Immunohematology

Journal of Group Serology and Education Celebrating

2 Ye5ars Volume 25, Number 4, 2009

Immunohematology Journal of Blood Group Serology and Education Volume 25, Number 4, 2009 Contents

Invited Review: Milestones Series 147 Historic milestones in the evolution of the crossmatch S.G. Sandler and M.M. Abedalthagafi

Review 152 Lutheran G. Daniels

Case report 160 New serologic findings in a patient with ulcerative colitis and a warm autoantibody T.G. Lightfoot and L. Delia VanThof

Original Report 165 A simple screening assay for the most common JK*0 alleles revealed compound heterozygosity in Jk(a–b–) probands from Guam E.S. Wester, J. Gustafsson, B. Snell, P. Spruell, Å. Hellberg, M.L. Olsson, and J.R. Storry

Original Report 170 Cost-effectiveness of FDA variance for blood collection from individuals with hereditary hemochromatosis at a 398-bed hospital-based donor center D.M. Gribble, D.J. Chaffin, and B.J. Bryant

Original Report 174 Characterization of three novel monoclonal anti-Oka M.H. Tian and G.R. Halverson

Original Report 179 The American Red Cross Hemovigilance Program: advancing the safety of and transfusion A.F. Eder, B.A. Dy, J. Barton, J.M. Kennedy, and R.J. Benjamin

Communication 186 Thank you to the contributors to the 2009 issues

187 Index Volume 25, Nos. 1, 2, 3, 4, 2009

191 Announcements

192 Advertisements

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

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

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

Editorial Board

Patricia Arndt, MT(ASCP)SBB Brenda J. Grossman, MD Joyce Poole, FIBMS Pomona, California St. Louis, Missouri Bristol, United Kingdom

James P. AuBuchon, MD W. John Judd, FIBMS, MIBiol Mark Popovsky, MD Seattle, Washington 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

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

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Copyright 2009 by The American National Red Cross ISSN 0894-203X Invited Review: Milestones Series

Historic milestones in the evolution of the crossmatch

S.G. Sandler and M.M. Abedalthagafi

The introduction of the serologic crossmatch—first proposed by contributed to replacing the serologic crossmatch with im- Hektoen (1907) and first performed by Ottenberg (1908)—made proved antibody screens as serologic surrogates and com- it possible to transfuse blood without fear of unpredictable and puter crossmatches as electronic surrogates. As this review potentially disastrous acute hemolytic reactions, most of which encompasses more than a century’s history in only a few were attributable to direct agglutinating (IgM) anti-A, anti-B, or pages, the authors were obliged to be selective in highlight- anti-A,B. Previously transfused or previously pregnant recipients ing important changes. We recognize this limitation and continued to experience sporadic hemolytic transfusion reactions as a result of “incomplete” (IgG) blood group antibodies. Coombs’ direct readers who seek more information to the original introduction of the antiglobulin test (1945) made it possible to articles cited in the references. detect “incomplete” (IgG) antibodies and to develop laboratory methods to identify and transfuse serologically compatible RBCs. Historical Origins of the Crossmatch During the past 50 years, the antibody screen has evolved to be In 1907, Ludwig Hektoen, a pathologist at Chicago’s In- more effective than the crossmatch for detecting the presence of stitute for Infectious Diseases, suggested, for the first time potential serologic incompatibility and has, in fact, replaced the on record, that direct matching of donors’ and recipients’ crossmatch as the key step in pretransfusion compatibility testing. blood could prevent the potential adverse effects that had The antibody screen has become the serologic surrogate for the been observed after many blood transfusions.1,2 Comment- crossmatch and, currently, the computer crossmatch is becoming the electronic surrogate for the crossmatch. In historical perspec- ing on Landsteiner’s observation that the serum of some tive, the past 100 years witnessed the rise and fall of the cross- persons agglutinated the RBCs of certain others, Hektoen match. Today, hemolytic transfusion reactions are more likely to speculated that “under special conditions, homologous be the result of misidentifying the intended transfusion recipient transfusion might prove dangerous by leading to agglutina- than of failure of routine compatibility testing to detect serologic tion within the vessels of the subject transfused.”1 Although incompatibility. The introduction of the serologic crossmatch—100 Hektoen did not perform actual serologic crossmatches, years ago—had a major positive impact on all aspects of clinical historians credit him as being the first physician to propose medicine. As a result of these advances, we are quickly approach- direct pretransfusion donor-recipient crossmatching.3–5 Be- ing the limits of improving the safety and efficacy of blood trans- fore Hektoen’s suggestion, transfusions of animal or human fusions by conventional serologic methods. Future improvements are more likely to be the result of applications of molecular geno- blood had been conducted without pretransfusion sero- typing—which could not have become available at a more perfect logic testing and, as might be anticipated, some transfu- time. When Immunohematology publishes its 50th-anniversary sions appeared to be beneficial, but others were disastrous.6 issue, the improvements in transfusion science are more likely to Only a few years before Hektoen’s proposal, Landsteiner be based on molecular methods than on conventional had succeeded in differentiating human RBCs into three serology. Immunohematology 2009;25:147–151. serologic groups (A, B, O),7 but neither Landsteiner nor other contemporary laboratory investigators linked the Key Words: compatibility testing, crossmatch, blood serologic differences between human RBCs to the cause of transfusion sporadic hemolytic transfusion reactions. In 1908, Otten- berg performed the first serologic crossmatch on record, he pretransfusion serologic crossmatch as we know mixing the blood of a wife (donor) and her husband (re- it—direct mixing of a donor’s RBCs and the intended cipient) and observing the absence of before an Trecipient’s serum or plasma—is fading into history. uneventful 17-minute direct donor-recipient transfusion.8 The development of increasingly sensitive antibody screen- Ottenberg did not provide details of how he performed the ing methods, ready availability of commercially marketed first pretransfusion donor-patient crossmatch, but he pro- reagent RBC screening panels, and acceptance of computer posed “that a thing much to be desired is, that a convenient crossmatches by accreditation and regulatory authori- clinical test for this purpose be devised.”8 Physicians were ties have significantly diminished the role of the serologic slow to recognize the potential importance of pretransfu- crossmatch in compatibility testing. The following review sion serologic testing for preventing hemolytic transfusion highlights historic milestones that contributed to the devel- reactions. Among the skeptics was George Crile, a surgeon opment of the crossmatch as a key step in routine compat- in Cleveland, who had pioneered direct patient-to-recipient ibility testing. More recently, other scientific events have transfusions.9 Crile wrote “contrary to common belief,

IMMUNOHEMATOLOGY, Volume 25, Number 4, 2009 147 S.G. Sandler and M.M. Abedalthagafi normal blood of one individual does quite as well as that of The Antibody Screen as a Surrogate for the Serologic Cross- another. Kinship apparently is of no special advantage.”10 match Commercially marketed panels of reagent RBCs for The Indirect Antiglobulin Test and Its Modifications screening for blood group antibodies began to appear in the Ottenberg’s approach of pretransfusion serologic United States in the late 1950s and early 1960s. In 1962, for matching of donor’s RBCs and recipient’s serum was slowly the first time, Standards (3rd edition) recognized a limited accepted by surgeons and obstetricians, who performed role for reagent RBCs for the detection of antibodies in pa- most transfusions at that time. During the next four tients’ sera.24 The Standards’ acceptance of reagent RBCs as decades, pretransfusion serologic crossmatching was grad- a surrogate for the direct crossmatching of donor RBCs and ually introduced in hospitals in the United States. By elimi- patients’ sera was not without controversy. Grove-Rasmussen nating those donor-recipient incompatibilities that were communicated his concern in Transfusion, commenting detectable by direct hemagglutination, i.e., ABO incompat- that stored reagent RBCs may not be “in as good condition” ibilities, the room-temperature direct crossmatch eliminated as freshly donated RBCs and may not express all pertinent most acute and disastrous hemolytic reactions. Neverthe- antigens.25 less, sporadically, a previously transfused or previously pregnant transfusion recipient experienced an acute or Alternatives to Testing for Hemagglutination in Test Tubes delayed hemolytic transfusion reaction. In 1945, Coombs From the outset, antibody screens and crossmatches in et, al. published their observation that rabbit anti-human US hospitals were performed mostly in test tubes and, less serum could identify “incomplete” antibodies, and the anti- frequently, by rapid slide method. Alternative formats have human globulin (AHG) phase of donor RBC–recipient serum been introduced, primarily to decrease the volume of re- matching was added to procedures for antibody screen- agents. These methods include capillary tubes,26 microtiter ing and crossmatching.11 The addition of an AHG phase to plates,27 solid-phase plates,28 and gel agglutination.29 How- compatibility testing was a major advance; and the AHG ever, only the microplate method has been used extensively phase remains the most effective of all laboratory methods as an alternative for crossmatching.30, 31 for ensuring serologic compatibility of ABO-matched RBC transfusions. Soon, other investigators proposed modifica- The Rise of the Antibody Screen and the Fall of the Serologic tions of the antibody screen to increase its sensitivity for Crossmatch detecting blood group antibodies. Among the more notable As methods to improve the sensitivity of the antibody modifications were the addition of albumin,12,13 trypsin,14,15 screen improved, the importance of a direct serologic cross- papain,16 dextran,17 ficin,18 bromelin,19 low-ionic-strength match of donor’s RBCs and recipient’s plasma became less solution (LISS),20 polyethylene glycol (PEG),21 and hexadi- critical. In fact, from the first proposal of the pretransfu- methrine bromide.22 Editors of the first edition of the Amer- sion crossmatch until the present, direct serologic match- ican Association of Blood Banks’ (AABB) Standards for a ing of donor’s RBCs and patient’s serum or plasma has Service (1958) required a major cross- been recognized as a highly desirable, but not absolutely match, but the minor crossmatch was optional.23 For the required, step in issuing RBCs for transfusion. Among the major crossmatch, “at least two methods shall be used, one clinical situations in which the crossmatch is often omitted to demonstrate optimally the presence of serum or saline are life-threatening hemorrhages,32 after massive transfu- active antibodies, and another to detect the presence of in- sions when most of the recipient’s blood has been replaced complete (blocking) antibodies.”23 The acceptable methods by donor’s blood,32 and for infants younger than 4 months were as follows:23 of age.33 In 1980, Winn and colleagues proposed that an ex- 1. A saline or serum crossmatch with 2% donor RBCs, tended antibody screen could replace the crossmatch if the read at room temperature and after an indirect anti- results of ABO typing and antibody screen were verified and globulin test. documented.34 In 2000, Kuriyan and Fox reported their ex- 2. A saline or serum crossmatch in addition to a high pro- perience transfusing RBCs without a serologic crossmatch tein (bovine albumin) slide test, read on a heated slide if the antibody screen was negative and if the ABO was resting on a view box. confirmed by two technologists.35 In their hospital, elimi- 3. Both a saline or serum crossmatch and a tube technic nating the crossmatch decreased the cost of compatibility (recipient’s serum, albumin, and donor’s RBCs incu- testing 40 percent ($32,968); retaining the immediate- bated for 10 to 20 minutes, centrifuged, and read). spin crossmatch saved 30 percent ($24,748). In a larger The third edition of Standards (1962) recognized “enzyme hospital, eliminating the AHG crossmatch after a negative systems” as an option for antibody screening for the first antibody screen saved $70,000 per year.36 In 1985, Shul- time and introduced the requirement that the “antiglobulin man and colleagues reported the effect of switching from system [for crossmatching] shall utilize antihuman serum a conventional crossmatch (readings after immediate spin, meeting N.I.H. standards….”24 37°C incubation, and AHG phase) to an abbreviated crossmatch

148 IMMUNOHEMATOLOGY, Volume 25, Number 4, 2009 Evolution of the crossmatch

(immediate spin only) for patients without blood group al- Judd, and colleagues at Blood Bank at the University of loantibodies.36 During an 8.5-month experience, they per- Michigan Hospital initiated a procedure to confirm donor- formed 27,742 crossmatches and avoided 46,959 unnec- recipient compatibility by electronic verification, i.e., by a essary crossmatches, decreasing costs by at least $49,300 computer crossmatch.42 In 1994, these investigators report- annually. In 1992, Judd and colleagues published a study ed their experience using a computer crossmatch for pre- entitled “Can the reading for serological reactivity follow- transfusion tests for 14,021 patients between February 1992 ing 37°C incubation be eliminated?”37 Before conducting and March 1993. Their report included a detailed standard the study, they had the impression that the 37°C reading operating procedure, consisting of specific requirements for contributed minimally to transfusion safety. However, af- (1) RBC unit processing, (2) patient sample processing, (3) ter their review identified approximately 25 patients with crossmatch for ABO compatibility, and (4) release of RBC 37°C-only antibodies with Rh, Kell, or Kidd specificity an- units for transfusion. As suggested by Judd’s 1991 editorial, nually, they decided to retain the 37°C reading. Seven years the key remaining function of the serologic crossmatch was later, Judd and colleagues revisited the issue.38 Using data ensuring ABO compatibility for RBC transfusions. The func- from prior publications, they estimated the risk that elimi- tion of detecting antibodies in the recipient’s serum against nating different components of the compatibility test would antigens on donors’ RBCs had been co-opted by the anti- introduce in their hospital. The result was that the risk for body screen. Although AABB Standards allowed computer transfusing incompatible blood by eliminating the direct crossmatching to replace the immediate-spin crossmatch by AHG test, the indirect AHG crossmatch, and 37°C read- the 15th edition (1993),43 the logistics of writing software for ing would be approximately 1:13,000, 1:2,000, and 1:2,400 individual hospital information systems limited widespread units transfused.38 Given this history of scientific and tech- implementation.42 In 1998, Judd reported to attendees of nical events, what is the current state of the art? Not all pro- the International Society of Blood Transfusion’s congress in posed variations of the serologic crossmatch have survived Hong Kong that there were electronic crossmatching proce- the test of time. In 2008, the College of American Patholo- dures in use in at least 10 North American facilities, Scandi- gists distributed a proficiency survey that consisted of a navia, Hong Kong, and Australia.44 Progress in widespread donor RBC sample (J-13R) to be crossmatched with a pa- implementation of the computer crossmatch has been slow, tient’s serum sample (J-10S) containing an unknown anti- but the historic milestone has been established as many body (anti-Jka).39 Of 2,572 laboratories reporting results for blood banks have moved forward. antibody screening, 874 (34.0%) used a tube method; 94 (3.7%), solid-phase red cell adherence (SPRCA); and 1604 Impact of Serologic Methods on Clinical Practice (62.3%), gel agglutination. For those using a tube method, We now change gears, moving from an objective his- 27 (3.0%) used saline AHG; 54 (6.2%), albumin AHG; 561 tory of the serologic crossmatch to more subjective opin- (64.1%) used LISS AHG; and 234 (26.7%) used PEG AHG. ions about how these historic milestones have impacted the Having identified (or misidentified) the anti-Jka, 1821 labo- practice of medicine. Surely, the starting point is Hektoen’s ratories reported performing an AHG crossmatch. Of these, suggestion that Landsteiner’s newly described serologic 107 (5.9%) used saline AHG; 137 (7.6 %), albumin AHG; blood groups may be related etiologically to observations of 1201(65.9%), LISS AHG; and 376 (20.6%) PEG AHG).39 unpredictable acute hemolytic reactions after certain blood Notably, the number of hospitals using enzyme-treated re- transfusions. When Ottenberg moved Hektoen’s sugges- agent RBCs for routine antibody screening was too few to tion forward and performed the first serologic crossmatch, be listed as a separate category. he initiated the era of safe blood transfusion. It was now possible to perform surgery and treat hemorrhage with- Automated Crossmatching out the fear of an unpredicted, acute, and potentially fatal The first widely used automated serologic analyzer hemolytic reaction. Coombs’ development of antihuman performed ABO grouping and Rh(D) typing, but not cross- globulin provided the next major impact, significantly de- matching.40 The first automated analyzer that did incorpo- creasing the risk of hemolytic reactions attributable to non- rate the option for a serologic donor RBC–recipient plasma ABO IgG blood group antibodies. By 1945, transfusions of crossmatch was the ABS2000 (Immucor, Norcross, GA).30, 31 RBCs could be administered without fear of an ABO- Most hospitals that acquired an ABS2000, including our related acute hemolytic reaction, and severe reactions from hospital, did not take advantage of the crossmatch option, non-ABO antibodies were relatively uncommon. With the preferring a manual immediate-spin crossmatch for pa- risk of acute and delayed hemolytic reactions greatly de- tients with negative AHG antibody screens as a more prac- creased, surgeons were not only able to manage trauma, tical alternative. obstetrical hemorrhage, and gastrointestinal bleeding, but also to develop procedures for previously impossible car- Eliminating the Crossmatch: the Computer Crossmatch diovascular and cancer surgeries requiring large numbers In 1991, Judd published an editorial asking the ques- of blood transfusions. Subsequent refinements in methods tion, “Are there better ways than the crossmatch to dem- for antibody screening and identification, although a major onstrate ABO incompatibility?”41 Shortly thereafter, Butch, focus in the blood banking community, have had only an

IMMUNOHEMATOLOGY, Volume 25, Number 4, 2009 149 S.G. Sandler and M.M. Abedalthagafi incremental impact on the practice of medicine. Even the 10. Crile GW. Further observations on transfusion with a introduction of the computer crossmatch—clearly a major note on hemolysis. Surg Gynecol Obstet 1909;9:16–8. advance for efficient management of pretransfusion labo- 11. Coombs RR, Mourant AE, and Race RR. A new test for ratory testing—has only a minor impact, if any, on clinical the detection of weak and “incomplete” Rh agglutinins. practice. Br Exp Pathol 1945;26: 255-66. After 100 years of some broad strides and many in- 12. Dodge OG. A comparative study of direct matching tech- cremental improvements, the serologic crossmatch has niques in blood transfusion. J Clin Pathol 1952;5:102– evolved to a standardized, highly sensitive, and safe labora- 7. tory practice. The heyday of discovering new blood group 13. Stroup M, MacIlroy M. Evaluation of the albumin an- antigens and developing incrementally more sensitive se- tiglobulin technic in antibody detection. Transfusion rologic methods may be passing, but there are new and 1965;5:184–91. challenging opportunities for transfusion science and blood 14. Morton JA, Pickles MM. The proteolytic enzyme test banking. The availability of molecular genotyping to fur- for detecting incomplete antibodies. J Clin Pathol ther define blood group antigens has opened new vistas for 1951;4:189–99. blood group science.45 Molecular science offers the promise 15. Rosenfield RE, Vogel P. The identification of hemagglu- of improved efficacy of transfusions of RBCs, particularly tinins with red cells altered with trypsin. Trans N Y Acad for those chronically transfused patients for whom con- Sci 1951;13:213–20. ventional compatibility testing does not ensure satisfactory 16. Goldsmith K. Papain-treated red cells in the detection of outcomes. As we look back and honor the contributions of incomplete antibodies. Lancet 1955;268:76–7. our colleagues in this 25th anniversary issue of Immunohe- 17. Munk-Andersen G. A dextran serum medium for the matology, we can only speculate on what further advances demonstration of incomplete anti-A and anti-B; a di- will be published in future issues of Immunohematology rect conglutination test for the demonstration of in-vivo and featured in the next historical review. sensitization with incomplete ABO antibody of the red cells of newborn infants. Acta Pathol Microbiol Scand References 1956;38:259-72. 1. Hektoen L. Isoagglutination of human corpuscles with 18. Haber G, Rosenfield R. Ficin treated red cells for he- respect to demonstration of opsonic index and to trans- magglutination studies. P.H. Andresen, Papers in dedi- fusion of blood. JAMA 1907;48:1739–40. cation of his 60th birthday, Munksgaard, Copenhagen, 2. Hektoen L. Isoagglutination of human corpuscles. J In- 1957:45–9. fect Dis 1907;4:297–303. 19. Pirofsky B, Mangum ME Jr. Use of bromelin to dem- 3. Rosenfield RE. Early twentieth century origins of onstrate erythrocyte antibodies. Proc Soc Exp Biol Med modern blood transfusion therapy. Mt Sinai J Med 1959;101:49–52. 1974;41:626–35. 20. Hughes-Jones NC, Polley MJ, Telford R, et al. Optimal 4. Lederer SE. Flesh and blood: organ transplantation conditions for detecting blood group antibodies by the and blood transfusion in twentieth-century America. antiglobulin test. Vox Sang 1964;9:385–95. New York: Oxford University Press, 2008. 21. Nance SJ, Garratty G. A new potentiator of red blood 5. Diamond LK. The story of our blood groups. In: Win- cell antigen-antibody reactions. Am J Clin Pathol trobe MM, ed. Blood pure and eloquent: a story of dis- 1987;87:633–5. covery, of people, and of ideas. New York: McGraw-Hill 22. Lalezari P. The manual hexadimethrine bromide (poly- Book Company, 1980:691-717. brene) test: effects of serum proteins and practical ap- 6. Oberman H. The history of . In: plications. Transfusion 1987;27:295–301. Petz LA, Swisher SN, eds. Clinical practice of transfu- 23. Strumia MM, Jenkins ER, eds. Standards for a blood sion medicine. 2nd ed. New York: Churchill Living- transfusion service. 1st ed. Washington, DC: American stone, 1989:9–30. Association of Blood Banks, 1958:17. 7. Garratty G, Dzik W, Issitt PD, et al. Terminology for 24. Strumia MM, Lesses MF, eds. Standards for a blood blood group antigens and genes—historical origins transfusion service. 3rd ed. Washington, DC: American and guidelines in the new millennium. Transfusion Association of Blood Banks, 1962:13. 2000;40:477–89. 25. Grove-Rasmussen M. Routine compatibility testing: 8. Ottenberg R. Transfusion and arterial anastomosis: standards of the AABB as applied to compatibility tests. some experiments in arterial anastomosis and a study Transfusion 1964;4:200–5. of transfusion with presentation of two clinical cases. 26. Chown B. A rapid, simple and economical method for Ann Surg 1908;47:486–505. Rh agglutination. Am J Clin Pathol 1944;14:144-50. 9. Crile GW. The technique of direct transfusion of blood. Ann Surg 1907;46:329–32.

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27. Crawford MN, Gottman FE, Gottman CA. Microplate 38. Judd WJ, Fullen DR, Steiner EA, et al. Revisiting the system for routine use in blood bank laboratories. issue: can the reading for serologic reactivity follow- Transfusion 1970;10:258–63. ing 37 degrees C incubation be omitted? Transfusion 28. Beck ML, Rachel JM, Sinor LT, Plapp FV. Semiauto- 1999;39:295–9. mated solid phase assays for pretransfusion testing. 39. Transfusion Medicine Resource Committee. Surveys Med Lab Sci 1984;41:374–81. 2008 and Anatomic Pathology Education Programs. 29. Lapierre Y, Rigal D, Adam J, et al. The gel test: a new J-B Transfusion Medicine (Comprehensive) and Edu- way to detect red cell antigen-antibody reactions. cational Challenge. Northfield, IL: College of American Transfusion 1990;30:109–13. Pathologists, 2008. 30. Sandler SG, Langeberg A, Avery N, Mintz PD. A fully 40. McNeil C, Helmick WM, Ferrari A. A preliminary in- automated blood typing system for hospital transfusion vestigation into automatic blood grouping. Vox Sang services. Transfusion 2000;40:201–7. 1963;8:235–41. 31. Sandler SG, Langeberg A, Rumsey AH, Novak SC. A 41. Judd WJ. Are there better ways than the crossmatch solid phase and microtiter hemagglutination method to demonstrate ABO incompatibility? Transfusion for pretransfusion compatibility testing. Haematologia 1991;31:192–4. (Budapest) 2000;30:149–57. 42. Butch SH, Judd WJ, Steiner EA, et al. Electronic veri- 32. Oberman HA, Barnes BA, Friedman BA. The risk of ab- fication of donor-recipient compatibility: the computer breviating the major crossmatch in urgent or massive crossmatch. Transfusion 1994;34:105–9. transfusion. Transfusion 1978;18:137–41. 43. Widmann, FK, ed. Technical manual. 15th ed. Bethesda, 33. DePalma L. Review: red cell alloantibody formation MD: AABB, 1993:25. in the neonate and infant: considerations for current 44. Judd WJ. Requirements for the electronic crossmatch. immunohematologic practice. Immunohematology Vox Sang 1998;74(Suppl 2):409–17. 1992;8:33–7. 45. Westhoff CM, Sloan SR. Molecular genotyping in trans- 34. Winn LC, Svoda R, Hafleight EB, Grument FC. An extended fusion medicine. Clin Chem 2008;54:1948–50. antibody screen to replace the crossmatch. Presented at the 33rd Annual Meeting of the American Association S. Gerald Sandler, MD (corresponding author), Professor of Blood Banks, Washington, DC, 1980. of Medicine and Pathology, Director, Transfusion Medi- 35. Kuriyan M, Fox E. Pretransfusion testing without sero- cine, Department of Laboratory Medicine, Room M-1306, logic crossmatch: approaches to ensure patient safety. Georgetown University Hospital, Washington, DC 20007, Vox Sang 2000;78:113–8. and Malak M. Abedalthagafi, MD, Chief Resident, Depart- 36. Shulman IA, Nelson JM, Kent DR, et al. Experi- ment of Pathology, Georgetown University Medical Cen- ence with a cost-effective crossmatch protocol. JAMA ter, Washington, DC. 1985;254:93–5. 37. Judd WJ, Steiner EA, Oberman HA, Nance SJ. Can the reading for serologic reactivity following 37 degrees C incubation be omitted? Transfusion 1992;32:304–8.

IMMUNOHEMATOLOGY, Volume 25, Number 4, 2009 151 Review

Lutheran

G. Daniels

The Lutheran blood group system consists of 19 antigens: four Lunull phenotype, Lu(a−b−), with complete absence of pairs of antithetical antigens—Lua/Lub, Lu6/Lu9, Lu8/Lu14, and Lutheran antigens and a recessive mode of inheritance, was Aua/Aub—and 11 antigens of very high frequency. These antigens found in an English woman in 19605; its molecular back- are located on four of the five immunoglobulin-like domains of ground was resolved 47 years later.6 A phenotype called both isoforms of the Lutheran . The LU gene is on Lu(a b ), but often subsequently referred to as In(Lu) phe- and comprises 15 exons. The two glycoprotein − − isoforms differ in the length of their cytoplasmic tails as a re- notype, was discovered in 1961 by Dr. Mary (Polly) Craw- ford, MD, in herself and in three generations of her fam- sult of alternative splicing of intron 13. Lunull phenotype arises from homozygosity for inactivating mutations in the LU gene. ily.7 This phenotype, in which Lutheran antigens are barely

The dominantly inherited Lumod phenotype, In(Lu), results from detectable and there is also weakened expression of some heterozygosity for inactivating mutations in KLF1, the gene for the other blood group antigens outside of the Lutheran system, erythroid transcription binding factor EKLF. Clinically, antibod- has a dominant mode of inheritance. The molecular basis ies of the Lutheran system are relatively benign. When hemolytic, of In(Lu), heterozygosity for inactivating mutations in a they generally cause only mild, delayed hemolytic transfusion re- gene for an erythroid transcription factor, was identified in actions or hemolytic disease of the fetus and newborn that can be 2008.8 treated by phototherapy. The Lutheran , which are members of the immunoglobulin superfamily of adhesion mol- Immunochemical analyses revealed that the Lutheran 9,10 ecules and receptors, bind isoforms of laminin with α5 chains, antigens were located on two isoforms of a glycoprotein. components of the extracellular matrix abundant in vascular Cloning and sequencing of the Lutheran gene in 1995,11 and endothelia. The primary function of the Lutheran glycoproteins subsequent disclosure of its organization,12,13 demonstrated on RBCs could involve the transfer of maturing RBCs from the to the peripheral Table 1. Antigens of the Lutheran system circulation. They could also be involved in vascu- Antigen Molecular basis of antigen-negative phenotype lar occlusion and thrombotic events as complica- tions of and polycythemia vera, Anti- respectively. No Name Frequency thetical Nucleotides Exon Amino acids Immunohematology 2009;25:152–159. LU1 Lua Polymorphic Lub 230A>G 3 His77Arg LU2 Lub High Lua 230G>A 3 Arg77His Key Words: blood groups, Lutheran, antigens, antibodies, molecular genetics, EKLF, immuno- LU3 Lu3 High Various globulin superfamily, laminin, sickle cell disease LU4 Lu4 High 1. 524G>A 5 1. Arg175Gln 2. 524G>T 5 2. Arg175Leu History LU5 Lu5 High 326G>A 3 Arg109His utheran was the fifth blood group sys- LU6 Lu6 High Lu9 824C>T 7 Ser275Phe tem to be discovered, after ABO, MN LU7 Lu7 High Not known (but before S), P, and Rh. Anti-Lua, L LU8 Lu8 High Lu14 611T>A 6 Met204Lys the first Lutheran antibody, was identified in 1945 in a serum that also contained the first LU9 Lu9 Low Lu6 824T>C 7 Phe275Ser examples of anti-Cw and anti-Levay (which LU11 Lu11 High Not known became anti-Kpc), plus anti-c and anti-N.1,2 LU12 Lu12 High 1. 99-104del 2 1. delArg34+Leu35 It was another 10 years before the antibody 2. 419G>A 3 2. Arg140Gln to the antithetical antigen, Lub, was identi- LU13 Lu13 High 1340C>T, 11, Ser447Leu, fied.3 Lutheran is now a complex system 1742A>T 13 Gln581Leu comprising four pairs of antithetical anti- LU14 Lu14 Low Lu8 611A>T 6 Lys204Met gens plus 11 antigens of very high frequency LU16 Lu16 High 679C>T 6 Arg227Cys (Table 1). In 1951 the first demonstration of LU17 Lu17 High 340G>A 3 Glu114Lys linkage between human autosomal genes LU18 Aua Polymorphic Aub 1615A>G 12 Thr529Ala was between the Lutheran blood group gene b a and the gene originally believed to govern LU19 Au Polymorphic Au 1615G>A 12 Ala529Thr the Lewis blood group, but which turned out LU20 Lu20 High 905C>T 7 Th302Met to be the ABO secretor gene.4 LU21 Lu21 High 282C>G 3 Asp94Glu Obsolete: LU10, previously Singleton; LU15, AnWj (now 901009).

152 IMMUNOHEMATOLOGY, Volume 25, Number 4, 2009 Lutheran that the Lutheran (Lu) glycoproteins are members of the immunoglobulin superfamily of receptors and adhesion molecules and differ from each other in the length of their cytoplasmic tails as a result of alternative splicing. Elucida- tion of the molecular bases of the Lutheran polymorphisms and variants soon followed.12–14 Clinically Lutheran is not very important, but it is a very interesting system from genetic and functional perspectives.

The Lutheran Gene (LU) and the Lutheran Glycopro- teins (CD239) LU is located on the long arm of chromosome 19, at 19q13.2, as part of the linkage group that includes the genes for the H (FUT1), secretor (FUT2), and Lewis (FUT3) gly- cosyltransferases and for the LW blood group (ICAM4).15,16 Lutheran cDNA was cloned from a human placental cDNA library by using an amino acid sequence derived from Lu- glycoproteins purified by immunoaffinity chromatography with a monoclonal antibody, BRIC 221.11 Immunoblotting of RBC membranes with monoclonal anti-Lub or with alloanti-Lua, -Lub, -Lu3, -Lu4, -Lu6, -Lu8, -Lu12, -Aua, or -Aub revealed components of apparent mo- lecular weights of 85 and 78 kDa.9,10,17 Immunoprecipitation experiments with a rabbit antiserum prepared to an ami- no acid sequence of the cytoplasmic domain showed that the 78-kDa structure lacks part of the cytoplasmic domain present in the 85-kDa isoform.11 The Lu-glycoproteins belong to the immunoglobulin superfamily (IgSF),11 a large collection of glycoproteins that contain repeating extracellular domains with sequence ho- mology to immunoglobulin variable (V) or constant (C1, C2, or I) domains. IgSF glycoproteins mostly function as receptors and adhesion molecules and may be involved in signal transduction.18 The extracellular domain of the Lu- glycoproteins is organized into two V and three C2 IgSF domains,11 with a distinctive bend and flexible junction be- Fig. 1 Model of the Lu-glycoproteins showing the five IgSF do- tween domains 2 and 3 (Fig. 1).19,20 There are five potential mains, the location of the Lutheran antigens, and the cytoplasmic domains in the two isoforms. N-glycosylation sites, one in the third domain and the other four in the fourth domain. The LU gene is organized into 15 exons: exon 1 encodes by exon 13.21 The 78-kDa transcript had previously been the signal peptide; exons 2 through 12, the five IgSF do- identified as an epithelial cancer antigen, and is often re- mains; exon 13, the transmembrane domain and the cyto- ferred to by its earlier name, B-CAM,22 or as Lu(v13).21 plasmic domain common to both isoforms; and exons 14 and 15, the C-terminal 40 amino acids of the 85-kDa iso- The Lutheran Antigens form.12,13 LU transcripts of 2.5 kb and 4.0 kb encode the 85- Lua (LU1) and Lub (LU2) kDa and 78-kDa Lu-glycoprotein isoforms, respectively.21 Lua and Lub are inherited as codominant allelic charac- The two transcripts differ as a result of alternative splicing ters.15 Lua is widely distributed among Europeans, Africans, of intron 13: in the 2.5-kb transcript intron 13 has been and North Americans, with a frequency of around 6 to 8 removed by splicing and exons 14 and 15 encode the C- percent, but it is very rare in or absent from all other in- terminal 40 amino acids of the larger isoform; in the 4.0-kb digenous populations studied.23 In Caucasian populations transcript intron 13 is retained and contains a translation- the incidence of Lu(b−) is about one in a thousand. Typi- stop codon, so the unspliced intron 13 and exons 14 and 15 cal frequencies were obtained from tests with anti-Lua and are not translated and the protein product has a cytoplas- -Lub on about 1500 white Canadians: Lua 6.9 percent; Lub mic domain consisting only of the 19 amino acids encoded 99.9 percent; Lu(a−b+) 93.1 percent; Lu(a+b+) 6.8 percent;

IMMUNOHEMATOLOGY, Volume 25, Number 4, 2009 153 G. Daniels

Lu(a+b−) 0.1 percent; Lua allele 3.5 percent; Lub allele 96.5 the antithetical antigen, Aub, was not found until 1989. percent.24,25 All of 1102 Chinese genotyped were homozy- For many years the Auberger antigens were considered gous for Lub.26 to be independent of Lutheran, despite being absent from The Lutheran antigens are very variable in strength, In(Lu) RBCs, mainly because of results on one family, but the antigenic strength usually remains roughly constant which showed recombination between Aua and Lua.34 After within families. There is also heterogeneity of Lutheran the demonstration that Aua and Aub are located on the Lu- antigen strength among individual RBCs within a person, glycoproteins,17 the family was retested for Aua and tested which accounts for the characteristic mixed field agglutina- for Aub; errors in the original testing were discovered and tion patterns often seen with Lutheran antisera, especially the family then supported linkage between Auberger and anti-Lua. Occasionally adsorption and elution tests are re- Lutheran.35 Family studies confirmed that Auberger and quired to detect weak Lub on Lu(a+b+) cells. RBCs from Lutheran antigens are controlled by the same gene.36 Aua cord samples and from infants in the first year of life have has an incidence of between 80 percent and 90 percent in markedly weakened expression of Lua and Lub compared European populations.33,37 Aub has an incidence of about with those from adults.15 50 percent in a European population and 68 percent in an Lua/Lub results from a single nucleotide polymorphism African American population.38 Genotyping in Chinese pre- (SNP) in exon 3 of LU, encoding an amino acid substitution dicted antigen frequencies for Aua and Aub of 98 percent in the first IgSF domain of the Lu-glycoproteins: Lua A230, and 24 percent, respectively.26 His77; Lub G230, Arg 77 (Table 1).12,13 This SNP is associated Aua and Aub represent an SNP in exon 12 encoding an with an AciI restriction-site polymorphism.13 amino acid substitution in the fifth IgSF domain.12

Lu6 (LU6) and Lu9 (LU9) Other Lutheran Antigens Lu6 and Lu9, Lutheran antigens of high and low fre- Lu3, Lu4, Lu5, Lu7, Lu11, Lu12, Lu13, Lu16, Lu17, Lu20, quency, respectively, have an antithetical relationship and and Lu21 are antigens of very high incidence.15,39 All are ab- represent an SNP and CfoI restriction polymorphism in LU sent from Lunull RBCs and absent from, or extremely weakly encoding an amino acid substitution in the third IgSF do- expressed on, In(Lu) cells. All except Lu11 have been shown main (Table 1).14 The original anti-Lu9 was found, together to be located on the Lu-glycoproteins by immunoblotting, with anti-Lua, in the serum of a white woman.27 RBCs of monoclonal antibody-specific immobilization of eryth- her husband were Lu(a+b+) and Lu:9. Study of his family rocyte antigens assay, or flow cytometry with K562 cells showed that Lu9 expression was controlled by the LU lo- transfected with LU cDNA,10,12,39,40 or their absence has been cus, although it did not represent an allele of Lua and Lub. associated with a mutation in the LU gene (Table 1).6,14,39 The only other example of anti-Lu9 was found in a multiply Lu11 has not been shown to be inherited29 and the evidence transfused woman and tests on 200 RBC samples unearthed that Lu11 belongs to the Lutheran system is limited, so Lu11 only one Lu:9 sample (0.5%).28 Tests with the original anti- should be referred to as a para-Lutheran antigen. Lu9 suggested a higher frequency of 1.7 percent,27 but that Absence of most of these high-frequency antigens is as- serum also contained anti-HLA-B7 (-Bga).29 sociated with a single nucleotide change in LU, encoding RBCs of the original Lu:–6 propositus and those of her an amino acid substitution, but there are some exceptions two Lu:–6 siblings were strongly Lu:9, suggesting homozy- (Table 1). Lu3 is defined by antibodies produced by immu- 30 gosity. All other Lu:–6 individuals (who are not Lunull) nized individuals with Lunull phenotype and is described have also been Lu:9. later. Homozygosity for two different mutations within the same codon has been responsible for the rare phenotype in Lu8 (LU8) and Lu14 (LU14) the only two known unrelated Lu:−4 individuals: 524G>A Lu8 and Lu14, Lutheran antigens of high and low fre- encoding Arg175Gln and 524G>T encoding Arg175Leu.14,41 quency, respectively, have an antithetical relationship and Lu:−12 also had two different molecular backgrounds in two represent an SNP and FatI and Nla restriction polymor- individuals: (1) a six-nucleotide deletion in exon 2 encoding phisms in LU, encoding an amino acid substitution in the a deletion of Arg34 and Leu35; (2) 419G>A in exon 3 en- second IgSF domain (Table 1).14 The original anti-Lu8 was coding Arg140Gln.14 Although the mutations are in differ- reported as an antibody to a high-frequency antigen absent ent exons, when mapped to a three-dimensional schematic 31 from Lunull cells. An antibody in the serum of a multiply presentation of the Lu-glycoprotein the amino acid changes transfused dialysis patient, which reacted with RBCs of appeared to be located in close spatial proximity because of 2.4 percent of random white donors, reacted strongly with the protein folding.14 It is likely that the mutations altered three examples of Lu:–8 and was numbered anti-Lu14.32 the same epitope on the Lu-glycoprotein, affecting binding of the anti-Lu12. Lu:−12 was associated with weak expres- Aua (LU18) and Aub (LU19): the Auberger Antigens sion of Lub in the original family and in an Lu:−12 individual The first anti-Aua was identified in 1961 in the serum found by screening 1050 Canadian donors with anti-Lu12.42 of a multiply transfused woman.33 The antibody to Lu12 and Lub are both located on the first IgSF domain, so

154 IMMUNOHEMATOLOGY, Volume 25, Number 4, 2009 Lutheran the Lub weakening may result from conformational changes Lutheran system antibody has been detected in the serum caused by the amino acid changes arising from the Lu:−12 of any person with an In(Lu) gene, presumably because of mutations. the weak expression of Lutheran system antigens on their RBCs. Recombinant Lutheran Antigens A large survey of about 250,000 London blood donors Lutheran antigens have been used as models for the with anti-Lub and -Lua revealed 79 Lu(a−b−) donors, an in- application of recombinant proteins in antibody identifi- cidence of about 0.03 percent.49 Most of these were prob- cation.12,43,44 Recombinant proteins containing all or some ably In(Lu). A similar incidence was found by screening of the IgSF domains of the Lutheran protein have been African Americans in Detroit.50 Screening of US donors in expressed in eukaryotic or prokaryotic cells. The purified Houston and Portland with monoclonal anti-CD44 or with protein was then used in agglutination inhibition tests, anti-AnWj, which would be more specific for In(Lu), gave attached to polystyrene plates for detection by an ELISA frequencies of 0.02 percent and 0.12 percent, respective- procedure, or coupled to superparamagnetic particles for ly.51,52 detection in a particle gel immunoassay.43–45 Alloanti-Lua The term In(Lu) is not really appropriate as the In(Lu) or -Lub, as specified by the cDNA transfected, was detected phenotype is also characterized by weakened expression of with high sensitivity and specificity. RBC antigens belonging to other blood group systems: • P1, although the effect is less obvious than that of the 49,53 Lunull and Lumod Phenotypes Lutheran antigens ; 49,53 Lunull • i antigen as judged by selected anti-i ; b The only true Lunull phenotype is extremely rare and has • CD44, and consequently the high-frequency In (IN2), a recessive mode of inheritance. No antigens of the Lu- INFI (IN3), and INJA (IN4) antigens located on CD44, theran system can be detected on the RBCs, and individuals although these determinants are still easily detected on 54,55 with the Lunull phenotype may make an antibody, anti-Lu3, In(Lu) RBCs ; which reacts with all RBCs apart from those with the Lunull • AnWj, an antigen of very high incidence, which may be phenotype. Lunull RBCs have normal expression of those an- associated with the CD44 glycoprotein, is not expressed, tigens, such as AnWj, that are expressed weakly on In(Lu) or at least is expressed only very weakly56; RBCs. • Kna, McCa, Sla, and Yka of the Knops system (CD35),57 a 57 15 The molecular background of Lunull has been identified Cs , and MER2 (CD151), although the effect is slight in five individuals. All are either homozygous or doubly and family studies are required. heterozygous for inactivating mutations in the LU gene. Individuals with an In(Lu) gene are generally healthy 1. English woman.5 Heterozygosity for a nonsense muta- with no obvious anemia or reticulocytosis, although a de- tion 691C>T in exon 6, encoding Arg231STOP, and for gree of acanthocytosis has been associated with In(Lu).58 a deletion of exons 3 and 4.6 Singleton et al.8 demonstrated that In(Lu) resulted from 2. Japanese blood donor. Homozygosity for a nonsense heterozygosity for mutations in KLF1, in the presence of a mutation 711C>A in exon 6 encoding Cys237STOP.6 normal KLF1 allele. KLF1 encodes the erythroid transcrip- 3. German woman of Czech origin. Homozygosity for a tion factor erythroid Kruppel-like factor (EKLF), which nonsense mutation 361C>T in exon 3 encoding Arg- is essential for terminal differentiation of erythroid cells. 121STOP.6 The following mutations were detected: two nonsense, two 4. Japanese blood donor. Homozygosity for a 27-kb dele- frameshift, four encoding single amino acid substitutions, tion encompassing exons 3 to 15 of LU.46 and one single nucleotide change in the promoter region. 5. Pregnant Dutch Caucasian woman. Homozygosity for

a dinucleotide deletion, 123GG, in exon 2, converting Lumod of the X-linked Type 47 42Glu-Val-Met to 42Gly-Arg-STOP. In another type of Lumod, found in just one large Austra- lian family, the mode of inheritance showed the character- In(Lu) istics of resulting from a recessive X-borne inhibitor gene.59

In(Lu) was the name given for a rare, dominant sup- All the Lumod members were males, and although the Lumod pressor of the Lutheran antigens and has subsequently also phenotype occurred in successive generations, there was no been used to describe the phenotype. RBCs of most individ- example of transmission of the phenotype from parent to uals with an In(Lu) gene appear to be Lu(a−b−) and Lunull child. The regulator locus is called XS; XS1 is the common by agglutination tests, but will bind selected Lutheran anti- allele and XS2 the rare inhibitor allele. The Lumod RBCs were bodies, as determined by adsorption and elution tests. Ad- Lu(a–b–) and lacked the other Lutheran antigens, yet anti-Lub sorption and elution tests with anti-Lua and -Lub permitted could be adsorbed and eluted from the RBCs. The RBCs were the determination of the Lutheran genotype in some In(Lu) AnWj+, appeared to have slightly enhanced i antigen, and had members of families, which demonstrated that the In(Lu) weak P1 antigens, although this may have been attributable to suppressor gene is not inherited at the LU locus.48 No the presence of a weak P1 gene in the family.

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Lutheran Antibodies and Their Clinical Significance process, however, is associated with the Lunull or Lumod phe- If Lutheran antibodies are implicated in hemolytic notypes.15 transfusion reactions, they are almost always mild and de- The IgSF glycoproteins expressing Lutheran and LW layed,15 although there could be exceptions.60 Lutheran an- blood group activity are overexpressed on SS RBCs in sickle tibodies have not been reported to have caused hemolytic cell disease. Enhanced binding of the Lu-glycoproteins to disease of the fetus and newborn severe enough to require laminin 511/521 on the endothelia of inflamed or damaged any treatment beyond phototherapy.15 Lua antibodies may blood vessels could contribute to blockage of the vessels be naturally occurring or immune and are often IgM, but and the painful episodes of vaso-occlusion often suffered by may also be IgG and IgA. They are usually reactive by direct sickle cell patients.67 Although laminin 511 and 521 are usu- agglutination of Lu(a+) RBCs, but often also react by an in- ally considered unique ligands of the Lu-glycoproteins, the direct antiglobulin test (IAT). Antibodies to Lub and other α4β1 (VLA-4) on SS reticulocytes may also bind Lu- Lutheran antigens are most often IgG, predominantly IgG1, glycoproteins on endothelial cells, contributing to the vaso- although IgM or IgA may be present. Most anti-Lub RBCs occlusion.68 Phosphorylation of Lu-glycoprotein serines react best by IAT, but some are directly agglutinating with a 596, 598, and 621 in RBCs, stimulated by the physiologic temperature optimum of 20°C. stress mediator epinephrine, could induce conformational Monoclonal anti-Lub has been produced from mice im- changes to the external domains of these proteins, modu- munized with Lu(b+) RBCs,9 and a single-chain variable lating their attraction to their corresponding ligands on fragment (scFv) with Lua specificity has been produced by endothelial cells.67,69 phage display and recombinant DNA technology.61 Polycythemia vera (PV) is a chronic myeloproliferative Lutheran antibodies react with papain-treated RBCs, disease in which clonal proliferation of multipotent he- but not with trypsin- or α-chymotrypsin-treated RBCs. mopoietic cells results in an increase in the RBC mass. It is RBCs treated with 6 percent 2-aminoethylisothiouronium usually associated with a somatic mutation in the gene for bromide (AET) or with 200 mM dithiothreitol (DTT) do JAK2 tyrosine kinase and is often complicated by thrombotic not usually react with Lutheran antibodies. This should be events. The Lu-glycoproteins are phosphorylated in PV, but expected considering that Lutheran antigens are located not in normal cells under the same conditions. Expression in disulfide-bonded IgSF domains and sulfhydryl reducing in an erythroid cell line of recombinant JAK2 containing the agents, such as AET and DTT, break disulfide bonds, un- PV mutation potentiated Lu-glycoprotein phosphorylation. folding the protein. As phosphorylation of the Lu-glycoproteins increases RBC adhesion, this led to the proposal that increased RBC adhe- Functional Aspects of the Lutheran Glycoproteins siveness may be a factor promoting thrombosis in PV.70 In addition to RBCs, the Lu-glycoproteins are present Both Lu-glycoprotein isoforms interact directly with on vascular endothelial cells and epithelial cells of multiple spectrin of the cytoskeleton, through Arg-Lys at positions tissues.11,21 The Lu-glycoproteins bind laminin, a compo- 573 and 574 of their cytoplasmic tails.71 This interaction with nent of the extracellular matrix (ECM) abundant in base- spectrin appears to modulate the adhesive activity of the ment membranes and also present in vascular endothelia. Lu-glycoproteins as disruption of the interaction resulted The 15 known types of laminin are composed of different in weakened linkage to the cytoskeleton and enhanced ad- isoforms of three protein chains (α, β, γ). Lu-glycoproteins hesion of RBCs to laminin.72 An et al.72 speculate that phos- bind specifically and with high affinity to the two isoforms phorylation of the cytoplasmic tail of the Lu-glycoproteins of laminin that contain α5 chains (511 and 521).62,63 The weakens its interaction with spectrin, enabling the freely laminin binding site is formed by Asp312 and a surround- floating transmembrane molecules to cluster and generate ing group of negatively charged residues in the region of a larger adhesive force. the flexible linker between IgSF domains 2 and 3 of the Lu- It is becoming clear that the Lutheran blood group, glycoproteins (Fig. 1).19 which is of minor clinical importance in transfusion medi- During in vitro , the Lu-glycoproteins cine, may play a much more substantial role in the pathol- appear on the erythroid cells at about the orthochromatic ogy of sickle cell disease or PV. erythroblast stage.64,65 This late appearance correlates with binding of the cells to laminin.66 The presence of lami- References nin 511/521 on the subendothelium basement membrane 1. Callender S, Race RR, Paykoc ZV. Hypersensitivity to of bone marrow sinusoids has led to speculation that the transfused blood. Br Med J 1945;2:83. Lu-glycoproteins are involved in facilitating movement of 2. Callender ST, Race RR. A serological and genetical maturing erythroid cells from the erythroblastic islands of study of multiple antibodies formed in response to the bone marrow, across the sinusoidal endothelium, to the blood transfusion by a patient with lupus erythemato- peripheral circulation.62,64 The Lu-glycoproteins may also sus diffusus. Ann Eugen 1946;13:102–17. play a role in the migration of erythroid progenitors from 3. Cutbush M, Chanarin I. The expected blood-group an- the fetal liver to the bone marrow.11 No obvious pathologic tibody, anti-Lub. Nature 1956;178:855–6.

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4. Mohr J. Estimation of linkage between the Lutheran 18. Isacke CM, Horton MA. The adhesion molecule Facts and the Lewis blood groups. Acta Pathol Microbiol Book. 2nd ed. London: Academic Press, 2000. Scand 1951;29:339–44. 19. Mankelow TJ, Burton N, Stefansdottir FO, et al. The 5. Darnborough J, Firth R, Giles CM, Goldsmith KLG, laminin 511/521 binding site on the Lutheran blood Crawford MN. A ‘new’ antibody anti-LuaLub and two group glycoprotein is located at the flexible junction of further examples of the genotype Lu(a–b–). Nature Ig domains 2 and 3. Blood 2007;110:3398–406. 1963;198:796. 20. Burton NM, Brady RL. Molecular structure of the ex- 6. Karamatic Crew V, Mallinson G, Green C, et al. Differ- tracellular region of Lutheran blood group glycoprotein ent inactivating mutations in the LU genes of three and location of the laminin binding site. Blood Cells individuals with the Lutheran-null phenotype. Trans- Mol Dis 2008;40:446–8. fusion 2007;47:492–8. 21. Rahuel C, Le Van Kim C, Mattei MG, Cartron JP, Colin 7. Crawford MN, Greenwalt TJ, Sasaki T, Tippett P, Y. A unique gene encodes spliceoforms of the B-cell ad- Sanger R, Race RR. The phenotype Lu(a–b–) together hesion molecule cell surface glycoprotein of epithelial with unconventional Kidd groups in one family. Trans- cancer and of the Lutheran blood group glycoprotein. fusion 1961;1:228–32. Blood 1996;88:1865–72. 8. Singleton BK, Burton NM, Green C, Brady RL, Anstee 22. Campbell IG, Foulkes WD, Senger G, Trowsdale J, DJ. Mutations in EKLF/KLF1 form the molecular ba- Garin-Chesa P, Rettig WJ. Molecular cloning of the sis of the rare blood group In(Lu) phenotype. Blood B-CAM cell surface glycoprotein of epithelial cancers: 2008;112:2081–8. a novel member of the immunoglobulin superfamily. 9. Parsons SF, Mallinson G, Judson PA, Anstee DJ, Tan- Cancer Res 1994;54:5761–5. ner MJA, Daniels GL. Evidence that the Lub blood group 23. Mourant AE, Kopec AC, Domaniewska-Sobczak K. The antigen is located on red cell membrane glycoproteins distribution of human blood groups and other poly- of 85 and 78 kd. Transfusion 1987;27:61–3. morphisms. 2nd ed. London: Oxford University Press, 10. Daniels G, Khalid G. Identification, by immunoblotting, 1976. of the structures carrying Lutheran and para-Lutheran 24. Chown B, Lewis M, Kaita H. The Lutheran blood blood group antigens. Vox Sang 1989;57:137–41. groups in two Caucasian population samples. Vox Sang 11. Parsons SF, Mallinson G, Holmes CH, et al. The Lu- 1966;11:108–10. theran blood group glycoprotein, another member of 25. Chown B, Lewis M, Kaita H, Philipps S. Some blood the immunoglobulin superfamily, is widely expressed group frequencies in a Caucasian population. Vox Sang in human tissues and is developmentally regulated in 1963;8:378–81. human liver. Proc Natl Acad Sci USA 1995;92:5496– 26. Zeng JQ, Deng ZH, Yang BC, et al. Polymorphic analy- 500. sis of the Lutheran blood group system in Chinese. Ann 12. Parsons SF, Mallinson G, Daniels GL, Green CA, Clin Lab Sci 2009;39:38–42. Smythe JS, Anstee DJ. Use of domain-deletion mutants 27. Molthan L, Crawford MN, Marsh WL, Allen FH. Lu9, to locate Lutheran blood group antigens to each of the another new antigen of the Lutheran blood-group sys- five immunoglobulin superfamily domains of the Lu- tem. Vox Sang 1973;24:468–71. theran glycoprotein: elucidation of the molecular basis 28. Champagne K, Moulds M, Schmidt J. Anti-Lu9: the of the Lua/Lub and the Aua/Aub polymorphisms. Blood finding of the second example after 25 years. Immuno- 1997;88:4219–25. 1999;15:113–16. 13. El Nemer W, Rahuel C, Colin Y, Gane P, Cartron JP, Le 29. Crawford MN. The Lutheran blood group system: se- Van Kim C. Organization of the human LU gene and rology and genetics. In: Pierce SR, Macpherson CR, molecular basis of the Lua/Lub blood group polymor- eds. Blood group systems: Duffy, Kidd and Lutheran. phism. Blood 1997;89:4608–16. Arlington: American Association of Blood Banks, 14. Karamatic Crew V, Green C, Daniels G. Molecular bas- 1988:93–117. es of the antigens of the Lutheran blood group system. 30. Marsh WL. Anti-Lu5, anti-Lu6 and anti-Lu7. Three an- Transfusion 2003;43:1729–37. tibodies defining high frequency antigens related to the 15. Daniels G. Human blood groups. 2nd ed. Oxford: Black- Lutheran blood group system. Transfusion 1972;12:27– well Science, 2002. 34. 16. Lögdberg L, Reid ME, Lamont RE, Zelinski T. Human 31. MacIlroy M, McCreary J, Stroup M. Anti-Lu8, an an- blood group genes 2004: chromosomal locations and tibody recognizing another Lutheran-related antigen. cloning strategies. Transfus Med Rev 2005;19:45–57. Vox Sang 1972;23:455–7. 17. Daniels G. Evidence that the Auberger blood group an- tigens are located on the Lutheran glycoproteins. Vox Sang 1990;58:56–60.

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32. Judd WJ, Marsh WL, Øyen R, et al. Anti-Lu14: a Lu- 47. Karamatic Crew V, Thornton N, Poole J, Liberman M, theran antibody defining the product of an allele at the Daniels G. A novel example of the recessive Lutheran- Lu8 blood group locus. Vox Sang 1977;32:214–19. null phenotype: a serological and molecular study (ab- 33. Salmon C, Salmon D, Liberge G, André R, Tippett P, stract). Transfus Med 2009; in press. Sanger R. Un nouvel antigène de groupe sanguin éryth- 48. Taliano V, Guévin R-M, Tippett P. The genetics of a rocytaire présent chez 80% des sujets de race blanche. dominant inhibitor of the Lutheran antigens. Vox Sang Nouv Rev Fr Hematol 1961;1:649–61. 1973;24:42–7. 34. Salmon C, Rouger P, Liberge G, Streiff F. A family dem- 49. Shaw MA, Leak MR, Daniels GL, Tippett P. The rare onstrating the independence between Lutheran and Lutheran blood group phenotype Lu(a–b–): a genetic Auberger loci. Rev Franc Transfus Immuno-Hematol study. Ann Hum Genet 1984;48:229–37. 1981;24:339–43. 50. Winkler MM, Hamilton JR. Previously tested donors 35. Daniels GL, Le Pennec PY, Rouger P, Salmon C, Tip- eliminated to determine rare phenotype frequencies. pett P. The red cell antigens Aua and Aub belong to the Abstracts, Joint Congr Int Soc Blood Transfus and Am Lutheran system. Vox Sang 1991;60:191–2. Assoc Blood Banks, 1990:158. 36. Zelinski T, Kaita H, Coghlan G, Philipps S. Assignment 51. Udden MM, Umeda M, Hirano Y, Marcus DM. New ab- of the Auberger red cell antigen polymorphism to the normalities in the morphology, cell surface receptors, Lutheran blood group system: genetic justification. Vox and electrolyte metabolism of In(Lu) erythrocytes. Sang 1991;61:275–6. Blood 1987;69:52–7. 37. Drachmann O, Thyme S, Tippett P. Serological charac- 52. Lukasavage T. Donor screening with anti-AnWj. Im- teristics of the third anti-Aua. Vox Sang 1982;43:259– munohematology 1993;9:112. 62. 53. Crawford MN, Tippett P, Sanger R. Antigens Aua, i and

38. Frandson S, Atkins CJ, Moulds M, Poole J, Crawford P1 of cells of the dominant type of Lu(a–b–). Vox Sang MN, Tippett P. Anti-Aub: the antithetical antibody to 1974;26:283–7. anti-Aua. Vox Sang 1989;56:54–6. 54. Spring FA, Dalchau R, Daniels GL, et al. The Ina and 39. Karamatic Crew V, Poole J, Banks J, Reed M, Daniels Inb blood group antigens are located on a glycoprotein G. LU21: a new high-frequency antigen in the Lutheran of 80,000 MW (the CDw44 glycoprotein) whose ex- blood group system. Vox Sang 2004;87:109–13. pression is influenced by the In(Lu) gene. Immunology 40. Reid ME, Hoffer J, Øyen R, Tossas E, Sadjadi M, Mes- 1988;64:37–43. sina G. The second example of Lu:–7 phenotype: serol- 55. Poole J, Tilley L, Warke N, et al. Two missense muta- ogy and immunochemical studies. Immunohematology tions in the CD44 gene encode two new antigens of the 1996;12:66–8. Indian blood group system. Transfusion 2007;47:1306– 41. Karamatic Crew V, Warke N, Ahrens N, Poole J, Daniels 11. Correction: Transfusion 2007;47:1741. G. The second example of LU:−4: a serological and mo- 56. Poole J, Levene C, Bennett M, Sela R, van Alphen L, lecular study. Transfus Med 2006;16(Suppl 1):40–1. Spruell PJ. A family showing inheritance of the Anton 42. Sinclair M, Buchanan DK, Tippett P, Sanger R. Another blood group antigen AnWj and independence of AnWj antibody related to the Lutheran blood group system from Lutheran. Transfus Med 1991;1:245–51. (Much.). Vox Sang 1973;25:156–61. 57. Daniels GL, Shaw MA, Lomas CG, Leak MR, Tippett P. 43. Ridgwell K, Dixey J, Parson SF, Green CA, Scott ML. The effect of In(Lu) on some high-frequency antigens. Screening human sera for anti-Lu antibodies using sol- Transfusion 1986;26:171–2. uble recombinant Lu antigens (abstract). Transfus Med 58. Ballas SK, Marcolina MJ, Crawford MN. In vitro stor- 2003;13(Suppl):32. age and in vivo survival studies of red cells from persons 44. Seltsam A, Grüger D, Blasczyk R. Prokaryotic versus eu- with the In(Lu) gene. Transfusion 1992;32:607–11. karyotic recombinant Lutheran blood group protein for 59. Norman PC, Tippett P, Beal RW. An Lu(a–b–) pheno- antibody investigation. Transfusion 2007;47:1630–6. type caused by an X-linked recessive gene. Vox Sang 45. Seltsam A, Agaylan A, Grueger D, Meyer O, Blasczyk 1986;51:49–52. R, Salama A. Rapid detection of anti-Lub with recom- 60. Puca KE, Feuger JT, Crew VK, Daniels G, Johnson ST. binant Lub protein and the particle gel immunoassay. Acute transfusion reaction (HTR) associated with anti- Transfusion 2008;48:731–4. Lu8 (abstract). Transfusion 2006;46(Suppl):130A. 46. Ogasawara K, Tsuneyama H, Uchikawa M, Suto Y, 61. Richard M, Perreault J, Gane P, El Nemer W, Cartron Okazaki H, Tadokoro K. An example of Lutheran-null J-P, St-Louis M. Phage-derived monoclonal anti-Lua. phenotype in a Japanese individual with 27-kb deletion Transfusion 2006;46:1011–17. from intron 2 of the LU genes (abstract). Transfusion 62. Parsons SF, Spring FA, Chasis JA, Anstee DJ. Erythroid 2008;48(Suppl):210A. cell adhesion molecules Lutheran and LW in health and disease. Baillière’s Clin Haematol 1999;12:729–45.

158 IMMUNOHEMATOLOGY, Volume 25, Number 4, 2009 Lutheran

63. Kikkawa Y, Sasaki T, Nguyen MT, Nomizu M, Mitaka 69. Gauthier E, Rahuel C, Wautier MP, et al. Protein kinase T, Miner JH. The LG1–3 tandem of laminin α5 har- A-dependent phosphorylation of Lutheran/basal cell bors the binding sites of Lutheran/basal cell adhe- adhesion molecule glycoprotein regulates cell adhesion sion molecule and α3β1/α6β1 . J Biol Chem to laminin α5. J Biol Chem 2005;280:30055–62. 2007;282:14853–60. 70. Wautier M-P, El Nemer W, Gane P, et al. Increased ad- 64. Southcott MJG, Tanner MJA, Anstee DJ. The expres- hesion to endothelial cells of erythrocytes from patients

sion of human blood group antigens during erythropoi- with polycythemia vera is mediated by laminin α5 chain esis in a cell culture system. Blood 1999;93:4425–35. and Lu/BCAM. Blood 2007;110:894–901. 65. Bony V, Gane P, Bailly P, Cartron J-P. Time-course ex- 71. Kroviarski Y, El Nemer W, Gane P, et al. Direct inter- pression of polypeptides carrying blood group antigens action between the Lu/B-CAM adhesion glycoproteins during human erythroid differentiation. Br J Haematol and erythroid spectrin. Br J Haematol 2004;126:255– 1999;107:263–74. 64. 66. Zen Q, Cottman M, Truskey G, Fraser R, Telen MJ. 72. An X, Gauthier E, Zhang X, Anstee DJ, Mohan- Critical factors in basal /Lu- das N, Chasis JA. Adhesive activity of Lu glycopro- theran-mediated adhesion to laminin. J Biol Chem teins is regulated by interaction with spectrin. Blood 1999;274:728–34. 2008;112:5212–8. 67. Eyler CE, Telen MJ. The Lutheran glycoprotein: a multifunctional adhesion receptor. Transfusion Geoff Daniels, PhD, FRCPath, Bristol Institute for Transfu- 2006;46:668–77. sion Sciences, National Health Service, Blood and Trans- 68. El Nemer W, Wautier M-P, Rahuel C, et al. Endothelial plant, North Bristol Park, Bristol BS34 7QH, UK Lu/BCAM glycoproteins are novel ligands for red blood

cell α4β1 integrin: role in adhesion of sickle red blood cells to endothelial cells. Blood 2007;109:3544–51.

Manuscripts The editorial staff of Immunohematology welcomes manuscripts pertaining to blood group serology and educa- tion for consideration for publication. We are especially interested in case reports, papers on 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].

IMMUNOHEMATOLOGY, Volume 25, Number 4, 2009 159 Case Report New serologic findings in a patient with ulcerative colitis and a warm autoantibody

T.G. Lightfoot and L. Delia VanThof

IgG-RBC sensitization associated with serine proteases is the cur- clotted serum is attributed to the generation of thrombin (a rent prevailing hypothesis used to explain an uncommon phe- serine protease in its activated form) in the coagulation pro- nomenon in which a positive DAT is obtained using the RBCs from cess. The blood group specificity of the antibodies involved a patient’s clotted blood sample but a negative DAT is obtained in this phenomenon is usually undetermined. There have when testing RBCs from the patient’s unclotted sample. Similarly, been three instances in which antibodies reacting in serum the patient’s serum but not plasma will also be reactive by IAT against all RBCs tested. The majority of patients demonstrating but not in plasma have been determined to have blood 4 this phenomenon have had a history of ulcerative colitis but no group specificities, and they are autoanti-Sc1, autoanti- signs of hemolytic anemia. A case of IgG-RBC sensitization associ- Kpb,5 and autoanti-c.6 Ulcerative colitis is also associated ated with serine proteases and a warm autoantibody in a 14-year- with a positive DAT as a result of warm autoantibodies. The old Hispanic girl with ulcerative colitis is reported. The patient association of a positive DAT with ulcerative colitis was first was admitted for severe anemia (Hb, 6.9 g/dL). On admission, described by Lorber et al. in 1955.7 Autoimmune hemolytic pretransfusion testing of the patient’s serum and RBCs showed anemia (AIHA) is an infrequent but serious complication an ABO/Rh discrepancy between the forward typing and reverse of ulcerative colitis with a reported frequency of 0.7 per- grouping. The phenomenon of IgG-RBC sensitization associated cent.8 The prevalence of a positive DAT in ulcerative colitis with serine proteases was considered in the differential evaluation of the serum versus plasma typing discrepancy. To confirm the patients without progression to AIHA is unknown. To our presence of the phenomenon of IgG-RBC sensitization associated knowledge, we report the first case in which both the phe- with serine proteases, the plasma was clotted and converted to se- nomenon of IgG-RBC sensitization associated with serine rum by the addition of thrombin. The initially nonreactive plasma proteases and a warm autoantibody are present concur- was 2+ reactive when converted to serum. A warm autoantibody rently in a pediatric patient with ulcerative colitis. was also detected during the course of serologic evaluation. The patient was transfused with 2 units of incompatible RBCs with no Case Report adverse reaction observed. The patient is a 14-year-old Hispanic girl who initially Immunohematology 2009;25:160–164. presented in August 2006 at the age of 12 with an 8-month history of bloody stools, abdominal pain, and weight loss. Key Words: serine proteases, serum, plasma, ulcerative She was hospitalized and a clinical workup was initiated. colitis On physical examination she was pale and lethargic. Abnor- mal laboratory findings included a Hb of 4.0 g/dL (normal Background range, 11.5–16 g/dL); Hct, 13.4% (37–47%); MCV, 54.4 fL n 1980, Garratty and colleagues1 described and inves- (80–96 fL); MCH, 16.4 pg (27–32 pg); MCHC, 30.2 g/dL tigated an unusual phenomenon in which a patient’s (32–36 g/dL); erythrocyte sedimentation rate, 35 mm/h blood had a positive DAT in clotted samples but a nega- I (0–20 mm/h); reticulocyte count, 3.2% (0.5–1.5%); LDH, tive DAT in anticoagulated samples. Since it was first recog- 233 U/L (105–333 U/L ); bilirubin, 0.7 mg/dL (0.3–1.9 nized, there have been several theories put forward to ex- mg/dL); C-reactive protein, 1.61 mg/dL (1.0–3.0 mg/dL); plain this phenomenon. The current prevailing hypothesis positive anti-cardiolipin antibodies (negative); ferritin < 2 used to explain the phenomenon is one proposed by Garrat- ng/mL (7–140 ng/mL); iron, 9 μg/dL (50–170 μg/dL); al- ty in 1993.2 Garratty suggested that the observed phenom- kaline phosphatase, 54 IU/L (20–140 IU/L); and positive enon was attributable to IgG-RBC sensitization associated atypical perinuclear antineutrophil cytoplasmic antibodies with serine proteases. In 1993, Garratty performed a re- (P-ANCA) with a titer of 1:1280 (negative). Stool culture view of case reports for patients with this phenomenon. He for Clostridium difficile toxin was positive. Other labora- found that 71 percent of patients had a history of ulcerative tory findings included a WBC of 6.9 × 103/μL (4.8–10.8 × colitis or other inflammatory bowel diseases.2 The proposed 103/μL) with a differential of 25% segmented neutrophils, mechanism for the phenomenon of IgG-RBC sensitization 28% bands, 30% lymphocytes, 13% monocytes, 2% eosino- associated with serine proteases is that an antibody in the phils, and 2% atypical lymphocytes. Platelet count was 344 patient’s serum is directed against an epitope present on × 109/L (150–450 × 109/L); prothrombin time, 15.3 sec- most serine proteases.3 Theoretically, the generated com- onds (< 10–15 seconds); activated partial thromboplastin plexes would then bind to RBCs. The reactivity observed in

160 IMMUNOHEMATOLOGY, Volume 25, Number 4, 2009 IgG-RBC sensitization associated with serine proteases

time, 28.9 seconds (< 35 seconds); INR, 1.20 (0.8–1.2); DAT result obtained with anti-C3 could also be attribut- and fibrinogen, 395 mg/dL (200–400 mg/dL). A CT scan able to the presence of a cold autoagglutinin. To determine of her abdomen showed inflammation of the entire colon. whether a cold autoagglutinin was indeed present, the pa- No abnormalities of the small bowel were seen on an up- tient’s serum was treated with 0.01 M DTT (Sigma-Aldrich, per gastrointestinal series. She underwent a colonoscopy, St. Louis, MO) to abolish IgM activity. No change in reac- which showed inflammation and friability of the mucosa. tivity was noted when the patient’s DTT-treated serum was Colonic biopsies showed cryptitis and crypt abscesses to tested against the reverse-grouping cells. The patient’s neat the hepatic flexure. The colonoscopic findings in conjunc- serum and reverse-grouping cells were then warmed to 37°C tion with her elevated atypical P-ANCA laboratory findings before being added to the appropriately labeled test tubes. confirmed a diagnosis of ulcerative colitis. She was treated The test tubes were incubated at 37°C for 1 hour before be- with intravenous methylprednisolone, oral mesalamine, ing examined for agglutination. Using the prewarmed pa- metronidazole, and 6-mercaptopurine. The patient had no tient serum and reverse-grouping cells, there was no change signs or symptoms of AIHA and this was not considered noted in reactivity when retested; as such the presence of a as a cause of her anemia. The patient did have significant cold autoagglutinin was excluded. The results obtained us- GI bleeding, which her treating physician determined to be ing these testing methods are shown in Table 1. the cause of her anemia. During this hospitalization, she received 4 units of packed RBCs to correct her anemia. During the next year she re- Table 1. ABO/Rh discrepancy detected using patient’s serum quired several courses of steroids and she Anti- Anti- Anti- Anti- Rh A1 A2 B O was started on infliximab (Remicade) ap- A B A,B D control cells cells cells cells proximately 14 months after her initial di- Patient’s 4+ 0 4+ 4+ 0 4+ 4+ 4+ 4+ agnosis. She was observed to develop hives RBC/serum that worsened on subsequent dosing. The DTT-treated NT NT NT NT NT 4+ 4+ 4+ 4+ infliximab therapy was discontinued and serum adalimumab (Humira) was instituted. Prewarmed NT NT NT NT NT 4+ 4+ 4+ 4+ In February 2008, when the patient serum was 14 years old, she was found to have a NT= not tested. Hb of 6.9 g/dL on routine monthly blood work. She was subsequently admitted to the hospital for further workup and evaluation. Initial se- An additional patient sample was requested to confirm rologic evaluation showed a positive DAT. Serum reactiv- that the discrepant reactivity was in fact sample related and ity also was detected with all RBCs tested at antiglobulin not attributable to a sample tube mix-up. The reactivity de- phase using standard tube technique. The serologic evalu- tected with the additional sample was identical to that of ation was unable to be completed by the hospital, and the the original sample submitted, excluding the possibility of a samples were sent to the immunohematology reference sample tube mix-up. laboratory (American Red Cross, West Henrietta, NY) for Owing to the initial reactivity detected with the reverse- evaluation of a positive DAT and antibody identification. grouping cells, a selected cell panel was tested in a saline tube at 22°C to determine whether the reactivity was at- Immunohematologic Evaluation and Results tributable to an IgM antibody. No antibody reactivity, how- The results of initial pretransfusion testing showed ever, was detected when this testing was performed using an ABO discrepancy between the forward typing and the the patient’s plasma. The same selected cell panel was then reverse grouping when testing the patient’s RBCs and se- retested using the patient’s serum, and all cells were noted rum using standard tube technique according to manu- to be 4+ reactive. Because discrepant reactivity was detect- facturer’s instructions. The patient’s RBCs forward typed ed between the patient’s serum and plasma, the patient’s as group A and the Rh type was D+. Test results obtained plasma was tested against the reverse-grouping cells, and with the reverse-grouping cells were inconclusive owing to no ABO discrepancy was detected. Using this method, the

4+ reactivity obtained with all reverse-grouping cells (A1, patient’s was determined to be A positive, which

A2, B, O cells; Referencells, Immucor, Inc., Norcross, GA). was consistent with the submitting hospital’s historical re- Pretransfusion testing using RBCs from an EDTA sample cords. showed a positive DAT, reactive with both anti-IgG and During the process of evaluating the serum versus plas- anti-C3 (Immucor, Inc.). DAT testing was not performed ma discrepancy, the possibility of IgG-RBC sensitization as- on RBCs from a clotted sample as our reference laboratory sociated with serine proteases was considered. To further does not routinely perform DAT testing on such samples. evaluate this hypothesis, the patient’s plasma was clotted A cold autoagglutinin was initially thought to be present using thrombin (Sigma-Aldrich) to convert the plasma to on the patient’s RBCs because of the additional reactivity serum. The plasma was initially nonreactive when tested detected with all reverse-grouping cells tested. The positive against the group O reverse-grouping cells. Using the same

IMMUNOHEMATOLOGY, Volume 25, Number 4, 2009 161 T.G. Lightfoot and L. Delia VanThof group O reverse-grouping cells, the converted serum was 1. Patient’s positive DAT, with anti-IgG, detected using found to be 2+ reactive (see Table 2 for the test results ob- tube technique tained using the patient’s plasma). 2. Positive autocontrol, which consisted of the patient’s As stated earlier, there have been three instances in RBCs treated to DAT negative tested against the pa- which antibodies reacting in serum but not in plasma have tient’s plasma in LISS-AHG been determined to have blood group specificities.4–6 The On February 14, 2008, the patient had a pretransfu- patient’s RBCs tested positive for the Sc1 antigen; however, sion Hb of 6.9 g/dL and was transfused with 2 units of Sc1-negative RBCs were 4+ reactive when tested against least-incompatible packed RBCs, attaining a posttransfu- the patient’s serum in Micro Typing System (MTS) IgG gel sion Hb of 10.2 g/dL. There was no clinical or serologic evi- (Ortho-Clinical Diagnostics, Raritan, NJ), thus excluding dence of an adverse reaction after transfusion. a possible IgG autoantibody with anti-Sc1 specificity. Au- toanti-c was excluded based on the patient’s phenotype; the Discussion patient’s RBCs tested negative for the c antigen. Autoanti- This report describes the presence of the phenomenon Kpb was not evaluated as this possibility was not known at of IgG-RBC sensitization associated with serine proteases the time of testing and was later identified during a further in a patient with ulcerative colitis. In this case, a positive literature search after the completion of this workup.5 DAT owing to the presence of a warm autoantibody was also present. To our knowledge this is the first published case in which both entities are concurrently present in a Table 2. ABO/Rh testing using patient’s plasma patient. Both of these entities may have a respec- Anti- Anti- Anti- Anti- Rh A1 A2 B O tive association with ulcerative colitis. In this case, A B A,B D control cells cells cells cells the antibodies involved in both the phenomenon of Patient’s 4+ 0 4+ 4+ 0 0 0 4+ 0 IgG-RBC sensitization associated with serine pro- RBC/ teases and the presence of a warm autoantibody plasma were of the IgG class. The mechanism of the phe- Thrombin- NT NT NT NT NT NT NT NT 2+ nomenon of IgG-RBC sensitization associated with treated plasma serine proteases is not completely understood. The NT= not tested. initial hypothesis proposed for the phenomenon was that the patient had an IgG antibody to an acti- Because the patient’s plasma was nonreactive when vated coagulation factor that would not normally be pres- tested in a saline tube at 22°C, antiglobulin testing was ent in plasma and that IgG immune complexes formed and performed using the patient’s plasma. Initial testing was attached to RBCs in vitro.1 In 1993, Garratty,2 performed performed in both MTS IgG gel and LISS-AHG (Immucor, a review of 28 patients with ulcerative colitis who mani- Inc.). The patient’s plasma was 3+s reactive in both MTS fested the IgG-RBC sensitization phenomenon associated IgG gel and LISS-AHG with three of three screening cells with in vitro clotting as initially reported in 1980. In this (Surgiscreen; Ortho-Clinical Diagnostics, Inc.) and two review, the author observed that 10 of 11 IgG antibodies phenotypically similar cells. Autologous cells treated tested were of the IgG3 subclass. Further, the author found with EDTA-glycine acid (EGA) (Immucor, Inc.) to remove that the IgG-RBC sensitization phenomenon was not di- the IgG coating were also 3+ reactive. The patient’s serum rectly related to clotting. Garratty observed that a nonreac- was also tested and found to be 4+ reactive with the same tive plasma could be made reactive without clotting occur- two phenotypically similar cells when tested in MTS IgG ring by adding a variety of serine proteases (e.g., trypsin) gel. to the plasma. Therefore the hypothesis was subsequently The pattern of reactivity detected when testing the pa- modified to suggest that the antibody in the patient’s se- tient’s plasma suggested the presence of an IgG autoanti- rum was directed against an epitope present on most serine body. We were unable to determine whether the reactivity proteases. This would include but would not be restricted detected when testing the serum sample at antiglobulin to activated coagulation factors.3 phase was also attributable to a possible IgG autoantibody In humans there are four broad classes of proteases, or interference of the serine protease antibody. A 3× alload- based on the mechanism they use to hydrolyze peptide bonds. Respectively, they are serine, cysteine, aspartic, and sorption was performed onto a phenotypically similar, R1R1 K– Jk(b–), glutaraldehyde-treated RBC to remove the IgG metalloproteases.9 Of these classes, the serine proteases are autoantibody reactivity and assess for the presence of un- the largest group, and their functions range from those at- derlying alloantibodies. No underlying alloantibodies were tributable to nonspecific digestive enzymes such as trypsin, detected when testing the 3× alloadsorbed plasma in LISS- to more highly regulated functions such as blood coagula- AHG. Alloantibodies to the major blood group antigens tion (thrombin) and immune response (complement sys- were ruled out by this testing. An IgG autoantibody was tem). Nearly all serine proteases have amino acid sequence concluded to be in the patient’s plasma because of: homology with pancreatic serine proteases. As a result,

162 IMMUNOHEMATOLOGY, Volume 25, Number 4, 2009 IgG-RBC sensitization associated with serine proteases serine proteases have very similar tertiary structures.10 The hemolytic anemia and will require the appropriate transfu- maintenance of structural homology within the serine pro- sion recommendations. In such cases, incompatible units tease class would be consistent with Garratty’s hypothesis screened negative for possible underlying alloantibodies regarding an antibody to an epitope commonly shared by will need to be selected, as crossmatch units will appear serine proteases and his observation that a variety of serine incompatible because of the presence of the IgG autoanti- proteases and not only those generated during the coagula- body. The patient will need to be monitored closely for signs tion process were capable of inducing the phenomenon of and symptoms of hemolysis after transfusion. In summary, IgG-RBC sensitization associated with serine proteases.3 although the phenomenon of IgG-RBC sensitization associ- Ulcerative colitis is a chronic inflammatory bowel dis- ated with serine proteases does not appear to be associated ease that may be associated with extraintestinal immune with accelerated RBC destruction, it is important that this features. Approximately 23 to 42 percent of patients with phenomenon be considered and recognized to avoid need- inflammatory bowel disease exhibit extraintestinal immu- less delay in serologic workup and the provision of blood for nologic manifestations.11 Immune hematologic features the patient. such as idiopathic thrombocytopenic purpura and AIHA have been reported in patients with ulcerative colitis.8,12 References Recently, the acquired form of thrombotic thrombocy- 1. Garratty G, Davis J, Myers M, Nelson A, Pierce A, eds. topenic purpura (TTP) has also been described in patients IgG red cell sensitization associated with in vitro clot- with ulcerative colitis.13 The most favored hypothesis for the ting in patients with ulcerative colitis (abstract). Trans- pathophysiology of acquired TTP is that of an autoimmune fusion 1980:20:646. process caused by autoantibodies that inhibit the activity 2. Garratty G. In vitro IgG sensitization of red cells asso- of the von Willebrand factor–cleaving metalloprotease AD- ciated with ulcerative colitis and serine proteases (ab- AMTS13.14 stract). Transfusion 1993;33(Suppl):22S. As the majority of the patients in whom the phenom- 3. Garratty G. In vitro reactions with red blood cells that enon of IgG-RBC sensitization associated with serine pro- are not due to blood group antibodies: a review. Immu- teases has been observed have a history of ulcerative colitis nohematology 1998;14:1–15. or other inflammatory bowel disease, the phenomenon of 4. Tregellas WM, Holub MP, Moulds JJ, Lacey PA. An ex- IgG-RBC associated with serine proteases may similarly ample of autoanti-Sc1 demonstrable in serum but not reflect another autoimmune extraintestinal immunologic in plasma (abstract). Transfusion 1979;19:650. manifestation of inflammatory bowel disease. The autoim- 5. Moses BH, South SF, Ubbelohde M. Serum reactive, mune mechanism previously described in the pathogenesis plasma nonreactive autoanti-Kel4 (Kpb) (abstract). of acquired TTP (autoantibody to a protease) would also be Transfusion 1991;31(Suppl):26S. a plausible model for the phenomenon of IgG-RBC sensiti- 6. South SF, Davis K. Serum-reactive, plasma-nonreactive zation associated with serine proteases. autoanti-c (abstract). Transfusion 1993;33(Suppl):22S. A positive DAT in association with ulcerative colitis may 7. Lorber M, Schwartz LI, Wasserman LR. Association of occur as a result of the phenomenon of IgG-RBC sensitiza- antibody-coated red blood cells with ulcerative colitis. tion associated with serine proteases or may be attributable Am J Med 1955;19:887–94. to a warm autoantibody with possible autoimmune hemo- 8. Gumaste V, Greenstein AJ. Meyers R, Sachar DB. lytic anemia. The presence of both of these distinct entities Coombs-positive autoimmune hemolytic anemia in ul- in the same patient is a rare occurrence. Serologic investi- cerative colitis. Dig Dis Sci 1989;34:1457–61. gation of the phenomenon of IgG-RBC sensitization associ- 9. Hart JP, Pizzo SV. α-Macroglobulins and kunins. In: ated with serine proteases may be quite challenging, and in- Colman RW, Clowes AW, Goldhaber SZ, Marder VJ, formation regarding the patient’s history of ulcerative colitis George JN, eds. Hemostasis and Thrombosis: Basic is beneficial. The clinical significance of the phenomenon of Principles and Clinical Practice. 5th ed. Philadelphia, IgG-RBC sensitization associated with serine proteases is Pa: Lippincott Williams & Wilkins; 2006:395-407. unknown. However, as best we are able to determine, it is 10. Perona JJ, Craik CS. Structural basis of substrate not associated with accelerated RBC destruction in vivo. specificity in the serine proteases: a review. Protein Sci For those patients who have a positive serum and the phe- 1995:337–60. nomenon of IgG-RBC sensitization associated with serine 11. Greenstein AJ, Janowitz HD, Sachar DB. The extra- proteases is believed to be present, serologic testing may be intestinal complications of Crohn’s disease and ul- repeated with plasma. If the plasma is subsequently nega- cerative colitis. A study of 700 patients. Medicine tive then compatibility testing may be performed using the 1976;55:401–12. plasma as per the Standards of the American Association 12. Zlatanic J, Korelitz BI, Wisch N, et al. Inflamma- of Blood Banks, which allows plasma to be used for com- tory bowel disease and immune thrombocytopenic patibility testing.3,15 Ulcerative colitis with a positive DAT purpura: is there a correlation? Am J Gastroenterol and warm autoantibody is on rare occasion associated with 1997;92:2285–8.

IMMUNOHEMATOLOGY, Volume 25, Number 4, 2009 163 T.G. Lightfoot and L. Delia VanThof

13. Baron BW, Jeon HR, Glunz C, et al. First two patients Thomas G. Lightfoot, MD (corresponding author), Associ- with ulcerative colitis who developed classical throm- ate Medical Director, American Red Cross Blood Services, botic thrombocytopenic purpura successfully treated NY-Penn Region, and Laurie Delia VanThof, MT (ASCP) with medical therapy and plasma exchange. J Clin SBB, Manager, Immunohematology Reference Lab/Stem Apher 2002;17:204–6. Cell Lab, NY-Penn and NEPA Regions, 825 John Street, 14. Klaus C, Plaimauer B, Studt JD, et al. Epitope mapping West Henrietta, NY 14586. of ADAMTS13 autoantibodies in acquired thrombotic thrombocytopenic purpura. Blood 2004;103:4514–19. 15. Friday J, ed. Standards for blood banks and transfusion services. 25th ed. Bethesda, MD: American Association of Blood Banks, 2008.

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164 IMMUNOHEMATOLOGY, Volume 25, Number 4, 2009 Original Report

A simple screening assay for the most common JK*0 alleles revealed compound heterozygosity in Jk(a–b–) probands from Guam

E.S. Wester, J. Gustafsson, B. Snell, P. Spruell, Å. Hellberg, M.L. Olsson, and J.R. Storry

The Jk(a–b–) phenotype results from alterations in the JK gene used as an efficient screening test in populations where the and is characterized by absence of the RBC urea transporter in the Jk(a–b–) phenotype is more prevalent.4–8 cell membrane. The frequency of Jk(a–b–) varies among popula- The Jka and Jkb antigens depend on the amino acid tions, but this phenotype is most commonly found in people of at position 280, aspartic acid for Jka and asparagine for Polynesian and Finnish descent. Although rare, Jk(a–b–) individ- Jkb.9 The Jk(a–b–) phenotype derives from homozygosity uals present a clinical challenge because anti-Jk3 is produced read- ily in response to transfusion and pregnancy, and Jk(a–b–) blood or compound heterozygosity for mutations in the JK gene is not routinely available. Identification of Jk(a–b–) patients and (SLC14A1; synonyms: UT-B1, HUT11, RACH1), so-called donors is most often performed serologically. However, ten JK*0 JKnull or JK*0 alleles. A rare dominant Jk(a–b–) phenotype alleles have been identified, and this information can be used in named In(Jk) was reported in two Japanese individuals DNA-based typing. We selected five JK*0 alleles that had been identified by 2 M urea lysis screening,10 although the mo- encountered by our reference laboratory in two or more samples lecular basis has not been identified. from unrelated individuals and designed an allele-specific primer The JK locus comprises 11 exons carried on chromo- PCR assay for use as an initial screening tool. After in-house vali- some 18, and several different genetic alterations have been dation, we tested genomic DNA from a family: a mother and her described to cause the Jk(a–b–) phenotype. The two most two sons referred to us for genetic investigation of their Jk(a–b–) phenotypes. Two different nucleotide substitutions, –1g>a in in- frequent JK*0 alleles are found in people of Polynesian 11–13 tron 5 (IVS5) and 956C>T in exon 10, originally associated with (IVS5–1g>a) and Finnish (871T>C) descent, in which Polynesian and Indian/African populations respectively, were the incidence of the Jk(a–b–) phenotype was shown to be identified in the family. The mother and one son were compound 0.27 percent and 0.03 percent, respectively.5,7 Other JK*0 heterozygotes, and the second son was homozygous for IVS5– alleles have been only sporadically detected in other popu- 1g>a. We conclude that the effort to design and validate such a lations.14–19 In this report, we have named silencing JK al- screening assay was cost-efficient when compared with DNA se- leles generally as JK*0; however, when the JK*01 or JK*02 quencing costs. Furthermore, selection of the more common JK*0 backbone is known, this is indicated as JK*01N or JK*02N mutations was a practical approach that resulted in rapid identifi- in line with current proposals under consideration by the cation of the genetic bases behind the Jk(a–b–) phenotypes in this unusual family. Although an obvious target for eventual inclusion ISBT committee on terminology for RBC surface antigens into high-throughput genotyping platforms for clinical diagnostic (http://ibgrl.blood.co.uk/). Although rare in the general services, current systems are very limited. Our approach provides population, Jk(a–b–) individuals present a clinical chal- a simple and inexpensive method for the identification of these lenge because anti-Jk3 may be produced in response to rare alleles. Immunohematology 2009;25:165–169. transfusion and pregnancy, and Jk(a–b–) blood is not rou- tinely available. Limited resources of anti-Jk3 make large- Key Words: JK blood group system, null phenotypes, mo- scale phenotype screening programs for Jk(a–b–) blood lecular basis, PCR-ASP impractical. Screening methods for blood group polymorphisms us- he antigens of the Kidd (JK; ISBT009) blood group ing genomic DNA are becoming widespread in many labora- system are carried on the RBC urea transporter (Kidd tories. In instances in which a rare null allele is found more Tglycoprotein, SLC14A1),1 a multipass membrane- commonly in one population than in others, it is practical spanning glycoprotein. Individuals who lack the Kidd gly- and cost-efficient to test only for that mutation, and large coprotein on their RBCs express the Jk(a–b–) phenotype, numbers of samples can be tested at a time. We sought to also known as Jknull. This phenotype is associated with a mild investigate the usefulness of developing an allele-specific insufficiency in urine concentration (which goes largely un- primer PCR (PCR-ASP) screen for those JK*0 alleles that detected in normal conditions), but may often be identified had been found in two or more unrelated individuals as a by the presence of anti-Jk3 in the plasma of immunized in- primary analysis for samples referred to the Nordic Refer- dividuals. The absence of the RBC urea transporter renders ence Laboratory for Blood Group Genomic typing for iden- Jk(a–b–) RBCs resistant to lysis by 2 M urea,2,3 which is tification or confirmation of their Jk(a–b–) phenotype. Our

IMMUNOHEMATOLOGY, Volume 25, Number 4, 2009 165 E.S. Wester et al. algorithm was such that samples in which a causative poly- Table 1. Primers used in the JK*0 assay morphism was identified could be reported directly. Ex- A. Primers for detection of the mutations clusion by the assay of these five mutations would prompt Ampli- DNA sequence analysis of exons 1 to 11 of the JK gene, a PCR Primer con size much more time-consuming and labor-intensive under- target name Nucleotide sequence (5´→3´) (bp) taking. ∆exons JK-i3-F2 AGTCTTCTCAACTAGACTGAAC 252 4+5 Materials and Methods JK-i5-R10 CCCTCCCTAAAGGAACTGGC JK*0 mutations that fulfilled our criteria for inclu- IVS5–1a JK-i5-F2b CTGTGTCTCTTGCCCCACAA 149 sion are listed as follows (shown in Fig. 1): deletion of JK-470-R13 CAAGTCATGGACATAGCACA exon 4 and 5 (∆exons 4+5),14,15 IVS5–1g>a,11,12,17 582C>G,20 582G JK-582G-F CTTTCAGCCACAGGACATTAGA 274 871T>C,12,13 and 956C>T.18 Allele-specific primers (Table 1) were designed to detect both the normal and mutant alleles JK-i7-RI GACCCTAATGCTCTGGAGTAG according to the HUT11A sequence (GenBank accession 871C JK-780-F3 CATGCTGCCATAGGATCATTGC 332 no. NM_015865) and were synthesized by DNA Technolo- JK-871C-R TTGCAATGCAGGCCAGAGG gies A/S (Aarhus, Denmark). A mismatch was introduced 956T JK-in9-F ACAGAGCCCATGGAGCTCC 211 in two primers to enhance allele specificity (underlined in JK-956T-R2 CCGACTCCAAGATAGGCCA Table 1). We performed the new assay in parallel with our routine in-house PCR-ASP for JK*01/JK*02.21 B. Primers for the detection of consensus nucleotides Ampli- PCR Primer con size target name Nucleotide sequence (5´→3´)* (bp) Exon 4 JK-i3-F2 AGTCTTCTCAACTAGACTGAAC 534 JK-i4R CCAAAGCTACTTCTCCTGTC IVS5–1g JK-i5-F1 TGTGTCTCTTGCCCCACAG 148 JK-470-R13 CAAGTCATGGACATAGCACA 582C Jk-582C-F CTTTCAGCCACAGGACATTAC 271 JK-i7-RII TTAGACCCTAATGCTCTGGAG 871T JK-780-F3 CATGCTGCCATAGGATCATTGC 333 JK-871T- ATTGCAATGCAGGCCATAGA Rmis2 956C JK-in9-F ACAGAGCCCATGGAGCTCC 211 Fig. 1 Cartoon of an RBC bearing the erythrocyte urea transporter framed by the JK gene of 11 exons. The coding exons 4–11 are JK-956C- CCGACTCCAAGATAGGACG shown in dark gray (white text), and the noncoding part, exons Rmis 1–3, are light gray (black text). JK*0 mutations, included in the *Mismatched nucleotides are underlined. assay, are indicated on the gene as well as the polymorphism a b encoding Jk and Jk . C. Control primers For the PCR reaction 100 ng of DNA was used in a total Primer name Nucleotide sequence (5´→3´) Amplicon size (bp) volume of 10 μL, using 0.2 mM dNTPs, 0.25 pmol of each primer, 0.25 pmol of control HGH primers, and 0.5 U of HGH-F TGCCTTCCCAACCATTCCCTTA 434 AmpliTaq Gold (Perkin Elmer, Roche Molecular Systems, HGH-R CCACTCACGGATTTCTGTTGTGTTTC Inc., Branchburg, NJ) in the buffer supplied. Amplification was performed in the GeneAmp PCR system 2700 or 2720 (Perkin Elmer Cetus, Norwalk, CT) as follows: 7 minutes at 97°C, then 35 cycles of 30 seconds at 94°C, 30 seconds at 62°C, 40 seconds at 72°C, and a final elongation of 2 minutes at 72°C. PCR products were separated by electro- after quality control. Samples carrying the JK*0 mutations phoresis for 25 minutes at 150 V using 3 percent agarose and samples of normal Jk type were from our in-house DNA gel (SeaKem. FMC BioProducts, Rockland, MA) containing collection. ethidium bromide (0.56 μg/mL). Genomic DNA was prepared either by a salting-out Once optimized, PCR reagent mixes for each muta- method modified from Miller et al. and diluted in sterile tion and for the corresponding consensus nucleotide were water to 100 ng/μL,22 or by the Qiagen blood and body fluid prepared to a final volume of 500 µL, and frozen at –20°C protocol (Qiagen Inc., Valencia, CA).

166 IMMUNOHEMATOLOGY, Volume 25, Number 4, 2009 PCR method for JK*0 detection

The numbering of nucleotides in this paper starts with homozygous for IVS5–1g>a (Fig. 3). The three individuals the A of the initiation codon in exon 4 as nucleotide number typed JK*02 by the in-house PCR-ASP.21 The pedigree of 1. The same codon encodes methionine at position number 1. this unusual family could be constructed to explain the phe- notypic and genetic results (Fig. 3). Results After optimization of each PCR-ASP to the conditions listed above, the new screening tool readily detected both mutation and consensus single-nucleotide polymorphisms (SNPs) for each tested allele (Fig. 2), and homozygote and heterozygote controls gave the expected results. Once the assay had been established and the expected bands shown to be robust and specific, DNA analysis of the individuals included in the case report was performed.

Fig. 3 The family pedigree together with the results of the PCR-ASP performed on samples from a Jk(a–b–) mother and her two Jk(a–b–) sons. The ASP-PCR kit is arranged as follows: lane 1: exon 4/Δexon 4+5; lane 2: IVS5–1g/a; lane 3: 582C/G; lane 4: 871T/C; lane 5: 956C/T. Bands amplified by allele-specific primers corresponding to IVS5–1g/a and 956C/ T are indicated by the arrows. An internal Fig. 2 Representative PCRs showing the (A) specificJK*0 and (B) control is run in parallel in all PCR reactions, and the ΦX174 HaeIII consensus bands for the five selected mutations. TheΦ X174 HaeIII DNA ladder (Advanced Biotechnologies Ltd., UK) was used as a DNA ladder (Advanced Biotechnologies Ltd., UK) was used as a size marker. The pedigree shows the IVS5–1a allele in black and size marker. P indicates a positive reaction and N a negative. In every the 956T allele in gray. White indicates that the allele is unknown. Samples from the boys’ father were not tested (n.t.), but one allele is set a PCR reaction with H2O instead of DNA was run in parallel to ensure that the PCR reagents were not contaminated. presumed to carry the IVS5–1a mutation.

Case Report Discussion Samples from a woman, originating from Guam, and With the more widespread use of genotyping for pre- her two sons were received for serologic investigation. An dicting blood group phenotypes, null alleles must be taken earlier investigation of the woman’s plasma performed by into consideration even if they are uncommon. Different ap- the referring hospital demonstrated an anti-Jkb although proaches for DNA-based typing permit different levels of in- she had not been transfused. The sample at the time of clusiveness. In a more high-throughput automation setting, the current investigation demonstrated anti-Jk3. Her sons in which space on a chip or bead is not overly restricted, all were tested together with other family members to locate known null mutations can be included. On the other hand, potential compatible donors. in a manual setting, cost-effectiveness of any assay lies in the The RBCs from all three samples typed Jk(a–b–) by se- relative frequency of a null mutation in a given population rology and showed no hemolysis by the urea hemolysis test. or in the expected cohort of referred samples. Based on this, Genomic DNA from these samples was tested by the HEA the PCR-ASP described was designed to include JK*0 muta- BeadChip Kit (Immucor Inc., Norcross, GA), and all three tions that had occurred in two or more unrelated individuals were identified as JK*02 homozygotes. and to be used as a primary screening tool in the investiga- However, the screening assay results showed that the tion of cases with a suspected Jk(a–b–) phenotype. Thus, mother and one of her sons carried two different mutations only samples that are unresolved by the screening assay will and thus were compound heterozygotes for the IVS5–1g>a be subjected to costly and labor-intensive DNA sequencing mutation and 956C>T in exon 10. The other son was analysis of all 11 exons in the JK gene.

IMMUNOHEMATOLOGY, Volume 25, Number 4, 2009 167 E.S. Wester et al.

Because these samples are uncommon even in a major References reference laboratory, a simple one-mutation-in-one-PCR- 1. Olives B, Mattei MG, Huet M, et al. Kidd blood group and tube approach was chosen, and no attempt to multiplex the urea transport function of human erythrocytes are car- assay was performed. In multiplex assays, competition for ried by the same protein. J Biol Chem 1995;270:15607– DNA (often of varying or unknown quality when referred 10. from another laboratory) can lessen the robustness of the 2. Heaton DC, McLoughlin K. Jk(a–b–) red blood cells re- individual allele-specific PCR reactions. We have tried this sist urea lysis. Transfusion 1982;22:70–1. approach previously but found that the reproducibility of 3. Edwards-Moulds J, Kasschau MR. The effect of 2 molar the assay was poor when performed infrequently.21 How- urea on Jk (a–b–) red cells. Vox Sang 1988;55:181–5. ever, our current approach is balanced by the advantage 4. Woodfield DG, Douglas R, Smith J, Simpson A, Pinder that novel SNPs of sufficient frequency are easily incor- L, Staveley JM. The Jk(a–b–) phenotype in New Zea- porated into the assay. As a matter of fact, the two CE- land Polynesians. Transfusion 1982;22:276–8. marked products approved for blood group diagnostic use 5. Henry S, Woodfield G. Frequencies of the Jk(a–b–) in Europe include either no JK*0 markers (BAGene, BAG phenotype in Polynesian ethnic groups (letter). Trans- Health Care GmbH) or detection only of the Finnish and fusion 1995;35:277. Polynesian variants (BLOODChip+, Progenika Biopharma 6. Okubo Y, Tomita T, Nagao N, Yamaguchi H, Tanaka M. SA). (The CE mark indicates that the product has met the Mass screening donors for –D– and Jk (a–b–) using the relevant quality requirements from the appropriate Euro- Groupamatic-360 (letter). Transfusion 1983;23:362–3. pean directive.) A third commercial blood group genotyp- 7. Sareneva H, Pirkola A, Sistonen S, Sistonen P. Excep- ing product (BeadChip, Immucor, Inc.), not yet approved tional high frequency of a gene for recessive JK blood for diagnostic use, also includes detection of JK*01/JK*02 group null phenotype among Finns. Abstract book from only. the 6th Regional Congress of the ISBT, Jerusalem, Is- We were fortunate enough to be able to test the PCR- rael, 1999;96. ASP assay by analyzing samples from an especially inter- 8. McDougall DC, McGregor M. Jk:-3 red cells have a de- esting family: a mother and two sons, all of whom fect in urea transport: a new urea-dependent lysis test carried the Jk(a–b–) phenotype. Somewhat expected (letter). Transfusion 1988;28:197–8 (published erra- from the ethnic background, we identified the Polynesian tum appears in Transfusion 1988;28:585). SNP (IVS5–1g>a) on a JK*02-allele (JK*02N) in all three 9. Olives B, Merriman M, Bailly P, et al. The molecular ba- individuals. One of the sons was homozygous for this SNP, sis of the Kidd blood group polymorphism and its lack thus the father (not included in this investigation) must at of association with type 1 diabetes susceptibility. Hum least be heterozygous. The IVS5–1g>a mutation has the Mol Genet 1997;6:1017–20. highest population frequency and has previously been de- 10. Okubo Y, Yamaguchi H, Nagao N, Tomita T, Seno T, scribed in the general overall Asian area in which Guam is Tanaka M. Heterogeneity of the phenotype Jk(a–b–) situated.19,23 The other allele, 956C>T, detected in samples found in Japanese. Transfusion 1986;26:237–9. from the mother and the other son, was more surprising 11. Lucien N, Sidoux-Walter F, Olives B, et al. Character- because this SNP has been described previously in com- ization of the gene encoding the human Kidd blood bination with a JK*02 allele in two samples only, which group/urea transporter protein. Evidence for splice originated from the Indian subcontinent.18 However, null site mutations in Jknull individuals. J Biol Chem genes are underdiagnosed in general and especially so in 1998;273:12973–80. populations in which more sophisticated DNA techniques 12. Irshaid NM, Henry SM, Olsson ML. Genomic charac- are not in common practice. We anticipate from our stud- terization of the Kidd blood group gene: different mo- ies and from screening studies being performed in other lecular basis of the Jk(a–b–) phenotype in Polynesians laboratories (Connie Westhoff, personal communication) and Finns. Transfusion 2000;40:69–74. that the latter mutation is more common than we first 13. Sidoux-Walter F, Lucien N, Nissinen R, et al. Molecu- thought. This highlights the importance of understanding lar heterogeneity of the Jk(null) phenotype: expression the genetic variation within a regional population under analysis of the Jk(S291P) mutation found in Finns. study, and shows that it can be worthwhile to incorporate Blood 2000;96:1566–73. null mutations into any strategy for genotype and pheno- 14. Lucien N, Chiaroni J, Cartron JP, Bailly P. Partial dele- type testing. tion in the JK locus causing a Jk(null) phenotype. Blood 2002;99:1079–81.

168 IMMUNOHEMATOLOGY, Volume 25, Number 4, 2009 PCR method for JK*0 detection

15. Irshaid NM, Eicher NI, Hustinx H, Poole J, Olsson ML. 22. Miller SA, Dykes DD, Polesky HF. A simple salting out Novel alleles at the JK blood group locus explain the procedure for extracting DNA from human nucleated absence of the erythrocyte urea transporter in Europe- cells. Nucleic Acids Res 1988;16:1215. an families. Br J Haematol 2002;116:445–53. 23. Yan L, Zhu F, Fu Q. Jk(a–b–) and Kidd blood group gen- 16. Meng Y, Zhou X, Li Y, et al. A novel mutation at the JK otypes in Chinese people. Transfusion 2003;43:289– locus causing Jk null phenotype in a Chinese family. Sci 91. China C Life Sci 2005;48:636–40. 17. Ekman GC, Hessner MJ. Screening of six racial groups Elisabet Sjöberg Wester, MS, and Julia Gustafsson, BS, for the intron 5 G→A 3′ splice acceptor mutation re- Nordic Reference Laboratory for Blood Group Genomic sponsible for the Polynesian Kidd (a–b–) phenotype: Typing, Clinical Immunology and Transfusion Medicine, the null mutation is not always associated with the JKB University and Regional Laboratories, SE-22185 Lund, allele (letter). Transfusion 2000;40:888–9. Sweden; Beverly Snell, MT(ASCP)SBB, and Peggy Spruell, 18. Wester ES, Johnson ST, Copeland T, et al. Erythroid MT(ASCP)SBB, Reference Laboratory, Gulf Coast Region- urea transporter deficiency due to novel JKnull alleles. al Blood Center, Houston, TX; Åsa Hellberg, PhD, Nordic Transfusion 2008;48:365–72. Reference Laboratory for Blood Group Genomic Typing, 19. Liu HM, Lin JS, Chen PS, Lyou JY, Chen YJ, Tzeng Clinical Immunology and Transfusion Medicine, Univer- CH. Two novel Jk(null) alleles derived from 222C>A in sity and Regional Laboratories, SE-22185 Lund, Sweden; exon 5 and 896G>A in exon 9 of the JK gene. Transfu- Professor Martin L. Olsson, MD, PhD, Department of Lab- sion 2009;49:259–64. oratory Medicine, Division of Hematology and Transfu- 20. Irshaid NM, Hustinx H, Olsson ML. A novel molecular sion Medicine, Lund University, SE-22185, Lund, Sweden, basis of the Jk(a–b–) phenotype in a Swiss family. Vox and Jill R. Storry, PhD (corresponding author), Nordic Sang 2000;78(Suppl 1):0019. Reference Laboratory for Blood Group Genomic Typing, 21. Irshaid NM, Thuresson B, Olsson ML. Genomic typ- Clinical Immunology and Transfusion Medicine, Univer- ing of the Kidd blood group locus by a single-tube sity and Regional Laboratories, SE-22185 Lund, Sweden. allele-specific primer PCR technique. Br J Haematol 1998;102:1010–14.

For information concerning For information concerning the National Immunohematology, Journal of Blood Reference Laboratory for Blood Group Group Serology and Education, or Serology, including the American Rare the Immunohematology Methods and Donor Program, please contact Sandra Procedures manual, contact us by e-mail at Nance, by phone at (215) 451-4362, by fax at [email protected] (215) 451-2538, or by e-mail at snance@usa. redcross.org

IMMUNOHEMATOLOGY, Volume 25, Number 4, 2009 169 Original Report

Cost-effectiveness of FDA variance for blood collection from individuals with hereditary hemochromatosis at a 398-bed hospital- based donor center

D.M. Gribble, D.J. Chaffin, and B.J. Bryant

Hereditary hemochromatosis (HH) is treated by therapeutic phlebot- to death. Iron toxicity can affect most organs, but the liver, omy to reduce excess body iron. This 398-bed, hospital-based donor pancreas, and heart are particularly affected. HH is treated center wanted to determine whether there was a financial advantage by phlebotomy, which reduces body iron by removing the to requesting FDA approval to allow transfusion of blood components hemoglobin found in the RBCs. Regular phlebotomy thera- from eligible individuals with HH. Donor center records from 2008 py safely reduces the iron stores in most patients. Early ini- were reviewed to identify all therapeutic phlebotomy patients with a di- agnosis of HH. HH patients were contacted and asked to complete the tiation of therapeutic phlebotomy procedures before organ AABB Uniform Donor History Questionnaire (UDHQ) to determine damage has occurred may restore normal life expectancy their eligibility as potential allogeneic blood donors. Financial ramifica- and improve symptoms. tions attributable to loss of revenue from the therapeutic phlebotomies On the basis of the currently understood molecular ($100/collection) were compared with the potential gain in revenue mechanisms of the disease, there is no evidence HH can be from collecting units for transfusion ($429/collection) in a 12-month transmitted by means of blood transfusion. Additionally, period. Nineteen HH patients were identified and screened for allo- there is no evidence to suggest that blood donated from in- geneic eligibility. Seventeen patients (89%) met the eligibility criteria dividuals with HH would carry any added risk for the trans- for allogeneic donors, and two patients (11%) did not. Retrospective fusion recipient provided the donor meets all the current review of donor records indicated that a total of 60 units were collected from these HH patients from January 2008 through December 2008. standards for allogeneic blood donation. Blood from HH Fifty-five of the 60 units collected (92%) were eligible for allogeneic patients has been used for transfusion in the United States use, potentially generating gross revenue of $23,595. After deducting and other countries without reports of adverse effects in re- expenses for infectious disease testing and loss of revenue for the non- cipients.1,2 qualified therapeutic phlebotomies, the net revenue from the collection Currently, the US Food and Drug Administration (FDA) of 55 RBC units that could have potentially been used for allogeneic does not prohibit the use of blood from therapeutic collec- transfusion was $20,345. In contrast, the current revenue generated tions from otherwise eligible donors, but it requires that by the collection of 60 therapeutic phlebotomies was only $6,000. In blood intended for transfusion be labeled with the donor’s 2008, using eligible HH individuals as allogeneic blood donors would disease (21 CFR 640.3(d)). The Code of Federal Regulations have resulted in an increase in revenue of $14,345 for our blood center. This study demonstrates that even at a medium-size, hospital-based requires that blood withdrawn to promote the health of a donor center, obtaining a variance from the FDA to establish an HH donor shall not be used as a source of , unless blood donor program is a cost-effective endeavor, which does not com- the container label conspicuously indicates the donor’s dis- promise donor or patient safety. ease necessitating the withdrawal of blood. This labeling Immunohematology 2009;25:170–173. requirement represents an almost insurmountable obstacle to the use of the blood in clinical transfusion settings. In Key Words: hereditary hemochromatosis (HH), FDA vari- addition, regulations state that a person may donate a unit ance, HH donor program of blood more than once in 8 weeks only after a physical examination and certification of health by a physician (21 ereditary hemochromatosis (HH) is an autosomal CFR 640.3(f)). Labeling and regulatory requirements are recessive genetic disorder of iron metabolism and the primary reasons that blood from HH individuals is not His one of the most commonly inherited disorders in routinely collected for transfusion. Caucasians of northern European descent. A single nucle- The FDA approved a final variance for collecting alloge- otide mutation in the HFE gene resulting in a C282Y sub- neic blood from individuals with a diagnosis of HH in Au- stitution is responsible for most of the classic cases of HH. gust 2001 that addresses the regulatory issues and labeling When hemochromatosis is present, iron absorption in the requirements.3 In this variance, the FDA allows a blood col- gastrointestinal tract is increased and iron stores are gradu- lection facility to include an alternative procedure to 21 CFR ally saturated over a prolonged period. There is progressive 640.3(d) to distribute blood and blood components collect- deposition of excess iron in body tissues, which leads to tox- ed from individuals with diagnosed HH without indicating ic levels of iron and organ damage and can eventually lead the donor’s disorder on the container label as long as they

170 IMMUNOHEMATOLOGY, Volume 25, Number 4, 2009 Cost-effectiveness of HH donor program met all other allogeneic criteria for blood donations. This scheduled therapeutic phlebotomy. Written informed con- guidance document also addresses concerns that individu- sent for study participation was documented on the UDHQ. als with HH would have an incentive to seek blood dona- The completed UDHQ was used to screen the HH individu- tion as a cost-free alternative to their therapeutic (medical) als to determine their eligibility as allogeneic donors as part phlebotomy and possibly deny risk-associated behaviors of this study. They were categorized into two groups: (1) that might preclude cost-free donations. individuals who qualified as allogeneic donors by meeting all FDA and other standard eligibility criteria, and (2) indi- Material and Methods viduals who did not qualify as allogeneic donors by current This study was designed to evaluate the financial im- eligibility criteria. The donor eligibility determined from the pact on our 398-bed hospital-based donor center of collect- UDHQ and frequencies of donation were recorded into an ing blood for transfusion from HH patients who met the Excel (Microsoft Corp., Redmond, WA) spreadsheet from eligibility requirements for allogeneic donations. Our donor January 2008 through December 2008. center has provided therapeutic phlebotomy services to the community for more than 15 years and serves patients with Results a broad range of diagnoses in the various stages of treat- Records of all patients presenting to our facility for ment. Monthly statistics from the donor center were used therapeutic phlebotomies in 2008 were reviewed, and 41 to categorize all therapeutic collections into two groups: percent (60 of 146) were from patients diagnosed with HH (1) individuals with a diagnosis of HH, and (2) individuals (Table 1). Nineteen individuals were eligible for the study with all other diagnoses. This was necessary to determine based on documentation of their diagnosis of HH from the number and percentage of HH therapeutic donations written requests to perform therapeutic phlebotomies by annually. physician prescriptions. All gave informed consent to par- Loss of revenue from providing free therapeutic ticipate in the study. phlebotomy collections for individuals with HH ($100/ The nineteen HH patients were screened for allogeneic collection) was compared with the potential gain in rev- eligibility using the AABB UDHQ V1.1. Seventeen patients enue generated by collecting additional RBC units ($429/ (89%) met the eligibility criteria for allogeneic donors as collection) from eligible HH donors in a 12-month period. defined by the UDHQ, and two patients (11%) did not. For Collection costs related to staffing were not considered rele- the purpose of this study, it was assumed that the 17 do- vant as it would cost the same to collect a unit that would be nors meeting eligibility requirements for allogeneic dona- further processed or discarded. It was also determined that tion would have met the requirements for the preceding any cost increase related to supplies (collection bags) and 12-month study timeframe. Retrospective review of donor additional processing time would be offset by a decrease in records indicated that a total of 60 units were collected costs associated with biohazardous waste disposal. The two from HH patients from January 2008 through December factors that would affect the potential net revenue in col- 2008. The average collection was 5 units per month. As il- lecting HH individuals as allogeneic donors were the addi- lustrated by the donation frequencies of the HH donors in tional expense of infectious disease testing and the expense Table 2, donors were in the various stages of therapeutic of providing therapeutic phlebotomies free of charge to all phlebotomy treatments (induction versus maintenance) HH patients regardless of their ability to meet eligibility cri- during this study. Fifty-five of the 60 units collected (92%) teria for allogeneic donations as required by the FDA vari- were eligible for allogeneic use as defined by the UDHQ ance guidance. and could be potentially introduced into the blood supply HH patients undergoing therapeutic phlebotomy treat- (Table 2). The remaining 5 units (8%) were not eligible for ments were contacted by telephone and asked whether they allogeneic use as defined by UDHQ donor criteria and were would be willing to participate in the study. Those express- discarded. Introduction of 55 RBC units into the blood bank ing interest in participating were asked to complete the inventory would have potentially generated gross revenue AABB “Uniform Donor History Questionnaire” (UDHQ) of $23,595. V1.1 when they presented to the donor center for their next

Table 1. Therapeutic phlebotomy collections in 2008 Diagnoses Jan Feb Mar Apr May June July Aug Sept Oct Nov Dec Total HH 5 6 8 4 4 5 3 4 6 5 4 6 60

Non-HH 10 9 13 9 3 4 4 4 8 8 10 4 86

Total 15 15 21 13 7 9 7 8 14 13 14 10 146

IMMUNOHEMATOLOGY, Volume 25, Number 4, 2009 171 D.M. Gribble et al.

Table 2. Donor eligibility and donation frequency for hereditary hemochromatosis donors in 2008 Donor No. UDHQ Eligibility Jan Feb Mar Apr May Jun Jul Aug Sept Oct Nov Dec Total 1 Y 1 1 1 1 1 1 0 1 1 0 0 1 9 2 Y 1 0 0 0 1 0 0 0 1 0 0 0 3 3 Y 0 1 1 0 0 1 1 1 1 1 0 0 7 4 N 0 1 0 1 0 0 0 0 0 0 0 1 3 5 Y 1 1 0 0 1 0 0 1 0 1 1 0 6 6 Y 0 1 0 0 0 0 0 0 0 0 0 0 1 7 Y 0 1 0 0 0 0 0 0 0 0 0 0 1 8 Y 0 0 1 0 0 0 0 0 0 0 0 1 2 9 Y 0 0 1 0 0 0 1 0 0 1 0 0 3 10 Y 0 0 1 0 1 0 0 0 0 0 0 0 2 11 Y 0 0 1 0 0 0 0 0 0 0 0 0 1 12 Y 0 0 0 1 0 0 0 0 0 0 0 0 1 13 Y 0 0 0 0 0 1 0 0 0 0 0 0 1 14 Y 0 0 1 0 0 1 0 0 1 0 0 1 4 15 Y 1 0 1 1 0 1 1 0 1 0 0 1 7 16 Y 0 0 0 0 0 0 0 1 0 0 0 0 1 17 N 1 0 0 0 0 0 0 0 1 0 0 0 2 18 Y 0 0 0 0 0 0 0 0 0 2 3 1 6 19 Y 0 0 0 0 0 0 0 0 0 0 0 0 0 Total 5 6 8 4 4 5 3 4 6 5 4 6 60

Discussion The collection and disposal costs associ- ated with the 5 units that could not be used for allogeneic transfusion would have totaled $500. Infectious disease testing would have been required on the remaining 55 units at $50 per unit, for a total expense of $2,750. Net revenue from the collection of an addi- tional 55 RBC units that could have poten- tially been used for allogeneic transfusion was $20,345. By comparison, the current revenue generated by the collection of 60 therapeutic phlebotomies was $6,000 (Fig. 1).

Conclusion This study was conducted at a 398-bed community hospital–based donor center located in a predominantly Caucasian com- munity (55% Caucasian, 35% Hispanic, 2% African American) of approximately 100,000 residents. Although the number of HH indi- viduals and the total number of units they donated as part of this study was relatively Fig. 1 Potential and current net revenues from hereditary hemochromatosis small, the potential to generate additional donors in 2008. revenue from the currently discarded collec- tions was substantial. In 2008, using eligible

172 IMMUNOHEMATOLOGY, Volume 25, Number 4, 2009 Cost-effectiveness of HH donor program

HH individuals as allogeneic blood donors would have re- 3. FDA Guidance. Variances for blood collec- sulted in an increase in revenue of more than $14,345 for tion from individuals with hereditary hemo- our blood center. The data from this study demonstrate that chromatosis (HH). August 2001. Available at: even at a medium-sized, hospital-based donor center, ob- (http://www.fda.gov/BiologicsBloodVaccines/ taining a variance from the FDA to collect and use blood GuidanceComplianceRegulatoryInformation/ from HH donors is a cost-effective endeavor, which does Guidances/Blood/ucm076719.htm) not compromise donor or patient safety. Dee M. Gribble, MT (ASCP)SBB (corresponding author), Acknowledgments Blood Services QA/QI Coordinator, North Colorado Medi- The authors would like to acknowledge the support of cal Center, and DJ Chaffin, MD, Medical Director, Blood the donor room staff and management at North Colorado Services, Summit Pathology, North Colorado Medical Cen- Medical Center Blood Bank, Greeley, Colorado, and the ter, 1801 16th Street, Greeley, CO 80631, and Barbara J. willingness of the hereditary hemochromatosis patients to Bryant, MD, Associate Professor of Pathology, Associate participate in this study. Director, Blood Bank Division, Medical Director, Specialist in Blood Banking Technology Program, Associate Direc- References tor, Pathology Residency Training Program, University of 1. Leitman SF, Browning JN, Yau YY, et al. Hemochroma- Texas Medical Branch, Galveston, TX. tosis subjects as allogeneic blood donors; a prospective study. Transfusion 2003;43:1538–44. 2. Newman B. Hemochromatosis blood donor programs: marginal for the supply but potentially good for patient care. Transfusion 2004;44:1537–8.

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IMMUNOHEMATOLOGY, Volume 25, Number 4, 2009 173 Original Report

Characterization of three novel monoclonal anti-Oka

M.H. Tian and G.R. Halverson

Anti-Oka was first described by Morel and Hamilton in 1979. The Oka secreting antibody after 6 to 12 months.9-10 As our work in antigen has a very high incidence, and only eight probands that are the production of Mabs to blood group antigens developed, Ok(a–) have been found; all are of Japanese heritage. In this study, so too did the use of novel approaches to achieve an immune we describe the generation and characterization of three novel mono- response in the mouse that would lead to the development clonal antibodies (Mabs), MIMA-25, MIMA-144, and MIMA-149. The of murine-based Mabs to human blood group antigens.11–13 reactivity of these three Mabs was compared with the original human polyclonal anti-Oka. Mice were immunized with transfected HEK cells to induce an immune response, and the spleen B lymphocytes were The OK Blood Group System fused with mouse myeloma X63-Ag8.653 cells to form antibody- The antibody Oka was named for the family name of the secreting hybridomas. The resulting Mabs were tested serologically, by proband (S.Ko.G) whose plasma reacted with all normal flow cytometry, and by immunoblotting. The specificity of each anti- RBCs.14 Born of consanguineous parents on a small island body was determined after excluding specificities to common antigens off the coast of Onomichi, Honshu, Japan, the proband also in the Rh, Kell, Duffy, Kidd, MNS, Lewis, Lutheran, P1, Colton, Diego, had two siblings who were Ok(a–). Anti-Oka reacted by the a Xg , and Dombrock blood group systems. In each case only the Ok(a–) antiglobulin test with blood samples from more than 4000 RBC sample was nonreactive. The Mabs and the original human anti- Japanese donors. Since its discovery there have been only Oka each have a unique pattern of reactivity when tested with enzyme- treated cells; however, none were reactive by immunoblotting. We eight Ok(a–) probands identified, all in persons of Japanese 15 have generated three novel anti-Oka Mabs: MIMA-144 is an indirectly heritage. Of more than 12,000 blood donors of various agglutinating IgG2b antibody, and MIMA-25 and MIMA-149 are di- ethnic backgrounds tested in the United States, no other rectly agglutinating antibodies (IgM and IgA, respectively), underscor- Ok(a–) individuals have been found. ing their usefulness as typing reagents for the clinical laboratory. The Oka antigen is the only antigen in the OK blood Immunohematology 2009;25:174–178. group system and was assigned the ISBT # 024001 after it was located on CD147 glycoprotein and thereby was eli- Key Words: anti-Oka, CD147, blood groups, hybridoma, gible for blood group system status. The OK glycoprotein monoclonal antibodies, transfection, flow cytometry is a single-pass type I membrane glycoprotein with two Ig superfamily (IgSF) domains and a short transmembrane ommercial reagents for antigen typing RBCs have and cytoplasmic domain. Sequence analysis showed the N- commonly been derived from polyclonal human terminal 30 amino acids are identical to the predicted se- Cserum collected from hyperimmunized donors. quence of the M6 leukocyte activation antigen, which is a However, the direct immunization of human subjects is no member of the IgSF. The OK gene consists of seven exons longer recommended by the FDA, thus the supply of human distributed over 1.8 kbp of gDNA, and it was localized using polyclonal reagents is rapidly diminishing and becoming in- somatic cell genetics to chromosome 19, subregion 19p13.2- creasingly more expensive to maintain. Kohler and Milstein pter.16 The rare Ok(a–) phenotype is the result of an amino first reported the successful development of a monoclonal acid substitution in the Ok glycoprotein (Ok(a+) > Ok(a–); antibody–producing hybridoma cell line.1 Monoclonal anti- E92K) encoded by exon 4 of OK.17 bodies (Mabs) are now commonly used as blood typing re- The Ok glycoprotein (CD147) is the most common pro- agents, replacing the need for human polyclonal reagents.2 tein found in the membranes of leukocytes having a role Although producing antibodies to carbohydrate anti- in cell-cell interaction and in signal transduction, and it gens (ABO, Lewis) was relatively easy, developing Mabs to is thought to act as a tumor growth factor stimulating the other types of antigens has proven to be much more diffi- production of collegenase and other cell invasion inducers. cult.3–5 Furthermore, the early methods applied only to the This glycoprotein is almost ubiquitous in that it is found murine model. The production of Mabs for human antigens on blood cells (RBCs, WBCs, and ) and in tissues requires a highly immunized donor, the viral transforma- of the renal cortex, liver, pancreas, colon, cervix, testes, tion of human B lymphocytes, and a successful fusion with smooth muscle, and brain. a mouse myeloma cell line.6 Transformation was needed The first monoclonal anti-Oka was produced by Andrews to expand the number of human B lymphocytes for fusion, et al. in 1983.18 in this paper we will describe the production and, fortunately, it was found to improve the susceptibility and characterization of three new murine antibodies to the of B lymphocytes for somatic cell hybridization by up to 25- high-incidence Oka antigen. They were evaluated serologically fold.7-8 Unfortunately, heterohybridoma cell lines have been for specificity, titer and score, immunoglobulin isotype, and found to be highly unstable, and thus more likely to stop reactivity pattern when tested with enzyme-treated RBCs.

174 IMMUNOHEMATOLOGY, Volume 25, Number 4, 2009 Serologic and flow cytometry evaluation of monoclonal anti-Oka

They were also tested by flow cytometry and by immuno- was then added to each well. The trays were again mixed, blotting. allowed to settle, and observed for indirect agglutination.

Materials and Methods Hemagglutination in Tube and Gel Cards Immunization Protocol Standard hemagglutination assays were done in test Two mice were immunized biweekly for 6 weeks with tubes using one drop of a 3 percent RBC suspension in PBS 2 million human embryonic kidney (HEK) cells that had plus two drops of Mab. MIMA-25 was incubated at room been transfected with human cDNA along with a CpG temperature for 30 minutes with Lo-Ion (Immucor/Gam- adjuvant specific for murine Mab production.18 After the ma, Norcross, GA) added to enhance reactions, then spun immune response was verified by serum hemagglutina- and read. MIMA-149 was incubated at room temperature tion, the spleen was removed and splenocytes obtained by for 30 minutes, spun, and read. MIMA-144 was incubated pulverizing the spleen with sterile glass slides. for 30 minutes at room temperature and tested by IAT us- ing anti-mouse IgG (The Binding Site, San Diego, CA) that Cellular Fusions had been diluted 1:50 in 6% bovine serum albumin (BSA) in Fusions between the splenocytes and the mouse my- PBS. eloma cell line (X63-Ag8.653) were done using polyethyl- For the IgG gel card (DiaMed ID-PNH, Cressier, Swit- ene glycol (PEG, mol. wt., 3450) in a ratio of 3:1 spleen zerland), 50 μL of a 1 percent suspension of Oka(+) RBCs to myeloma cells, respectively. Fusions were performed by and 25 μL of the Mab were added to each column, incu- drop-wise addition of 1.0 mL of PEG to the washed, dry bated at 37°C for 15 minutes, and then centrifuged and read cells in a conical centrifuge tube with constant mixing for for agglutination. 60 seconds. This was followed immediately by submerging the centrifuge tube into a 37°C water bath for 90 seconds Cloning by Limiting Dilution with constant mixing, after which a drop-wise addition of When a colony of hybrid cells is found to be producing 1 mL of sterile phosphate buffered saline (PBS, pH 7.3, the antibody of interest, the hybrids are counted and dilut- Gibco, Grand Island, NY) without calcium and magne- ed such that there will be a finite number of cells in a 20-mL sium was added for 60 seconds. This step was repeated, dilution. This dilution is done so that there will statistically and then followed by the addition of 2 mL of PBS for 120 be a limited number of cells per well when they are pipetted seconds. This was continued until the total volume was into 96-well flat-bottom cloning trays. Three clonings were approximately 15 mL. The now fused sample was rested done to ensure that the final product is from a monoclonal for 15 minutes in the 37°C CO2 incubator, and then gently cell line. The initial cloning was done at 10, 5, and 2 cells per centrifuged. The PBS was removed and the cells were sus- well to promote the development of a stable hybridoma cell pended in culture media (HAMS/DF-12; Sigma Chemicals, line. The next two clonings were done at a dilution of 5, 2, St. Louis, MO) that had been supplemented with 10 per- and 1 cell per well. The final antibody was selected from the cent fetal calf serum and hypoxanthine-adenine-thymidine last set of cloning trays. (HAT) media (Sigma). The fused cells were gently mixed, then spread over 96-well flat-bottom culture trays at 100 Enzyme Treatment of RBCs μL per well. After 1 week, fresh culture media supplemented Human RBCs that had been pretreated with a variety of with hypoxanthine-thymidine and gentamicin, but without enzymes or reducing chemicals were tested by standard he- aminopterin (StemCell Technologies, Vancouver, BC), was magglutination methods to determine whether the Oka an- added as a selection media. After another week, the result- tigen detected by the antibodies was resistant or sensitive to ing hybrids were screened by hemagglutination in v-well the enzyme effect on the protein.19 This included treatment microtiter trays for antibody production. with papain, trypsin, α1-chymotrypsin, pronase, neuramini- dase, and 0.2 M dithiothrietol (DTT). The original human Hemagglutination Assays of 96-Well V-Well Trays anti-Oka was tested in parallel. The supernatant culture fluid was assayed for reactiv- ity with selected RBCs by 96-well v-bottom microtiter trays Flow Cytometric Analysis (Greiner Labs, Germany, www.GBO.com). For v-well as- A 1% RBC suspension of test RBCs was first incubated says, 50 μL of supernatant fluid (SNF) was removed from with 500 μL of each Mab and incubated at 37°C for 30 min- each well of the fusion trays and placed in the correspond- utes, and then washed with PBS. The antibody-sensitized ing well on the v-well assay tray. To this, 25 μL of a 1% RBCs were then stained with fluoresceinisothiocyanate suspension of human Ok(a+) RBCs in PBS was added, (FITC)-tagged anti-mouse antiglobulin (IgG from Vector followed by mixing on a mechanical plate mixer (Labline Labs, Burlingame, CA; IgA and IgM from Southern Bio- Instruments Inc., Melrose Park, IL). The reactions were al- technology Associates, Birmingham, AL) by incubating in lowed to settle for 30 minutes and then observed for direct the dark at 4°C for a minimum of 15 minutes with frequent agglutination. The cells were washed in PBS, and 50 μL of mixing to ensure adequate staining. The analytes were anti-mouse IgG diluted 1:100 in 6 percent BSA and PBS brought up to a final volume of 250 μL with PBS. Data were

IMMUNOHEMATOLOGY, Volume 25, Number 4, 2009 175 M.H. Tian and G.R. Halverson acquired on a FACSCaliber flow cytometer (BD Biosciences, San Jose, CA) and analyzed with Cell Quest Pro software (BD Biosciences).

Results MIMA-25, MIMA-144, and MIMA-149 are specific for the Oka antigen (Fig. 1).21 MIMA-144 is an indirectly agglu- tinating Mab, while MIMA-25 and MIMA-149 are directly agglutinating Mabs. Their isotype, titer, score, and reactions Ok(a+) Ok(a–) Ok(a+) Ok(a–) Ok(a+) Ok(a–) with enzyme and chemically pretreated Ok(a+) RBCs are shown in Table 1. When tested in parallel with the original MIMA-25 MIMA-144 MIMA-149 human anti-Oka , they were found to have different reactivi- ty patterns when tested with enzyme or chemically modified Fig. 1 Gel card photo of Mab reactivity with Ok(a+) and Ok(a–) RBCs. RBCs. MIMA-25 was strongly reactive with Ok(a+) RBCs that were treated with trypsin, pronase, and neuraminidase, and weaker with papain, α1-chymotrypsin, and 0.2 M DTT. MIMA-144 was weakly reactivity with Ok(a+) cells treated with papain, trypsin, and α1-chymotrypsin. It was sensitive to treatment with pronase and neuraminidase, but resistant to treatment with 0.2M DTT. MIMA-149 was resistant to much weaker, with only a slight right shift, probably due to treatment with trypsin and pronase and weakly reactive the isotype and low titer of this antibody. Flow cytometry of a with treatments of papain, α1-chymotrypsin, neuramini- the native HEK cells shows the presence of the Ok antigen dase, and 0.2M DTT. The reactivity of the polyclonal anti- on untransfected cells, and the expression seems to be stron- Oka with Ok(a+) RBCs was unaffected by treatment with ger than the expression on normal human RBCs (Fig. 3). trypsin, α1-chymotrypsin, pronase, and 0.2M DTT, weak- None of the Mabs were reactive by immunoblotting (re- ened when treated with papain, and sensitive to treatment sults not shown). with neuraminidase. By flow cytometry each antibody demonstrated a posi- Discussion tive change in fluorescence when compared with the negative We had previously reported that the HEK cells, wheth- control (Fig. 2). MIMA-149 demonstrated the best inten- er transfected with human cDNA or not, had a high enough sity and specificity, with a sharp, narrow peak suggesting expression of CD147 to initiate an immune response, and that its epitope is a short sequence of amino acids. MIMA- this is the reason why we were able to produce so many dif- 144 showed a broader pattern of reactivity indicative of ferent Mabs to Oka. MIMA-25, -144, and -149 are new anti- an epitope of a wider amino acid sequence. MIMA-25 was Oka Mabs as demonstrated by their specific reaction with

Table 1. Isotype, titer (score), and effect of enzymes and chemicals on Ok(a+) RBCs Titer 0.2 M

Antibody Isotype (score) Papain Trypsin α1-Chymotrypsin Pronase Neuraminidase Dithiothreitol MIMA-25 IgM 8 (29) Weak Resistant Weak Resistant Resistant Weak

MIMA-144 IgG2b 128 (77) Weak Weak Weak Sensitive Sensitive Resistant

MIMA-149 IgA 32 (55) Weak Resistant Weak Resistant Weak Weak

Human anti- Not tested Not tested Weak Resistant Resistant Resistant Sensitive Resistant Oka

176 IMMUNOHEMATOLOGY, Volume 25, Number 4, 2009 Serologic and flow cytometry evaluation of monoclonal anti-Oka

Ok(a+) RBCs, and their nonreactivity with six different Ok(a–) RBC samples (M. Uchikawa, personal communica- tion). The reactivity pattern of each antibody is unique in the way it is affected when RBCs are modified by 0.2M DTT or various enzymes. All three antibodies react well in test tubes and gel cards. With our flow cytometry data, it is not surprising that MIMA-25 was much weaker, as it is an IgM Mab requiring very careful dilution controls for demonstra- tion of the reactivity by flow cytometry. Serologically this antibody works best by hemagglutination in the presence of Lo-Ion to enhance the reactions. None of these Mabs were reactive by Western blotting, suggesting that the epitope(s) they recognize may be con- formation dependent.

Conclusions The three Mabs are unique antibodies that will be use- ful for testing patients and screening blood donors to find Ok(a–) blood. MIMA-144 is an indirect agglutinating anti- body, and MIMA-25 and MIMA-149 are directly agglutinat- ing antibodies, and as such, are ideal reagents for rapidly typing RBCs.

Acknowledgments We thank Robert Ratner for the preparation of the manuscript, tables, and figures. We thank Wu He for his as- sistance in performing the flow cytometry experiments, and Makoto Uchikawa for additional testing with Ok(a) RBC samples. We thank Karina Yazdanbakhsh for her review of and comments on the manuscript. This work was funded in part by a grant from the MetLife Foundation.

Oka is Expressed at High a Levels on 293T Cells Ok Expression on RBCs

Fig. 2 Flow cytometry results with three Mabs against Ok(a+) and Ok(a–) RBCs. On the x-axis is the fluorescent intensity of the signal and on the y-axis is the number of cells counted. The shaded gray area represents the FITC-positive cell population. The unshaded graph represents the negative control cell population. Fig. 3 Flow cytometry of transfected 293T cells and human Ok(a+) RBCs.

IMMUNOHEMATOLOGY, Volume 25, Number 4, 2009 177 M.H. Tian and G.R. Halverson

References 12. Yazdanbakhsh K, Chaudhuri A, Lee S, Reid ME. High 1. Kohler G, Milstein C. Continuous cultures of fused cells level expression of blood group antigens in erythroid secreting antibody of predefined specificity. Nature cells (abstract). Mol Biol Cell 1997;8S:292a. 1975;256:495–7. 13. Halverson G, Chaudhuri A, Huang T, et al. Immuniza- 2. Voak D, Lennox ES. Monoclonal antibodies for labora- tion of transgenic mice for production of MoAbs direct- tory aspects of transfusion practice. In: Cash JD, ed. ed at polymorphic blood group antigens. Transfusion Progress in transfusion medicine. Edinburgh: Churchill 2001;41:1393–6. Livingstone, 1986:1–18. 14. Morel PA, Hamilton HB. Oka: an erythrocytic antigen of 3. Hughes-Jones NC. Monoclonal antibodies in haema- high frequency. Vox Sang 1979;36:182–5. tology (review). Blood Rev 1989;3:53–8. 15. Daniels G. Blood group gene mapping. In: Daniels G, 4. Anstee DJ, Edwards PA. Monoclonal antibodies to hu- ed. Human blood groups. Oxford: Blackwell Science man erythrocytes. Eur J Immunol 1982;12:228–32 Ltd., 1995:693–710. 5. Anstee D. Characterization of human blood group al- 16. Williams BP, Daniels GL, Pym B, et al. Biochemical and loantigens. In: McMichael AJ, Fabre JW, eds. Monoclo- genetic analysis of the OKa blood group antigen. Immu- nal antibodies in clinical medicine. London: Academic nogenetics 1988;27:322–9. Press, 1982:237–49. 17. Spring FA, Holmes CH, Simpson KL, et al. The Oka 6. Andrzejewski C, Young PJ, Goldman J, et al. Produc- blood group antigen is a marker for the M6 leukocyte tion of human warm-reacting red cell monoclonal au- activation antigen, the human homolog of OX-47 anti- toantibodies by Epstein-Barr virus transformation. gen, and neurothelin, an immunoglobulin su- Transfusion 1989;29:196–200. perfamily molecule that is widely expressed in human 7. Kozbor D, Lagarde AE, Roder JC. Human hybrido- cells and tissues. Eur J Immunol 1997;27:891–7. mas constructed with antigen-specific Epstein-Barr 18. Andrews PW, Goodfellow PN, Damjanov I. Human virus-transformed cell lines. Proc Natl Acad Sci USA teratocarcinoma cells in culture. Cancer Surveys 1982;79:6651–5. 1983;2:41-73. 8. Thompson KM, Hough DW, Maddison PJ, et al. The 19. Krieg AM, Yi AK, Matson S, et al. CpG motifs in bac- efficient production of stable, human monoclonal an- terial DNA trigger direct B-cell activation. Nature tibody-secreting hybridomas from EBV-transformed 1995;374:546–9. lymphocytes using the mouse myeloma X63-Ag8.653 20. Judd WJ. Methods in Immunohematology. 2nd ed. as a fusion partner. J Immunol Meth 1986;94:7–12. Durham, NC: Montgomery Scientific Publications, 9. Nowinski R, Berglund C, Lane J, et al. Human mono- 1994. clonal antibody against Forssman antigen. Science 21. Halverson G, Velliquette RW, Schawalder A. Serological 1980;210:537–9. evaluation of two new monoclonal anti-Oka (abstract). 10. Carson DA, Freimark BD. Human lymphocyte hy- Transfusion 2004;44(Suppl):117A–18A. bridomas and monoclonal antibodies. Adv Immunol 1986;38:275–311. Mary H. Tian, BS, and Gregory R. Halverson, BS, 11. Chu T, Reid ME, Øyen R, Yazdanbakhsh K. Production MT(ASCP)SBB (corresponding author), DLM, Laboratory of novel murine monoclonal antibodies against blood of Immunochemistry, New York Blood Center, 310 East group antigens using a high level expression system 67th Street, New York, NY 10065. (abstract). Blood 1997;90(Suppl 1):435a.

178 IMMUNOHEMATOLOGY, Volume 25, Number 4, 2009 Original Report

The ARC Hemovigilance Program: advancing the safety of blood donation and transfusion

A.F. Eder, B.A. Dy, J. Barton, J.M. Kennedy, and R.J. Benjamin

ince 2005, the American Red Cross (ARC) Hemovigi- prevent transfusion of other components from the involved lance Program has systematically evaluated adverse or prior donations, to temporarily defer the individual from Sreactions and complications after blood donation donation, and to address any suspected problems with the and transfusion, which has led to improvements in safety manufacturing process or procedures. for both donors and patients. After establishing base- After completing the investigation, the ARC physician line estimates of the risk of transfusion reactions such as evaluates all the information in the case and assigns a prob- transfusion-related acute lung injury (TRALI) and sepsis ability score on a 6-point scale, to classify the likelihood from bacterially contaminated platelet components, the that the transfusion caused the reaction (Table 2). All cases program has demonstrated that the preventive measures are counted on a tally in the month that the investigation is that were implemented to reduce their occurrence were closed and reported to the ARC Hemovigilance Program. effective.1–4 Reports of transfusion-transmitted infections, The ARC Hemovigilance Program maintains a national most commonly babesiosis linked to RBC components, database of investigated transfusion reactions to monitor, have identified the need for targeted interventions.5 The track, and analyze trends in donor and recipient complica- program has also described the spectrum of adverse reac- tions at each region and across the system. The number of tions experienced by healthy volunteers after whole blood transfusion reactions investigated by the 35 ARC regions in or donation, including systemic (e.g., vasovagal), calendar year 2007 is shown in Figure 1. phlebotomy-related, and other complications.6,7 The infor- Table 1. ARC Hemovigilance Program goals mation about donor reactions has led to several initiatives to reduce the already low rates of complications among the • To improve safety for recipients of blood components most susceptible groups and improve the donors’ experi- • To minimize procedure risk for blood donors ence.8,9 In this report, we present annual data on donation • To identify significant trends that emerge from analysis of reports of and transfusion complications in the ARC in 2007 and dis- uncommon events cuss the strengths and limitations of our national hemo- • To identify strategies to reduce the risk of complications in vigilance program. susceptible patient and donor groups • To monitor the effectiveness of the interventions introduced to improve safety Overview: Complications of Blood Transfusion To meet safety goals (Table 1), the ARC Hemovigilance Program compiles and analyzes data from the 35 ARC re- gional blood centers across the United States and Puerto Table 2. Probability scores for transfusion reactions Rico. The regional blood centers investigate complications P6 – The clinical information is consistent with a transfusion reaction, of transfusion reported by the hospitals and transfusion and a transfusion source is confirmed or highly probable (e.g., a services that may be related to the blood donor or to the donor or component was implicated). manufacture of the blood components.10 Common transfu- sion reactions, such as allergic and febrile nonhemolytic re- P5 – The clinical information is supportive of a transfusion reaction, but a actions, as well as acute or delayed hemolytic reactions, are transfusion source is not identified. not usually reported, unless the blood centers also provide P4 – The clinical information suggests that a transfusion reaction is transfusion services to hospitals or a donor- or product- posible or a transfusion reaction cannot be ruled out, but the case is related issue is suspected to have caused the reaction. When not typical and a transfusion source is not identified. a complication of transfusion is confirmed to be fatal, the facility that performed the compatibility testing must re- P3 – The clinical information suggests that a transfusion reaction is possible but other etiologies are present and equally or more likely port the death to the U.S. Food and Drug Administration the cause of the reaction. (FDA).10 In these cases, the ARC Hemovigilance Program also provides a voluntary, supplemental report to the FDA P2 – The clinical information does not support the diagnosis of a with a medical assessment of the transfusion reaction along specified transfusion reaction. with the results of our donor and manufacturing investiga- P1 – There is insufficient information to investigate the case or case is tion. When any transfusion reaction is reported, the blood rescinded by the reporting institution. centers take immediate action, if needed, to gain control and

IMMUNOHEMATOLOGY, Volume 25, Number 4, 2009 179 A.F. Eder et al.

250 45

40

200 35

30 150 25

20 100 15 Number of cases Number of cases 10

50 5

0 TRL ALL HEM SEP ORX BAB HCV HBV 0 Complication Type TRL SEP ORX ALL HEM HCV HBV BAB HIV OTI Complication Type Fig. 2. The number of cases with complications after transfusions in Fig. 1. The number of cases with complications after transfusions 2007 reviewed by the ARC Hemovigilance Program is shown for all reported to the ARC Hemovigilance Program in 2007 is shown for complication types, except hepatitis C (HCV). The number of cases all complication types. ALL = allergic; BAB = babesiosis; HBV = open in 2007 is shown for HCV, with the year of transfusion in each hepatitis B; HCV = hepatitis C; HEM = hemolytic; ORX = other case indicated on the graph. ALL = allergic; BAB = babesiosis; transfusion reaction; OTI = other infection; SEP = septic; TRL = HBV = hepatitis B; HEM = hemolytic; ORX = other transfusion transfusion-related acute lung injury. P1-P6, see probability scores, reaction; SEP = septic; TRL = transfusion-related acute lung injury. Table 2. P5-P6, see probability scores, Table 2.

In addition to the monthly tallies of transfusion reac- 2003, to reduce TRALI by the preferential use of plasma tions, the ARC regions convey complete information to the from male donors. In the ensuing 5 years, their Serious ARC Hemovigilance Program on all suspected transfusion- Hazards of Transfusion program (SHOT) registered fewer related fatalities and adverse reactions determined to have TRALI cases and no fatalities associated with plasma trans- a probable or confirmed transfusion source (i.e., cases as- fusion.12 Although 2008 saw three cases of TRALI that im- signed probability codes P5 or P6). These “high-probability” plicated a female donor of fresh-frozen plasma, SHOT cases are reviewed by the national medical director of the reported no TRALI cases in 2005, 2006, or 2007.12 ARC Hemovigilance Program and further characterized. Similar to the United Kingdom’s experience, the ARC This evaluation affords the advantage of having one phy- Hemovigilance Program found that plasma components sician review all high-probability cases to confirm that ap- were responsible for the majority (63%) of probable TRALI propriate actions were taken and to achieve consistency in fatalities and that a female antibody-positive donor was coding cases across the system. Moreover, the ARC Hemov- identified in 75 percent of these cases.3 The number of re- igilance Program has developed more-specific case ported TRALI cases increased each year between 2003 and definitions for certain transfusion complications (e.g., 2006. In late 2006, the ARC began shifting plasma com- Babesia infection) in an effort to more accurately describe ponents collected from female donors to further pharma- the current transfusion risk, as described in greater detail ceutical manufacturing so that the plasma distributed for in the following sections. The number of high-probability transfusion to patients was collected predominantly from transfusion reactions reviewed by the ARC Hemovigilance male donors. The proportion of plasma components col- Program in 2007 are shown in Figure 2. Focusing on the lected from male donors progressively increased from 55 transfusion reactions most consistently reported to our percent in 2006, to 78 percent in 2007, and to more than blood centers, the ARC Hemovigilance Program has con- 95 percent in 2008. Concurrently, the number of probable ducted in-depth analysis that has advanced transfusion TRALI cases among reported fatalities that involved only safety for patients and identified the need for further inter- plasma transfusion was significantly decreased in 2008 (0 ventions to improve clinical outcomes of transfusion. cases) compared with 2006 (6 cases) or 2007 (5 cases; p < 0.05).4 Moreover, probable TRALI was more likely to be Transfusion-Related Acute Lung Injury associated with plasma than RBC transfusion in 2006 and Transfusion-related acute lung injury (TRALI) emerged 2007, but not in 2008.4 as a leading cause of morbidity and mortality associated Both the ARC Hemovigilance Program and the United with blood component therapy, accounting for most of the Kingdom’s SHOT organization acknowledge the limitations deaths reported to the FDA since 2004.11 The United King- inherent to passive surveillance and retrospective review, dom was the first to introduce precautionary measures, in including incomplete hospital and regional blood center

180 IMMUNOHEMATOLOGY, Volume 25, Number 4, 2009 The ARC Hemovigilance Program participation, and potential investigational, reporting, and analytical bias. Regardless, the encouraging reports from SHOT and the corroborative data from the ARC Hemo- vigilance Program suggest that limiting transfusion of leu- kocyte antibody–containing plasma components reduces the morbidity and mortality associated with TRALI. In formulating a rational, and incremental, approach to TRALI prevention in the United States, blood centers are currently planning measures to reduce the risk of TRALI associated with apheresis , while bal- ancing the effect of any measures on availability of compo- nents for transfusions. Data on the effectiveness of these precautionary measures are not yet available but are being Reactions per million distributed collected by the ARC Hemovigilance Program and other centers as the measures are introduced.

Septic Transfusion Reactions In response to a new AABB Standard in 2004, most Fig. 3. The rate of septic transfusion reactions (per million aphere- blood centers introduced methods to limit and detect bac- sis platelet components created) before and after introducing teria in platelet components, which decreased but did routine quality control bacterial culture in Period 1 (39 percent of not eliminate the risk of sepsis after apheresis platelet collection procedures with sample diversion; 4-mL sample volume transfusion (Fig. 3).1,2,13 The ARC Hemovigilance Program cultured) and Period 2 (100 percent collection procedures with subsequently demonstrated incremental improvements in sample diversion, 8-mL sample volume cultured).1,2,13 Reported limiting contamination and increasing the sensitivity of fatalities are shown in red. The rate of septic reactions significantly bacterial testing to further reduce the risk of septic reac- decreased with time (Before Culture vs. Period 2, odds ratio, 0.33; tions to platelet transfusion. First, an association was ob- 95 percent confidence interval, 014 to 0.76). served between a method to collect apheresis platelets and subsequent transfusion-related sepsis. The increased risk diversion decreased bacterial contamination during two- with apheresis platelet donations from two-arm (double- arm collections by more than 46 percent. Second, doubling needle) procedures compared with one-arm (single-needle) the sample volume was associated with a 54 percent relative procedures was both clinically apparent as reported sep- increase in culture sensitivity, which corresponded to the tic transfusion reactions and detected by routine quality- predicted absolute increase of 25 percent.2,14 Similar benefit control bacterial culture of apheresis platelet donations.1 of initial sample diversion was observed for whole-blood– The data implicated skin bacteria in the vast majority of derived platelet pools when bacterial culture was intro- clinical reactions and all reported fatalities. The most no- duced.15 Ongoing surveillance by the ARC Hemovigilance table difference between the two procedure types was that Program and operational trials will continue to focus on the two-arm collection sets did not discard or divert the initial residual risk of septic reactions to platelet transfusion, to 10 to 20 mL of blood collected from the draw line into a identify additional opportunities for improvement. separate container or tubes during two-arm procedures, unlike the one-arm procedures. The lack of this feature on Infectious Complications of Transfusion the collection set, commonly referred to as sample diver- The ARC Hemovigilance Program also monitors all sion, may have contributed to the higher amount of skin suspected transfusion-related infections reported to the 35 flora contamination of platelet components collected with ARC blood centers that are investigated by regional physi- the two-arm procedures. cians. Not surprisingly, transfusion-transmitted infectious To address this possibility, the ARC converted all diseases for which the blood supply is screened are rarely apheresis collection procedures to use inlet-line sample di- identified through passive surveillance even though version in 2006. Concomitantly, the sample volume taken hundreds of cases of suspected transfusion-transmitted for quality-control bacterial culture was increased from infections are considered each year. Since the introduc- 4 mL to 8 mL from each apheresis platelet donation.2 This tion of infectious disease testing more than 20 years ago change to double the sample volume was predicted to in- and nucleic acid amplification testing (NAT) in 1999, the crease culture sensitivity by about 25 percent.13 The ARC risk of infection with human immunodeficiency virus (HIV) Hemovigilance Program reported the outcome after intro- or hepatitis C virus (HCV) through a blood transfusion has ducing the two operational changes and demonstrated that been reduced to an estimated 1 in 2 million.16 FDA-man- each measure contributed to a substantial improvement in dated “lookback” procedures, which are initiated after a safety for apheresis platelet transfusion.2 First, inlet-line returning donor has a confirmed-positive HIV test result,

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have identified only four reported breakthrough HIV cases Table 3. Transfusion-transmitted Babesia classification from three implicated donations (nonreactive by minipool Cases in which the patient had a proven B. microti infection after transfu- NAT) since 1999 in the United States.16,17 HCV lookback is sion that was not related to known prior infection were classified as also required by the FDA; similarly, recipient-tracing is of- • “definite” if the patient was not a resident in a Babesia-endemic ten performed for HBV and other infections, even though state (CT, NJ, NY, MN, WI), had no known risk factors for Babesia not required by FDA, when a donor is subsequently found infection, was diagnosed with clinical disease within 3 months of to have confirmed positive test results. In addition, patients transfusion, and a donor was identified and tested positive forBabe - suspected of contracting infection through transfusion are sia antibodies or had evidence of recent infection; reported and investigated by blood centers in an effort to • “probable” if the patient was a resident in a Babesia-endemic state (CT, NJ, NY, MN, WI) or had another risk factor for Babesia infection determine whether any of the involved donations, which and a donor tested positive for Babesia antibodies or had evidence tested negative for all markers at the time of donation, were of recent infection; or from donors who subsequently had positive test results. The • “possible” if the donor was not tested but was a resident of an ARC Hemovigilance Program has identified cases of trans- endemic area or had a recent travel history to an endemic area. fusion-transmitted HCV and HIV; however, all transfusions occurred before 1999, and the implicated donors had pre- as mild symptoms (e.g., lightheadedness, dizziness) that viously triggered lookback, although the notification at the resolve promptly but are still unpleasant for the donor.6,7 time had not identified the infected recipient. As expected, Serious injury is rare, but typically follows from a loss of the rarity of definite cases of transfusion-transmitted infec- consciousness, either at the donation site or after leaving tions identified through passive surveillance supports the the premises, or from nerve injury after the phlebotomy.6 known low risk of infection with viruses for which the blood All adverse reactions occurring at ARC collection sites supply is screened. are managed by collection staff and documented on the More importantly, the ARC Hemovigilance Program blood donation record according to the standard proce- has delineated the risk of infections for which blood donors dures. All donors are also instructed to contact the donor are not tested.5 Babesia microti accounts for most of the center if they experience problems or have concerns about infections investigated by the ARC, with five to ten definite their health after donation. Donor complications are classi- or probable cases of transfusion transmission each year. fied according to the reaction type (systemic, phlebotomy- Because of the difficulty in assessing whether infection was related, other) and severity of the symptoms (minor, major; acquired through a blood transfusion or other means (e.g., Table 4). The reactions coded on the blood donation record tick exposure) in endemic areas, more specific definitions by collections staff are captured in a centralized database; than the standard six-point imputability scale were devel- reactions that are called back to donor centers and cases oped by the ARC Hemovigilance Program to further define that received outside medical care are captured in a sepa- the likelihood of a transfusion source of infection (Table rate database. The ARC Hemovigilance Program compiles 3). The value in having detailed information on suspected and analyzes data on complications after whole blood do- cases is that it allows further characterization, which is not nation that occurred at the collection site or that were re- possible in other hemovigilance systems that do not capture ported to the blood center after the donor left the site and primary data about the event. Although passive surveillance on all cases involving donors who were referred for outside will not identify all cases, the scope of the problem defined medical care by staff or later reported that they sought or by the ARC Hemovigilance Program supports the need for received care from another health-care provider (Table 5). targeted interventions to reduce the risk of transfusion- Most syncopal-type reactions occurred at the collection site, transmitted Babesia infection.18 but many cases involving loss of consciousness (LOC), pro- longed recovery, or syncope-related injury were recognized Donation-Related Complications or classified after the donation. About 15 percent of synco- Although public attention, government regulation, and pal reactions result in injury (e.g., head trauma, lacerations, the industry effort are traditionally focused on patient safe- contusions), with about a third of these incidents requiring ty, blood centers also have an obligation to make blood do- outside medical care (0.57 per 10,000 donations). Phleboto- nation as safe as possible for healthy volunteers. Each year, my-related adverse events (e.g., large hematoma, suspected the ARC evaluates about 7.8 million individuals who pres- nerve irritation) more often become apparent after the do- ent to donate whole blood or apheresis components and nor leaves the collection site. Taking these reactions into provides about 40 percent of the blood for transfusion in account, the rate of suspected nerve irritation was 3.2 cases the United States. The blood supply depends entirely on the per 10,000 donations; only 13 percent of donors with pos- daily commitment of volunteers, who gain only intangible sible nerve irritation reported receiving outside medical care personal benefit from blood donation but are exposed to (0.43 per 10,000 donations). The ARC Hemovigilance Pro- potential risk of discomfort or, in rare cases, injury result- gram has evaluated the risks to certain groups of donors who ing from the collection procedure. About 2 to 12 percent of are more susceptible to reactions after donation (e.g., young, all presenting donors experience an adverse reaction that first-time, female donors).8 Future study will analyze the risk is documented at the collection site, most being classified factors associated with delayed reactions and cases of out-

182 IMMUNOHEMATOLOGY, Volume 25, Number 4, 2009 The ARC Hemovigilance Program

Table 4. Definitions of donor complications in the ARC Table 5. Complications after whole blood donation, ARC Hemovigilance Program Systemic (syncopal-type): Complications after 12,033,323 whole blood donations Minor category Major category At collection site All major Outside medical Complica- Presyncopal (prefaint) reactions care tion type Pallor, weakness, light-headedness, (subset, minor)* dizziness, diaphoresis, nausea/vom- Number † Number † Number † iting, no loss of consciousness Rate Rate Rate Systemic (syncopal-type) Loss of consciousness (LOC), LOC, long: Minor categories short: lasting 1 minute or more or compli- Presyncopal 324,129 269 (766) (0.64) 69 0.06 lasting less than 1 minute cated by seizures or convulsions or (prefaint) loss of bladder or bowel control LOC 11,081 9.21 (733) (0.61) 107 0.09 (< 1 min) Prolonged recovery: Presyncopal symptoms, loss of Major categories consciousness or other reaction LOC 1,839 1.53 2,050 1.70 251 0.21 that does not resolve within ap- (≥ 1 min) proximately 30 minutes Prolonged 2,623 2.18 4,228 3.51 829 0.69 Injury recovery associated with symptoms of LOC with 1,239 1.03 2,181 1.81 680 0.57 prefaint or LOC (e.g., head injury, injury fractures, abrasions, lacerations) Subtotal‡ 340,911 283 9,958 8.28 1,936 1.61 Phlebotomy-related: Phlebotomy-related Minor category Major category Minor categories Hematoma, small: Hematoma, large: Small 125,082 104 (2,625) (2.18) 87 0.07 Involved area measures 2 × 2 Involved area measures more than hematoma inches or less 2 × 2 inches Major categories Suspected nerve irritation: Large 792 0.66 4,932 4.09 556 0.46 Suggested by pain, tingling, numb- hematoma ness, or sharp shooting pains after phlebotomy Suspected 1,021 0.85 3,858 3.20 513 0.43 nerve Suspected arterial puncture: irritation Suggested by rapid (< 3 min) bleed time, pulsatile flow, or bright Suspected 1,302 1.08 1,644 1.37 112 0.09 red blood arterial puncture Other Reaction Types Subtotal‡ 128,197 107 13,059 10.9 1,268 1.05 Minor category Major category Citrate reactions*: Citrate reactions*: Other Reaction Types Perioral or peripheral tingling or Symptoms of minor citrate plus Minor categories numbness that does not resolve prolonged or exaggerated muscle Allergic 123 0.10 (166) (0.14) 19 0.02 with reduced flow rate or oral cal- spasm (tetany), vomiting, chest (minor, local) cium supplementation (e.g., Tums) tightness or when accompanied by additional Other 458 0.38 (1,153) (0.96) 141 0.12 symptoms such as nausea, muscle Major categories tightness, or cramping; other symptoms Allergic 10 0.01 17 0.01 11 0.01 (systemic) Allergic reaction, localized: Allergic reaction, systemic: Other 252 0.21 1,208 1.00 352 0.29 Itching, rash, or redness of skin; Symptoms of minor allergic reac- Subtotal‡ 843 0.70 2,544 2.11 523 0.43 hives tions, plus swelling of the face, neck, or throat, wheezing, or respi- Total (all 469,951 391 25,561 21.2 3,727 3.10 ratory difficulty categories) * This column includes all major events documented at the collection Other reaction: Other reaction: site and reactions reported after donation, which are reviewed by an Symptom profile different from es- Symptom profile different from ARC physician. The subset of minor reactions reviewed by an ARC phy- tablished categories (e.g., anxious- established categories (e.g., chest sician, typically identified through callbacks, are included in parentheses. ness, hyperventilation, headache) pain, thrombophlebitis) † Rate per 10,000 donations (successful and unsuccessful [QNS] col- lections). ‡ Subtotals are of minor and major categories. *Automated procedures only. LOC = loss of consciousness.

IMMUNOHEMATOLOGY, Volume 25, Number 4, 2009 183 A.F. Eder et al. side medical care to identify or refine preventive strategies 3. Eder AF, Herron R, Strupp A, et al. Transfusion- for these more medically severe reactions or other symptoms related acute lung injury surveillance (2003–2005) and after donation that lead to additional medical attention. the potential impact of the selective use of plasma from male donors in the American Red Cross. Transfusion Conclusions 2007;47:599–607. The ARC Hemovigilance Program is one aspect of a 4. Eder AF, Dy BA, Herron RM, Strupp A, Dodd RY, Ben- continuous improvement effort at the ARC to advance the jamin RJ. Effective reduction of TRALI risk with plasma safety of blood donation and transfusion. Data from the collected predominantly from male donors. Transfusion program have informed policy and have prompted changes 2009; 49(Suppl):45A. in procedures to reduce the risk to both transfused patients 5. Tonnetti L, Eder AF, Dy BA, et al. Transfusion-transmit- and blood donors. The program’s strength lies in its abil- ted Babesia microti identified through hemovigilance. ity to analyze uncommon or rare events and to recognize Transfusion 2009; 49:2557–63. trends that may be associated with certain donor selection 6. Eder AF, Dy BA, Kennedy J, et al. The American Red or blood component manufacturing practices. Another ad- Cross Donor Hemovigilance Program: complica- vantage offered by a blood center–driven hemovigilance tions of blood donation reported in 2006. Transfusion program is the ability to capture detailed information about 2008;48:1809–19. each reported transfusion event and every donation-related 7. Benjamin RJ, Dy BA, Kennedy JM, Notari EP, Eder AF. complication, rather than summary information derived The relative safety of automated two-unit red blood cell from the final assignment of reaction codes or aggregate procedures and manual whole-blood collection in young data to estimate denominators. Challenges that the pro- donors. Transfusion 2009; 49:1874-83. gram faces are the inherent limitation of passive surveil- 8. Eder AF, Hillyer CD, Dy BA, Notari EP, Benjamin RJ. lance and the likelihood that hospitals do not report all Adverse reactions to allogeneic whole blood donation by transfusion reactions to the blood center. Regardless, the 16- and 17-year-olds. JAMA 2008;299:2279–86. sample size is adequate to estimate the scope of TRALI, 9. Eder AF, Dy BA, Ralston J, et al. Effective reduction of septic transfusion reactions, and transfusion-transmitted donor reactions on high school drives with standard babesiosis. Finally, blood centers are typically focused on work guidance. Transfusion 2009;49(Suppl):51A. issues related to blood component manufacturing and do- 10. Code of Federal Regulations Title 21 CFR 606.170(a) and nor selection; consequently, their hemovigilance programs (b), 2009 Washington, DC. US Government Printing Of- are not designed to capture common, idiosyncratic transfu- fice. sion reactions (e.g., allergic) or problems associated with 11. FDA/Centers for Biologics Evaluation and Research. medical errors or near misses, such as ABO incompatible Fatalities reported to FDA following blood collec- transfusion, overtransfusion, or inappropriate transfu- tion and transfusion: annual summary for fiscal year sion practice. In this regard, the US Biovigilance Network, 2008. Available at http://www.fda.gov/Biologics a unique public-private collaboration between the federal BloodVaccines/SafetyAvailability/ReportaProblem/ government (e.g., CDC, FDA) and organizations involved in TransfusionDonationFatalities/ucm113649.htm. Accessed blood collection, transfusion, and tissue and organ trans- August 12, 2009. plantation (e.g., AABB) promises to address this need in a 12. Taylor C, Cohen H, Mold D, et al., on behalf of the Seri- national hemovigilance program for the United States.19 In ous Hazards of Transfusion (SHOT) Steering Group. The conclusion, blood centers have a dual responsibility to pro- 2008 Annual SHOT Report (2009). www.shotuk.org. vide an adequate supply of blood components to the com- 13. Fang CT, Chambers LA, Kennedy J, et al. Detection of munity and to protect the safety of volunteer donors. We bacterial contamination in apheresis products: American have made our data available to highlight our continued Red Cross experience, 2004. Transfusion 2005;45:1845–52. focus on improving donor and patient safety. 14. Wagner SJ, Eder AF. A model to predict the improve- ment of automated blood culture bacterial detec- References tion by doubling platelet sample volume. Transfusion 1. Eder AF, Kennedy JM, Dy BA, et al. Bacterial screen- 2007;47:430–3. ing of apheresis platelets and the residual risk of septic 15. Benjamin RJ, Kline L, Dy BA, et al. Bacterial contami- transfusion reactions: the American Red Cross experi- nation of whole blood-derived platelets: the introduc- ence (2004–6). Transfusion 2007;47:1134–42. tion of sample diversion and prestorage pooling with 2. Eder AF, Kennedy JM, Dy BA, et al. Limiting and detect- culture testing in the American Red Cross. Transfusion ing bacterial contamination of apheresis platelets: inlet- 2008;48:2348–55. line diversion and increased culture volume improve 16. Stramer SL. Current risks of transfusion-transmitted component safety. Transfusion 2009; 49:1554-63. agents. Arch Pathol Lab Med 2007;131:702–7.

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17. Code of Federal Regulations 21 CFR 610.46 Human im- Anne F. Eder, MD, PhD; Beth A. Dy; Jessica Barton; Jean munodeficiency virus (HIV) “lookback” requirements; 21 M. Kennedy, RN, and Richard J. Benjamin, MD, PhD, CFR 610.47, Hepatitis C virus (HCV) “lookback” require- American Red Cross, National Headquarters, Biomedical ments; 2009 Washington, DC. US Government Printing Services, Medical Office, 15601 Crabbs Branch Way, Rock- Office. Available at http://frwebgate2.access.gpo.gov/ ville, MD 20855 cgi-bin/PDFgate.cgi?WAISdocID=s9imto/3/2/0&WAI Saction=retrieve. Accessed August 12, 2009. 18. AABB Association Bulletin 09-06. Transfusion- transmitted Babesia. Bethesda, AABB. 19. The US Biovigilance Network. Available at http://www. .org/Content/Programs_and_Services/Data_Center/ US_Biovigilance_Network. Accessed December 6, 2009.

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

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

IMMUNOHEMATOLOGY, Volume 25, Number 4, 2009 185 Communication

Thank You to the Contributors of the 2009 Issue

To Contributors to the 2009 Issues

The journal depends on readers, authors, editorial board, peer reviewers, and our Penn-Jersey staff. We wish we could thank all of you personally, but doing so is not practical. Instead, we thank each of you as members of an honored group. First and foremost, we thank the authors for their reviews, scientific articles, case reports, book reviews, and letters to the editors that come not only from the United States but from many countries of the world. This has given the journal an international flavor. Our editorial board is a prestigious one and we depend on them not only for peer reviews, but for guidance in policy and suggestions for improvements. Special thanks go to our medical editors, who review every article for medical content, and to our technical editors, who read every article for technical content. The current board is listed by name in the front of each issue of the journal. Our peer reviewers did a wonderful job in 2009. In each December issue we list them by name with thanks to each.

Roy A. Alther Jr., MT(ASCP)SBB Joann Moulds, PhD, MT(ASCP)SBB Nicholas Bandarenko, MD John Moulds, MT(ASCP)SBB Richard Benjamin, MD, PhD Maria Muniz, MD Martha Rae Combs, MT(ASCP)SBB Theresa Nester, MD Richard Davey MD Frank Nizzi, MD Greg Denomme, PhD Lawrence Petz, MD Joan Gibble, MD Joyce Poole, FIBMS Michelle Henry William Reed, MD A. J. Hibbard, MD S. Gerald Sandler, MD W. John Judd, FIBMS, MIBiol Randy Schuller, MT(ASCP) Maryann Keeshan-Schnell Chelsea Sheppard, MD Barbara LeChien, MT(ASCP)SBB Susan Shirey Douglas Lublin, MD Jayanna Slayten, MS,MT(ASCP)SBB Jeff McCullough, MD Elizabeth Smart David Moolten, MD Jill R. Storry, PhD, FIBMS Yvette Miller, MD Ralph R. Vassallo, MD Monique Mohamed Sunitha Vege, MS Gary Moroff, PhD

We also want to thank the office staff at Penn-Jersey, Marge Manigly and Judy Abrams, for their help in preparing the journal for press. They manage the manuscript submissions, keep up with subscriptions, and many other behind-the-scenes tasks. We also thank Mary Tod, our copy editor, Lucy Oppenheim, our proofreader; and Wilson Tang, our electronic pub- lisher. Finally, thanks go to our readers, whose enthusiasm and interest in the journal make it all worthwhile.

Sandra Nance Connie M. Westhoff Editors-in-Chief

Cindy Flickinger Managing Editor

186 IMMUNOHEMATOLOGY, Volume 25, Number 4, 2009 Subject Index

Subject Index

Immunohematology Volume 25, Nos. 1, 2, 3, 4, 2009

ABO blood group system Blood group systems/antigens ABO blood group system nomenclature 25(2):48 Antigens of the Lewis blood group system 25(3):112 Biochemistry of the ABO blood group system 25(2):48 Antigens of the MNS blood group system, 25(3):95 Inheritance and molecular genetics of ABO 25(2):48 ISBT numbers and prevalence Pathogen interactions of the ABO system 25(2):48 Case report of JK*0 in a family originating 25(4):165 The ABO blood group system: review and update 25(2):48 from Guam Lewis antigens and renal transplantation 25(3):112 Autoantibodies Lewis antigen distribution within the human 25(3):112 Alloantibody binding to transfused RBCs causing 25(1):9 body conformational changes in antigenic epitopes Lewis antigens in pregnancy and infancy 25(3):112 and stimulation of autoantibody The ABO blood group system: review and 25(2):48 IgM plus IgG auto anti-It leads to diagnosis 25(2):60 update of B-cell lymphoproliferative disease The OK blood group system 25(4):174 Review of the Lutheran blood group system 25(4):152 Autoimmune hemolytic anemia (AIHA) Simple screening assay for JK*0 25(4):165 Bystander immune hemolysis after 25(1):9 Southeast Asian ovalocytosis associated with 25(2):63 alloimmunization increased expression of the Duffy antigen

Biochemistry Case report Lewis antigen biosynthesis 25(3):112 Anti-Kpa–induced severe delayed hemolytic 25(2):44 Primers—PCR-ASP analysis of JK*0 25(4):165 transfusion reaction Primers—PCR-RFLP analysis of SC 25(1):18 Autoantibody formation with hemolysis after 25(1):9 alloimmunization Blood components Clinical evaluation for lymphoproliferative 25(2):60 D+ platelet transfusions in D– patients 25(1):5 disease prompted by finding of IgM warm autoanti-It in two cases Blood components—manufacturing practices New serologic findings in patient with 25(4):160 FDA variance for collection of allogeneic 25(4):170 ulcerative colitis and a warm autoantibody blood from donors with hereditary Nonhemolytic passenger lymphocyte 25(1):20 hemochromatosis syndrome: donor derived Anti-M in In vitro quality of cryopreserved and 25(1):13 M+ transplant recipient deglycerolized RBCs over ten years old using ACP 215 instrument Disease associations Case report of IgG-RBC sensitization associated 25(4):160 Blood group antibodies with serine proteases in a patient with Antibodies and clinical significance of the 24(3):112 ulcerative colitis Lewis system Clinical evaluation for lymphoproliferative 25(2):60 Antibodies and clinical significance of the 25(4):152 disease prompted by finding of IgM warm Lutheran system autoanti-It in two cases Anti-Kpa–induced severe delayed hemolytic 25(2):44 Lewis (a–b) phenotype and cardiovascular 25(3):112 transfusion reaction disease Anti-M in M+ multi-organ recipient 25(1):20 Lewis (a–b–) Bombay phenotype and 25(3):112 associated with passenger leukocyte adhesion deficiency syndrome lymphocyte syndrome Lewis antigens: receptors for pathogenic 25(3):112 Clinical significance of ABO blood group 25(2):48 bacteria system antibodies Lu-glycoproteins and chronic myeloproliferative 25(4):152 Clinical significance of MNS blood group 25(3):95 disease system antibodies Lu-glycoproteins and sickle cell disease 25(4):152

IMMUNOHEMATOLOGY, Volume 25, Number 4, 2009 187 Subject Index

Donors Tissue tracking records and management of 25(3):102 Donor-derived anti-M in an M+ multiorgan 25(1):20 recalls transplant recipient Hereditary hemochromatosis donor program 25(4):170 Methods Retaining and transfusing frozen rare donor 25(1):13 Deglycerolization of rare RBCs older than 10 25(1):13 cells older than 10 years years using the ACP 215 instrument Table: summary of literature review of 25(1):20 Development, validation, and clinical 25(1):24 passenger lymphocyte syndrome involving performance of a fluorescent microsphere non-ABO blood group antigens in HPC and immunoassay for IgG anti-IgA solid-organ transplant recipients Use of antigen-capture ELISA in detection of 25(3):125 platelet-specific antibodies Genetics Flow cytometric analysis of Duffy antigen 25(2):63 Consortium for Blood Group Genes: 2008 report 25(2):75 expression on Southeast Asian ovalocytes DNA analysis of Caucasians and African 25(1):18 Flow cytometric analysis of Oka monoclonal 25(4):174 Americans for the SC nt76 change antibodies Functional aspects of Lutheran glycoproteins 25(4):152 Historic milestones in the evolution of the 25(4):147 Graphic: organization of the ABO locus 25(2):48 crossmatch GYP gene deletions of the MNS blood group 25(3):95 Milestones in laboratory procedures 25(2):39 Inheritance of hemoglobin S 25(2):67 and techniques Lewis phenotypes, prevalence, and inherited 25(3):112 Use of flow cytometry in the investigation of 25(3):125 alleles immune platelet disorders Lewis phenotypes and disease associations 25(3):112 Use of HPLC in diagnosis of sickle cell disease 25(2):67 Platelet glycoproteins and antigens 25(3):125 Use of IgA coupled covalently to fluorescent 25(1):24 Mutations within the ABO system 25(2):48 microspheres in fluorescent microsphere Southeast Asian ovalocytosis and the expression 25(2):63 immunoassay of the Duffy antigen: how they relate to Use of isoelectric focusing in diagnosis of sickle 25(2):67 malarial infection cell disease Use of monoclonal antibody immobilization of 25(3):125 Hemolytic disease of the fetus and newborn (HDFN) platelet antigens in detection of platelet- HDFN as it applies to the ABO blood group 25(2):48 specific antibodies system Use of PF4 ELISA in detection of platelet 25(3):125 antibodies Hemolytic transfusion reactions (HTR) Use of platelet aggregation assays in detection 25(3):125 Severe delayed hemolytic reaction to anti-Kpa 25(2):44 of platelet antibodies with severe hepatotoxicity Use of protein bead arrays in detection of 25(3):125 platelet-specific antibodies Historic reviews—Milestones series Use of serotonin-release assays (SRA) in 25(3):125 Historic milestones in the evolution of the 25(4):147 platelet antibody detection crossmatch Use of solid-phase RBC adherence in the 25(3):125 Milestones in Immunohematology 1984–2009 25(1):2 detection of platelet antibodies Milestones in laboratory procedures and 25(2):39 Use of solubility test in diagnosis of sickle 25(2):67 techniques cell disease Pioneers in blood group serology in the 25(3):90 United States: 1950–1990 Molecular Consortium for Blood Group Genes 25(2):75 Immune hemolysis (CBGG): 2008 report Bystander immune hemolysis after 25(1):9 Consortium for Blood Group Genes target 25(2):75 alloimmunization alleles and guidelines for molecular testing Graphic representation of ABO alleles currently 25(2):48 Laboratory management listed in the dbRBC database Adverse event reporting in tissue services 25(3):102 Graphic: organization of the ABO locus 25(2):48

FDA HCT/P registration: who must register 25(3):102 Lunull and Lumod phenotypes 25(4):152 and the process Methods for molecular platelet antigen typing 25(3):125 Qualification and selection of tissue suppliers 25(3):102 Milestones in laboratory procedures 25(2):39 Quality activities associated with hospital 25(3):102 and techniques tissue services

188 IMMUNOHEMATOLOGY, Volume 25, Number 4, 2009 Subject Index

Molecular bases of glycophorin variants and 25(3):95 Milestones in laboratory procedures and 25(2):39 antigens of the MNS blood group system techniques Molecular basis of the Lewis system 25(3):112 MNS blood group system 25(3):95 Molecular basis of the Lutheran system 25(4):152 Pioneers in blood group serology in the 25(3):90 PCR-RFLP analysis for the prevalence of the 25(1):18 United States: 1950–1990 SC nt76 change in Caucasians and Quality activities associated with hospital 25(3):102 African Americans tissue services Proficiency program of the Consortium for 25(2):75 Sickle cell disease review 25(2):67 Blood Group Genes The American Red Cross Hemovigilance 25(4):179 Simple screening assay for the most common 25(4):165 Program JK*0 alleles Use of DNA-based assays in platelet antigen 25(3):125 genotyping D+ platelet transfusion in D– patients 25(1):5

Platelets/white cells Serology Detection and identification of platelet 25(3):125 Consideration for inclusion of low-frequency 25(2):44 antibodies and antigens in the antigens in screening cells clinical laboratory Serologic workup of patient with bystander 25(1):9 D+ platelet transfusion in D– patients 25(1):5 immune hemolysis Human platelet antigens and nomenclature 25(3):119 Serologic workup of patient with ulcerative 25(4):160 Investigating the possibility of drug-induced 25(3)136 colitis and warm autoantibody platelet antibodies Three novel monoclonal Oka antibodies for 25(4):174 Prevalence of human platelet antibodies 25(3):119 screening blood and testing of patients Providing HPA-compatible platelets 25(3):119 Recognition and management of antibodies to 25(3):119 Transfusion Practices human platelet antigens—refractory patients Benefits of simple transfusion in sickle cell 25(2):67 patients Quality Benefits of in sickle 25(2):67 Consortium for Blood Group Genes: 2008 report 25(2):75 cell patients Cost-effectiveness of FDA variance for blood 25(4):170 Causes of platelet transfusion refractoriness 25(3):119 collection from individuals with hereditary Complications of blood transfusions and 25(4):179 hemochromatosis the ARC Hemovigilance Program Logistical aspects of human tissue management 25(3):107 Donation-related complications and the 25(4):179 in a hospital setting ARC Hemovigilance Program Personnel requirements of a hospital-based 25(3):107 Hospital tissue bank acquisition and storage 25(3):107 tissue bank Indications for transfusion in sickle cell disease 25(2):67 Quality activities associated with hospital 25(3):102 Logistical aspects of human tissue management 25(3):107 tissue services in a hospital setting The American Red Cross Hemovigilance 25(4):179 Matched platelet strategies 25(3):119 Program Necessity of RhIG in transfusion of D+ 25(1):5 platelets in D– patients Reviews Probability scores for transfusion reactions 25(4):179 ABO blood group system 25(2):48 Providing HPA-compatible platelets 25(3):119 Detection and identification of 25(3):125 Recognition and management of antibodies to 25(3):119 platelet antibodies and antigens human platelet antigens in platelet in the clinical laboratory transfusion—refractory patients Historic milestones in the evolution of the 25(4):147 Tissue orders, transportation to surgical areas, 25(3):107 crossmatch and management of returns Investigating the possibility of drug-dependent 25(3):136 Transfusion of rare frozen units older than 10 25(1):13 platelet antibodies years and the French hemovigilance Lewis blood group system 25(3):112 program Lutheran blood group system 25(4):152 Transfusion recommendations for sickle cell 25(2):67 Management of platelet antibodies in 25(3):119 patients refractory patients Importance of identification of IgA-deficient 25(1):24 Milestones in Immunohematology 1984–2009 25(1):2 persons to aid in optimal use of blood components from IgA-deficient donors

IMMUNOHEMATOLOGY, Volume 25, Number 4, 2009 189 Contributors

Immunohematology Volume 25, Nos. 1, 2, 3, 4, 2009

A H R

Abedalthagafi, M.M. 25(4):147 Halverson, G.R. 25(4):174 Reeder, J.C. 25(2):63 Alden, B.M. 25(3):107 Hamerschlak, N. 25(1):9 Reid, M.E. 25(1):18 Aravechia, M.G. 25(1):9 Harrison, J.S. 25(2):44 25(2):39 AuBuchon, J.P. 25(3):89 Hellberg, A. 25(4):165 25(2):75 25(3):136 Hillberry, C.M. 25(3):102 25(3):95 Homburger, H.A. 25(1):24 Roseff, S.D. 25(2):67 B Hue-Roye, K. 25(1):18 Rouger, P. 25(1):13 Hutchinson, P. 25(2):63 Rumilla, K.M 25(1):24 Bartley, A.N. 25(1):5 Barton, J. 25(4):179 I,J,K S, T Benjamin, R.J. 25(4):179 Berg, M.P. 25(1):5 Jedynak, E.A. 25(1):24 Sakashita, A. 25(1):9 Bley, C. 25(1):9 Kazura, J.W. 25(2):63 Sandler, S.G. 25(1):20 Bryant, B.J. 25(4):170 Kennedy, J.M. 25(4):179 25(4):147 Koshy, P. 25(2):44 Schlueter, A.J. 25(3):102 Kutner, J.M. 25(1):9 25(3):107 C Snell, B. 25(4):165 L Spruell, P. 25(4):165 Carpenter, J.B 25(1):95 Storry, J.R. 25(2):48 Castilho, L 25(1):9 Langeberg, A. 25(1):20 25(4):165 25(2):75 Leach, M.F. 25(3):136 Chaffin, D.J. 25(4):170 Leger, R.M. 25(2):60 V, W, Y Chen, W. 25(2):60 Le Pennec, P.Y. 25(1):13 Combs, M.R. 25(3):112 Lightfoot, T.G. 25(4):160 Vassallo, R.R. 25(3):119 Cortés, A. 25(2):63 Lomas-Francis, C 25(1):18 Wester, E.S. 25(4):165 Curtis, B.R. 25(3):125 Lowder, F. 25(2):60 Westhoff, C.M. 25(2):75 Winters, J.L. 25(1):24 Woolley, I.J. 25(2):63 D, E M

Daniels, G. 25(3):125 Makuria, A.T. 25(1):20 Delia VanThof, L. 25(4):160 Mallory, D. 25(1):2 Denomme, G.A. 25(2):75 Mason, H.M. 25(2):60 Dungo, M.C. 25(2):60 McFarland, J.G. 25(3):125 Dy, B. A. 25(4):179 Mota, M. 25(1):9 Eder, A. F. 25(4):179 N, O F Olsson, M.L. 25(2):48 Fishbein, T.M. 25(1):20 25(4):165 Fuchisawa, A. 25(1):18 P, Q G Patel, B. 25(2):44 Garratty, G 25(2):60 Peterman, J.M. 25(1):24 Gribble, D.M. 25(4):170 Peyrard, T. 25(1):13 Gustafsson, J. 25(4):165 Pham, B.N. 25(1):13 Pierce, S.R. 25(3):90

190 IMMUNOHEMATOLOGY, Volume 25, Number 4, 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 2010 and will last for 1 year. A part-time option lasting 2 or 3 years is also available. There may also be opportunities to continue studies for PhD or MD following the MSc. The syllabus is organized jointly by The Bristol Institute for Transfusion Sciences and the University of Bristol, Department of Pathology and Microbiology. It includes:

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

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

The course is accredited by the Institute of Biomedical Sciences.

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

For further details and application forms please contact:

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

IMMUNOHEMATOLOGY, Volume 25, Number 4, 2009 191 Announcements, cont., and advertisements

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 www. nybloodcenter.org >research >immunochemistry >current list of monoclonal antibodies available.

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

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

192 IMMUNOHEMATOLOGY, Volume 25, Number 4, 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 4, 2009 193 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.

194 IMMUNOHEMATOLOGY, Volume 25, Number 4, 2009 Advertisements, cont. Becoming a Specialist in Blood Banking (SBB)

What is a certified Specialist in Blood Banking (SBB)? • What is a certified Specialist in Blood Banking (SBB)? • Someone with educational and work experience qualifications that successfully passes the American Society for Clinical Pathology (ASCP) board of certification (BOC) examination for 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 nonconformances • Design and present educational programs • Provide technical and scientific training in transfusion medicine • Conduct research in transfusion medicine

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

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

How does one become an SBB? CAAHEP accredited SBB Technology program or grandfather the exam based on ASCP education and experience criteria.

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

Which approach are you more compatible with?

Contact the following programs for more information Additional Information can be found by visiting the following Web sites: www.ascp.org, www.caahep.org and www.aabb.org

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 Revised August 2009 IMMUNOHEMATOLOGY, Volume 25, Number 4, 2009 195 ImmunohematologyImmunohematology JournalJOURNAL ofOF BloodBLOOD GroupGROUP SEROLOGY Serology AND and EDUCATION Education Instructions to the 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 215 196 IMMUNOHEMATOLOGY, Volume 25, Number 4, 2009

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