles are constantly being translational ORF and predicts a shortened protein product identified. Figure 4 shows a breakdown of the 181 alleles with no transferase activity. This large group of O alleles in the dbRBC categorized by their association with normal includes four principally important evolutionary lineages: or altered phenotypes: these include 65 different A alleles, O1 [O01], O1v [O02], Otlse09 or O1(467T;318T) [O09], and O1bantu 47 B alleles, 58 O alleles, and 11 “AB” alleles. The remain- [O54].33,34 Numerous minor variants of these main alleles35 ing 34 sequences listed in the current dbRBC consist of 20 as well as hybrid O alleles combining A2 or B with different sequences encompassing the 5′ noncoding region including 261delG-containing O sequences36 have also been found, the repetitive and polymorphic CCAAT box binding fac- most notably in individuals of African ancestry. Of the re- tor/nuclear factor Y (CBF/NF-Y) motif and 14 intronic or maining 13 “nondeletional” alleles, 3 suffer similar fates to overlap sequences. Of the A alleles, 6 and 11 encode nor- that caused by 261delG because they contain nonsense mu- mal A1 or A2 phenotypes on RBCs, respectively, whereas 48 tations resulting in altered ORFs caused by nucleotide in- describe mutated A alleles (Aweak) associated with different sertions. Another 3 have nonsense mutations introducing variants of weak A antigen expression (Table 1); the B al- immediate stop codons, thereby truncating the ORF at the leles include 9 normal and 38 weak alleles; and 11 alleles same codon where they occur. Finally, 7 recorded nonde- 261261 467 467 526526 703703 * * 10601060 letional O alleles are crippled by at least one missense mutation each, giving rise to critical amino acid substitutions, A 1 the most famous example of which is 802G>A resulting in

2 268Arg. For a recent review about this latter group of inter- A esting O alleles designated O2 [O03], see Yazer and Olsson.37 What is most intriguing about these nondeletional O alleles B is that they are all attributable to mutations in A allele back- bone sequences. This has two main practical consequences: O 1 first, virtually all ABO genotyping methods will signal the

O 2 70

Fig. 2 Schematic representation of predicted open reading frames 60 for some common ABO alleles. White rectangles represent trans- 13 lated A1 [A101] consensus; black rectangles represent translated 50 non-A1 consensus (nonsense). Vertical bars indicate nucleotide 40 48 (nt) positions (given above bars) of mutations leading to amino acid UUnusual/weaknusual/weak p hphenotypeenotype CCommo/expectedommon/expected pphenotypehenotype changes. * indicates B [B01] allele is mutated at nt 796 and 803; 30 O2 [O03] allele at nt 802. 38 Number of alleles 45 Number of alleles

Number of alleles 20

10 17 9 11

0 A B O AB BloodBlood blood grou pspecificity spe cificity

Fig. 4 Graphic representation showing the number of ABO alleles currently listed in the dbRBC database. The black bars represent al- Fig. 3 Organization of the ABO gene. A: The seven exons and six leles that encode a glycosyltransferase with normal activity; the white introns are not drawn to scale. The numerals above boxes represent bars represent alleles encoding glycosyltransferases that have altered the first and last nucleotides (nt) of the coding region in each exon, activity or specificity. The AB bar includes alleles encoding glycosyl- and those below boxes show the corresponding amino acid (a.a.) transferases capable of synthesizing both A and B antigens, i.e., cisAB numbers. The size of each intron is indicated with a thin oblique bar. and B(A). The group O bar has been divided into the 45 O alleles B: The ABO gene drawn to scale (except intron 1); exons are black that contain the mutation 261delG (black) and the 13 “nondeletional” and introns gray. alleles without it (white). The latter group is often associated with weak expression of A antigen.

50 IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009 ABO blood group system presence of A1 or A2 alleles unless specifically designed cal phenotypes can have more than one molecular genetic to detect rare O alleles, which will result in the potentially background. catastrophic prediction of group A in a group O person. The regulatory mechanism of the ABO gene has been Second, the phenotype actually conveyed by inheritance of investigated extensively. An enhancer element located ap- one of these so-called O alleles is not always O but often proximately 4 kbp upstream of exon 150 was found to con- weak A or O-like but without anti-A present in plasma. The tain four 43-bp repeats in all alleles except A1 [A101] and the reason for this is currently unclear, but there has recently infrequent O2 [O03], which have only one copy.51 This may been a series of papers investigating the serologic pattern play a role in expression.52 The enhancer region contains and mechanisms behind this interesting phenomenon.38–42 a CBF/NF-Y binding site; mutations in this site decrease There is currently no O allele described that is based on a B enhancer activity in a gastric cancer cell line,50 and altera- sequence, but we predict that when such an allele is found, tions in this region may even cause rare B subgroup pheno- it will show the opposite serology, i.e., either weak B expres- types.53 However, it was recently shown that A1 [A101] or A2 sion or O-like without anti-B in plasma. [A201] transcripts are virtually undetectable in peripheral It is the molecular genetics that make this system so fas- blood whereas B and O (including O2 [O03]) mRNA is read- cinating because mutations ranging from single nucleotide ily found.54,55 This appears to speak against a critical role for changes to more complex hybrid gene formations can alter CBF repeats in erythroid ABO regulation, but more work is the specificity of the enzyme, the efficacy of the enzyme, or required. There is also an Sp1-binding site in the proximal both; changes that, at the phenotypic level, are manifested promoter that may be important for erythroid ABO regula- simply by altered antigen expression. Good examples are tion.56 the various molecular bases of the Ax phenotype, which can Although much is understood regarding the cause of either result from a single missense mutation in exon 7 of weak A/B antigen expression on RBCs when it comes to in- an A1 [A101] allele or be based on different hybrids, for inherited weak ABO subgroups,5,6 less is known about altered stance between the 5′ end of B and the 3′ end of O alleles.43,44 ABO expression in hematologic disorders. Erythrocytes los-

The B3 phenotype in the Taiwanese population has also ing A, B, or H antigen have been noted in patients with he- been shown to result from different molecular backgrounds, matologic malignancy, especially in the myeloid lineage.57,58 one a missense mutation but more commonly, a principally Very recently it was suggested that methylation of the ABO interesting mutation that was the first one shown to affect proximal promoter is the reason for such leukemia-associated splicing of ABO mRNA and cause exon skipping.45 In ad- downregulation.59 Reduced A and B antigen expression in dition to altered specificity or enzymatic efficacy, aberrant bladder and oral carcinomas is partially attributable to loss intracellular trafficking of ABO transferase may cause weak of heterozygosity or hypermethylation.60,61 Furthermore, subgroup phenotypes.46 urothelial tumor tissue contains decreased amounts of A/B As alluded to previously, this added layer of complexity, antigens that correlated to decreased levels of ABO mRNA in which mutations affect enzyme activity and not the anti- compared with cells from normal tissue.62 Transient depres- gen directly, provides difficulties in designing genotyping sion of A antigens has also been observed in some pregnant assays that will detect rare variants, which if misinterpreted women,44 but the reason for this is still unknown. can have serious consequences in ABO group determination.47 Biochemistry There are problems associated with all the more than The biochemistry of the A and B antigens was elucidat- 30 ABO genotype screening methods published or commer- ed by the astonishingly early and brilliant work from the cially available so far (reviewed in Olsson and Chester48). groups of Morgan and Watkins, and Kabat (reviewed by The vast majority is only designed to determine between Watkins2 and Kabat63). The A, B, and H determinants were three and six of the common alleles, although additional hypothesized to reside on water-soluble glycoproteins able alleles can often surface if their polymorphisms interfere to inhibit agglutination of RBCs by antibodies or lectins. with the detection system. However, virtually all methods A precursor substance, H, was hypothesized as a building will fail to predict the phenotype of a sample in the pres- structure for A and B, and the terms O- (or H-) substance ence of most A/B subgroup alleles, rare nondeletional null and anti-H were introduced in 1948.64 alleles, cisAB and B(A) alleles, or hybrid alleles, which can Owing to difficulties in obtaining sufficient quantities of lead to serious consequences. Because this is particularly blood group active material after extraction from RBCs, most dangerous if blood or transplant recipients are typed (as of the early studies were performed on ovarian cyst or animal suggested, e.g., by Procter et al.49) we recently developed mucin, rich in secreted blood group substances. Before isola- and implemented an improved ABO genotype screening tion and chemical identification of these substances, inhibi- method that addresses these problems and can be used in a tion with simple sugars indicated N-acetyl-d-galactosamine clinical laboratory setting.47 In summary, the complex genet- (GalNAc), d-galactose (Gal), and l-fucose (Fuc) as the defin- ics of ABO is a major challenge for ABO genotyping efforts, ing sugars in blood groups A, B, and O, respectively.65,66 not least because of the disturbing fact that a certain allele Independent of the blood group of the individual can lead to more than one phenotype and seemingly identi- investigated, a surprisingly similar composition of carbo-

IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009 51 J.R. Storry and M.L. Olsson hydrate residues (mainly Fuc, Gal, GalNAc and N-acetyl- epithelial cells that line the lumen of the gastrointestinal, d-glucosamine [GlcNAc]) was found. This was taken as a respiratory, and reproductive tracts as well as in salivary sign of large similar precursor molecules carrying small glands and skin. This wide distribution is a common fea- residues that differentiate blood groups.67 The minimal ture for many of the carbohydrate blood groups, which has determinant structures were subsequently shown to be resulted in the term histo-blood group often being used to trisaccharides as shown in Figure 1. The important concept reflect this wide distribution. A and B antigen synthesis of precursor-product relationship between H and A/B was occurs during normal glycosylation of proteins and lipids formulated,67,68 and the critical role of nucleotide-bound in the Golgi compartment.77 The precursor H substance is sugars as substrates in oligosaccharide synthesis was appre- synthesized by one of two fucosyltransferases depending on ciated.69 The biosynthetic pathway of the ABO blood group the acceptor substrate used. The FUT1 gene that encodes structures is as outlined in Figure 5, and the presence of the 2-α-fucosyltransferase (α2FucT1) is responsible mainly A and B glycosyltransferases was first predicted70 and then for the synthesis of the H antigen on type 2 (and type 4) car- experimentally established.71–73 Thus, the A and B glycosyl- bohydrate precursors found on RBCs.78 The closely related transferases use UDP-GalNAc and UDP-Gal, respectively, FUT2 gene encodes a very similar 2-α-fucosyltransferase as substrates. Both require the H determinant as acceptor. (α2FucT2) that is expressed in epithelial cells and synthe- The O protein is nonfunctional, leaving the H-defining sizes H antigen mainly on type 1 and type 3 chains.77 The terminal Fuc unaltered. β α major precursor types present in different tissues and

UDP - GaINAc UDP secretions are shown in Table 3. U D P - G a lN A c U D P GaINAc α 3Galβ - R GDPG D P - -FucF u c GGDPD P G a lN A c α 3 G a l β - R Table 3. Peripheral core structures and their principal tissue A antigen α2α GGala lββ -- R- R A transferase A a n t ig e n 2 Fuc 3 A tr a n s fe r a s e F u c distribution (modified from Clausen and Hakomori ) H Htr transferasea n s fe r a s e α 2 G aGall ββ --R R α2 F uFucc BB ttransferaser a n s fe r a s e B antigen Galα3Galβ - R Peripheral core type Structure Distribution B a n t ig e n G a l α 3 G a l β - R H antigen Precursor α P r e c u r s o r H a n t ig e n α2 Type 1 Galβ1→3GlcNAcβ1→R Endodermal, secretions, UPD - Gal UDP FFucu c U D P - G a l U D P plasma Type 2 Gal 1 4GlcNAc 1 R Ecto- and mesodermal Fig. 5. Schematic depiction of the biosynthetic pathways for con- β → β → (e.g., erythrocytes) version of H determinants to A or B determinants. R represents the core structure. See text for enzyme abbreviations. Type 3 Galβ1→3GalNAcα1→R O-linked mucin-type, repetitive A Type 4 Galβ1→3GalNAcβ1→R Glycolipids in kidneys (and erythrocytes) Although the composition of the A and B antigens is R = inner core structure or linkage. apparently straightforward, the biochemistry behind the shared A,B antigen recognized by many group O plasmas In the Bombay phenotype (Oh), a silenced FUT1 gene is has only recently been elucidated experimentally. Bovin present together with a silenced FUT2 gene. Because H an- and his colleagues16 synthesized a deacetylated A trisaccha- tigen is the precursor substrate for both A and B antigens, ride structure and bound it to the precursor in such a way neither antigen can be synthesized without α2FucT activity, as to expose what they hypothesized would be the common independent of the ABO genotype. The para-Bombay phe- A,B epitope. They were able to demonstrate binding of both notype results from either (1) a silenced FUT1 gene present monoclonal and polyclonal anti-A,B to the synthetic struc- together with an active FUT2 gene, which permits the syn- ture. thesis of H type 1 (and therefore A/B antigens) that may be Although it is well known that there are approximately adsorbed onto the RBC from the plasma; or (2) a mutated five times fewer A antigens on A2 than on A1 RBCs, the un- FUT1 gene in which the encoded enzyme activity is greatly derlying basis for the qualitative differences between them diminished, so that very low amounts of H antigen (and is less well defined. The A2 transferase is 10 times less ef- A/B antigen) are produced. It may be present with or with- ficient than the 1A transferase and has a different pH opti- out an active FUT2 gene. In both cases, H antigen (and A/B 74,75 mum and pI. The A2 enzyme is also less able to use chains antigen) is very weakly expressed and is often only detected other than type 1 or 2 carbohydrate precursors like the ex- by adsorption and elution tests with the appropriate blood tended type 3 (repetitive A) and type 4 (globo-A) chains on group reagents. The H blood group system (ISBT 018) will RBC glycolipids.17,76 More recently, Svensson et al.18 have be the subject of another review later in this Immunohematology produced somewhat contradictory data indicating that al- series. though the A2 transferase can readily use H type 3 chains to The A and B glycosyltransferases are type II membrane synthesize A antigen, the low levels of type 4 chains remain proteins located in the Golgi compartment,79,80 although unconverted. soluble forms are found in plasma and other body fluids. The ABH sugars are found on glycolipids (approximate- The enzyme consists of a short transmembrane domain, a ly 10%) and glycoproteins (approximately 90%) on the RBC stem region, and a catalytic domain that extends into the as well as on many different tissues and cell types, including Golgi lumen (Fig. 6). The crystal structure elucidated by

52 IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009 ABO blood group system

The authors concluded that the absence of dietary exposure to bacteria prevented the production of anti-B despite normal immunoglobulin levels in the patients’ sera. Recogni- tion of self prevents an individual from making antibodies to antigens shared by the bacteria and may also partially explain disease susceptibility of one blood group over another (discussed briefly in the next section). Anti-A and anti-B are predominantly IgM antibodies although IgG or IgA components are often found.85,86 Class- switching to IgG does not occur unless there is a “hyperim- munizing” event such as an ABO-incompatible pregnancy or transfusion. Absence of the expected antibodies occurs rarely, although antibody titers vary considerably among individuals and have been shown to diminish with age. Pa- Fig. 6 A: The topology of the transferase is shown as a Golgi-local- tients who have immunoglobulin deficiencies will also lack ized, membrane-bound enzyme with a cytoplasmic N-terminus and anti-A or anti-B. Otherwise, most often the absence of an catalytic C-terminal domain (modified from Paulson and Colley79). agglutinin in a healthy person should be taken as an indi- B and C: The three-dimensional surface model (B) was created cation that there may be a weak antigen or even (micro) with the Deep View Swiss Pdb Viewer version 3.7, and the three- chimerism present, perhaps detectable by adsorption and dimensional ribbon structure (C) was generated using SETOR and elution or by flow cytometry. SetoRibbon (unpublished). The vertical bar to the left illustrates Individuals of the A2 and A2B phenotypes as well as the approximate exon usage for the different glycosyltransferase those whose RBCs carry certain A subgroups (Table 1) can domains. also produce anti-A1, generally reactive at room temperature and below. Although ABO antibodies of IgG type can Patenaude and colleagues81 showed that the catalytic site cross the placenta, severe ABO-related hemolytic disease is divided into two domains; the N-terminal domain rec- of the newborn is not common. The mechanisms behind ognizes the nucleotide sugar donor substrate (UDP-Gal or this are discussed in more detail in the section on clinical UDP-GalNAc), whereas the acceptor substrate is held by consequences. the C-terminal domain. The DXD motif (DVD in these enzymes) that serves to capture Mn2+, essential to the reac- Pathogen Interactions tion because of its ability to bind the UDP part of the donor Bacteria, viruses, and parasites have been proposed as substrate, lies between the two domains. In addition, two important driving forces for the geographic distribution of so-called disordered loops have been identified by the crys- ABO blood group phenotypes because different pathogens tal structural studies. One is located at the C-terminal of the demonstrate blood group–identical or –cross-reactive mol- enzyme. The other disordered loop lies close to the catalytic ecules on their surfaces. These are the probable targets for site of the enzyme. Its role is unclear4; however, mutations “blood group” antibodies, and their existence is the lead- in this region have been shown to reduce enzyme activity ing hypothesis as to why we make naturally occurring an- and are often associated with weak subgroup phenotypes.82 tibodies against the carbohydrate blood groups we lack. In addition, many pathogens show selective binding to blood Antibodies in the System group carbohydrate moieties via lectins (reviewed by Gar- Anti-A and anti-B are naturally occurring antibodies ratty87). More recent but perhaps anecdotal evidence for that are produced by immunocompetent individuals from this theory was provided after severe hemolytic transfusion the age of approximately 6 months. The widely held dogma reactions in two group B patients receiving apheresis plate- is that these antibodies are in fact mimicking antibodies lets from the same group A platelet donor.88 The donor had produced against terminal carbohydrates on bacterial cell donated regularly for a period of 20 years, during which walls as a response to our normal intestinal microbial flora, time no adverse effects of transfusion had been observed, and that these glycotopes share structural homology with A but had recently begun to supplement his diet with high- and B antigens.83 In a modern follow-up to this original con- dose probiotics and his anti-B titer was shown to be greater cept, three children with different congenital immune de- than 8000. Inhibition studies performed with plasma from fects were studied after apparent ABO grouping discrepan- random group A donors and solubilized probiotic tablets cies.84 These patients (age range, 1.7 to 8 years) were limited demonstrated a reduction in titer, hinting at a role for ABO to total parenteral nutrition and tube feeding. Their RBCs antibodies in neutralizing pathogens. This line of thinking typed as group A, but each child lacked the expected anti-B is supported further by data from studies on viral glycosy- by routine testing, although anti-B was weakly detectable in lation in host cells. HIV cultured in vitro with peripheral the serum of one of them after prolonged incubation at 4°C. blood mononuclear cells (PBMC) from donors of different

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ABO groups demonstrated specific neutralization with 59 percent. Surprisingly, however, many reports show that anti-A of those isolates grown in group A PBMCs but not approximately 50 percent of patients who are inadvertently those cultured with group B or group O cells.89 Measles vi- transfused with a major ABO-mismatched unit of blood tol- rus, when cocultured in a system expressing ABH glycosyl- erate the blood without any apparent signs of a transfusion transferases (to mimic an in vivo environment), expressed reaction.101 The mechanisms underlying the ability of some A or B epitopes, or not, according to the enzymes expressed. individuals to tolerate ABO-mismatched blood are not well Viral particles could then be neutralized by normal poly- understood but are very interesting as identification of clonal anti-A or anti-B sera in the presence of complement resistance markers could be exploited in transplantation if the corresponding glycans were present.90 Conversely, therapy. Despite our relative sophistication in providing ap- the A and B (and H) antigens are also used as receptors for propriately ABO-matched blood for patients, there is recent pathogen invasion by attachment to host cells as mentioned clinical evidence that even the concept of ABO-compatible previously, so the equilibrium between invasion and eva- (as opposed to ABO-identical) products may not be as safe sion is a fine balance on both sides. as it would appear. A study that followed the posttransfu- Furthermore, growing evidence for a leading role of sion mortality among more than 86,000 patients receiving Plasmodium falciparum as the major force shaping the hu- plasma showed that exposure to ABO-compatible but non- man genome including the distribution of the blood groups ABO-identical plasma was associated with an increased risk has emerged.91,92 The parasite-induced RBC surface protein of death.102 Whether this is associated with immune com- pfEMP1 is known to bind to the A antigen trisaccharide,93 plexes between anti-A/B and soluble A/B antigen in AB and it has been shown that the severity, including mortality, plasma, for instance, remains to be studied. Furthermore, of malarial disease is significantly lower in group O children reports of the usage of platelet concentrates in cardiac than in other groups, mainly through the mechanism of re- surgery has also demonstrated less favorable outcomes in duced rosetting.94,95 It is the focus on pediatric disease that those patients receiving ABO-mismatched products.103 The is thought to make this pathogen particularly effective in authors hypothesized that the formation of immune com- exerting a selection pressure on our genome. plexes may trigger cellular and inflammatory changes that The general theme here is the concept of herd immu- adversely affect patient outcome. nity, i.e., the fact that differences in the population will keep Hemolytic disease of the fetus and newborn (HDFN) as at least a fraction of all individuals protected from most if a result of ABO incompatibility between mother and baby not all pathogens for humanity to survive. ABO differences is a relatively common event in group O mothers carrying a as part of our innate immunity appear to be one of the bet- group A or group B fetus. However, the disease is generally ter examples of this idea.96 mild and rarely requires treatment, most often by photo- therapy,97 although inhibition of antibodies by administra- Clinical Significance tion of soluble A or B trisaccharides has been used success- Of all antibodies to RBC blood group antigens, anti-A fully.104 Mild HDFN is a consequence of the comparatively and anti-B are arguably the most clinically important. On low levels of IgG ABO antibodies (which are often mainly the other hand anti-A1 and anti-H are very seldom found IgG2 or IgG4) capable of crossing the placenta and also is to correlate with hemolytic adverse events but often show related to the immaturity of the glycosylated structures on lower thermal optima. Before the discovery of ABO, trans- fetal RBCs. In addition, ABH antigens are present on many fusion between humans had been attempted with mixed other cell types so the antibody concentration on RBCs is success. In some cases, the patient survived and got bet- limited and the DAT often only weakly positive. ter. In others the patient died rapidly, which we now re- The clinical significance of anti-A and anti-B extends alize was attributable to rapid intravascular hemolysis of beyond transfusion medicine and is important in both solid ABO-incompatible RBCs (reviewed by Mollison et al.97). organ and hematopoietic transplantation. Although ABO- It has been reported that transfusion of as little as 30 mL mismatched hematopoietic stem cell transplantation is of ABO-mismatched blood can result in a rapid fatal in- standard practice with a favorable outcome in most cases, travascular transfusion reaction (reviewed in Issitt and transplantation of ABO-incompatible solid organs has been Anstee98). Together with transfusion-related acute lung established only relatively recently with moderately suc- injury (TRALI) and bacterial contamination of blood com- cessful outcome.105 Children younger than 3 years of age ponents, ABO incompatibility is still one of the main risks have been shown to tolerate mismatched organs better, and for major morbidity and mortality associated with transfu- West and colleagues have demonstrated good posttrans- sions, after erroneous transfusions.99,100 The latest FDA re- plant survival in infants undergoing ABO-incompatible port on transfusion-associated fatalities (found at http:// heart transplants,106 showing that the relatively immature www.fda.gov/cber/blood/fatal08.htm) shows that blood B-cell response in these patients can be exploited.107 In a group incompatibility was judged as the cause of death in study of 46 patients undergoing ABO-mismatched renal 37 percent of all cases during 2008, with TRALI at 35 per- transplants, Tobian et al.108 demonstrated that therapeu- cent. Among the hemolytic fatalities ABO was the cause in tic apheresis to reduce anti-A and anti-B titers to less than

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16 together with standard immunosuppression protocols we recently introduced the first microarray-based ABO typ- results in successful and stable engraftment. Progress ing system that takes the first steps toward this goal on a continues in this field, driven by a shortage of appropriate higher throughput system.110 Efforts to renew methods for organs for transplant. Yazer and Triulzi109 conclude that serologic typing are also ongoing. For instance, the young immune hemolysis remains a major complication in ABO- field of microfluidics holds promise for alternative blood mismatched transplantation of solid organs and to a lesser grouping technology being more rapid, using smaller vol- extent hematopoietic progenitor cells. However, passenger umes of reactants, and being applicable to broader testing lymphocyte syndrome attributable to ABO or other blood platforms.111 group antibodies can now often be ameliorated by mono- The temptation to create ABO-universal blood for clonal antibody therapy, once clinicians realize the prob- transfusion purposes has kept scientists busy since the lem. early 1980s when the first successful report of deliberate transfusion with modified RBCs across the ABO barrier was Summary and Future Perspectives published.112 Progress from those early days, using a poorly Despite the relative simplicity of the A and B antigens, effective B-converting exoglycosidase from green coffee perhaps especially considering the minor biochemical dif- beans, has continued with the aims of eliminating ABO- ference between them, the ABO blood group system remains associated hemolytic transfusion reactions and simplify- one of the most interesting, both clinically and scientifi- ing blood logistics and inventory management. Not only cally, dividing the world’s population including patients has the process taken important strides toward becoming and donors into four groups irrespective of origin or creed. a reality with both A- and B-degrading enzymes recombi- Figure 7 summarizes the four principal levels at which the nantly available and necessary clinical trials in progress or ABO system can be considered and shows how immunohe- being designed, but the project has also brought with it the matologists have been able to exploit the steadily increasing scientifically exciting discovery of a large new class of bac- knowledge of this system by devising tests taking advan- terially derived exoglycosidase enzymes unknown hitherto tage of each of the different levels. It also shows some of the and without clear homology or resemblance to any other natural consequences generated and their relation to the group of molecules.113,114 It is unknown at this point why so microbes surrounding us. many different bacterial species have developed these blood group–converting enzymes. Speculation includes that it Analytic modalities Natural consequences Analytic modal ities Natural conse quences would simply be a means of digesting potential nutrient GenotypinGenotypingGenotypingg Genetic level Polymorphic population saccharides, but it also may be yet another way for microbes (DNA, mRNA ) to make sure they are able to attach to the host cell surface, Counteracting enzymes EnzymEnzymicic actiactivityvity Enzyme level Counteracting(exoglycosidases)enzymes developed(exoglyco - even if the histo-blood group of the current host happens (glycosyltransferase) (glyco syltransferase) sidasby bacteriaes) developed by bacteria not to fit the bacterial lectins initially. It is actually quite ForwardForward typing typing likely that yet other glycosidase specificities will be found Glycoconjugate level Cell surface receptor variation Adsorption/elutionAdsorption/ elution Cell surface receptor variation ( carbohydrate antigens) Neutralization by secreted antigens in this extended arsenal of newly discovered enzymes. A Flow Flow cytomery cytometry Neutralization by secreted antigens SalivaSaliva inhibition inhibi tion recently exploited example is the Galα3Gal (also known as Reverse typing Immunological level the straight B, fucose-less B, or Galili antigen) -degrading Reverse typing NeutralizationNeutralizatioNeutralizatiomn by by immunoglobu immunoglobulinslins Titration ( antibodies ) Titration enzyme subfamily of galactosidases that can, for instance, Fig. 7 Principles of the ABO system at four molecular levels and be used to degrade xenograft antigenicity in pig tendons for 115 their modes of interaction are schematically represented by arrows use in knee surgery. in black. On the left side, the laboratory analytical modalities cur- Finally, it is easy to make the mistake of sitting back rently exploited at each level are listed; the right side shows some of and looking at the ABO system as close to complete when the innate mechanisms used between host and pathogen. it comes to knowledge and discovery. We predict that both the antigenic diversity and the way we look at antibody specificities in this and related systems will change dramat- A whole array of ABO-related research and other proj- ically during the next few years based on recent and future ects is currently in progress. Even if a surprising number findings made possible by new technologies. ABO has for a of ABO alleles has been discovered already, no doubt there long time served as a great model for genetics, enzymology, are more to come. ISBT is currently working to facilitate and biochemistry thanks to our deep understanding of the communication and reporting by introducing an official al- interindividual variation in this system, and there is noth- lele nomenclature. Although ABO genotyping cannot yet be ing to suggest that it will stop now when high-throughput used as a stand-alone analysis to support clinical decisions, genetics and glycotope arrays are here to help us. On the improvement of the current status is highly desirable for contrary, it should be expected that there are still more sur- its use as an independent addition to serology in the refer- prises waiting around the corner. ence laboratory. Together with colleagues across Europe,

IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009 55 J.R. Storry and M.L. Olsson

Acknowledgments 16. Korchagina EY, Pochechueva TV, Obukhova PS, Some of the content and figures in this review have been Formanovsky AA, Imberty A, Rieben R, Bovin NV. modified from the doctoral theses of M.L.O. (Molecular Ge- Design of the blood group AB glycotope. Glycoconj J netic Studies of the Blood Group ABO Locus in Man; Lund 2005;22:127–33. University, 1997) and Dr. Bahram Hosseini-Maaf (Genetic 17. Clausen H, Levery SB, Dabelsteen E, Hakomori S. Characterisation of Human ABO Blood Group Variants with Blood group ABH antigens: a new series of blood group a Focus on Subgroups and Hybrid Alleles; Lund University, A-associated structures (genetic regulation and tissue 2007). distribution). Transplant Proc 1987;19:4408–12. 18. Svensson L, Rydberg L, de Mattos LC, Henry SM. Blood References group A and A revisited: an immunochemical analysis. 1. Camp FR, Ellis FR. Selected contributions to the litera- Vox Sang 2009;96:56–61. ture of blood groups and immunology. Fort Knox, KY: 19. Daniels GL, Fletcher A, Garratty G, et al. Blood group US Army Medical Research Laboratory, 1966. terminology 2004: from the International Society of 2. Watkins WM. Biochemistry and genetics of the ABO, Blood Transfusion committee on terminology for red Lewis and P blood group systems. In: Harris H, cell surface antigens. Vox Sang 2004;87:304–16. Hirschhorn K, eds. Advances in human genetics. New 20. Blumenfeld OO, Patnaik SK. Allelic genes of blood York: Plenum Press, 1980:1–136. group antigens: a source of human mutations and cS- 3. Clausen H, Hakomori S. ABH and related histo-blood NPs documented in the Blood Group Antigen Gene group antigens; immunochemical differences in carrier Mutation Database. Hum Mutat 2004;23:8–16. isotypes and their distribution. Vox Sang 1989;56:1– 21. Epstein AA, Ottenberg R. Simple method of performing 20. serum reactions. Proc N Y Pathol Soc 1908;8:117–23. 4. Yazer MH, Palcic MM. The importance of disordered 22. von Dungern E, Hirszfeld L. Concerning hered- loops in ABO glycosyltransferases. Transfus Med Rev ity of group specific structures of blood. Transfusion 2005;19:210–16. 1962;2:70–2. 5. Yamamoto F. Molecular genetics of ABO. Vox Sang 23. Bernstein F. Zusammenfassande betrachtungen über 2000;78:91–103. die erblichen Blutstrukturen des Menschen. Z Indukt 6. Chester MA, Olsson ML. The ABO blood group gene: Abstamm u VererbLehre 1925;37:237–70. a locus of considerable genetic diversity. Transfus Med 24. Crow JF. Felix Bernstein and the first human marker Rev 2001;15:177–200. locus. Genetics 1993;133:4–7. 7. Landsteiner K. Zur Kenntnis der antifermentativen, 25. Ferguson-Smith MA, Aitken DA, Turleau C, de Grouchy lytischen und agglutinierenden Wirkungen des Blutse- J. Localisation of the human ABO: Np-1: AK-1 linkage rums und der Lymphe. Zbl Bakt 1900;27:357–62. group by regional assignment of AK-1 to 9q34. Hum 8. Landsteiner K. Über Agglutinationserscheinungen nor- Genet 1976;34:35–43. malen Menschlichen Blutes. Wien Klin Wochenschr 26. Yamamoto F, Clausen H, White T, Marken J, Hakomori 1901;14:1132–4. S. Molecular genetic basis of the histo-blood group ABO 9. Decastello A, Sturli A. Ueber die isoagglutinine im ge- system. Nature 1990;345:229–33. sunder und kranker menschen. Munch Med Wochen- 27. Yamamoto F, Marken J, Tsuji T, White T, Clausen H, schrift 1902;49:1090–5. Hakomori S. Cloning and characterization of DNA 10. Race RR, Sanger R. Blood groups in man. 6th ed. Ox- complementary to human UDP-GalNAc:Fuc-alpha1– ford, UK: Blackwell Scientific Publications, 1975. 2Gal alpha1–3GalNAc transferase (histo-blood group 11. von Dungern E, Hirzsfeld L. Uber die gruppens- A transferase) mRNA. J Biol Chem 1990;265:1146–51. pezifische strukturen des blutes III. Immun Forsch 28. Clausen H, White T, Takio K, et al. Isolation to homo- 2191;8:526–62. geneity and partial characterization of a histo-blood 12. Mourant AE, Kope AC, Domaniewska K. The distribu- group A defined Fuc-alpha1–2Gal alpha1–3-N-acetyl- tion of human blood groups and other polymorphisms. galactosaminyltransferase from human lung tissue. J New York: Oxford University Press, 1976. Biol Chem 1990;265:1139–45. 13. Roychoudhuri AK, Nei M. Human polymorphic genes 29. Yamamoto F, McNeill PD, Hakomori S. Human histo- world distribution. Oxford: Oxford University Press, blood group A2 transferase coded by A2 allele, one of 1988. the A subtypes, is characterized by a single base deletion 14. Lalueza-Fox C, Gigli E, de la Rasilla M, Fortea J, Rosas in the coding sequence, which results in an additional A, Bertranpetit J, Krause J. Genetic characterization of domain at the carboxyl terminal. Biochem Biophys Res the ABO blood group in Neandertals. BMC Evol Biol Commun 1992;187:366–74. 2008;8:342. 30. Yamamoto F, McNeill PD, Hakomori S. Genomic orga- 15. Crainic K, Durigon M, Oriol R. ABO tissue antigens of nization of human histo-blood group ABO genes. Gly- Egyptian mummies. Forensic Sci Int 1989;43:113–24. cobiology 1995;5:51–8.

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31. Bennett EP, Steffensen R, Clausen H, Weghuis DO, van 45. Yu LC, Twu YC, Chou ML, Chang CY, Wu CY, Lin M. Kessel AG. Genomic cloning of the human histo-blood Molecular genetic analysis for the B(3) allele. Blood group ABO locus. Biochem Biophys Res Commun 2002;100:1490–2. 1995;206:318–25. 46. Seltsam A, Gruger D, Just B, et al. Aberrant intracellu- 32. Kominato Y, Hata Y, Takizawa H, et al. Alternative lar trafficking of a variant B glycosyltransferase. Trans- promoter identified between a hypermethylated up- fusion 2008;48:1898–905. stream region of repetitive elements and a CpG island 47. Hosseini-Maaf B, Hellberg A, Chester MA, Olsson ML. in human ABO histo-blood group genes. J Biol Chem An extensive PCR-ASP strategy for clinical ABO blood 2002;277:37936–48. group genotyping that avoids potential errors caused 33. Hosseini-Maaf B, Smart E, Chester MA, Olsson ML. by null, subgroup and hybrid alleles. Transfusion The Abantu phenotype in the ABO blood group system 2007;47:2110–25. is due to a splice-site mutation in a hybrid between a 48. Olsson ML, Chester MA. Polymorphism and recombi- new O1-like allelic lineage and the A2 allele. Vox Sang nation events at the ABO locus: a major challenge for 2005;88:256–64. genomic ABO blood grouping strategies. Transfus Med 34. Calafell F, Roubinet F, Ramirez-Soriano A, Saitou N, 2001;11:295–313. Bertranpetit J, Blancher A. Evolutionary dynamics of 49. Procter J, Crawford J, Bunce M, Welsh KI. A rapid molec- the human ABO gene. Hum Genet 2008;124:123–35. ular method (polymerase chain reaction with sequence- 35. Roubinet F, Kermarrec N, Despiau S, Apoil PA, Dug- specific primers) to genotype for ABO blood group and oujon JM, Blancher A. Molecular polymorphism of O secretor status and its potential for organ transplants. alleles in five populations of different ethnic origins. Tissue Antigens 1997;50:475–83 [published erratum Immunogenetics 2001;53:95–104. appears in Tissue Antigens 1998;51:319]. 36. Olsson ML, Guerreiro JF, Zago MA, Chester MA. Mo- 50. Kominato Y, Tsuchiya T, Hata N, Takizawa H, Yama- lecular analysis of the O alleles at the blood group ABO moto F. Transcription of human ABO histo-blood group locus in populations of different ethnic origin reveals genes is dependent upon binding of transcription fac- novel crossing-over events and point mutations. Bio- tor CBF/NF-Y to minisatellite sequence. J Biol Chem chem Biophys Res Commun 1997;234:779–82. 1997;272:25890–8. 37. Yazer MH, Olsson ML. The O2 allele: questioning the 51. Irshaid NM, Chester MA, Olsson ML. Allele-related phenotypic definition of an ABO allele. Immunohema- variation in minisatellite repeats involved in the tran- tology 2008;24:138–47. scription of the blood group ABO gene. Transfus Med 38. Hosseini-Maaf B, Irshaid NM, Hellberg A, et al. New 1999;9:219–26. and unusual O alleles at the ABO locus are implicated 52. Yu LC, Chang CY, Twu YC, Lin M. Human histo-blood in unexpected blood group phenotypes. Transfusion group ABO glycosyltransferase genes: different enhanc- 2005;45:70–81. er structures with different transcriptional activities. 39. Seltsam A, Das GC, Wagner FF, Blasczyk R. Nondele- Biochem Biophys Res Commun 2000;273:459–66. tional ABO*O alleles express weak blood group A phe- 53. Seltsam A, Wagner FF, Grüger D, Gupta CD, Bade-Do- notypes. Transfusion 2005;45:359–65. eding C, Blasczyk R. Weak blood group B phenotypes 40. Lee HJ, Barry CH, Borisova SN, et al. Structural basis may be caused by variations in the CCAAT-binding fac- for the inactivity of human blood group O2 glycosyl- tor/NF-Y enhancer region of the ABO gene. Transfu- transferase. J Biol Chem 2005;280:525–9. sion 2007;47:2330–5. 41. Wagner FF, Blasczyk R, Seltsam A. Nondeletional 54. Twu YC, Hsieh CY, Yu LC. Expression of the histo- ABO*O alleles frequently cause blood donor typing blood group B gene predominates in AB-genotype cells. problems. Transfusion 2005;45:1331–4. Transfusion 2006;46:1988–96. 42. Yazer MH, Hult AK, Hellberg A, Hosseini-Maaf B, Palcic 55. Thuresson B, Chester MA, Storry JR, Olsson ML. ABO MM, Olsson ML. Investigation into A antigen expres- transcript levels in peripheral blood and erythropoietic sion on O2 heterozygous group O-labeled red blood cell culture show different allele-related patterns indepen- units. Transfusion 2008;48:1650–7. dent of the CBF/NF-Y enhancer motif and multiple 43. Olsson ML, Chester MA. Heterogeneity of the blood novel allele-specific variations in the 5′- and 3′-noncod- group Ax allele: genetic recombination of common al- ing regions. Transfusion 2008;48:493–504. leles can result in the Ax phenotype. Transfus Med 56. Hata Y, Kominato Y, Yamamoto FI, Takizawa H. Char- 1998;8:231–8. acterization of the human ABO gene promoter in eryth- 44. Olsson ML, Irshaid NM, Hosseini-Maaf B, et al. Ge- roid cell lineage. Vox Sang 2002;82:39–46. nomic analysis of clinical samples with serologic ABO 57. Bianco T, Farmer BJ, Sage RE, Dobrovic A. Loss of red blood grouping discrepancies: identification of 15 novel cell A, B, and H antigens is frequent in myeloid malig- A and B subgroup alleles. Blood 2001;98:1585–93. nancies. Blood 2001;97:3633–9.

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58. Hosoi E, Hirose M, Hamano S, Kuroda Y. Detection of 74. Schachter H, Michaels MA, Tilley CA, Crookston MC, histo-blood group ABO mRNA in human chronic my- Crookston JH. Qualitative differences in the N-acetyl-d- eloid leukemia cell lines using reverse transcription- galactosaminyltransferases produced by human A1 and polymerase chain reaction (RT-PCR). Cancer Lett A2 genes. Proc Natl Acad Sci U S A 1973;70:220–4. 1998;133:191–6. 75. Topping MD, Watkins WM. Isolectric points of the hu- 59. Bianco-Miotto T, Hussey DJ, Day TK, O’Keefe DS, Do- man blood group A-1, A-2 and B gene- associated gly- brovic A. DNA methylation of the ABO promoter under- cosyltransferases in ovarian cyst fluids and serum. Bio- lies loss of ABO allelic expression in a significant propor- chem Biophys Res Commun 1975;64:89–96. tion of leukemic patients. PLoS ONE 2009;4:e4788. 76. Clausen H, Levery SB, Nudelman E, Tsuchiya S, Hako- 60. Chihara Y, Sugano K, Kobayashi A, et al. Loss of blood mori S. Repetitive A epitope (type 3 chain A) defined group A antigen expression in bladder cancer caused by blood group A1-specific monoclonal antibody TH- by allelic loss and/or methylation of the ABO gene. Lab 1: chemical basis of qualitative A1 and A2 distinction. Invest 2005;85:895–907. Proc Natl Acad Sci U S A 1985;82:1199–203. 61. Dabelsteen E, Gao S. ABO blood-group antigens in oral 77. Varki A, Cummings R, Esko J, Freeze H, Hart G, Marth cancer. J Dent Res 2005;84:21–8. J. Essentials of glycobiology. Cold Spring Harbor, NY: 62. Orntoft TF, Meldgaard P, Pedersen B, Wolf H. The Cold Spring Harbor Laboratory Press, 1999. blood group ABO gene transcript is down-regulated in 78. Oriol R, Candelier JJ, Mollicone R. Molecular genetics human bladder tumors and growth-stimulated urothe- of H. Vox Sang 2000;78(Suppl 2):105–8. lial cell lines. Cancer Res 1996;56:1031–6. 79. Paulson JC, Colley KJ. Glycosyltransferases. Structure, 63. Kabat EA. Blood group substances. Their chemistry and localization, and control of cell type-specific glycosyla- immunohistochemistry. New York: Academic Press, tion. J Biol Chem 1989;264:17615–18. 1956. 80. Clausen H, Bennett EP, Dabelsteen E. Carbohydrates 64. Morgan WT, Watkins WM. The detection of a product of the cell surface: molecular aspects of glycosyltrans- of the blood group O gene and the relationship of the ferases and their genes. APMIS Suppl 1992;27:9–17. so-called O-substance to the agglutinates A and B. Br J 81. Patenaude SI, Seto NO, Borisova SN, et al. The struc- Exp Pathol 1948;29:159–73. tural basis for specificity in human ABO(H) blood group 65. Morgan WT, Watkins WM. The inhibition of the hae- biosynthesis. Nat Struct Biol 2002;9:685–90. magglutinins in plant seeds by human blood group sub- 82. Hosseini-Maaf B, Letts JA, Persson M, et al. Structural stances and simple sugars. Br J Exp Pathol 1953;34:94– basis for red cell phenotypic changes in newly identi- 103. fied, naturally occurring subgroup mutants of the hu- 66. Watkins WM, Morgan WT. Inhibition by simple sug- man blood group B glycosyltransferase. Transfusion ars of enzymes which decompose the blood-group sub- 2007;47:864–75. stances. Nature 1955;175:676–7. 83. Springer GF, Williamson P, Readler BL. Blood group 67. Watkins WM, Morgan WT. Possible genetical pathways active gram-negative bacteria and higher plants. Ann N for the biosynthesis of blood group mucopolysaccha- Y Acad Sci 1962;97:104–10. rides. Vox Sang 1959;4:97–119. 84. Cooling LW, Sitwala K, Dake LR, Judd WJ, Davenport 68. Ceppellini R. Physiological genetics of human blood R. ABO typing discrepancies in children requiring long- group factors. In: Wolstenholme GEW, O’Connor CM, term nutritional support: it is the gut after all! Transfu- eds. Ciba Foundation symposium on biochemistry of sion 2007;47:10A. human genetics. London: Churchill, 1959:242–61. 85. Kunkel HG, Fudenberg H, Ovary Z. High molecular 69. Leloir LF. Nucleoside diphosphate sugars and saccha- weight antibodies. Ann NY Acad Sci 1960;86:966-73.

ride synthesis. Biochem J 1964;91:1–8. 86. Kunkel HG, Rockey JH. β2A and other immunoglobu- 70. Watkins WM. Gene-enzyme relationships of the A, B, lins in isolated anti-A antibodies. Proc Soc Exp Biol H and Le blood group genes (abstract). Transfusion Med 1963;113:278. 1967;7:367. 87. Garratty G. Blood group antigens as tumor markers, 71. Hearn VM, Smith ZG, Watkins WM. An a-N-acetyl-D- parasitic/bacterial/viral receptors, and their associa- galactosaminyltransferase associated with the human tion with immunologically important proteins. Immu- blood-group A character. Biochem J 1968;109:315–17. nol Invest 1995;24:213–32. 72. Race C, Ziderman D, Watkins WM. An alpha-d-galacto- 88. Daniel-Johnson J, Lee-Stroka A, Schechterly C, et al. syltransferase associated with the blood-group B char- Probiotic-associated high-titer anti-B in a group A acter. Biochem J 1968;107:733–5. platelet donor as a cause of severe hemolytic transfu- 73. Schenkel-Brunner H, Tuppy H. Enzymes from human sion reactions. Transfusion 2008;48S(Suppl 2):260A. gastric mucosa conferring blood-group A and B specificities upon erythrocytes. Eur J Biochem 1970;17:218– 22.

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89. Arendrup M, Hansen JE, Clausen H, Nielsen C, Math- 104. Romano EL, Soyano A, Montano RF, et al. Treatment of iesen LR, Nielsen JO. Antibody to histo-blood group A ABO hemolytic disease with synthetic blood group trisac- antigen neutralizes HIV produced by lymphocytes from charides. Vox Sang 1994;66:194–9. blood group A donors but not from blood group B or O 105. Rydberg L. ABO-incompatibility in solid organ transplan- donors. AIDS 1991;5:441–4. tation. Transfus Med 2001;11:325–42. 90. Preece AF, Strahan KM, Devitt J, Yamamoto F, Gustafs- 106. West LJ, Pollock-Barziv SM, Dipchand AI, et al. ABO-in- son K. Expression of ABO or related antigenic carbohy- compatible heart transplantation in infants. N Engl J Med drates on viral envelopes leads to neutralization in the 2001;344:793–800. presence of serum containing specific natural antibod- 107. West LJ. B-cell tolerance following ABO-incompat- ies and complement. Blood 2002;99:2477–82. ible infant heart transplantation. Transplantation 91. Cserti CM, Dzik WH. The ABO blood group sys- 2006;81:301–7. tem and Plasmodium falciparum malaria. Blood 108. Tobian AA, Shirey RS, Montgomery RA, Tisch DJ, Ness 2007;110:2250–8. PM, King KE. Therapeutic plasma exchange reduces ABO 92. Uneke CJ. Plasmodium falciparum malaria and ABO titers to permit ABO-incompatible renal transplantation. blood group: is there any relationship? Parasitol Res Transfusion 2009 Feb 6 [Epub ahead of print]. 2007;100:759–65. 109. Yazer MH, Triulzi DJ. Immune hemolysis following ABO- 93. Chen Q, Heddini A, Barragan A, Fernandez V, Pearce mismatched stem cell or solid organ transplantation. SF, Wahlgren M. The semiconserved head structure of Curr Opin Hematol 2007;14:664–70. Plasmodium falciparum erythrocyte membrane pro- 110. Avent ND, Martinez A, Flegel WA, et al. The BloodGen tein 1 mediates binding to multiple independent host project: toward mass-scale comprehensive genotyping receptors. J Exp Med 2000;192:1–10. of blood donors in the European Union and beyond. 94. Loscertales MP, Owens S, O’Donnell J, Bunn J, Bosch- Transfusion 2007;47(Suppl):40S–6S. Capblanch X, Brabin BJ. ABO blood group phenotypes 111. Kline TR, Runyon MK, Pothiawala M, Ismagilov RF. and Plasmodium falciparum malaria: unlocking a piv- ABO, D blood typing and subtyping using plug-based otal mechanism. Adv Parasitol 2007;65:1–50. microfluidics. Anal Chem 2008;80:6190–7. 95. Rowe JA, Handel IG, Thera MA, et al. Blood group O 112. Goldstein J, Siviglia G, Hurst R, Lenny L, Reich L. protects against severe Plasmodium falciparum ma- Group B erythrocytes enzymatically converted to group laria through the mechanism of reduced rosetting. Proc O survive normally in A, B, and O individuals. Science Natl Acad Sci U S A 2007;104:17471–6. 1982;215:168–70. 96. Gagneux P, Varki A. Evolutionary considerations in re- 113. Liu QP, Sulzenbacher G, Yuan H, et al. Bacterial gly- lating oligosaccharide diversity to biological function. cosidases for the production of universal red blood Glycobiology 1999;9:747–55. cells. Nat Biotechnol 2007;25:454–64. 97. Mollison PL, Engelfriet CP, Contreras M. Blood trans- 114. Olsson ML, Clausen H. Modifying the red cell surface: fusion in clinical medicine. 10th ed. London: Blackwell towards an ABO-universal blood supply. Br J Haematol Scientific, 1997. 2008;140:3–12. 98. Issitt PD, Anstee DJ. Applied blood group serology. 4th 115. Liu QP, Yuan H, Bennett EP, et al. Identification of a ed. Miami: Montgomery Scientific Publications, 1998. GH110 subfamily of alpha 1,3-galactosidases: novel en- 99. Sazama K. Reports of 355 transfusion-associated deaths: zymes for removal of the alpha 3Gal xenotransplanta- 1976 through 1985. Transfusion 1990;30:583–90. tion antigen. J Biol Chem 2008;283:8545–54. 100. Stainsby D, Jones H, Asher D, et al. Serious hazards of transfusion: a decade of hemovigilance in the UK. Transfus Med Jill R. Storry, PhD, FIBMS (corresponding author), Coor- Rev 2006;20:273–82. dinator, Special Serology, Department of Clinical Immu- 101. Linden JV, Wagner K, Voytovich AE, Sheehan J. Trans- nology and Transfusion Medicine, University and Regional fusion errors in New York State: an analysis of 10 years’ Laboratories, and Martin L. Olsson, MD, PhD, Division of experience. Transfusion 2000;40:1207–13. Haematology and Transfusion Medicine, Department of 102. Shanwell A, Andersson TM, Rostgaard K, et al. Post-trans- Laboratory Medicine, Lund University, Lund, Sweden. fusion mortality among recipients of ABO-compatible but non-identical plasma. Vox Sang 2009;96:316–23. 103. Blumberg N, Heal JM, Hicks GL Jr, Risher WH. Asso- ciation of ABO-mismatched platelet transfusions with morbidity and mortality in cardiac surgery. Transfusion 2001;41:790–3.

IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009 59 Case Report

Clinical evaluation for lymphoproliferative disease prompted by finding of IgM warm autoanti-IT in two cases

R.M. Leger, F. Lowder, M.C. Dungo, W. Chen, H.M. Mason, and G. Garratty

Anti-IT is an unusual specificity originally described as a naturally Case Reports occurring cold agglutinin. The antibody reacts strongly with cord Case 1 RBCs, weakly with adult I RBCs, and most weakly with the rare A 69-year-old Hispanic woman presented with pancy- T adult i RBCs. IgG anti-I in patients with hemolytic anemia has topenia (WBC = 2.3 × 109/L; Hb = 6.9 g/dL; Hct = 19.1%; been associated with Hodgkin’s lymphoma. Difficulties in blood platelets = 64,000/µL), splenomegaly but no lymphade- grouping tests and the presence of a warm reactive agglutinin in samples from two patients with hemolytic anemia led to further nopathy, and hemolytic anemia (spherocytosis; LDH = 672 serologic studies and the identification of anti-IT. In both cases, U/L [normal 355–630]; total bilirubin = 2.8 mg/dL [normal the anti-IT was a rarely encountered IgM warm reactive aggluti- 0.1–1.2]; direct bilirubin = 0.7 mg/dL [normal 0–0.4]). The nin; in one case, the IgG component was also anti-IT, whereas in direct antiglobulin test (DAT) was positive. Lymphoma was the second case the IgG antibody was broadly reactive. The un- not suspected until after the anti-IT was identified. Comput- usual serologic finding of anti-IT prompted further clinical evalu- ed tomography (CT) scans showed evidence of large lym- ation for lymphoproliferative disease in these two patients. phoid infiltrates composed of a mixture of small and large Immunohematology 2009;25:60–62. lymphocytes. Bilateral bone marrow aspiration showed increased lymphocytes but was not diagnostic of lymphoma. Key Words: IgM warm autoantibody, autoanti-IT, The patient was empirically treated with steroids and had hemolytic anemia improved hemoglobin and sense of well-being. Steroids were tapered. Two and a half months later the patient re- nti-IT was originally described as a benign, naturally turned for a splenectomy, which was pathologically nega- occurring, IgM cold agglutinin in Melanesians1 and tive for lymphoma. Sections showed lymphoid hyperplasia Alater in Venezuela Indians.2 The anti-IT agglutinin with a benign phenotype consisting of T and B cells with no did not demonstrate classical I or i specificity but reacted aberrant expression of CD5, CD23, or bcl-2. Serologic stud- strongly with cord RBCs, weakly with normal adult I RBCs, ies at that time showed similar reactivity of anti-IT. Three and most weakly with the rare adult i RBCs. It was thought months later, the patient exhibited mild thrombocytopenia the agglutinin recognized a transition state of i to I, thus the with slightly elevated LDH. A repeat bone marrow showed designation IT (T for transition).1 Subsequent data do not lymphocytosis suggestive of low-grade B-cell lymphopro- support this hypothesis; IT is expressed nearly as well or as liferative disease. A year later, after rituximab and other well on fetal RBCs ranging in age from 11 to 16 weeks as on chemotherapy, the bone marrow showed atypical lymphoid cord RBCs.3 The biochemical structure of IT is still unclear. infiltrates. The infiltrates were composed primarily of small The first four examples of IgG anti-IT were in sera of mature lymphocytes with occasional larger lymphocytes patients with autoimmune hemolytic anemia (AIHA) and noted, most likely representing a lymphoproliferative dis- Hodgkin’s lymphoma.3,4 Others reported a similar associa- order. Flow cytometry showed an entire B-cell population tion.5 Hafleigh et al.6 found three examples of IgG autoanti- that did not express CD20. Less than 3 years after original IT in patients who did not have Hodgkin’s disease or AIHA. presentation, the patient was transfusion-dependant and One example of IgM warm anti-IT and one IgM cold plus diagnosed with aplastic anemia. She was discharged to hos- IgG anti-IT in patients with AIHA were not associated with pice care with end-stage liver disease, lymphoproliferative Hodgkin’s disease, but one of the cases was associated with disorder, and hemolytic anemia. non-Hodgkin’s lymphoma.7,8 We report an unusual 37°C reactive IgM agglutinin plus an IgG anti-IT9 and a 37°C re- Case 2 active IgM agglutinating anti-IT plus broadly reactive IgG A previously healthy 19-year-old Korean man presented autoantibody in two patients with hemolytic anemia who to the emergency room with insidious onset of fatigue, short- were subsequently evaluated for lymphoproliferative dis- ness of breath, dyspnea on exertion, and pallor. Laboratory ease owing to the specificity of the antibodies reported. data revealed thrombocytopenia and hemolytic anemia:

60 IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009 Two cases of IgM warm autoanti-IT

WBC = 6.8 × 109/L; Hb = 4.6 g/dL; Hct = 12%; platelets Results = 20,000/µL; spherocytes on the peripheral smear; total Case 1 bilirubin = 2.9 mg/dL; direct bilirubin = 0.4 mg/dL; and The DAT on DTT-treated RBCs was 3+ with anti-IgG LDH = 539 U/L. The haptoglobin was less than 5.8 mg/ and anti-C3; the 6% albumin control was nonreactive. The dL. There was no splenomegaly or lymphadenopathy. The initial titer and thermal amplitude studies showed a 37°C DAT was positive. An autoanti-IT agglutinin plus a broadly agglutinin that reacted to a higher titer with cord RBCs than reactive IgG autoantibody were identified. After a few days with adult I RBCs. Titration results with adult I, cord i, and on pulse dexamethasone (Decadron) therapy the hemoglo- adult i RBCs (to determine specificity) are shown in Table 1. bin improved only mildly, and the patient was started on Anomalous results (unexplained high titer) were obtained a 4-day course of IVIG with recovery of his hemoglobin to with DTT-treated autologous RBCs: titers of 512 at 37°C, 9.3 g/dL. Because of the association of lymphoma with an- 1024 at 30°C, 2048 at 22°C, and 8000 at 4°C. An apparent ti-IT the patient underwent CT scans, bone marrow biopsy, anti-I was detected at 4°C (adult I RBC titer of 256, cord and positron emission tomography. There was no evidence RBC titer of 64). An acid eluate prepared from the patient’s of malignancy or lymphoproliferative process. During the RBCs reacted more strongly with cord RBCs than with adult period of a year, the patient had multiple relapses of the I RBCs at 37°C. After treatment with DTT, the patient’s se- hemolytic anemia (but not thrombocytopenia), necessi- rum and the eluate did not agglutinate cord RBCs, but re- tating further treatment with steroids. Rituximab, given acted 1+ and 4+, respectively, at the antiglobulin test with for the hemolytic process, had no sustained benefit. The anti-IgG. The dilution control (for the abolished aggluti- patient underwent a splenectomy, and both the hemoglo- nation) reacted 3½ to 4+ with the serum and 1½ + with bin and platelet count remain normal. There has been no the eluate. Thus, IgM plus IgG anti-IT was demonstrated in evidence of Hodgkin’s disease, lymphoma, or other malig- both the serum and the eluate. In the AIHA serum screen, nancy in several years of follow-up. agglutination was observed at 22°C and 37°C similar to that in the titration and thermal amplitude studies; a hemolysin Materials and Methods was also observed with enzyme-treated RBCs at both tem- The DAT was performed with anti-human IgG (Ortho peratures, but without a clear preference for either temper- Clinical Diagnostics, Raritan, NJ) and anti-human C3 (in- ature. house reagent). A 6% bovine albumin control was tested in parallel. The in-house anti-C3 was prepared by inject- Case 2 ing rabbits with purified proteins and standardized as The DAT on DTT-treated RBCs was 4+ with anti-IgG previously described.10 Before testing, the patients’ RBCs and anti-C3; the 6% albumin control was nonreactive. In the were treated with 0.01 M dithiothreitol (DTT) to break initial titer and thermal amplitude studies, an agglutinin re- IgM bonds that cause spontaneous agglutination.11 Eluates acted at 37°C with cord RBCs; adult I RBCs did not react at were prepared from the patients’ RBCs using a commercial 37°C or 30°C. Titration results with adult I, cord i, and adult i acid elution kit (Gamma Elu-Kit II, Gamma Biologicals, RBCs are shown in Table 1. Sufficient autologous RBCs were Houston, TX); cold LISS was substituted for the kit wash not available. DTT treatment of the patient’s serum abolished solution.12 agglutination with cord RBCs; however, dilutions of DTT- For immunoglobulin classification, samples were treat- treated serum tested against adult I and cord RBCs did not ed with 0.01 M DTT. The agglutinin titer and thermal am- show a difference in reaction strength of the IgG component. plitude was determined at 37°C (prewarmed), 30°C, 22°C, Thus, an IgM anti-IT plus a broadly reactive IgG antibody was and 4°C with group O adult I, cord, and adult i RBCs,10 and demonstrated in the serum. The eluate contained an IgG an- for Case 1, with DTT-treated autologous RBCS. The tests at tibody suggestive of anti-IT (titer/score versus adult I RBCs each temperature were set up separately (i.e., using sepa- was 32/42 and versus cord RBCs was 128/55). In the AIHA rate sets of dilutions) to eliminate potential carryover of serum screen, untreated RBCs were weakly agglutinated at agglutination. Patients’ sera were also tested by a previous- 22°C and weakly sensitized at 37°C; enzyme-treated RBCs ly described serum screen method10 used to characterize were agglutinated and hemolyzed at both temperatures. antibodies in AIHA. Briefly, serum Discussion was tested with and without acidification and the addition Table 1. Titer and thermal amplitude of two examples of anti-IT of fresh normal serum as a RBCS Case 1 titer (score) at: Case 2 titer (score) at: source of complement, against 37°C 30°C 22°C 4°C 37°C 30°C 22°C 4°C untreated and enzyme-treated Adult I 1 (4) 1 (6) 2 (11) 256 (78) 0 0 1 (4) 8 (28) RBCs at 37°C (prewarmed) and 20°C. Agglutination results Cord i 32 (37) 128 (61) 256 (68) 64 (56) 64 (54) 64 (52)-128 64 (49) 32 (48) (60) were graded and scored as previously described.10 Adult i 1 (6) 2 (10) 2 (10) NT* 0 1 (4) 1 (5) NT *NT = not tested

IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009 61 R.M. Leger et al.

IgM warm autoagglutinins reactive at 37°C are an References unusual subcategory of warm autoimmune hemolytic ane- 1. Booth PB, Jenkins WJ, Marsh WL. Anti-IT: a new an- mia.10,13 Typically, the patient’s RBCs are spontaneously tibody of the I blood group system occurring in certain agglutinated, requiring treatment with a sulfhydryl reagent Melanesian sera. Br J Haematol 1966;12:341–4. such as DTT to resolve ABO and Rh typing and to interpret 2. Layrisse Z, Layrisse M. High incidence cold autoag- the DAT. The two cases presented here were referred to the glutinins of anti-IT specificity in Yanomama Indians of immunohematology research laboratory for further diag- Venezuela. Vox Sang 1968;14:369–82. nostic testing and characterization of the agglutinin when 3. Garratty G, Petz LD, Wallerstein RO, Fudenberg HH. these characteristics were noted by our reference labora- Autoimmune hemolytic anemia in Hodgkin’s disease tory. In both cases the warm agglutinin demonstrated IT associated with anti-IT. Transfusion 1974;14:226–31. specificity. Both of our cases also demonstrated an IgG au- [Correction note: Transfusion 1974;14:630.] toantibody component; in Case 1, the IgG autoantibody was 4. Garratty G, Hafleigh B, Dalziel J, Petz LD. An IgG an- anti-IT. ti-IT detected in a Caucasian American. Transfusion We have no explanation for the high titers obtained af- 1972;12:325–9. ter DTT-treatment of the group O autologous RBCs in Case 5. Levine AM, Thornton P, Forman SJ, et al. Positive 1. This may indicate that the patient’s RBCs had greatly Coombs test in Hodgkin’s disease: significance and im- increased IT expression. When group O untreated, DTT- plications. Blood 1980;55:607–11. treated, and ficin-treated cord RBCs were tested in parallel 6. Hafleigh EB, Wells RF, Grumet FC. Nonhemolytic IgG with dilutions of this patient’s anti-IT and another sample anti-IT. Transfusion 1978;18:592–7. of anti-IT, no enhancement of reactivity was observed with 7. Freedman J, Newlands M, Johnson CA. Warm IgM DTT treatment whereas reactivity with ficin-treated RBCs anti-IT causing autoimmune haemolytic anaemia. Vox was increased as expected by three to four dilutions (data Sang 1977;32:135–42. not shown). Thus, the DTT treatment should not have af- 8. Freedman J, Cheng LF, Murray D, Myers R. An unusual fected the autologous RBC test results. The previous sample autoimmune hemolytic anemia in a patient with immu- of warm IgM anti-IT associated with AIHA did not aggluti- noblastic sarcoma. Am J Hematol 1983;14:175–84. nate that patient’s untreated autologous RBCs at 37°C (the 9. Leger RM, Nguyen N, Garratty G, Lowder F, Feldman optimal temperature of reactivity).7 N. An unusual IgM warm + IgG autoanti-IT associated Anti-IT is an unusual specificity and may not even be with lymphoma, pancytopenia and hemolytic anemia suspected unless dilutions of the sera are tested with cord i (abstract). Transfusion 2003;43:100A. and adult i RBCs in parallel with adult I RBCs. In our cases, 10. Petz LD, Garratty G. Immune hemolytic anemias. 2nd additional titrations to identify the specificity were only ed. Philadelphia: Churchill Livingstone, 2004. pursued after the initial increased reactivity with cord RBCs 11. Reid ME. Autoagglutination dispersal utilizing sulphy- was observed. Examples of anti-IT have been reported to be dryl compounds. Transfusion 1978;18:353–5. IgM cold agglutinins or IgG antibodies optimally reactive 12. Leger RM, Arndt PA, Ciesielski DJ, Garratty G. False- at 37°C. We are only aware of one reported example of IgM positive eluate reactivity due to the low-ionic wash so- warm anti-IT, and that case was not associated with Hodg- lution used with commercial acid-elution kits. Transfu- kin’s disease or lymphoma.7 In more than 20 years, we have sion 1998;38:565–72. encountered only two other examples of IgM warm reactive 13. Arndt PA, Leger RM, Garratty G. Serologic findings anti-IT; neither had an IgG component, but both were as- in autoimmune hemolytic anemia associated with im- sociated with a history of lymphoma before the serologic munoglobulin M warm autoantibodies. Transfusion workup. 2009;49:235–42. Determining the specificity of autoantibodies is typically of academic interest only. In general, serologic results do Regina M. Leger, MSQA, MT(ASCP)SBB, Research Associ- not necessarily prompt a clinical evaluation for lymphoma. ate, American Red Cross Blood Services, Southern Califor- In both of these cases, the unusual RBC antibody and the nia, 100 Red Cross Circle, Pomona, CA 91768; Frederick historic association of this specificity with lymphoma, to- Lowder, CLS,MT(ASCP)SBB, Olive View-UCLA Medical gether with the presenting findings of AIHA, led to the lym- Center, Sylmar, CA; Maria C. Dungo, MD, Assistant Clini- phoma workup. For patients who do not progress to lym- cal Professor, David Geffen School of Medicine at UCLA, phoma, one might speculate that rituximab (anti-CD20) Harbor-UCLA Medical Center, Torrance, CA; Weip Chen, given for the AIHA could have eradicated a malignant clone MD, Harbor-UCLA Medical Center, Torrance, CA; Holli of B cells. M. Mason, MD, Assistant Clinical Professor of Pathol- ogy, David Geffen School of Medicine at UCLA, Director of Transfusion Medicine and Serology, Harbor-UCLA Medical Center, Torrance, CA; and George Garratty, PhD, FRCPath, Scientific Director, American Red Cross Blood Services, Southern California, Pomona, CA.

62 IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009 Original Report

Southeast Asian ovalocytosis is associated with increased expression of Duffy antigen receptor for chemokines (DARC)

I.J. Woolley, P. Hutchinson, J.C. Reeder, J.W. Kazura, and A. Cortés

The Duffy antigen receptor for chemokines (DARC or Fy glyco- RBC morphology.3 SAO is widespread in several popula- protein) carries antigens that are important in blood transfusion tions of Papua New Guinea (PNG), where its prevalence and is the main receptor used by Plasmodium vivax to invade correlates with malaria endemicity and altitude.4 In these reticulocytes. Southeast Asian ovalocytosis (SAO) results from populations, homozygosity is apparently incompatible with an alteration in RBC membrane protein band 3 and is thought to full development of the fetus inasmuch as no persons ho- mitigate susceptibility to falciparum malaria. Expression of some RBC antigens is suppressed by SAO, and we hypothesized that mozygous for the band 3 mutation have yet been described, SAO may also reduce Fy expression, potentially leading to reduced but heterozygosity confers strong protection against cere- susceptibility to vivax malaria. Blood samples were collected from bral malaria.5,6 Although early studies had suggested that individuals living in the Madang Province of Papua New Guinea. the SAO trait may afford some protection against vivax or Samples were assayed using a flow cytometry assay for expres- falciparum parasitemia,7–9 other studies did not support sion of Fy on the surface of RBC and reticulocytes by measuring this hypothesis.10 The mechanism of protection against the attachment of a phycoerythrin-labeled Fy6 antibody. Reticu- cerebral malaria is not completely understood,11,12 but the locytes were detected using thiazole orange. The presence of the specific protection against cerebral malaria suggests that it SAO mutation was confirmed by PCR. There was a small (approxi- may operate by means of alterations in sequestration of in- mately 10%) but statistically significant (p=0.049, Mann-Whitney U test) increase in Fy expression on SAO RBC compared with RBC fected RBC. However, SAO confers limited or no protection from individuals without this polymorphism: mean Fy expres- against placental malaria, which is also linked to sequestra- sion (mean fluorescence intensity [MFI]) was 10.12 ± 1.22 for SAO tion of infected RBCs.13,14 heterozygotes versus an MFI of 8.95 ± 1.1 for individuals without Individuals whose RBCs are negative for expression of SAO. For reticulocytes the MFI values were 27.61 ± 19.12 for SAO Duffy antigens illustrate homozygote advantage for vivax heterozygotes and 16.47 ± 3.81 for controls. SAO is associated with malaria in that their RBCs are completely refractory to in- increased and not decreased Fy6 expression so that susceptibility vasion by Plasmodium vivax.15 Historic serologic data sug- to P. vivax infection is unlikely to be affected. gest expression of some antigens on human RBCs was sup- Immunohematology 2009;25:63–66. pressed by the SAO trait, but expression of Duffy antigens did not appear suppressed as determined by the less sensi- Key Words: Duffy, Southeast Asian ovalocytosis, antigen tive serologic instruments available at that time.16 In those expression studies, antibodies of varying specificities were placed in tubes using serial dilution, and results were read macro- n 1949 Haldane proposed that malaria had a dramatic scopically with the aid of an eyepiece using a standard- influence on the human genome, by selecting forge- ized scoring system. In this study, we used molecular and netic polymorphisms that were known or suspected to I cytometric techniques to diagnose SAO and to reexamine protect against malaria.1 Although some of these polymor- whether there was a difference in expression of Duffy an- phisms can be detrimental when individuals are homozy- tigen receptor for chemokines (DARC) between SAO and gous, e.g., sickle cell anemia, their apparent selection in hu- control individuals in the context of possible differential man populations living in malaria-endemic areas is evidence susceptibility to infection with different Plasmodium spe- that they offer some protection against death from malaria, cies an infectious disease that continues to kill more than 1 million persons annually.2 Critical to this theory is the concept Materials and Methods of heterozygote advantage. That is, carriage of the mutant Blood was collected by venipuncture in CPD anticoag- gene on one member of a chromosomal pair is beneficial ulant or by finger prick using a BD Microtainer (Becton whereas homozygosity is deleterious. An extreme example Dickinson, San Jose, CA) with EDTA anticoagulant from of heterozygote advantage in Melanesian populations is healthy volunteers from two villages, Sempi and Bemlon, Southeast Asian ovalocytosis (SAO), a hereditary form of on the north coast of the Madang Province, PNG. Oral in- ovalocytosis that is caused by a 27-base pair deletion in the formed consent was obtained after approval for the study gene encoding the major integral RBC membrane protein by the Medical Research Advisory Committee of the PNG band 3 (AE1, SLC4A1), leading to a distinctive change in

IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009 63 I.J. Woolley et al.

Ministry of Health. After collection, samples were kept on Table 1. Mean fluorescence intensity (± standard deviation) of Fy6 ice or refrigerated at 4°C. Within 1 week, flow cytometric antibody binding to erythrocytes and reticulocytes studies were undertaken with antibody to Fy6 (kindly pro- All Subjects All Subjects vided by John Barnwell, CDC, Atlanta, GA). This is a mouse (Reticulocytes) (Erythrocytes) monoclonal antibody that attaches to the first extracellular SAO heterozygotes (N = 19) 27.61 ± 19.12 10.12 ± 1.22 domain of the Fy glycoprotein, in an area thought to cor- Non-SAO individuals (N = 20) 16.47 ± 3.81 8.95 ± 1.1 17,18 respond to the binding site of P. vivax merozoites. The P value for difference (Mann- 0.65 0.0487 antibody was conjugated directly to a phycoerythrin (PE) Whitney U test) label (Prozyme, San Leandro, CA) according to the manufacturer’s instructions. A 5-µL aliquot of blood was washed three times in 50 µL of PBS and pelleted. RBCs were incu- Discussion bated with 50 µL of a 1 in 50 dilution of Fy6 antibody for 15 The main finding of this study was that the SAO trait minutes at 37°C. Cells were then washed twice in PBS and does not result in reduced expression of Fy antigen. In fact, resuspended in thiazole orange (TO) solution according to we observed a 10 percent increase (p=0.049) in Fy antigen the manufacturer’s specifications (Becton Dickinson). The expression by SAO RBCs when compared with RBCs from samples were analyzed on a flow cytometer (Mo-Flo, Da- non-SAO individuals from the same area. This difference koCytomation, Fort Collins, CO) equipped with a 70-mW was significant when expression by all RBC populations air-cooled argon ion laser at 488 nm. TO fluorescence was was evaluated but not when only SAO reticulocytes were read with a 525-nm bandpass optical filter and PE fluores- compared with non-SAO reticulocytes (although a sugges- cence with a 570-nm bandpass filter. Compensation was tive trend existed). As anticipated, reticulocyte expression applied, and negative fluorescence signals were adjusted of Fy6 antigen exceeded RBC expression.20 The reasons for until they were orthogonal. Forward-scatter, side-scatter, the increased RBC Fy6 antigen expression in SAO are un- and fluorescence data were analyzed. A count of 5000 TO- clear at present, but it may reflect either a decreased rate of positive cells was taken per sample. This was approximately loss of Fy6 antigen from the surface of SAO RBC relative to 1 to 2 percent of the total number of cells assayed for each control RBC, perhaps in relation to the increased rigidity of individual. Beads of known immunofluorescence (Immu- the SAO cell wall, or differences in the surface area of ovalo- no-Brite, Coulter, Puerto Rico) were used to standardize cytes versus non-SAO RBC. Because there was only a small experiments undertaken at different times. Detection of the increase in Fy6 antigen expression on SAO RBC and there SAO mutation was undertaken using previously described was no statistically significant increase in the reticulocyte PCR methods.19 Experienced microscopists from the PNG subpopulation, which is the only subpopulation susceptible Institute of Medical Research performed light microscopic to infection by P. vivax, it is unlikely that this alteration af- inspection of Giemsa-stained blood films for malaria para- fects susceptibility to this parasite, although there is other sites. Statistical analysis was undertaken by using SPSS in vitro evidence that alterations in numbers of Fy antigens (Statview 4.51, Abacus Concepts, Berkeley, CA). We have expressed may alter that susceptibility.21 It is not surprising used nonparametric testing because we do not have evi- that the difference in Fy levels was not shown in the paper dence for normal distribution of DARC abundance in the of Booth et al.16 because they were using less sensitive se- surface of SAO erythrocytes. rologic methods or because Fy6 antigen is more available or more expressed in SAO ovalocytosis over the antigens Results tested in the previous study. However, their findings with Samples from 19 SAO heterozygotes and 20 non-SAO respect to other antigens, including Rh polypeptides, have donors were examined. Mean age of donors did not dif- subsequently been confirmed by molecular techniques.22 fer significantly between the two groups (mean age, 29.32 Fy antigen negativity caused by homozygosity for a pro- ± 13.70 years for SAO heterozygotes versus 26.91 ± 11.80 moter mutation that silences transcription of the FY gene23 years for non-SAO individuals). Four individuals had ma- is observed in many ethnic groups in sub-Saharan Africa, laria infection based on inspection of blood smears. One and presumably accounts for the relative lack of vivax ma- SAO and one non-SAO individual had Plasmodium malari- laria in this part of the world. The apparently independent ae, and two non-SAO donors had Plasmodium falciparum. emergence of heterozygosity for this same promoter muta- SAO RBC showed increased expression of Fy glycoprotein tion has also been described in the Wosera area of PNG, relative to non-SAO RBC as measured by binding of the Fy6 where SAO is rare or nonexistent. There is a need for further monoclonal antibody (Table 1). The level of Fy6 antigen evaluation of whether other common RBC polymorphisms was also higher in SAO reticulocytes compared with control affect Fy antigen expression, especially those that are as- reticulocytes, but the difference was not statistically sig- sociated with changes in RBC shape and surface area. For nificant (Table 1). Subsequent analysis of the data did not example, α-thalassemia is common in PNG, and in many suggest the differences were caused by a systemic bias at- areas, including the Madang Province, nearly all individuals tributable to sex, village of origin, or method of obtaining have the typical α-globin mutation seen in Melanesians.24 It blood (data not shown).

64 IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009 SAO and DARC seems unlikely that this common polymorphism would act 9. Serjeantson S, Bryson K, Amato D, Babona D. Malaria as a confounding factor in our study on the grounds of bio- and hereditary ovalocytosis. Hum Genet 1977;37:161– logic plausibility, low likelihood of linkage disequilibrium, 7. and very high prevalence in the region (assuming therefore 10. Foo LC, Rekhraj V, Chiang GL, Mak JW. Ovalocytosis more or less equal distribution between SAO and control protects against severe malaria parasitemia in the Ma- individuals within the population studied). Finally, it will layan aborigines. Am J Trop Med Hyg 1992;47:271–5. be important to determine whether environmental vari- 11. Cortes A, Benet A, Cooke BM, Barnwell JW, Reeder JC. ables extant in malaria-endemic populations might change Ability of Plasmodium falciparum to invade Southeast Fy antigen expression. Another example would be iron defi- Asian ovalocytes varies between parasite lines. Blood ciency, which is prevalent in PNG and many other malaria- 2004;104:2961–6. endemic regions of the world. 12. Cortes A, Mellombo M, Mgone CS, Beck HP, Reeder JC, There is evidence that the SAO trait affects several steps Cooke BM. Adhesion of Plasmodium falciparum– of the biologic cycle of P. falciparum that may be relevant infected red blood cells to CD36 under flow is enhanced for the selective advantage conferred by this trait against by the cerebral malaria-protective trait South-East Asian malaria. In particular, SAO RBCs are partly resistant to in- ovalocytosis. Mol Biochem Parasitol 2005;142:252–7. vasion in culture by merozoites of Plasmodium knowlesi 13. O’Donnell A, Raiko A, Clegg JB, Weatherall DJ, Allen and several lines of P. falciparum,25,26 and the SAO trait SJ. Southeast Asian ovalocytosis and pregnancy in a also results in altered adhesion of infected RBCs to CD36. malaria-endemic region of Papua New Guinea. Am J As with ABO,27 it is likely that the evolutionary pressure ex- Trop Med Hyg 2007;76:631–3. erted by survival with SAO is related to survival of children 14. Benet A, Khong TY, Ura A, et al. Placental malaria in from cerebral malaria before reproductive age. Although Fy women with South-East Asian ovalocytosis. Am J Trop expression probably affects P. vivax infection,28 this evolu- Med Hyg 2006;75:597–604. tionary pressure is almost certainly caused by P. falciparum 15. Miller LH, Mason SJ, Clyde DF, et al. The resistance through its relationship to RBC adhesion. factor to Plasmodium vivax in blacks. The Duffy blood group genotype, FyFy. N Engl J Med 1976;295:302–4. Acknowledgment 16. Booth PB, Serjeantson S, Woodfield DG, Amato D. Se- This study was funded in part by a Covance Fellowship lective depression of blood group antigens associated from the Royal Australasian College of Physicians (I.W.). with hereditary ovalocytosis among Melanesians. Vox Sang 1977;32:99–110. References 17. Nichols ME, Rubenstein P, Barnwell J, et al. A new hu- 1. Haldane JBS. Disease and evolution. Ric Sci Suppl A man Duffy blood group specificity defined by a murine 1949;19:68–76. monoclonal antibody. Immunogenetics and association 2. Maitland K, Bejon P, Newton CR. Malaria. Curr Opin with susceptibility to Plasmodium vivax. J Exp Med Infect Dis 2003;16:389–95. 1987;166:776–85. 3. Jarolim P, Palek J, Amato D, et al. Deletion in erythro- 18. Wasniowksa K, Blanchard D, Janvier D, et al. Identifi- cyte band 3 gene in malaria-resistant Southeast Asian cation of the Fy6 epitope recognised by two monoclonal ovalocytosis. Proc Natl Acad Sci U S A 1991;88:11022– antibodies in the N-terminal extracellular portion of 6. the Duffy antigen receptor for chemokines. Mol Im- 4. Mgone CS, Koki G, Paniu MM, et al. Occurrence of the munol 1996;33:917–23. erythrocyte band 3 (AE1) gene deletion in relation to 19. Jarolim P, Palek J, Amato D, et al. Deletion in erythro- malaria endemicity in Papua New Guinea. Trans R Soc cyte band 3 gene in malaria-resistant Southeast Asian Trop Med Hyg 1996;90:228–31. ovalocytosis. Proc Natl Acad Sci U S A 1991;88:11022– 5. Genton B, al-Yaman F, Mgone CS, et al. Ovalocytosis 6. and cerebral malaria. Nature 1995;378:564–5. 20. Woolley IJ, Hotmire KA, Sramkoski RM, Zimmerman 6. Allen SJ, O’Donnell A, Alexander ND, et al. Prevention PA, Kazura JW. Differential expression of the Duffy of cerebral malaria in children in Papua New Guinea by antigen receptor for chemokines according to RBC age Southeast Asian ovalocytosis band 3. Am J Trop Med and FY genotype. Transfusion 2000;40:949–53. Hyg 1999;60:1056–60. 21. Michon P, Woolley I, Wood EM, Kastens W, Zimmer- 7. Fowkes FJ, Michon P, Pilling L, et al. Host erythrocyte man PA, Adams JH. Duffy-null promoter heterozygosi- polymorphisms and exposure to Plasmodium falcipar- ty reduces DARC expression and abrogates adhesion of um in Papua New Guinea. Malar J 2008;7:1. the P. vivax ligand required for blood-stage infection. 8. Cattani JA, Gibson FD, Alpers MP, Crane GG. Heredi- FEBS Lett 2001;495:111–14. tary ovalocytosis and reduced susceptibility to malaria in Papua New Guinea. Trans R Soc Trop Med Hyg 1987;81:705–9.

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22. Beckmann R, Smythe JS, Anstee DJ, Tanner MJ. Coex- 27. Cserti CM, Dzik WH. The ABO blood group sys- pression of band 3 mutants and Rh polypeptides: dif- tem and Plasmodium falciparum malaria. Blood ferential effects of band 3 on the expression of the Rh 2007;110:2250–8. complex containing D polypeptide and the Rh complex 28. Kasehagen LJ, Mueller I, Kiniboro B, et al. Reduced containing CcEe polypeptide. Blood 2001;97:2496– Plasmodium vivax erythrocyte infection in PNG Duffy- 505. negative heterozygotes. PLoS ONE 2007;2:e336. 23. Tournamille C, Colin Y, Cartron JP, Le Van Kim C. Dis- ruption of a GATA motif in the Duffy gene promoter Ian J. Woolley (corresponding author), Department of abolishes erythroid gene expression in Duffy-negative Infectious Diseases, Monash Medical Centre and Monash individuals. Nat Genet 1995;10:224–8. University, Clayton, Victoria, Australia, Paul Hutchinson, 24. Cockburn IA, Mackinnon MJ, O’Donnell A, et al. A Centre for Inflammatory Diseases, Monash Medical Cen- human complement receptor 1 polymorphism that re- tre, Clayton, Victoria, Australia, John C. Reeder, Papua duces Plasmodium falciparum rosetting confers protec- New Guinea Institute of Medical Research, Goroka, Papua tion against severe malaria. Proc Natl Acad Sci U S A New Guinea (current affiliation: Burnet Institute, Prah- 2004;101:272–7. ran, Victoria, Australia), James W. Kazura, Center for 25. Kidson C, Lamont G, Saul A, Nurse GT. Ovalocytic Global Health & Diseases, Case Western Reserve Univer- erythrocytes from Melanesians are resistant to invasion sity, Cleveland, Ohio, USA, and Alfred Cortés, Papua New by malaria parasites in culture. Proc Natl Acad Sci U S Guinea Institute of Medical Research, Madang, Papua A 1981;78:5829–32. New Guinea (current affiliation: ICREA and Institute for 26. Hadley T, Saul A, Lamont G, Hudson DE, Miller LH, Research in Biomedicine, Barcelona, Spain). Kidson C. Resistance of Melanesian elliptocytes (ovalocytes) to invasion by Plasmodium knowlesi and Plas- modium falciparum malaria parasites in vitro. J Clin Invest 1983;71:780–2.

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].

66 IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009 Review

Sickle cell disease: a review

S.D. Roseff*

ickle cell disease (SCD) is described as the first identi- commonly found in western Africa. About one in every 400– fied “molecular” disease since its manifestations stem 500 African Americans, or 80,000, has SCD. About 9000 Sfrom a substitution of valine for glutamic acid in the African Americans, or one in 12, have sickle cell trait.4 structure of the β chain hemoglobin molecule.1 As a result On the other hand, patients who have manifestations of this change, RBCs form characteristic “sickle” shapes and of their sickle hemoglobin are considered to have SCD. This the surface of these RBCs attract each other, polymerizing includes patients who are homozygous for Hgb SS, as de- when in a low oxygen environment. This seemingly “small” scribed previously. In addition, some patients who inherit variation in the structure of the RBC causing polymerization Hgb S from one parent and another abnormal hemoglobin leads to manifestations such as chronic occlusion of blood from the other parent can also have SCD. Common exam- vessels (vaso-occlusion), reduced blood flow to vital organs ples are designated as Hgb SC and Sβ-thalassemia. These (ischemia), and alterations of the immune system. In ad- individuals can have a milder clinical course than that of dition, the abnormal sickle cells are prematurely removed individuals who are homozygous for Hgb S. from circulation, resulting in hemolytic anemia. Transfu- Since RBCs with Hgb S are abnormal, they are removed sion is a vital component of the treatment of some of the from circulation in the spleen more rapidly than normal complications of SCD. It is also a modality used to prevent RBCs. This leads to a reduced life in the circulation of 16– some of these complications from occurring. Patients with 20 days in comparison to 120 days for normal RBCs.5 The SCD are unique because those who are transfused usually premature destruction of RBCs, with the accompanying require chronic transfusion, resulting in exposure to many decrease in hemoglobin, is classified as a hemolytic anemia. different blood donors over the course of treatment. In addition, most patients with SCD in the United States are Table 1. Composition of normal adult hemoglobin African American, and most donors are Caucasian from Hemoglobin Composition Percent (%) Western European descent. As a result of this difference, Hgb A α β 96–98 patients with SCD are exposed to RBC antigens that they 2 2 Hgb A α δ 1.5–3.5 lack, putting them at risk for forming alloantibodies, de- 2 2 2 2 fined as RBC alloimmunization. Therefore, it is important Hgb F α2γ2 <1 to understand the issues involved in the safe and effective transfusion of patients with SCD.

Table 2. Hemoglobin SS (-α β S) Defining Hemoglobinopathies 2 2 Hemoglobin (Hgb) is made up of iron (heme) and 4 Hemoglobin Percent (%) globin chains. The type of globin chains determines the type Hgb S 80 of hemoglobin (see Table 1).3 Hgb A2 2–4.5 Since SCD is characterized by the presence of an abnor- Hgb F 120 mal or variant hemoglobin, hemoglobin S, it is characterized as a hemoglobinopathy. The composition of hemoglobin for patients with homozygous SS is presented in Table 2. Typical Laboratory Findings in Sickle Cell Disease There are a variety of other abnormal hemoglobins that Patients with SCD are commonly diagnosed after new- may be present with or without Hgb S. The type of hemo- born screening. In the past, young children presented with globin a person has is based on patterns of inheritance. If painful swellings of the feet and hands, a consequence of each parent contributes hemoglobin S, the child can inherit vaso-occlusion (see discussion on Clinical Manifestations two copies and is designated as Hgb SS, or is homozygous of Disease). for hemoglobin S. If a child only inherits one copy from one Many patients with SCD have anemia, with hemoglobin parent and a copy of normal hemoglobin, Hgb A, they are levels between 6 and 8 g/dL. Characteristic sickle shaped designated as Hgb AS, or heterozygous. These individuals cells are seen on peripheral smear (Fig. 1). Patients do not are usually asymptomatic, and only develop manifestations always have symptomatic anemia because the body com- under rare circumstances, where they become hypoxic, pensates for the peripheral RBC destruction by increasing such as at high altitudes. Therefore, not every person with the rate of RBC production (erythropoiesis) in the bone mar- an abnormal hemoglobin develops or exhibits signs and row. Consequently, the bone marrow contains an increase symptoms. Although Hgb S is found worldwide, it is most in the number of RBC precursors. As a result, immature

*Reprinted with permission of the College of American Pathologists. IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009 67 S.D. Roseff precursors, suggestive of reticulocytes, are released from to refer these patients to specialists and provide state-of- the marrow prematurely. Therefore, the peripheral smear the-art patient care, identification of children with SCD at will also show these immature RBCs, which are larger than birth is an important public health intervention. Screening mature RBCs and have a slightly blue color since they are can be done in the neonatal period using fetal DNA during not fully hemoglobinized.3 The term reticulocytes will be pregnancy, or more commonly, performing DNA analysis used in this education activity, and refers to these RBCs. soon after birth. Prenatal testing can be done between 14– 16 weeks of gestation in pregnancies where there is a chance that the baby will inherit Hgb S from both parents.1,3,4 More commonly, newborn infants are screened through mandatory programs required by most states, soon after birth.1,3,4 Several methods exist for detecting abnormal hemoglobins. These include: • High performance liquid chromatography (HPLC)— Considered one of the best methods to be able to detect abnormal hemoglobins and to be able to differentiate them from normal hemoglobin. HPLC uses reactions in columns of reagents. This technique also measures the quantity of the various hemoglobins. • Hemoglobin electrophoresis—Separates different he- Fig. 1 The arrowed cell is a classic example of a sickle cell. Anoth- moglobins according to their characteristic migration er sickle cell is located in the upper left corner. There is a nucleated red blood cell in the lower right corner. The large cell in the center across a gel of either cellulose acetate at an alkaline is a polychromatophilic red cell or reticulocyte. (Source: Figure pH or citrate agar at an acid pH, based on charge. The HE-42. In: Glassy EF, ed. Color Atlas of Hematology. Northfield, IL: different types of hemoglobin will migrate at different College of American Pathologists, 1998:97.) rates, occupying a characteristic position on the gel. • Isoelectric focusing—If further separation is necessary, Another consequence of the high rate of hemolysis and then isoelectric focusing can be performed. An electric erythropoiesis, or increased RBC turnover, is the accumula- current is passed through the gel that has areas of dif- tion of unconjugated or indirect bilirubin. As a result, some ferent pH. The identity of the hemoglobin can be made patients will have yellowing of the white of the eyes, scleral by its pattern of migration. icterus, and skin, or jaundice. The increase in bilirubin also • Solubility test—Taking advantage of the fact that re- puts patients at increased risk for gallstones, and the pa- duced Hgb S is insoluble in certain solutions, a simple tient may have to have their gallbladder removed. Table 3 solubility test can be used to detect the presence of Hgb lists the common laboratory findings in SCD and their S. After lysis, RBCs are reduced by sodium hydrosulfite. etiologies. If Hgb S is present, the solution will be turbid; if not Early diagnosis is essential in order to treat and even present, then the hemoglobin remains in solution and prevent some of the complications of SCD. In the past, the suspension is clear. Since this test requires a certain approximately 25% of children between the ages of four amount of whole blood, it is not routinely used for new- months and five years with SCD died of pneumonia. The born screening. Note that this test will just detect the use of prophylactic penicillin in children with SCD in the presence of Hgb S, so patients who have sickle cell trait setting of comprehensive care reduced the risk of death in (heterozygous for Hgb S) will also be positive. childhood to less than 3%.6–8 Therefore, in order to be able When performing patient testing, it is always important to follow the package insert and ensure that proficiency test- Table 3. Common laboratory findings in sickle cell disease ing is performed in compliance with the Clinical Laboratory Laboratory value Etiology Improvement Amendments of 1988 (CLIA). CLIA regula- Low hemoglobin and hematocrit Hemolysis tions require that laboratories enroll in a Centers for Medi- High mean cellular volume (MCV) Reticulocytes care and Medicaid Services (CMS) approved proficiency testing (PT) program for all regulated tests that the labora- High white blood cell (WBC) Inflammation and increased marrow count production tory performs. High platelet count Increased marrow production Clinical Manifestations of Sickle Cell Disease High lactate dehydrogenase Hemolysis (LDH) The function of a normal RBC is dependent upon its ability to flow freely through blood vessels and to be flexible Low haptoglobin Hemolysis enough to move in and out of vessels and tissue where oxygen High total and indirect bilirubin Hemolysis delivery occurs. Due to the rigidity of the sickle RBC, these High alkaline phosphatase until Elevated bone marrow activity properties are lost. Interestingly, when a patient with SCD puberty has a higher percentage of hemoglobin F, the course of their

68 IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009 A review of topics in SCD disease is milder and their life expectancy is longer. This is blood flow through the internal carotid and middle cerebral probably the result of the lower percentage of Hgb S in these arteries of the brain, a study found that one could predict patients, and that RBCs with Hgb F do not form polymers.9 patients at risk for a first stroke.14 Chronic transfusion ther- These chronic physiologic changes can lead to problems in apy has been used successfully to prevent primary stroke all organ systems; heart, lungs, and kidneys. SCD can be and then subsequent stroke (see Treatment of Sickle Cell seen as a disease based on vaso-occlusion (VOC), chronic Disease).15,16 hemolytic anemia, and immunologic impairment. Acute Chest Syndrome Occlusive Disease Acute chest syndrome (ACS) is the leading cause of There are a variety of ways by which sickle cells cause death in adults with SCD and a major cause of morbidity obstruction. One minor mechanism is whereby irreversibly in patients of all ages. It is characterized by the onset of sickled cells obstruct small capillaries. More commonly, worsening respiratory status, fever, and new infiltrate on sickle cells are trapped in small venules and capillaries.1,10 chest x-ray. The inciting event can be respiratory infection; The process of sickling and occlusion involves a complex in- however, since a majority of cases of acute chest syndrome teraction between chemical mediators, as well as elements in adults follow pain crisis, it is believed that an embolus other than the RBC itself. There is increasing understanding of fatty tissue from the bone marrow (the site of the initial that the walls of blood vessels are involved. There appears to vaso-occlusion) into the lungs is the culprit. Patients need be increased adherence of sickle cells to the vascular walls, enhanced oxygenation, so supplemental oxygen is admin- due to properties of the RBCs, as well as changes in the en- istered. Transfusion is an essential component in the treat- dothelial cells of blood vessels. The lipid bilayer of RBCs is ment of acute chest syndrome, as will be discussed in more disrupted in sickle cells, causing their early removal from detail later.17 circulation. Additionally, changes in their interactions with Of interest, there are many investigators looking at the other cells within the blood stream, such as sticky white role of inflammatory chemicals in the pathophysiology of blood cells (WBCs) and activated platelets, contribute to SCD, as well as their potential for developing new treat- vaso-occlusion.10,11 These changes can also cause the acti- ments. For example, there is a reduction in levels of an ad- vation of coagulation, leading to an increased risk of deep hesion molecule (VCAM-1) in patients with SCD on chronic venous thrombosis.10,12 transfusion protocols.18 Therefore, chronic transfusion pro- Vaso-occlusion manifests itself as one of the most com- tocols might prevent vaso-occlusion. Another recent study mon findings in a patient with SCD, pain crisis. Prior to found that the increases in the inflammatory mediator, routine screening of newborns, children were commonly secretory phospholipase A2, can predict acute chest syn- diagnosed with SCD prior to the age of two, with occlu- drome. Transfusing when levels are elevated can prevent sion in the hands and feet presenting as painful swelling, acute chest syndrome.19 or dactylitis.4,5 This would not occur until six months of age because the higher level of fetal hemoglobin prior to six Hemolytic Anemia months is protective. Most patients will often have severe The abnormal shape and surface of sickle cells result pain due to occlusion of blood flow to bones, bone marrow, in their shortened life span in the circulation and lead to muscles, or organs, and in their arms, legs, back, abdomen, the characteristic anemia. As RBCs are cycled through the and chest. Treatment consists of analgesia (many patients spleen, the site of their ultimate destruction, they also injure with SCD require chronic medication) and hydration, since the tissue of the spleen. Their rigidity impairs their ability to dehydrated RBCs are more likely to sickle. The cause of cri- flow smoothly through the sinusoids, and their sharp edges sis can vary between patients and between episodes in the cause them to be stuck, and to damage splenic tissue. In same patient, but may be related to infection, extremes of children under the age of five, blood can pool in the spleen, temperature, medication, or any other physiologic changes. which becomes a site of sequestration of RBCs. When this Occlusion is also associated with bony infarcts leading to occurs, the child has a painful, enlarged spleen accompa- osteonecrosis, skin ulcers, organ occlusion (including the nied by a drop in hemoglobin. Splenic sequestration can be spleen, resulting in functional asplenia or autosplenecto- life-threatening since there is also a drop in blood volume in my), acute chest syndrome, and cerebrovascular accidents the vasculature, or hypovolemia. Transfusion management or strokes. is essential in these cases. Many of these patients eventually need to have their spleen removed. As a patient gets Stroke older, the damage to the spleen is chronic, and the spleen Stroke in any patient is a devastating event, with ap- actually becomes smaller, eventually shriveling and losing proximately 11 percent of patients with Hgb SS having a function.1,4,5 stroke by the age of 20.13 As blood vessels become occluded, In order to keep up with this chronic destruction, the their diameter gets smaller and the rate of blood flow in- body attempts to compensate by increasing RBC produc- creases, thereby increasing the risk of stroke. Using a type tion. This chronic hemolysis may also create an inflamma- of ultrasound, transcranial Doppler (TCD), to measure tory state.1,10,11 Also, the free hemoglobin that is released

IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009 69 S.D. Roseff from hemolyzed RBCs causes additional damage to the lin- The chemotherapeutic agent, hydroxyurea (HU), increases ing of blood vessels.10,13 The marrow is very active, as evi- the amount of Hgb F that is made by the bone marrow. denced by immature RBCs or reticulocytes in the peripheral RBCs with Hgb F lack the β chain, which is responsible for blood. As the marrow increases production of RBCs, plate- the sickling seen in Hgb S RBCs. Therefore, these RBCs let and WBC production also increases. As a consequence of do not sickle, nor can they polymerize. This is one of the increased erythropoiesis, many patients do not suffer from mechanisms considered to be responsible for the improved signs and symptoms of anemia, even though they may have outcomes. In addition, HU has been found to reduce WBC a low hemoglobin. As long as the individual can continue production, thereby reducing the number of circulating in- to compensate, the anemia does not present a problem. flammatory cells, capable of adhering to blood vessel walls. When the marrow cannot keep up with production (i.e., a Platelet counts also drop and this may inhibit one of the lack of folic acid), or has its production impaired by viral ill- factors responsible for vaso-occlusion. HU also influences ness, such as parvovirus B-19, an aplastic crisis may occur. nitric oxide metabolism. Nitric oxide leads to the dilation Parvovirus B-19 is a common childhood virus responsible of blood vessels and seems to reduce the adhesion between for Fifth’s Disease. This disease causes a rash, the classic RBCs and blood vessel walls.22 “slapped-cheek” appearance, and high fevers. Parvovirus HU has been found to reduce mortality due to an induc- can suppress bone marrow production by destroying the tion of Hgb F and a reduction in vaso-occlusive events. In RBC precursor cells in the bone marrow. In these cases of addition, it reduces the incidence of acute chest syndrome, severe anemia, transfusion therapy is necessary.5 transfusion requirements, episodes of hospitalization, pain crisis, and reduces blood flow through intracranial Immunologic and Infectious Manifestations blood vessels, as measured by transcranial Doppler in chil- The spleen can be affected in SCD due to occlusive forc- dren.22–24 Currently, there is controversy about whether or es, but it is also damaged by the pointed, inflexible sickle not HU can be used to prevent repeat strokes as effectively cells that travel through it and are stuck. The spleen is an as chronic transfusion in children.13,24,25 Since HU is a che- important immunologic organ, helping to fight infections motherapeutic agent, there is concern that it will have long with encapsulated organisms (S. pneumoniae, H. influ- term side effects. It is not used in women who are preg- enzae, and N. meningitidis). Therefore, it is important to nant or planning to become pregnant. In addition, some recognize children with SCD and immunize them. As stated patients have to discontinue therapy because their WBC earlier, the use of prophylactic penicillin has been found to count becomes too low. The effects of hydroxyurea are not reduce death in children.9,20 All infections should be treated immediate, generally taking a few months to be effective.23 aggressively. Therefore, it does not provide rapid response during acute illness. Treatment of Sickle Cell Disease Bone marrow transplant, peripheral blood stem cell Prevention of early complications, such as infection, is transplant, and umbilical cord blood transplant are all important in the overall treatment plan for patients with strategies that can cure SCD. Due to the morbidity and SCD. As more is being learned about the basic mechanisms mortality secondary to transplant, it is important to care- of the disease, new treatments are being developed. Anal- fully choose patients with severe disease with a high risk gesics, including opioids, are a mainstay of treatment for of morbidity and mortality, such as those with recurrent people suffering with pain crisis. In addition, it is impor- strokes. Therefore, trials have centered on treating children tant to give patients with SCD vitamins, such as folic acid, or young adults, and there are some encouraging results. which are essential to the production of RBCs. Hydration is Unfortunately, one impediment is the low availability of also key, since RBCs sickle when dehydrated. In addition, matched sibling donors.26–28 hydration improves the viscosity of the patient’s blood and allows the RBCs to move more easily. As hemoglobin rises, Transfusion Management the viscosity of the bloodstream increases and there is an Transfusion therapy should only be initiated in patients increased risk of occlusive disorders, as well as an increased with signs and symptoms of anemia. As mentioned, most risk of pain crisis.21 There is no data to show that transfu- patients with SCD, though anemic, do not generally have sion should be part of the routine treatment for crisis.5,20 daily signs and symptoms of anemia. Therefore, RBC trans- The role of nitric oxide is also being investigated as a thera- fusion should only be used for specific indications. RBC peutic modality, since nitric oxide plays an important role transfusion, when necessary in patients with SCD, provides in the physiology of RBCs.1,4 some additional benefits. While increasing the patient’s he- Patients of all ages who have higher concentrations moglobin, transfusion dilutes the Hgb S with Hgb A. The of Hgb F have milder disease and lower mortality than RBCs with Hgb A have longer survival than the RBCs with patients with lower levels of Hgb F. Increasing the pro- Hgb S and neither sickle nor polymerize. In addition, trans- duction of Hgb F seems like a reasonable therapeutic in- fusion will suppress the patient’s own erythropoiesis, or tervention. In the laboratory, Hgb F has been shown to RBC production. As a consequence, they will produce less interfere with the polymerization of deoxygenated Hgb S. of their own Hgb S RBCs.4,5,29

70 IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009 A review of topics in SCD

The decision to transfuse should take into account the Splenic Sequestration: When a child’s hemoglobin known benefits in the face of risks. All blood products are drops and they become symptomatic, transfusion is neces- capable of transmitting certain infectious diseases and sary, in this setting. Of interest, transfusion increases the causing transfusion reactions. In addition, the recipient is hemoglobin beyond what’s expected, so it is important to exposed to certain inflammatory mediators that accumu- transfuse slowly to avoid over-transfusion. Hepatic seques- late in transfused blood and to foreign antigens from the tration can also occur.5,29 donor. Exposure to foreign RBC antigens has important im- Pregnancy: In uncomplicated pregnancies, there is no im- plications for patients with sickle cell disease. provement in outcomes in women who are transfused. Pa- RBCs can be transfused as a simple transfusion or via tients with other complications of SCD should be transfused exchange transfusion (erythrocytopheresis).5,29 During accordingly.5,29,30 simple transfusion, one or two units of RBCs are transfused Priapism: Priapism is the painful engorgement of the through a peripheral IV. Exchange transfusion is usually penis, as a result of vaso-occlusion. Supportive therapy performed using an automated machine that is designed to consisting of hydration and analgesia is recommended, and remove whole blood from the patient, separate into its vari- a urologist should be consulted. When priapism does not ous components, and then discard the patient’s Hgb S RBCs. resolve, simple transfusion or exchange transfusion should RBCs from the blood bank are then used as replacement. be considered. There are no studies to direct transfusion Typically, an RBC exchange exchanges one to two RBC vol- therapy. In addition, exchange transfusion has been associ- umes (total blood volume × hematocrit = 1 blood volume). ated with adverse neurologic sequelae, such as seizures and A larger bore, stiff-walled central venous catheter is usually increases in intracranial pressure, so there is a reluctance required due to the flow requirements of the automated in- to perform exchange until the patient is refractory to other strument. In the absence of automated equipment, manual treatments.5,29,31 exchange transfusion can be done. This is not optimal, since Presurgical Prophylaxis: Patients with SCD are at high this can create blood pressure and volume changes during risk for complications when undergoing major surgery. the alternating removal of whole blood through a peripheral Currently, some practitioners recommend that patients vein with subsequent reinfusion of banked RBCs. Each type be transfused to a hemoglobin of 10 g/dL prior to surgery. of transfusion has its own risks and benefits, as outlined in A study done comparing exchange transfusion to simple Table 4 and in Table 5. transfusion found that exchange transfusion is unneces- Table 4. The benefits and risks of simple transfusion sary.5,29,32 There are investigations underway to determine Benefits Risks whether or not a hemoglobin lower than 10 g/dL is safe for certain surgeries.33 Technical ease—requires only peripheral Increases viscosity IV access Risk of iron overload Acute Chest Syndrome (ACS): Transfusion, either simple or exchange, implemented early in the course, improves Low donor exposure—only 1 or 2 units oxygenation and alleviates organ dysfunction. For patients of RBCs necessary who are stable, simple transfusion should be performed. If Dilution of Hgb S the patient deteriorates, does not improve, or has a rapidly evolving course, exchange transfusion is recommended.17,34 Simple transfusion should only be performed until the he- Table 5. The benefits and risks of exchange transfusion moglobin reaches about 10 g/dL. Beyond that, there is con- Benefits Risks cern for vaso-occulsion.5,29 Produces rapid reduction in Hgb S Requires large gauge IV, usually Stroke: Due to the ease of simple transfusion in pediat- No increase in viscosity central venous catheter ric patients, they usually undergo simple transfusion. For No risk of iron overload—some Requires expertise and special patients who have an initial stroke, exchange transfusion observe reductions in serum equipment—may require transfer of ferritin over time patient to another facility is used to rapidly reduce the amount of Hgb S that can be Higher donor exposure—at least 4 recruited and extend the immediate damage to the brain. units of RBCs for an adult, usually Once a patient has a stroke, they are at risk for additional more strokes. By performing a monthly transfusion, either sim- Higher expense ple or exchange, this risk of recurrence is reduced. A recent study showed that stopping monthly transfusion after the Indications for Transfusion in Sickle Cell Disease return of normal flow by transcranial Doppler results in Indications for transfusion in SCD include: recurrent strokes and the return of abnormal TCDs.13,15,16,35 Aplastic Crisis: When RBC production in the bone marrow Of interest, during studies to reduce the risk of first stroke, is interrupted, the delicate balance to maintain RBC pro- patients on a chronic transfusion protocol also had a reduc- duction during chronic hemolysis is disrupted. Therefore, tion in the risk of acute chest syndrome and a reduction in if the patient has a drop in their hemoglobin and becomes the number of pain crises.15,16 symptomatic, transfusion is required until the underlying process abates.5,29

IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009 71 S.D. Roseff

Adverse Consequences of Transfusion of Patients with vent this, medications are used to remove, or chelate, iron. Sickle Cell Disease Intravenous chelators have been used, but their efficacy Adverse consequences of transfusion of patients with is hindered by its half-life and poor patient compliance. SCD include: A new generation of oral chelators is effective since there Alloimmunization: Due to the disparity between do- is increased compliance, and mode of actions results in a nors and patients with SCD in the United States, patients more continuous chelation. Serum ferritin is monitored in with SCD are among those most frequently alloimmmu- patients on chronic transfusion protocols who have SCD.45 nized. Studies have shown that the most common antibod- The aim of treatment is to reduce serum ferritin and to ies formed in this population are C, E, and K1.2,36 There are remove iron from organs. also other antibodies that are formed due to donor–recipient disparity. Therefore, in order to prevent alloimmuniza- Transfusion Recommendations tion, some centers routinely perform RBC phenotypes on When a patient develops complications from SCD, it patients with SCD and only transfuse RBCs that lack C, E, also makes sense not to transfuse them with RBCs with ad- and K1, if the patient is negative for the antigen. This strat- ditional Hgb S. Therefore, many laboratories will do a sim- egy reduces the rate of antibody formation in these at-risk ple solubility test, as described earlier, and select units that patients.36 Despite these findings, practice is variable. lack Hgb S. Realize that individuals with SCD are anemic Analyzing the results of the 2003 J-C College of American and cannot serve as blood donors, however, there are active Pathologists Proficiency Testing Survey, Osby and - Shul donors with sickle cell trait.41 man found that only 37 percent of North American hospi- Leukoreduction (LR) of blood products has been prov- tals routinely perform antigen typing on the RBCs of non- en to reduce the risk of cytomegalovirus (CMV) transmis- alloimmunized patients with SCD. In the 439 laboratories sion, reduce the risk of febrile non-hemolytic transfusion that do perform RBC phenotyping, C, E, and K1 were the reactions, and reduce the risk of human leukocyte antigen most frequent antigens that were matched.37 In a survey of (HLA) alloimmunization.41 There are also studies, though 50 academic medical centers in the US and Canada, 73 per- controversial, that show there are deleterious immunologic cent of centers reported that they performed routine phe- sequelae of transfusion that are prevented by reducing the notyping with 89% of those centers also matching for C, E, load of WBCs in transfused blood products. Since patients and K1.38 There is controversy about providing RBCs that with SCD are chronically transfused, many believe that these lack additional antigens. As you match for additional anti- patients should receive LR blood products. Of note, there is gens, it becomes more difficult to find compatible units. It is also a study that shows reduced rates of RBC alloimmuniza- argued that it is a better use of resources to save those rare tion in patients who receive LR blood products.46 Transfu- units for patients with existing antibodies.39 Another strat- sion recommendations for patients with SCD are: egy has been to use RBCs from donors who are ethnically • Blood products that are sickle hemoglobin negative similar to the patient, thereby creating a better chance that • Blood products that are leukoreduced the donor and patient will match more closely.40 Whenever • Blood products that are negative for C, E, and K1 anti- a patient develops an antibody, they will also receive RBCs gens if the patient lacks these antigens lacking the corresponding antigen.41 • Blood products that are negative for any additional an- Hyperhemolytic Syndrome: A serious type of hemo- tigens against which the patient has antibody lytic transfusion reaction, called the “hyperhemolytic syn- • Blood products that are from African American donors, drome,” can occur in the setting of transfusion. During these if a program exists episodes, the patients are typically being transfused, and instead of rising, their hemoglobin falls with subsequent Summary transfusion. It is felt that there is a “bystander” hemolysis of The substitution of one amino acid in the hemoglobin the patient’s own RBCs, as well as destruction of transfused molecule results in sickle hemoglobin. As a result, RBCs RBCs. Of interest, the units transfused are crossmatch com- sickle in low oxygen states causing occlusion of blood ves- patible, and no new alloantibodies are identified at the time sels, increased viscosity, and inflammation. These RBCs of transfusion. Further transfusion compounds the prob- are prematurely removed from the circulation, resulting in lem. Therefore, it is important to recognize the syndrome a chronic hemolytic anemia. With newborn screening and and, if possible, stop transfusing. If transfusion is neces- early treatment, the death rate among children with SCD sary, intravenous immunoglobulin (IVIG) and intravenous has declined. In addition, a variety of treatments are being steroids have been found to be effective. If transfusion is introduced to help manage the various manifestations of required because of life-threatening anemia, it should be disease. Transfusion, simple or exchange, is a mainstay of done cautiously, using IVIG and steroids concurrently.42–44 therapy, since it reduces the amount of Hgb S in circulation Iron Overload: Each unit of transfused RBCs contains and suppresses erythropoiesis. Transfusion is indicated about 200–250 mg of iron. With chronic transfusion, this for symptomatic anemia and specifically to prevent stroke iron accumulates and can be deposited into organs such (first or recurrent), during acute stroke, and for acute chest as the heart, liver, and endocrine glands. In order to pre- syndrome. Unfortunately, transfusion carries risks

72 IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009 A review of topics in SCD for infectious disease transmission, as well as immunologic 14. Adams RJ, McKie VC, Nichols F, et al. The use of and inflammatory sequelae. For patients with SCD who may transcranial ultrasonography to predict stroke in sick- be chronically transfused, iron overload occurs frequently. le cell disease. N Engl J Med 1992;326:605–10. In addition, due to differences in RBC antigens between do- 15. Adams RJ, McKie VC, Hsu L, et al. Prevention of a first nors and recipients, these patients are at increased risk for stroke by transfusions in children with sickle cell ane- development of RBC alloantibodies, which can complicate mia and abnormal results on transcranial Doppler ultra- further transfusion. It is, therefore, important to prevent sonography. N Engl J Med 1998;339:5–11. alloimmunization by transfusing leukoreduced RBCs that 16. Adams RJ, McKie VC, Brambilla D, et al. Stroke pre- match the patient for the C, E, and K1 antigens. Human vention trial in sickle cell anemia. Control Clin Trials progenitor cell (from bone marrow, peripheral blood stem 1998;19:110–29. cells, or umbilical blood) transplant can cure the disease, 17. Vichinsky EP, Neumayr LD, Earles AN, et al. Causes and and is used for patients with severe disease for whom con- outcomes of the acute chest syndrome in sickle cell dis- ventional therapy may not be effective. ease. National Acute Chest Syndrome Study Group. N Engl J Med 2000;342:1855–65. References 18. Sakhalkar VS, Rao SP, Weedon J, Miller ST. Elevated 1. Frenette PS, Atweh GF. Sickle cell disease: old discov- plasma sVCAM-1 levels in children with sickle cell dis- eries, new concepts, and future promise. J Clin Invest ease: impact of chronic transfusion therapy. Am J He- 2007;117:850–8. matol 2004;76:57–60. 2. Vichinsky EP, Earles A, Johnson RA, et al. Alloimmu- 19. Styles LA, Abboud M, Larkin S, Lo M, Kuypers FA. nization in sickle cell anemia and transfusion of racially Transfusion prevents acute chest syndrome predicted

unmatched blood. N Engl J Med 1990;322:1617–21. by elevated secretory phospholipase A2. Br J Haematol 3. Elghetany MT, Banki K. Erythrocytic disorders. In: 2007;136:343–4. McPherson RA, Pincus MR, eds. Henry’s clinical diag- 20. Claster S, Vichinsky EP. Managing sickle cell disease. nosis and management by laboratory methods. 21st ed. BMJ 2003;327:1151–5. Philadelphia, PA: Saunders Elsevier, 2007:504–44. 21. Platt O, Thorington B, Brambilla D, et al. Pain in sickle 4. Ogedegbe HO. Sickle cell disease: an overview. Lab Med cell disease. N Engl J Med 1991;325:11–16. 2002;33:515–43. 22. Steinberg MH, Barton F, Castro O, et al. Effect of hy- 5. Ohene-Frempong K. Indications for red cell transfusion droxyurea on mortality and morbidity in adult sickle cell in sickle cell disease. Semin Hematol 2001;38:5–13. anemia. Risks and benefits up to 9 years of treatment. 6. Gaston MH, Verter JI, Woods G, et al. Prophylaxis with JAMA 2003;289:1645–51. oral penicillin in children with sickle cell anemia. A ran- 23. Charache S, Terrin ML, Moore RD, et al. Effect of hy- domized trial. N Engl J Med 1986;314:1593–9. droxyurea on the frequency of painful crises in sickle cell 7. Leikin SL, Gallagher D, Kinney TR, et al. Mortality in chil- anemia. N Engl J Med 1995;332:1317–22. dren and adolescents with sickle cell disease. Cooperative 24. Ware RE, Zimmerman SA, Shultz WH. Hydroxyurea as study of sickle cell disease. Pediatrics 1989;84:500–8. an alternative to blood transfusion for the prevention 8. Gill FM, Sleeper LA, Weiner SJ, et al. Clinical events in of recurrent stroke in children with sickle cell disease. the first decade in a cohort of infants with sickle cell dis- Blood 1999;94:3022–6. ease. Blood 1995;86:776–83. 25. Zimmerman SA, Schultz WH, Burgett S, et al. Hydroxyu- 9. Platt OS, Brambilla DJ, Rosse WF, et al. Mortality in rea therapy lowers transcranial Doppler flow velocities in sickle cell disease—life expectancy and risk factors for children with sickle cell anemia. Blood 2007;110:1043–7. early death. N Engl J Med 1994;330:1639–44. 26. Locatelli F, Rocha V, Reed W, et al. Related umbilical 10. Telen MJ. Role of adhesion molecules and vascular en- cord blood transplantation in patients with thalassemia dothelium in the pathogenesis of sickle cell disease. He- and sickle cell disease. Blood 2003;101:2137–43. matol Am Soc Hematol Educ Program 2007;84–90. 27. Bernaudin F, Socie G, Kuentz M, et al. Long-term results 11. Kupyers FA. Membrane lipid alterations in hemoglobin- of related myeloablative stem-cell transplantation to opathies. Hematology Am Soc Hematol Educ Program cure sickle cell disease. Blood 2007;110:2749–56. 2007;68–73. 28. Woodard P, Jeng M, Handgretinger R, et al. Sum- 12. Key NS, Slungaard A, Dandelet L, et al. Whole blood tis- mary of symposium: the future of stem cell transplan- sue factor procoagulant activity is elevated in patients tation for sickle cell disease. J Pediatr Hematol Oncol with sickle cell disease. Blood 1998;91:4216–23. 2002;34:512–17. 13. Wang WC. The pathophysiology, prevention, and treat- 29. Josephson CD, Lu LL, Killyer KL, Hillyer CD. Transfu- ment of stroke in sickle cell disease. Curr Opin Hematol sion in the patient with sickle cell disease: a critical review 2007;14:191–7. of the literature and transfusion guidelines. Transfus Med Rev 2007;21:118–33.

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30. Koshy M, Burd L, Wallace D, et al. Prophylactic red-cell 41. Brecher ME. Technical Manual. 15th ed. Bethesda, MD: transfusions in pregnant patients with sickle cell disease. American Association of Blood Banks, 2005. N Engl J Med 1988;319:1447–52. 42. King KE, Shirey RS, Lenkiewica MW, et al. Delayed 31. Rackoff WR, Ohene-Frempong K, Month S, et al. Neuro- hemolytic transfusion reactions in sickle cell disease: logic events after partial exchange transfusion for pria- simultaneous destruction of recipients’ red cells. Trans- pism in sickle cell disease. J Pediatr 1992;120:882–5. fusion 1997;37:376–81. 32. Vichinsky EP, Haberkern CM, Neumayr L, et al. A com- 43. Petz LD, Calhoun L, Shulman IA, et al. The sickle cell parison of conservative and aggressive transfusion regi- hemolytic transfusion reaction syndrome. Transfusion mens in the perioperative management of sickle cell dis- 1997;37:382–92. ease. N Engl J Med 1995;333:206–13. 44. Win N, Yeghen T, Needs, et al. Use of intravenous immuno- 33. Buck J, Casbard A, Llewelyn C, Johnson T, Davies SC, globulin and intravenous methylprednisolone in hyper- Williamson LM. Preoperative transfusion in sickle cell haemolysis syndrome in sickle cell disease. Hematology disease: a survey of practice in England. Eur J Haematol 2004;9:433–6. 2005;75:14–21. 45. Porter JB. Concepts and goals in the management of trans- 34. Platt OS. The acute chest syndrome of sickle cell disease. fusional iron overload. Am J Hematol 2007;82:1136–9. N Engl J Med 2000;342:1904–7. 46. Blumberg N, Heal JM, Gettings KF. Leukoreduction 35. Adams RJ, Brambilla D; Optimizing Primary Stroke of red cell transfusion is associated with a decreased Prevention in Sickle Cell Anemia (STOP 2) Trial Inves- incidence of red cell alloimmunization. Transfusion tigators. Discontinuing prophylactic transfusions used 2003;43:945–52. to prevent stroke in sickle cell disease. N Engl J Med 2005;353:2769–78. Additional Resources 36. Vichinsky EP, Luban NL, Wright E, et al. Prospective Mayo Clinic. Sickle cell anemia. Available at: http://www. RBC phenotype matching in a stroke-prevention trial in mayoclinic.com/health/sickle-cell-anemia/DS00324. Ac- sickle cell anemia: a multicenter transfusion trial. Trans- cessed July 20, 2009. fusion 2001;41:1086–92. Sickle Cell Disease Association of America, Inc. Available 37. Osby M, Shulman IA. Phenotype matching of donor red at: http://www.sicklecelldisease.org/. Accessed July 20, blood cells units for non alloimmunized sickle cell dis- 2009. ease patients: a survey of 1182 North American laborato- March of Dimes. Available at: http://www.marchofdimes. ries. Arch Pathol Lab Med 2005;129:190–3. com/. Accessed July 20, 2009. 38. Afenyi-Annan A, Brecher ME. Pre-transfusion pheno- Lab Tests On Line. Available at: http://www.labtestsonline. type matching for sickle cell disease patients. Transfu- org/. Accessed July 20, 2009. sion 2005;44:619–20. 39. Ness PM. To match or not to match: the question for Susan D. Roseff, MD, Medical Director, Transfusion Medi- chronically transfused patients with sickle cell anemia. cine, Professor, Virginia Commonwealth University, De- Transfusion 1994;34:558–60. partment of Pathology, PO Box 980662, Richmond VA 40. Hillyer KL, Hare VW, Josephson CD, et al. Partners for 23298-0662. life: the transfused program for patients with sickle cell disease offered at the American Red Cross Blood Services, Southern Region, Atlanta, Georgia. Immunohematology 2006;22:108–11.

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74 IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009 Report

Consortium for Blood Group Genes (CBGG): 2008 report

G. A. Denomme, C.M. Westhoff, L. Castilho, and M.E. Reid, on behalf of the CBGG Members*

The Consortium for Blood Group Genes is a worldwide organiza- of appropriately worded reports of molecular analyses for tion whose goal is to have a vehicle to interact, establish guide- both blood donors and transfusion recipients. Members lines, operate a proficiency program, and provide education for continue to support the current recommendation that blood laboratories involved in DNA and RNA testing for the prediction components should not be labeled with molecular test re- of blood group, platelet, and neutrophil antigens. sults as the sole means of antigen identification. Molecular Immunohematology 2009;25:75–78. data continue to be a source of information in the resolution of complex serologic problems, and are not intended as the Key Words: Blood group alleles, Consortium for Blood sole means for patient transfusion management decisions. Group Genes, proficiency, target alleles Target Alleles he Consortium for Blood Group Genes (CBGG) is a The list of target alleles prepared by the CBGG after nonprofit organization whose mission is “To estab- meeting in 2007 was adopted. The preferred current termi- lish guidelines, to provide education, and to provide T nology is listed in Table 1 of this report under the heading a proficiency exchange for laboratories involved in DNA or Target antigen (target allele). However, the final naming RNA testing for the determination of blood group, platelet, of alleles relies on the decision of the International Soci- and neutrophil antigens.” The consortium was established ety for Blood Transfusion. When referring to a particular at the inaugural meeting on October 23, 2004, in Balti- single-nucleotide polymorphism, nucleotide changes fol- more, Maryland, by a group of people with scientific or in- low the designated position, e.g., 125G>A; intronic nucle- dustry experience and interest in the field. The consortium otide changes represented in the lower case, e.g., –33t>c. is coordinated by Marion Reid with assistance of country The associated amino acid substitutions (not shown) flank coordinators: Lilian Castilho for Brazil, Gregory Denomme the designated position, e.g., Pro103Ser or P103S. Because (with Maryse St. Louis as successor in 2009) for Canada, gene numbering systems vary, and to avoid ambiguity in and Connie Westhoff for the United States. All members are the location of nucleotide changes, the CBGG has adopted expected to interact and participate. New members are en- GenBank gene reference sequences (RefSeqGene) and ref- couraged to refer to the previously published information erence SNP numbers (rs#) for blood group genes and nucle- for background and progress information.1–4 The exchange otides (Table 1). These numbers provide the set of common of information is mainly accomplished through electronic references used to communicate or report molecular testing mailings, proficiency evaluation exercises, and a yearly results. meeting. Guidelines for Molecular Testing The CBGG Document The CBGG has developed guidelines for free and gener- The CBGG Document outlines the function of the CBGG. al distribution. In 2007, the CBGG guidelines were shared This document contains information on the structure, or- with the AABB Molecular Testing Standards Program Unit ganizational rules and bylaws, regulatory compliance plan, (SPU). In addition, five CBGG members are committee preferred terminology, and progress on the working parties members of the AABB Molecular Testing SPU, and one including proficiency exchange program, guidelines of prac- member (MER) holds liaison status with the committee. tice, DNA repository, funding, forms and disclaimers, and The AABB Standards for Molecular Testing for Red Cell, proposed Web site. It is highly recommended that CBGG Platelet, and Neutrophil Antigens was published in 2008.5 members refer to this document for the aforementioned The intent of the CBGG guidelines is to maintain an inde- duties and activities. Meetings are held annually, in part, pendent forum and voice for proposed molecular testing for members to discuss outstanding issues and to provide standards in ISO format for use by international laborato- the opportunity to give input and accept amendments to ries. The members will update, modify, or otherwise amend the document. The CBGG Document is distributed to mem- the CBGG guidelines by process of discussion and consen- bers and is available to nonmembers on request. sus. The CBGG guidelines will not become standards as

such to reflect the fact that the CBGG is not responsible for Template Disclaimers laboratory inspection or accreditation. CBGG members continue to recognize the importance

* Full listing of the members of the CBGG can be found in the appendix.

IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009 75 G.A. Denomme et al.

Proficiency Program Serologic confirmation is not mandated by this group owing An exchange of samples among several blood centers, to the lack of regulated antisera. The first exchange for plate- transfusion services, and reference laboratories serves as let genotyping was coordinated by Vagner Castro (Platelet one mechanism to evaluate proficiency in molecular test- Immunology Laboratory of Hematology and Hemotherapy ing. The proficiency programs involve the sending out of a Center of the State University of Campinas, UNICAMP). single sample (either DNA or whole blood) by a member This sample was previously genotyped by PCR-RFLP and in the spring and fall of each year. In the fall of 2008, the PCR-SSP to the two HPA systems most frequently involved CBGG blood group program provided its first whole blood in platelet alloimmunization: HPA-1 and HPA-5. sample for proficiency exchange, which was coordinated through the Canadian Blood Services, Network Center for Conclusions Applied Development (NetCAD) laboratory. Before any dis- The CBGG is a self-help, not-for-profit organization tribution, the target antigen must be tested by a PCR-based designed as an interactive collaborative for members to method and be confirmed by serology, with the additional learn from each other and to strive to achieve excellence in caveat that the proficiency exercise will not involve rare al- molecular testing of blood group, platelet, and neutrophil leles. A limited number of antithetical blood group antigens antigens. Anyone interested and willing to contribute intel- are evaluated, which are based on the assays performed by lectually is welcome to join. To become a member contact all participating members. As of spring 2008, the alleles are Marion Reid ([email protected]), Lilian Castilho as follows: RHCE*E/RHCE*e, GYPB*S/GYPB*s, KEL*1/ ([email protected]), Maryse St. Louis (maryse.st-louis@ KEL*2, FY*A/FY*B, FY*–33C/T GATA, and JK*A/JK*B. hema-quebec.qc.ca), Greg Denomme (greg.denomme@ The cost of sample preparation and shipping the DNA bcw.edu), or Connie Westhoff ([email protected]. sample is borne in turn by each submitting laboratory. To org). participate in the sample exchange, proficiency program members must agree to provide a sample for distribution Acknowledgments in a subsequent year through a predetermined rotation. We thank Robert Ratner for help in the preparation of Although participation in the CBGG proficiency program this manuscript and Lakshmi Gaur and Tanya Kerrigan for mandates that samples are discarded after the results have their help assembling the GenBank gene and SNP reference been validated, proficiency program members must ensure accession codes. The findings and conclusions in the article they comply with regulatory bodies. Thus, before joining should not be construed to represent any agency determi- the proficiency exchange program and committing to sup- nation or policy. ply a sample for the exchange, new members should address their institutional requirements on informed consent. References To prevent communication errors because of different 1. Denomme G, Reid M. Inaugural meeting of the Consor- reporting mechanisms, a report form has been developed tium for Blood Group Genes (CBGG): a summary report. specifically for the CBGG proficiency program, a copy of Immunohematology 2005;21:129–31. which is contained in the CBGG 2008 Document. Commu- 2. Reid ME. Consortium for Blood Group Genes. Transfu- nication of results between the submitting and testing labo- sion 2007;47(Suppl):98S–100S. ratories is done by using this form. Presently, proficiency 3. Reid ME, Westhoff CM, Denomme G, Castilho L. Con- reports for FY or GYPB*S/s should include appropriate sortium for Blood Group Genes (CBGG): Miami 2006 disclaimers in the comment section for those laboratories report. Immunohematology 2007;23:81–4. that do not evaluate common modifying and silencing nu- 4. Reid ME, Westhoff C, Denomme G, Castilho L. Consor- cleotide changes for these genes. A summary of the results tium for Blood Group Genes (CBGG): 2007 report. from the participating laboratories for each proficiency ex- Immunohematology 2007;23:165–8. ercise is compiled by the submitting laboratory and submit- 5. Guidance for standards for molecular testing for red cell, ted to the New York Blood Center for archival purposes. platelet, and neutrophil antigens. Bethesda, MD: AABB The proficiency evaluations for platelets and neutro- Press, 2008. phils are distributed to a specific group of members.

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76 IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009 CBGG: 2008 report

Table 1. List of blood group target antigens and alleles ISBT system name Target antigen Gene* Target nucleotide number Controls and comments (symbol) number (target allele) (RefSeqGene) (GeneBank SNP rs#) Control each nt analysis with the three genotypic classes when appropriate † ABO (ABO) 001 A (ABO*A1) ABO nt 1061ΔC (rs56392308) Many A/B alleles have been A2 (ABO*A2) (NG_006669.1) described, multiple targets are necessary for identification. A (ABO*A1) nt 526C>G (rs7853989) Targets for non-261delG Group O B (ABO*B1) nt 703G>A (rs8176743) alleles not listed. nt 796C>A (rs8176746) nt 803G>C (rs8176747)

A (ABO*A1) nt 261G/ΔG (rs8176719) O (ABO*01) MNS (MNS) M (GYPA*M) GYPA nt 59C>T;71G>A;72T>G nt 72 is the 3rd nt of codon 24 002 N (GYPA*N) (NG_007470.2) (rs7682260;7687256;7658293)

S (GPB*S) GYPB (NG_007483.1) nt 143T>C (rs7683365) Disclaimer required if null alleles not s (GPB*s) tested.

S silenced nt 230C>T † (see note below) intron 5+5g>t

Rh (RH) D RHD (NG_007494) Exon 4 & 7 Targets may vary as there are many 004 RHDΨ Ex 4 37 bp insert approaches

C (RHCE*C) RHCE (NG_009208) intron 2 insert Insert present in RHCE*C c (RHCE*c) nt 307T>C (rs676785)

E (RHCE*E) nt 676C>G (rs609320) e (RHCE*e)

Cw nt 122A>G † Cx nt 106G>A † V & VS nt 733C>G (rs1053361) † V (VS-) nt 1006G>T † Lutheran (LU) Lua (LU*01) LU (NG_007480.1) nt 230A>G (rs28399653) 005 Lub(LU*02) Kell (KEL) K (KEL*01) KEL (NG_007492.1) nt 578T>C (rs8176058) 006 k (KEL*02) Kpa (KEL*03) nt 841T>C (rs8176059) Kpb (KEL*04) Jsa (KEL*06) nt 1790C>T (rs8176038) Jsb (KEL*07) Duffy (FY) Fya (FY*A) FY (NC_000001.9) nt 125G>A (rs12075) 008 Fyb (FY*B) Fyx nt 265C>T (rs34599082) † Fy null (RBC) nt -33t>c (rs2814778) Promoter GATA-1 box Kidd (JK) Jka (JK*A) JK (NC_000018.8) nt 838G>A (rs1058396) Testing for null alleles may be ap- 009 Jkb (JK*B) propriate. Diego (DI) Dia DI (NG_007498.1) nt 2561T>C (rs2285644) 010 Dib Yt (YT) Yta YT (NG_007474.1) nt 1057C>A (rs1799805) 011 Ytb Scianna (SC) Sc1 SC (NG_008749.1) nt 169G>A (rs56025238) † 013 Sc2

IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009 77 G.A. Denomme et al.

Dombrock (DO) Doa DO (NG_007477.1) nt 793A>G (rs11276) 014 Dob Hy nt 323G>T (rs28362797) Joa nt 350C>T (rs28362798) Colton (CO) Coa CO (NG_007475.1) nt 134C>T (rs28362692) † 015 Cob Landsteiner-Wiener (LW) LWa LW (NG_007728.1) nt 308A>G † 016 LWb Cromer (CR) Cra CROM (NG_007465.1) nt 679G>C (rs60822373) 021 Knops (KN) Kna KN (NG_007481.1) nt 4681G>A (rs41274768) 022 Knb McCa nt 4768A>G (rs17047660) † McCb Sla nt 4801A>G (rs17047661) † Vil Indian (IN) Ina IN (NG_008937.1) nt 252C>G 023 Inb OK (OK) Oka OK (NG_007468.1) nt 274G>A † 024 Heterozygote not required * NG_number.n represent the GenBank reference gene sequence (RefSeqGene) accession code for each blood group system gene. # The rs number in brackets represents the GenBank SNP reference sequence (SNP rs) accession code for the given nucleotide (nt). † Homozygous rare alleles are not required for molecular testing.

Appendix Daniel B. Bellissimo, PhD Members of CBGG at end of 2008: Molecular Diagnostics Laboratory BloodCenter of Wisconsin Coordinator: 638 N. 18th Street Marion E. Reid, PhD Milwaukee, WI 53233 Laboratory of Immunochemistry and Laboratory of Immunohematology Imelda M. Bromilow New York Blood Center DIAMED SA 310 East 67th Street Cressier-sur-Morat, Switzerland New York, NY 10065 Tony S. Casina Country Coordinators: Ortho-Clinical Diagnostics, Inc. Connie Westhoff, PhD 1001 Highway 202 Molecular Blood Group and Platelet Antigen Testing Laboratory Raritan, NJ 08869 American Red Cross-Penn-Jersey Region 700 Spring Garden Street Vagner Castro Philadelphia, PA 19123 Hematology and Hemotherapy Center Hemocentro-State University of Campinas (UNICAMP) Maryse St-Louis, PhD Rua Carlos Chagas 480 Barão Geraldo Operational Research, Hema-Quebec Campinas, São Paulo, 13081-970, Brazil 1070, Avenue des Sciences-de-la-Vie Quebec, Quebec G1V 5C3, Canada Laura Cooling, MD University of Michigan Hospitals Lilian Maria de Castilho, PhD 1500 East Medical Center Drive Laboratory of Immunohematology Ann Arbor, MI 48109-0054 Hemocentro-State University of Campinas (UNICAMP) Ana Paula Cozac, MD Rua Carlos Chagas 480 Barão Geraldo Centro Regional de Hemoterapia de Ribeirão Preto Campinas, São Paulo, 13081-970, Brazil Ribeirão Preto, S.P. CEP 14051-140, Brazil

Members: Daniel de la Vega, PhD Bai Yu, MD, PhD Servicio de Medicina Transfusional Department of Pathology and Laboratory Medicine Hospital Italiano Garibaldi University of Texas Health Science Center at Houston Virasoro 1249 Rosario (2000) 6431 Fannin Street, MSB 2.222 Argentina Houston, TX 77030

78 IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009 CBGG: 2008 report

Gregory Denomme, PhD Immunohematology Reference Laboratory Mariza A. Mota, PhD Blood Center of Wisconsin Departamento de Hemoterapia 638 N 18th Street Hospital Israelita Albert Einstein Milwaukee, WI 53201-72178 Av. Albert Einstein, 627 - 4° andar, Morumbi São Paulo, SP CEP 05651-901 Brazil Helene M. DePalma Immunohematology Laboratory Joann M. Moulds, PhD New York Blood Center Clinical Immunogenetics 310 East 67th Street LifeShare Blood Centers New York, NY 10065 8910 Linwood Avenue Shreveport, LA 71106 Lakshmi K. Gaur, MSc, PhD Puget Sound Blood Center John J. Moulds, MT(ASCP)SBB 921 Terry Avenue Scientific Support Services Seattle, WA 98104-1256 LifeShare Blood Centers 8910 Linwood Avenue Ghazala P. Hashmi, PhD Shreveport, LA 71106 Bioarray Solutions 35 Technology Drive, Suite 100 Sandra Nance, MS, MT(ASCP)SBB Warren, NJ 07059 Technical Services American Red Cross Penn-Jersey Region Kim Hue-Roye 700 Spring Garden Street Laboratory of Immunochemistry Philadelphia, PA 19123 New York Blood Center 310 East 67th Street Marcia Zago Novaretti, MD, PhD New York, NY 10065 Division of Immunohematology and Transfusion Fundaçao Pro-Sangue/Hemocentro de Sangue Susan T. Johnson, MSTM, MT(ASCP)SBB Av. Angélica, 2.261 Manager, Immunohematology Services Sao Paulo, Brazil BloodCenter of Wisconsin 638 North 18th Street Joseph Ong Milwaukee, WI 53233 Blood Centers of the Pacific 270 Masonic Avenue Hallie Lee-Stroka San Francisco, CA 94118 Department of Transfusion Medicine Clinical Center Jordão Pellegrino, Jr. National Institutes of Health Rua Sampaio Ferraz 750 ap 41 B Campinas, São Paulo, Brazil CEP 10 Center Drive MSC 1184 13024-431 Bethesda, MD 20892 Maria Rios, PhD Christine Lomas-Francis, MS, FIBMS DETTD/OBRR/CBER Immunohematology Laboratory 29 Lincoln Drive New York Blood Center NIH Campus Building 29, Room B24 45-01 Vernon Blvd. Bethesda, MD 20892 Long Island City, NY 11101 Dennis Roscetti Tanya Kanigan GTI Diagnostics BioTrove Incorporated 20925 Crossroads Circle, Suite 200 12 Gill Street Waukesha, WI 53186 Woburn, MA 01801 Dawn M. Rumsey, ART(CSMLT) Kirk D. Kitchen PEPFAR Team, Global Development Clinical Laboratories American Association of Blood Banks Blood Systems Laboratories 8101 Glenbrook Road 2424 West Erie Drive Bethesda, MD 20814 Tempe, AZ 85282 Gayle Teramura Gandhi J. Manish, MD Puget Sound Blood Center Laboratory Medicine and Pathology 921 Terry Avenue Mayo Clinic Seattle, WA 98104-1256 200 First Street, S.W. Rochester, MN 55905

IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009 79 G.A. Denomme et al.

Rebecca Thomas Liaison Members: Memorial Blood Centers Sheryl A. Kochman 737 Pelham Blvd Devices Review Branch St. Paul, MN 55114 Center for Biologics Evaluation and Research Office of Blood Research and Review Antonio Sergio Torloni, MD Division of Blood Applications Mayo Clinic Food and Drug Administration 13400 East Shea Boulevard 1401 Rockville Pike, HFM-390 Scottsdale, AZ 85259 Rockville, MD 20852-1448

Sunitha Vege, MS Jill Storry, PhD Molecular Blood Group and Platelet Antigen Testing Laboratory Blodcentralen Skåne-University Hospital American Red Cross SE-221 85 700 Spring Garden Street Lund, Sweden Philadelphia, PA 19123

Rita Fontão Wendel Instituto de Hemoterapia Sirio-Libanes Rua D. Adma Jafet, 91 - 2 Andar Sao Paulo, CEP 01308-050 Brazil

Mark Yazer, MD RBC Serology Reference Laboratory Centralized Transfusion Service University of Pittsburgh 3636 Boulevard of the Allies Pittsburgh, PA 15213

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80 IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009 Speical Dedication

W. John Judd, FIBMS, MIBiol

D. Mallory

. John Judd, NYABB; and Suzanne Leiden Memorial Lecturer, CABB FIBMS, MIBiol, Society. In North Carolina, the extra room over the garage Wis being honored is called the FROG (furnished room over garage); however, by Immunohematology, in John’s case, it is called a TROG (trophy room over ga- Journal of Blood Group rage) for all of his well-deserved awards! Serology and Education John received these awards for his many investigative for his many contribu- studies, research reports, review papers, presentations, and tions to the journal and to publications. In 2008, John coauthored the third edition of the field of blood banking. Judd’s Methods in Immunohematology, a major reference John served on the edito- methods manual for laboratory workers. This edition con- rial board of Immunohe- tains more than 150 methods and procedures documents. matology for 7 years, from During the span of his career, he has had more than 80 2002 through 2008. We scientific papers published in peer-reviewed journals. Ad- thank him for giving gen- ditionally, John is the author of review articles on lectins, erously of his time and polyagglutination, elution studies, pretransfusion studies, ideas to improve the scope Rh blood groups, MNS-system antibodies, and investiga- and concept of the journal. tion of the positive DAT. These are just some of the topics This included contributing nine articles published between that he studied and investigated in depth. 1979 and 2005 that were significant to the interest of the A popular speaker, John gave numerous presenta- readers of Immunohematology. He also was a dedicated, tions at local, state, national, and international meetings. knowledgeable, and responsive peer reviewer of submitted One could always count on John to talk! In fact, he and the articles from the beginning of the publication and during late John Case, of Gamma Biologicals, Inc., were famous the length of his very important career. for their “chats” on the AABB SSCC forum and were well John retired in 2008 after 34 years as director of a first- known for debating, in depth, many questions concerning rate AABB-accredited reference laboratory at the Univer- blood group serology and its complexities. sity of Michigan. His career is a tribute to his determination In addition to this impressive scientific career, John to investigate the unusual and the undiscovered. In 1972, also managed to be active on many committees of the AABB, he studied at the Sir John Cass College in London, England, MABB, and International Society of Blood Transfusion. He where he was elected a Fellow of the Institute of Biomedical was also on the board of the AABB for 4 years and was on Sciences (FIBMS). He came to the University of Michigan the board and president of the MABB. in 1974 and was the director of the reference laboratory and The editors, authors, and reviewers of Immunohematology, professor of immunohematology in the Department of Pa- Journal of Blood Group Serology and Education, would thology until his retirement in 2008. He is now emeritus like to thank John for his contributions to the field of blood professor of immunohematology, Department of Pathol- banking during his 34 years at the bench as an investigator, ogy, at the University of Michigan. John was awarded the and as an author, educator, and mentor. We would particu- usual blue and gold embossed chair at retirement to prove larly like to acknowledge his contributions during the past it! John and his wife, Jane, then moved to North Carolina to several years as an editor, peer reviewer, and contributing relax and watch the golfers go by. author to Immunohematology. John, you may be gone John has received many awards, including the Ivor from the field, but you cannot be forgotten. Like the impor- Dunsford Memorial Award and the John Elliott Memo- tant scientists before you, you have left a large and lasting rial Award from the American Association of Blood Banks legacy of knowledge and respect. Thank you, and may you (AABB); the Founders Award and Kay Beattie Lecture from and Jane have a long, healthy, and happy retirement. the Michigan Association of Blood Banks (MABB); the Pet- taway-Sheppard Award of the North Carolina Association Delores Mallory, MT(ASCP)SBB of Blood Banks; L. Jean Stubbins Memorial Lecture of the Emeritus Editorial Board UTMB Medical Branch; Ronald Dubin Memorial Lecturer, Immunohematology

IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009 81 Letter from the Editors

Blood Group Reviews—A New Feature

he editors of Immunohematology present a new feature that is extremely exciting. Starting with this issue, Immunohe- matology will publish a series of 28 “Blood Group Reviews” in upcoming issues. Experts in the field have agreed to put Ttheir considerable knowledge and energy into writing a thorough review of each of the 28 selected areas. Readers will then have comprehensive reviews of all the major blood group systems and other topics at their fingertips. Of additional value will be the references for each article to which the reader can refer if interested in reading the original work. The authors will present each blood group review in the following format: history, nomenclature, genetics, molecular basis, biochemistry, antibodies in system, and clinical significance. The topics that will appear in sequential issues are detailed in the table below.

When all the topics have been published, Immunohematology will publish the entire collection as a stand-alone resource. This series provides an excellent opportunity for experienced staff to obtain continuing education, and it will be an educational tool for new staff. The editors envision a copy of the collected reviews as a must-have for transfusion medicine facilities, libraries, and personal bookshelves everywhere.

ABO FY H CR JMH MNS JK DO KN P/Globoside Rh and RHAG DI CO IN Lows LU YT LW I GIL KEL and Kx XG Ch/Rg OK LE SC GE RAPH

Sandra Nance Connie M. Westhoff Editors-in-Chief Immunohematology

82 IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009 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 2009 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 25, Number 2, 2009 83 Announcements, cont. Meetings

September 25 Illinois Association of Blood Banks (ILABB) The Illinois Association of Blood Banks (ILABB) Fall Meeting will be held September 25, 2009, at the Lisle/Naperville Hil- ton in Lisle, Illinois. For additional details please visit the Web site at www.ilabb.org or contact Kristi Williams at (309) 745-8999 or [email protected].

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, 2009, for the July 2010 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. Advertisements 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

84 IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009 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 25, Number 2, 2009 85 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.

86 IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009 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 25, Number 2, 2009 87 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 88 IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009

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