Journal of Blood Group Serology and Molecular Genetics

Vo l u m e 35, N u m b e r 1, 2019 This issue of Immunohematology is supported by a contribution from

Grifols Diagnostics Solutions, Inc.

Dedicated to advancement and education in molecular and serologic immunohematology Immunohematology Journal of Blood Group Serology and Molecular Genetics Volume 35, Number 1, 2019 CONTENTS

B lo o d G r o u p R e v i e w 1 An update on the Augustine blood group system G. Daniels

S e r o lo g i c M e t h o d R e v i e w 3 Donath-Landsteiner Test M. Kilty and T.S. Ipe

B lo o d G r o u p R e v i e w 7 An update on the CD59 blood group system C. Weinstock, M. Anliker, and I. von Zabern

S e r o lo g i c M e t h o d R e v i e w 9 ZZAP treatment of red blood cells S.I. Marckwardt

B lo o d G r o u p R e v i e w 11 An update on the Duffy blood group system G.M. Meny

R e v i e w 13 Serologic problems associated with administration of intravenous immune globulin (IVIg) D.R. Branch

B lo o d G r o u p R e v i e w 16 An update on the Knops blood group system J.M. Moulds

S e r o lo g i c M e t h o d R e v i e w 19 Inhibition of blood group antibodies by soluble substances K.M. Byrne, C.M.C. Mercado, T.N. Nnabue, T.D. Paige, and W.A. Flegel

B lo o d G r o u p R e v i e w 23 An update on the Lutheran blood group system G. Daniels

25 33 37 41 A n n o u n c e m e n t s A dv e r t i s e m e n t s I n s t r u c t i o n s S u bs c r i p t i o n f o r A u t h o r s I n f o r mat i o n E d i to r - i n -C h i e f E d i to r i a l B oa r d Sandra Nance, MS, MT(ASCP)SBB Philadelphia, Pennsylvania Patricia Arndt, MT(ASCP)SBB Geralyn M. Meny, MD, MS Pomona, California Coppell, Texas M a n ag i n g E d i to r Cynthia Flickinger, MT(ASCP)SBB Barbara J. Bryant, MD Paul M. Ness, MD Wilmington, Delaware Galveston, Texas Baltimore, Maryland Lilian M. Castilho, PhD Thierry Peyrard, PharmD, PhD Tec h n i c a l E d i to r s Campinas, Brazil Paris, France Janis R. Hamilton, MS, MT(ASCP)SBB Detroit, Michigan Martha R. Combs, MT(ASCP)SBB S. Gerald Sandler, MD Durham, North Carolina Washington, District of Columbia Christine Lomas-Francis, MSc New York City, New York Geoffrey Daniels, PhD Ira A. Shulman, MD Bristol, United Kingdom Los Angeles, California Joyce Poole, FIBMS Bristol, United Kingdom Anne F. Eder, MD Jill R. Storry, PhD Washington, District of Columbia Lund, Sweden Dawn M. Rumsey, ART(CSMLT) Norcross, Georgia Melissa R. George, DO, FCAP, FASCP Nicole Thornton Hershey, Pennsylvania Bristol, United Kingdom Tiffany Walters, MT(ASCP)SBBCM Julie K. Karp, MD Charlotte, North Carolina Philadelphia, Pennsylvania E m e r i t u s E d i to r s S e n i o r M e d i c a l E d i to r Jose Lima, MD Delores Mallory, MT(ASCP)SBB David Moolten, MD Douglassville, Georgia Supply, North Carolina Philadelphia, Pennsylvania Christine Lomas-Francis, MSc Marion E. Reid, PhD, FIBMS A ss o c i at e M e d i c a l E d i to r s New York City, New York Bristol, United Kingdom P. Dayand Borge, MD Philadelphia, Pennsylvania Corinne L. Goldberg, MD Durham, North Carolina

M o l ec u l a r E d i to r Immunohematology is published quarterly (March, June, September, and December) by the Margaret A. Keller, PhD American Red Cross, National Headquarters, Washington, DC 20006. Philadelphia, Pennsylvania Immunohematology is indexed and included in Index Medicus and MEDLINE on the MEDLARS system. The contents are also cited in the EBASE/Excerpta Medica and Elsevier E d i to r i a l A ss i s ta n t BIOBASE/Current Awareness in Biological Sciences (CABS) databases. Linda Frazier The subscription price is $50 for individual, $100 for institution (U.S.), and P r o d u c t i o n A ss i s ta n t $60 for individual, $100 for institution (foreign), per year. Latasha Melvin Subscriptions, Change of Address, and Extra Copies:

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O n O u r C o v e r

Eugeen Van Mieghem grew up in Antwerp, Belgium, and in mood and subject his art never strayed far from the busy port’s austere setting. His laudatory yet realistic portrayals of the laborers and emigrant poor on the wharves and piers reflect his socially aware empathy and insight. At the start of his career, he used his wife Augustine Pautre as his primary model and first muse, and she predominates his early work. Tragically Augustine succumbed to tuberculosis after only 3 years of marriage, but Van Mieghem’s charged depictions of her during her illness have been compared with similar efforts by Ferdinand Hodler and Rembrandt. Van Mieghem completed De bloem van de voorstad (translation, Flower of the City) in 1901. This issue of Immunohematology includes an update of the Augustine blood group system. David Moolten, MD

ii IMMUNOHEMATOLOGY, Volume 35, Number 1, 2019 B l o o d G r o u p R e v i e w An update on the Augustine blood group system

G. Daniels

This update of the Augustine (AUG) blood group system (Daniels An antibody to a high-prevalence antigen (AUG4) in G. The Augustine blood group system, 48 years in the making. a white woman, who had been pregnant and transfused, Immunohematology 2016;32:100–3) describes two antigens was first found in 1995. Recent whole exome sequencing that have been added to the Augustine system (International Society of Blood Transfusion system 36), bringing the number of revealed homozygosity for a missense mutation in SLC29A1 antigens in the system to four. Further information on the clinical encoding p.Asn81Ser. The variant was present in 0.1 percent significance of Augustine system antibodies and the function of of individuals in the Exome Aggregation Consortium but was the Augustine glycoprotein, equilibrative nucleoside transporter 1, is presented. Immunohematology 2019;35:1–2. never found in a homozygous state. The AUG:–4 RBCs of the propositus were AUG:1,2, confirming that ENT1 is present, Key Words: Augustine, SLC29A1, adenosine transport, but flow cytometric analysis revealed an approximately erythropoiesis, ENT1 30 percent reduction in surface ENT1 relative to that on AUG:1,2,4 (common phenotype) RBCs. The antibody of the New Augustine Antigens propositus reacted with AUG:1,2 and AUG:1,–2 RBCs, but not with AUG:–1,–2 (null phenotype) RBCs.3 Since the publication of the original review,1 two new antigens have been added to the Augustine (AUG) blood group Clinical Significance of Augustine System system: AUG3 and AUG4 (Table 1). Antibodies An antibody to a low-prevalence antigen (AUG3) caused severe hemolytic disease of the fetus and newborn (HDFN) An acute hemolytic transfusion reaction caused by anti- requiring two exchange and four top-up transfusions. Targeted AUG2 (anti-Ata)4 and severe HDFN caused by anti-AUG32 exome sequencing for blood group genes revealed that the baby confirm previous reports that Augustine system antibodies was heterozygous for a novel variant in the Augustine gene, have the potential to be dangerously hemolytic. SLC29A1, encoding p.Thr387Pro in the fifth extracellular loop of equilibrative nucleoside transporter 1 (ENT1). The baby’s Functional Aspects of the Augustine Glycoprotein brother, father, two paternal aunts, and grandmother were all heterozygous for the variant allele, and their red blood cells Analysis of mature RBCs and developing erythroid (RBCs) were all AUG:3.2 cells from the three previously reported AUG:–1,–2 (null phenotype) siblings revealed macrocytosis Table 1. Antigens of the Augustine system and abnormal morphologies together with

Antigen Molecular basis a decreased level of proliferation during Number Name Prevalence Nucleotides Exon Amino acids Reference erythropoiesis and delayed erythroblast maturation. This finding suggests a role AUG1 High c.589+1G>C 6 Slice site 1 of ENT1-mediated adenosine transport in AUG2 Ata High c.1171G>A 12 Glu391Lys 1 erythroid differentiation.5 AUG3 ATML Low c.1159A>C 12 p.Thr387Pro 2 AUG4 ATAM High c.242A>G 3 p.Asn81Ser 3

IMMUNOHEMATOLOGY, Volume 35, Number 1, 2019 1 G. Daniels

References 4. Raval JS, Harm SK, Wagner B, Triulzi DJ, Yazer MH. Acute hemolytic transfusion reaction attributed to anti-Ata. 1. Daniels G. The Augustine blood group system, 48 years in the Immunohematology 2016;32:140–2. making. Immunohematology 2016;32:100–3. 5. Mikdar M, Le Van Kim C, Kinet S, et al. Lack of the nucleoside 2. Millard GM, McGowan EC, Wilson B, et al. A proposed new transporter ENT1 in individuals with the rare Augustine-null low-frequency antigen in the Augustine blood group system blood group is associated with an abnormal erythropoiesis and associated with a severe case of haemolytic disease of the fetus microcytosis (abstract). Vox Sang 2018;113(Suppl 1):64. and newborn. Transfusion 2018;58:1320–2. 3. Vrignaud C, Mikdar M, Raneri A, et al. Characterization of a Geoffrey Daniels, PhD, retired, previously Head of Diagnostics, novel high-prevalence red blood cell antigen in the Augustine blood group system (abstract). Vox Sang 2018;113(Suppl International Blood Group Reference Laboratory, Bristol, UK, geoff. 1):64–5. [email protected].

Manuscripts

The editorial staff of Immunohematology welcomes manuscripts submitting scientific articles, case reports, and review articles, pertaining to blood group serology and molecular genetics for see Instructions for Authors in every issue of Immunohematology consideration for publication. We are especially interested in or e-mail a request to [email protected]. Include fax and review articles, case reports, papers on platelet and white cell phone numbers and e-mail address with all manuscripts serology, scientific articles covering original investigations or new and correspondence. E-mail all manuscripts to immuno@ blood group alleles, papers on molecular testing, and papers on redcross.org. new methods for use in the blood bank. To obtain instructions for

2 IMMUNOHEMATOLOGY, Volume 35, Number 1, 2019 Donath-Landsteiner test

M. Kilty and T.S. Ipe

The Donath-Landsteiner (DL) test is a serologic test used to detect Reagents/Supplies the presence of a biphasic hemolysin. This autoantibody is seen in patients with paroxysmal cold hemoglobinuria. The test relies Reagents Supplies on the characteristic cold binding of an IgG autoantibody with Indirect DL test specificity to the P blood group antigen. This autoantibody causes • Fresh pooled normal sera complement-mediated red blood cell (RBC) lysis when warmed to • 50% suspension of washed P+ RBCs body temperature. In this review, we describe the various methods • Glass test tubes for performing the DL test—namely a direct test, an indirect test, an Enzyme-treated indirect DL test indirect test with modifications such as the use of enzyme-treated • 1% papain-treated P+ RBCs • 0°C ice bath RBCs and two stages, and an indirect antiglobulin DL test—and • 37°C heat block or Two-stage indirect DL test highlight the advantages and disadvantages of each. Our focus is incubator • Group O RBCs on the indirect testing method as it is most commonly used in • Centrifuge REVIEW METHOD SEROLOGIC blood bank laboratories. Immunohematology 2019;35:3–6. • Fresh pooled normal sera Anti-P specificity identification Key Words: Donath-Landsteiner test, paroxysmal cold • Washed ABO-compatible p or Pk RBCs hemoglobinuria, biphasic hemolysin, hemolysis, autoantibody DL = Donath-Landsteiner; RBCs = red blood cells.

The Donath-Landsteiner (DL) test is a serologic test used Procedural Steps for Indirect DL Test to detect the presence of a biphasic hemolysin, seen in patients with paroxysmal cold hemoglobinuria (PCH). The test relies • Label three sets of three test tubes as follows: 1A, 1B, 1C; 2A, 2B, 2C; 3A, 3B, 3C. on the characteristic cold binding of an IgG autoantibody • To tubes 1 and 2 of each set, add 10 drops of the patient’s serum. with specificity to the P blood group antigen, which causes • To tubes 2 and 3 of each set, add 10 drops of fresh pooled normal complement-mediated red blood cell (RBC) lysis when warmed sera. • To all tubes, add one drop of the 50 percent suspension of washed to body temperature. Julius Donath and Karl Landsteiner P+ RBCs,* and mix well. first described the antibody responsible for this hemolysis in • Place the three “A” tubes in a bath of melting ice for 30 minutes† 1904.1 DL antibodies are usually low titer (<32) autoantibodies and then at 37°C for 1 hour. • Place the three “B” tubes in a bath of melting ice for 90 minutes. 1,2 that have low thermal amplitude (<20°C). The biphasic • Place the three “C” tubes at 37°C for 90 minutes. hemolysis test was the first noted for the identification of this • Mix, and centrifuge all tubes. Examine for hemolysis. 1,2 autoantibody. *For enzyme-treated indirect DL test, add one drop of 1 percent PCH is a rare form of direct antiglobulin test (DAT)- papain-treated P+ RBCs; for anti-P specificity identification, add one drop of a 50 percent suspension of washed ABO-compatible p or Pk 3 positive autoimmune hemolytic anemia. According to Dacie, RBCs. there are three types of PCH: acute, chronic syphilitic, and †If performing the two-stage indirect DL test, replace the patient’s serum with ABO-compatible fresh pooled normal sera after the initial chronic non-syphilitic. Acute PCH occurs primarily in 30-minute ice bath incubation. children. Chronic syphilitic and non-syphilitic types can be DL = Donath-Landsteiner; RBCs = red blood cells. more difficult to diagnose and are often seen in adult patients.3,4 The DL test is the diagnostic test for PCH.5

Principle bind to RBCs, causing complement (C3) to irreversibly bind to the RBCs at the same time. As this complex is warmed to 37°C, The DL test exploits the biphasic nature of the DL activation of the complement cascade leads to intravascular autoantibody, which is usually IgG. Some case reports, hemolysis of RBCs when the autoantibody dissociates. This however, show these antibodies having IgM and IgA class test shows a positive result when hemolysis is visibly seen in specificities.6,7 Initially, at cold temperatures, the antibodies the test system (Fig. 1).

IMMUNOHEMATOLOGY, Volume 35, Number 1, 2019 3 M. Kilty and T.S. Ipe

Indications

The DL test should be considered when a patient presents with symptoms including recurrent fevers, shaking chills, abdominal pain, and laboratory findings of an intravascular hemolytic anemia with both hemoglobinemia and hemoglo- binuria. The onset of the disease is predominantly precipitated by a recent upper respiratory illness, either viral or bacterial. The patient’s RBC sample should also have a positive DAT due to C3 only and no demonstrable autoantibody activity by routine methods.

Procedure

The DL test was developed on the characteristic biphasic hemolysis that was seen in vivo when PCH was highly associated with syphilis, and hemoglobinemia was directly related to exposure to cold. The basic steps of the test are to incubate the patient’s sample at a cold temperature to allow the antibody to attach and then to incubate the sample at body temperature to activate complement and detect the presence of RBC lysis. An important aspect of the test is that the patient’s samples have to be kept at 37°C after collection. Historically, the test consisted of two different methods: a direct DL test and an indirect DL test.1,8 Modifications such as the indirect DL test with enzyme-treated RBCs, a two-stage test, and an indirect antiglobulin test (IAT) were developed and used in patients strongly suspected of having the DL antibody.1

Direct DL Test The direct DL test is performed by collecting two whole- blood samples into tubes without anticoagulant at 37°C.5 One sample is incubated at 37°C for 1.5 hours, and the other sample is incubated in melting ice (approximately 0°C) for 1 hour and then at 37°C for 30 minutes.8 Both samples are centrifuged, and the supernatants are observed for hemolysis. A positive direct DL test occurs when only the sample incubated at 0°C and then at 37°C shows hemolysis. This test is primarily used as an initial screening test in hospitals.1 When this test is negative, the indirect DL test is performed.

Indirect DL Test The indirect DL test was developed to increase the sensitivity and specificity of the test system. This test is performed on a fresh blood sample that is collected and allowed to clot at 37°C.5 The clotted sample is then centrifuged Fig. 1 Indirect Donath-Landsteiner test.

4 IMMUNOHEMATOLOGY, Volume 35, Number 1, 2019 Donath-Landsteiner test

at 37°C, and the serum is separated from the RBCs. Freshly that is present for the second half of the assay while decreasing collected pooled normal sera are used as a complement source. antibody inhibition. Label three sets of three tubes: 1A, 1B, 1C; 2A, 2B, 2C; and 3A, 3B, 3C (Fig. 1). Add 10 drops of the patient’s separated Indirect antiglobulin test serum to each of the tubes labeled 1A, 1B, 1C and 2A, 2B, The IAT can also be used for the detection of the DL 2C. Add 10 drops of normal pooled sera to each of the tubes antibody because the antibody is IgG.1,5,10,11 The IAT should be labeled 2A, 2B, 2C and 3A, 3B, 3C. Add 1 drop of a 50 percent performed with anti-IgG reagents after the low temperature suspension of washed P+ RBCs to all nine tubes. Place the three incubation. tubes labeled 1A, 2A, 3A in a bath of melting ice for 30 minutes and then at 37°C for 1 hour. Place the three tubes labeled 1B, Procedure Summary 2B, 3B in a bath of melting ice for 90 minutes. The three tubes The procedural method for the DL test suggested by the labeled 1C, 2C, 3C are kept at 37°C for 90 minutes. After the AABB is the described indirect DL test.5 This indirect testing completion of incubation, gently mix the tubes and centrifuge. method is widely accepted. Examine the supernatant for hemolysis. A positive indirect test occurs when tubes 1A and/or 2A show hemolysis and Limitations there is no hemolysis in any of the other tubes. When this test is negative but there is a strong suspicion for PCH, a modified The DL test has several limitations. The blood bank indirect test can be performed. The three modifications include staff performing the procedure need to be highly skilled and using enzyme-treated reagent RBCs, performing a two-stage meticulous about the temperature requirements of the DL test, and testing the DL antibody by the IAT.1 sample throughout collection, clotting, and testing. Given After identification of the biphasic antibody, one can these restrictions, the procedure requires considerable time further demonstrate the anti-P specificity by repeating the and resources and is expensive to perform at institutions with test steps described previously but adding washed ABO- limited resources and expertise. At institutions performing compatible p or Pk RBCs in place of the P+ RBCs. These tubes high-complexity testing, this procedure can be simple to should have no observable hemolysis, thus confirming the perform. The timing of the blood sample collection is also antibody specificity. important. The DL antibody is transient, and the in vivo titer rises and falls very quickly. The titer is at its highest Enzyme-treated indirect DL test concentration during the period of clinical hemolysis.4 The use of enzyme-treated reagent O RBCs is one means In addition, there are further limitations that depend of increasing the sensitivity of the indirect DL test.9 Various on the assay used. The direct DL test is more prone to false- enzymes can be used, including 1 percent papain. This negative results than the indirect DL test. This finding modified test is performed similarly to the indirect DL test. could be due to low antibody titer, low complement level, or Treating RBCs with enzymes causes increased exposure C3dg presence on the patient’s RBCs. C3dg is protective by of the P antigen on the RBC membrane.1,8–10 This increased preventing complement-mediated lysis.1,11–13 Low complement sensitivity allowed for detection of low-titer DL antibodies in levels in the patient due to consumption during the hemolytic two patients whose DL tests were only positive when papain- process can also cause a false-negative result. Also, additional treated P+ RBCs were used.10 sample volume is needed when performing the direct DL. The indirect DL test can be falsely negative when the Two-stage indirect DL test antibody titer is low. False-negative results can also occur Currently, the two-stage indirect DL test is not often because of autoadsorption of antibody (if serum separation is performed in blood bank laboratories. This test is performed not carried out strictly at 37°C) or neutralization of anti-P by by initially incubating the patient’s serum at 0°C with group globoside in fresh serum (added as a complement source).1,11 O RBCs. If hemolysis is present, the tube is centrifuged, and This latter scenario can be avoided by performing a two-stage the patient’s serum is removed and replaced with freshly DL test.10 collected pooled normal sera.1,9–11 This replacement increases The enzyme-treated indirect DL test makes the treated the sensitivity of the test because of the additional complement RBCs more prone to lysis and therefore comparison with controls is important. Use of the IAT can lead to a false-positive

IMMUNOHEMATOLOGY, Volume 35, Number 1, 2019 5 M. Kilty and T.S. Ipe

result due to carryover of direct agglutination by a cold IgM 3. Dacie J. The hemolytic anemias. Part II. Philadelphia, PA: antibody.1,11 Gruce and Stratton, 1962. Another limitation occurs when the patient’s serum is 4. Zeller MP, Arnold DM, Al Habsi K, et al. Paroxysmal cold hemoglobinuria: a difficult diagnosis in adult patients. 1,2 red (due to free hemoglobin) before the test. Preanalytical Transfusion 2017;57:137–43. analysis of hemolysis should be taken into account, and special 5. Leger RM. The positive direct antiglobulin test and immune- care should be taken to avoid hemolysis during the collection mediated hemolysis. In: Fung MK, Grossman BJ, Hillyer CD, Westhoff CM, Eds. Technical manual. Bethesda, MD: AABB, process. If the patient experiences in vivo hemolysis, the 2017:425–48. presence of free hemoglobin in the serum may be unavoidable. 6. Hayashi H, Yasutomi M, Hayashi T, et al. Paroxysmal cold Both the direct and indirect DL test can be falsely positive hemoglobinuria caused by an IgM-class Donath-Landsteiner when lysis occurs because of the presence of a cold-reacting antibody. Pediatr Int 2013;55:664–6. 7. Whipple NS, Moreau DA, Moulds JM, Hankins JS, Wang IgM autoantibody.2 This reactivity is usually seen in patients WC, Nottage KA. Paroxysmal cold hemoglobinuria due to with cold agglutinin disease. an IgA Donath-Landsteiner antibody. Pediatr Blood Cancer 2015;62:2044–6. Quality Control 8. Wolach B, Heddle N, Barr R, Zipursky A, Pai K, Blajchman M. Transient Donath-Landsteiner haemolytic anaemia. Br J Haematol 1981;48:425–34. The tubes labeled 3A, 3B, 3C serve as a negative control 9. Sokol RJ, Booker DJ, Stamps R. Paroxysmal cold for the indirect DL test. Other negative controls include the hemoglobinuria and the elusive Donath-Landsteiner antibody. tubes incubated only at 37°C (for both direct and indirect tests) Immunohematology 1998;14:109–12. 10. Heddle NM. Acute paroxysmal cold hemoglobinuria. Transfus and those incubated only in melting ice. Med Rev 1989;3:219–29. 11. Win NR, Stephen J. Acquired haemolytic anaemias. In: Summary Bain BJ, Bates I, Laffan MA, Eds. Dacie and Lewis practical hematology. 12th ed. Philadelphia, PA: Elsevier, 2017:254–81. The DL test is the diagnostic assay for detecting the 12. Win N, Stamps R, Knight R. Paroxysmal cold haemoglobinuria/ Donath-Landsteiner test. Transfus Med 2005;15:254. presence of a biphasic hemolysin. A high index of suspicion 13. Stowell SR, Winkler AM, Maier CL, et al. Initiation and is required when a patient presents with symptoms and regulation of complement during hemolytic transfusion laboratory findings consistent with PCH, given the variable reactions. Clin Dev Immunol 2012;2012:307093. titer of this antibody. The DL test can be performed in several ways: direct, indirect, or indirect with modifications. Each of Morgan Kilty, MHA, MLS(ASCP)CM, SBB, Product Manager, these assays has benefits and limitations. QualTex Laboratories, San Antonio, TX; and Tina S. Ipe, MD, MPH (corresponding author), Medical Director of Donor Services, Associate Medical Director of Transfusion Medicine, Houston References Methodist Hospital, Department of Pathology and Genomic 1. Eder AF. Review: acute Donath-Landsteiner hemolytic anemia. Medicine, 6565 Fannin Street, MS 205, Houston, TX 77030, tsipe@ Immunohematology 2005;21:56–62. houstonmethodist.org. 2. Petz LD. Review: evaluation of patients with immune hemolysis. Immunohematology 2004;20:167–76.

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6 IMMUNOHEMATOLOGY, Volume 35, Number 1, 2019 B l o o d G r o u p R e v i e w An update on the CD59 blood group system

C. Weinstock, M. Anliker, and I. von Zabern

This update of the CD59 blood group system (Weinstock C, Anliker reported (Table 1). Leading symptoms of CD59 deficiency M, von Zabern I. CD59: a long-known complement inhibitor are chronic inflammatory demyelinating neuropathy causing has advanced to a blood group system. Immunohematology pareses and muscular weakness, ischemic or hemorrhagic 2015;31:145–51) increases the number of reported patients with CD59 deficiency from 10 to 14. All of these 14 patients lesions in the central nervous system, and hemolytic episodes. suffered from severe illness. Recently, a new variant allele was Several of these 14 patients had been transfused; only one of found in heterozygosity. Flow cytometry data suggest that this them made an anti-CD59. variant was expressed on the red blood cells of the propositus. Although additional alleles have been found, the CD59 system (International Society of Blood Transfusion system 35) continues The CD59 Gene and Its Variations to have one antigen. Immunohematology 2019;35:7–8. The CD59 gene spans over 33 kbp.11,12 Eight transcript Key Words: CD59, MIRL, complement regulatory protein, variants have been deposited as reference sequences in the blood group. National Center for Biotechnology Information database (Fig. 1). The International Society of Blood Transfusion Red Introduction Cell Immunogenetics and Blood Group Terminology working party refers to the genomic sequence NG_008057 and the CD59 is a 20-kDa cell membrane glycoprotein, present on transcript variant NM_203330.13 a large number of cells, including red blood cells (RBCs), that In 2018, another CD59 allele was found14 (Table 2) and binds the complement components C8 and C9 and, thereby, was carried in heterozygosity together with the wild-type protects the cell from a complement attack.1 CD59 was assigned allele. When testing the propositus’ RBCs by flow cytometry a blood group system after a CD59-deficient child presented using fluorescence-labeled monoclonal anti-CD59, the authors with anti-CD59, reacting with all CD59-carrying RBCs.2 found the density of CD59 comparable to that of controls and In contrast to patients with paroxysmal nocturnal hemo- concluded that both wild-type CD59 and the variant CD59 globinuria, where an acquired mutation in a stem cell clone were expressed on RBCs. If this finding holds true, the binding causes the maturation of CD59-deficient RBCs, in this child, all site for the reagent anti-CD59 was conserved in this variant. cell types were CD59-deficient. The cause was homozygosity Any further investigation of the variant CD59 protein might be for a CD59 null allele.3 Since the first publications in 19904 and hampered by the presence of the wild-type CD59. 2013,5 a total of 14 children with CD59 deficiency have been

Table 1. Clinical presentation of patients with CD59 deficiency

Number of Deceased at Onset of Protein Clinical symptoms Ethnic origin patients time of report disease Report p.Val42Serfs*38 • Hemolysis Japanese 1 1 13 years Yamashina et al.4 p.Ala121Glnfs • Cerebral infarction Motoyama et al.6 p.Cys89Tyr • Chronic inflammatory demyelinating neuropathy North-African 5 1 3–7 months Nevo et al.5 • Flaccid pareses/muscular weakness Jewish 7 • Two out of seven with ischemic CNS lesions 2 2 3–5 months Ben-Zeev et al. • Acute and chronic hemolysis, transfusions required Asp49Valfs*31 • Chronic inflammatory demyelinating neuropathy Turkish 1 0 7 months Höchsmann et al.3 • Flaccid pareses/muscular weakness 8 • Ischemic or hemorrhagic CNS lesions 3 1 6–11 months Haliloglu et al. • Acute and chronic hemolysis, four patients 1 1 1 month Klemann et al.9 required transfusions 1 0 1 month Ardicli et al.10 CNS = central nervous system.

IMMUNOHEMATOLOGY, Volume 35, Number 1, 2019 7 C. Weinstock et al.

5. Nevo Y, Ben-Zeev B, Tabib A, et al. CD59 deficiency is associated with chronic hemolysis and childhood relapsing immune-mediated polyneuropathy. Blood 2013;121:129–35. 6. Motoyama N, Okada N, Yamashina M, Okada H. Paroxysmal nocturnal hemoglobinuria due to hereditary nucleotide deletion in the HRF20 (CD59) gene. Eur J Immunol 1992;22:2669–73. 7. Ben-Zeev B, Tabib A, Nissenkorn A, et al. Devastating recurrent brain ischemic infarctions and retinal disease in pediatric patients with CD59 deficiency. Eur J Paediatr Neurol 2015;19:688–93. 8. Haliloglu G, Maluenda J, Sayinbatur B, et al. Early-onset chronic axonal neuropathy, strokes, and hemolysis: inherited CD59 deficiency. Neurology 2015;84:1220–4. 9. Klemann C, Kirschner J, Ammann S, et al. CD59 deficiency presenting as polyneuropathy and Moyamoya syndrome with endothelial abnormalities of small brain vessels. Eur J Paediatr Neurol 2018;22:870–7. Fig. 1 CD59 gene structure and mRNA isoforms. Viewing the figure 10. Ardicli D, Taskiran EZ, Kosukcu C, et al. Neonatal-onset from top to bottom: NG_008057 is the reference sequence for the recurrent Guillain-Barre syndrome-like disease: clues for CD59 gene, and the black boxes represent the exons. The exons inherited CD59 deficiency. Neuropediatrics 2017;48:477–81. of the eight transcription variants deposited in the National Center for Biotechnology Information database are depicted under the 11. Tone M, Walsh LA, Waldmann H. Gene structure of human reference gene. The hatched boxes represent the coding sequence. CD59 and demonstration that discrete mRNAs are generated The numbers indicate the first and the last base pair of the exons by alternative polyadenylation. J Mol Biol 1992;227:971–6. or of the coding sequence, respectively. Numbering is according 12. Holguin MH, Martin CB, Eggett T, Parker CJ. Analysis of to the numbering of the reference gene NG_008057.1. n.a = not the gene that encodes the complement regulatory protein, applicable. membrane inhibitor of reactive lysis (CD59): identification of an alternatively spliced exon and characterization of the transcriptional regulatory regions of the promoter. J Immunol 1996;157:1659–68. Table 2. CD59 alleles 13. International Society of Blood Transfusion, Working Party Red Year of first Cell Immunogenetics and Blood Group Terminology. Names for Allele name DNA Exon† Protein description CD59 (ISBT 035) blood group alleles v1 0 160701 2018; http:// CD59*01N.01 c.146delA 5 p.Asp49Valfs*31 20133 www.isbtweb.org/working-parties/red-cell-immunogenetics- and-blood-group-terminology. CD59*01N.02 c.123delC 5 p.Val42Serfs*38 19904,6 c.361delG 6 not applicable 14. Li XF, Lin FQ, Li JP. Identification of c.238 A>G (p.Arg80Gly) of CD59 blood group gene. Transfusion 2018;58:3033–4. CD59*01N.03 c.266G>A 6 p.Cys89Tyr 20125 To be named c.238A>G 6 p.Arg80Gly 201814 Christof Weinstock, MD (corresponding author), Head of the †Exons were counted as in the reference sequence NG_008057.1. Immunohematology Laboratory, Institute for Clinical Transfusion Medicine and Immunogenetics, German Red Cross Blood Service Baden-Württemberg - Hessen, and University of Ulm, Germany, References Helmholtzstraße 10, 89081 Ulm, Germany, c.weinstock@blutspende. de; Markus Anliker, MD, Deputy Head of the Institute of Medical and 1. Weinstock C, Anliker M, von Zabern I. CD59: a long-known Chemical Laboratory Diagnostics (ZIMCL), University Hospital complement inhibitor has advanced to a blood group system. Immunohematology 2015;31:145–51. Innsbruck, Innsbruck, Austria; and Inge von Zabern, MD, PhD, retired, previously at the Institute for Clinical Transfusion Medicine 2. Anliker M, von Zabern I, Höchsmann B, et al. A new blood group antigen is defined by anti-CD59, detected in a CD59- and Immunogenetics, German Red Cross Blood Service Baden- deficient patient. Transfusion 2014;54:1817–22. Württemberg - Hessen, and University of Ulm, Ulm, Germany. 3. Höchsmann B, Dohna-Schwake C, Kyrieleis HA, Pannicke U, Schrezenmeier H. Targeted therapy with eculizumab for inherited CD59 deficiency. N Engl J Med 2014;370:90–2. 4. Yamashina M, Ueda E, Kinoshita T, et al. Inherited complete deficiency of 20-kilodalton homologous restriction factor (CD59) as a cause of paroxysmal nocturnal hemoglobinuria. N Engl J Med 1990;323:1184–9.

8 IMMUNOHEMATOLOGY, Volume 35, Number 1, 2019 ZZAP treatment of red blood cells

S.I. Marckwardt

ZZAP is a mixture of a sulfhydryl reagent (dithiothreitol) and a Reagents/Supplies proteolytic enzyme (papain or ficin). This reagent dissociates IgG and complement from red blood cells, allowing for phenotyping, Reagents Supplies enhanced adsorption, or denaturing of multiple blood group • Phosphate-buffered saline, • Calibrated serologic centrifuge pH 7.3 system antigens to aid in completing complex antibody workups. • 13 × 100, 16 × 100 mm test ZZAP treatment destroys all antigens in the Kell, Landsteiner- • 0.2 M DTT tubes Wiener, Cartwright, Dombrock, and Knops blood group systems a • 1% cysteine-activated • Transfer pipettes as well as antigens destroyed by proteases (e.g., M, N, S, Fy , and papain or 1% ficin Fyb). Immunohematology 2019;35:9–10. • Agglutination viewer • 37°C water bath Key Words: ZZAP, ficin, papain, DTT DTT = dithiothreitol. SEROLOGIC METHOD REVIEW METHOD SEROLOGIC

Principle Procedural Steps

• Prepare ZZAP. ZZAP is a mixture of a sulfhydryl reagent (dithiothreitol • Add to packed RBCs to be treated. [DTT]) and a proteolytic enzyme (papain or ficin). It was • Incubate at 37°C for 30 minutes. first described by Branch and Petz in their 1982 article in • Wash treated RBCs. the American Journal of Clinical Pathology.1 ZZAP is used to • Remove supernatant saline from packed RBCs or dissociate IgG and complement from red blood cells (RBCs), • Resuspend RBCs to appropriate concentration for testing. an action that neither reagent can achieve alone. According to RBCs = red blood cells. Branch and Petz, it is believed that ZZAP “reduces interchain disulfide linkages, increasing exposure of the IgG polypeptides to the surrounding medium, allowing the proteolytic enzyme increased accessibility to the peptide groups. The IgG molecule all antigens in blood group systems destroyed by ficin/papain may lose integrity and dissociate completely from the RBC or DTT; for example, it can be used to denature all Kell system 1 antigen with which it had reacted.” antigens, essentially creating K0 RBCs. ZZAP can also be used to treat reagent panel cells for use in serologic investigations, Indications particularly those showing panagglutination. By comparing the untreated panel results with ZZAP-treated panel results, ZZAP treatment is useful in several ways. It may eliminate the antibody specificity(ies) may be more rapidly identified.2 spontaneous RBC agglutination, allowing for antigen typing of RBCs with direct agglutinating antisera, i.e., resolving an Procedure ABO discrepancy or performing Kidd typings. Because ZZAP removes IgG from the RBCs, it may also allow for antigen Prepare ZZAP by mixing 0.5 mL of 1 percent cysteine- typing of RBCs using indirect antiglobulin test (IAT) methods activated papain with 2.5 mL of 0.2 M DTT and 2 mL of pH if the antigen is not destroyed by the reagent. Because ZZAP 7.3 phosphate-buffered saline (PBS) or 1.0 mL of 1 percent denatures many antigens determined in a common RBC ficin, 2.5 mL of 0.2 M DTT, and 1.5 mL of pH 7.3 PBS. Add phenotype (K, M, N, S, s, Fya, Fyb), this application has limited 2 volumes of ZZAP to 1 volume of packed RBCs. Incubate value. As a pretreatment before adsorption, ZZAP-treated the ZZAP/RBC mixture at 37°C for 30 minutes, mixing RBCs may remove autoantibody more quickly and completely occasionally. Lastly, wash the RBCs at least three times using than untreated RBCs. large volumes of isotonic saline before preparing the RBCs to Lastly, ZZAP treatment can be helpful in complex the concentration needed for testing.3 antibody investigations. It can be used to create RBCs lacking

IMMUNOHEMATOLOGY, Volume 35, Number 1, 2019 9 S.I. Marckwardt

Limitations • ZZAP treatment to disperse spontaneous agglutination: Test an inert control (6–8% albumin or antibody- ZZAP treatment destroys all antigens in the Kell, negative ABO-compatible plasma) with direct Landsteiner-Wiener, Cartwright, Dombrock, and Knops blood agglutinating antisera to verify that the spontaneous group systems as well as antigens destroyed by proteases (e.g., agglutination is no longer present. M, N, S, Fya, and Fyb).3 Therefore, ZZAP-treated RBCs cannot • ZZAP treatment to destroy a specific antigen: Known be used to antigen-type for these antigens. double-dose antigen-positive RBCs should be tested before and after treatment with the corresponding Quality Control antisera to verify that denaturation occurred.

The enzyme and DTT reagents used in preparation of Acknowledgments ZZAP should be qualified for use during individual preparation. Quality control (QC) of ZZAP-treated RBCs is specific for the Thank you to Jan Hamilton for assistance with this intended use. review. • ZZAP treatment for adsorption: QC is generally not performed before using ZZAP-treated RBCs for References adsorption. However, if an antibody is ruled out based 1. Branch DR, Petz LD. A new reagent (ZZAP) having multiple on the assumption that ZZAP destroyed the antigen applications in immunohematology. Am J Clin Pathol and would leave the antibody in the adsorbed plasma, 1982;78:161–7. then the treated adsorbing RBC must be tested for the 2. Branch DR. Investigation of panagglutinating sera. Transfusion 2010;50:739–40. antigen to prove that it was in fact destroyed [i.e., ruling 3. Fung MK, Ed. Method 4-8: Adsorbing warm-reactive a out anti-Fy after using an Fy(a+)-adsorbing RBC]. autoantibodies using autologous red cells. In: Technical • ZZAP treatment for antigen typing: Treated known manual. 19th ed. Bethesda, MD: American Association of antigen-positive and -negative control RBCs should be Blood Banks, 2017. tested in parallel with treated patient RBCs. Before using CM CM ZZAP-treated RBCs for antigen typing by IAT, a direct Sarah I. Marckwardt, BS, MLS(ASCP) SBB , IRL Technologist III, Immunohematology Reference Laboratory, American Red Cross antiglobulin test (DAT) with a saline control should be Southeastern Michigan Region, 1415 Trumbull Avenue, Detroit, MI performed to ensure that all IgG coating on the RBCs 48216, [email protected]. has been removed and the RBCs are now DAT-negative.

Notice to Readers Attention: State Blood Bank Meeting Organizers All articles published, including communications and book reviews, reflect the opinions of the authors and do If you are planning a state meeting and would like copies of not necessarily reflect the official policy of the American Immunohematology for distribution, please send a request, Red Cross. 4 months in advance, to [email protected].

10 IMMUNOHEMATOLOGY, Volume 35, Number 1, 2019 B l o o d G r o u p R e v i e w An update on the Duffy blood group system

G.M. Meny

This update of the Duffy (FY) blood group system (Meny GM. to increase as the use of molecular methods for blood typing The Duffy blood group system: a review. Immunohematology patients and donors becomes more widespread. The total 2010;26:51–6) includes additional variants to the Duffy system number of variants remains small (<50) when compared with (International Society of Blood Transfusion system 8; five anti- gens) identified through molecular studies. The most interesting the hundreds identified to date in other blood group systems clinical updates, however, include further evaluation of the roles such as Rh.3,4 The practical implications in clinical situations of the Duffy glycoprotein, also known as the atypical chemokine for blood donors and patients carrying these variants has yet receptor 1, in malaria and hematopoiesis. The transition to understanding the important role of blood group antigens in to be described. homeostasis and disease continues. Immunohematology 2019;35:11–12. ACKR1 and Malaria

Key Words: ACKR1, DARC, GATA, Duffy Plasmodium vivax is a major infectious species causing human malaria in Asia and Latin America but is an Introduction uncommon cause of malaria in Africa. Individuals with the Fy(a–b–) phenotype may have a selective advantage in that A description of Duffy (FY) blood group system antigens their red blood cells (RBCs) are resistant to P. vivax invasion. and antibodies and how this information is used in transfusion Liu et al.5 believe that human P. vivax likely arose from within management was featured prominently in the original Duffy the diverse strain of Plasmodium species infecting primates blood group review published in 2010.1 At that time, the in central Africa. They speculate that this ancestral African P. Duffy glycoprotein was also known to bind to a variety of vivax stock infected humans, gorillas, and chimpanzees until chemokines of the CXC and CC classes and was referred to as the GATA-1 [Fy(a–b–)] mutation appeared, which seemed to the Duffy antigen receptor for chemokines (DARC). Ongoing eliminate the infection in humans. research studies of FY, particularly at the molecular level, P. vivax infection was identified in Fy(a–b–) individuals continue to expand our understanding of the important role of in several countries, including Africa.6–9 For example, Menard blood group antigens in homeostasis and disease. Recently, a et al.6 identified clinical P. vivax infection in 4.9 percent of new nomenclature was adopted and approved; DARC is now individuals with the Fy(a–b–) phenotype, including the blood known as atypical chemokine receptor 1 (ACKR1).2 stage development of P. vivax confirmed by microscopic examination of blood smears. P. vivax polymerase chain Molecular Update reaction positivity was noted in 8.8 percent of asymptomatic individuals with the Fy(a–b–) phenotype. Mendes et al.7 As with other blood group systems, the number of showed two different strains of P. vivax infection in 10.9 variants identified in the Duffy blood group system continues percent of mosquitos and in 4.6 percent of individuals with

Table 1. Duffy blood group system variants

Phenotype Allele Nucleotide change Predicted amino acid change Number of other reported variants* Fy(a+) FY*01 125A>G Asp42Gly NA Fy(b+) FY*02 125G>A Gly42Asp NA Fy(a–b–) FY*01N.01 –67T>C;125A>G NA 8 Fy(a–b–) FY*02N.01 – 67T>C NA 5 Fy(a+w) FY*01W.01 125A>G;265C>T Asp42Gly;Arg89Cys 2 Fy(b+w) FY*02W.01 265C>T;298G>A Arg89Cys;Ala100Thr 3 *Table modified from Moller et al.3 Only variants approved by the International Society of Blood Transfusion are included. NA = not applicable.

IMMUNOHEMATOLOGY, Volume 35, Number 1, 2019 11 G.M. Meny

the Fy(a–b–) phenotype. Originally, Miller et al.10 concluded 4. Hoher G, Fiegenbaum M, Almeida S. Molecular basis of the that P. vivax (and Plasmodium knowlesi) invades RBCs by Duffy blood group system. Blood Transfus 2018;16:93–100. interacting with receptors on the RBCs, and Duffy antigens 5. Liu W, Li Y, Shaw KS, et al. African origin of the malaria parasite Plasmodium vivax. Nat Commun 2014;5:3346. were those receptors. However, P. vivax erythrocyte receptors 6. Menard D, Barnadas C, Bouchier C, et al. Plasmodium vivax involved in the invasion of Fy(a–b–) RBCs have yet to be clinical malaria is commonly observed in Duffy-negative identified.11 Malagasy people. Proc Natl Acad Sci U S A 2010;107:329–31. There is currently no effective vaccine forP. vivax, with 7. Mendes C, Dias F, Figueiredo J, et al. Duffy negative antigen is no longer a barrier to Plasmodium vivax: molecular evidences limited hope on the horizon as vaccine development remains from the African West Coast (Angola and Equatorial Guinea). in pre-clinical stages.12,13 PLoS Negl Trop Dis 2011;5:e1192. 8. Ngassa Mbenda HG, Das A. Molecular evidence of Plasmodium ACKR1 and Hematopoiesis vivax mono and mixed malaria parasite infections in Duffy- negative native Cameroonians. PLoS One 2014;9:e103262. 9. Abdelraheem MH, Albsheer MM, Mohamed HS, Amin M, Individuals of African ancestry can have neutropenia Abdel Hamid MM. Transmission of Plasmodium vivax in under physiological conditions, which is linked to a variant Duffy-negative individuals in central Sudan. Trans R Soc Trop Med Hyg 2016;110:258–60. of ACKR1 that also results in the Fy(a–b–) phenotype on 10. Miller LH, Mason SJ, Clyde DF, McGinniss MH. The resistance 14 RBCs. Duchene et al. examined the role that ACKR1 played factor to Plasmodium vivax in blacks. The Duffy-blood-group in murine hematopoiesis and found that nucleated erythroid genotype, FyFy. N Engl J Med 1976;295:302–4. cells highly express ACKR1, which facilitates contact with 11. Gunalan K, Lo E, Hostetler JB, et al. Role of Plasmodium vivax Duffy-binding protein 1in invasion of Duffy null Africans. Proc hematopoietic stem cells. The absence of erythroid ACKR1 Natl Acad Sci USA 2016;113:6271–6. ultimately gave rise to phenotypically distinct neutrophils, 12. Mueller I, Shakri AR, Chitnis CE. Development of vaccines for which left the circulation. It is possible that this phenomenon Plasmodium vivax malaria. Vaccine 2015;33:7489–95. provides a selective advantage to Duffy negative individuals by 13. Tham WH, Beeson JG, Rayner JC. Plasmodium vivax vaccine enhancing innate responses to invading microbial pathogens. research: we’ve only just begun. Int J Parasitol 2017;47:111–8. 14. Duchene J, Novitzky-Basso I, Thiriot A, et al. Atypical chemokine receptor 1 on nucleated erythroid cells regulates References hematopoiesis. Nat Immunol 2017;18:753–61.

1. Meny GM. The Duffy blood group system: a review. Immunohematology 2010;26:51–6. Geralyn M. Meny, MD, MS, Director, Medical Affairs, Diagnostics 2. Bachelerie F, Ben-Baruch A, Burkhardt AM, et al. International USA, Grifols Diagnostics, 201 Carlson Circle, San Marcos, TX 78666, Union of Basic and Clinical Pharmacology. [corrected]. [email protected]. LXXXIX. Update on the extended family of chemokine receptors and introducing a new nomenclature for atypical chemokine receptors. Pharmacol Rev 201366:1–79. 3. Moller M, Joud M, Storry JR, Olsson ML. Erythrogene: a database for in-depth analysis of the extensive variation in 36 blood group systems in the 1000 Genomes Project. Blood Adv 2016;1:240–9.

12 IMMUNOHEMATOLOGY, Volume 35, Number 1, 2019 R e v i e w Serologic problems associated with administration of intravenous immune globulin (IVIg)

D.R. Branch

Intravenous immune globulin (IVIg) is manufactured from autoimmune and inflammatory diseases.9,10 IVIg has now large pools of donor plasma and contains a high diversity of been approved by the U.S. Food and Drug Administration for antibodies, primarily IgG. For this reason, IVIg is routinely used use in ITP, Kawasaki disease (where it is most effective at an as antibody replacement therapy for patients having primary immunodeficiencies. In 1981, IVIg was also found to be a strong even higher dose of 4 g/kg), as well as chronic inflammatory immunomodulator of various inflammatory and autoimmune demyelinating polyradiculoneuropathy and other neurologic conditions. This observation has led to the exponential increase disorders. There are many other conditions where IVIg has in the use of IVIg throughout the world, with the United States 11 and Canada being the biggest users of IVIg. Although relatively been used “off label.” rare, adverse events, such as hemolytic anemia and thrombosis, Because IVIg is produced from donor plasma, the final can complicate the administration of IVIg. More frequently, product contains a great diversity of IgG antibodies, some of the administration of IVIg can cause serologic challenges for which may cause clinical complications. Anti-A and/or anti-B the transfusion service including ABO discrepancies, positive direct antiglobulin tests, positive antibody detection tests, and antibodies present in IVIg can cause immune-mediated incompatible crossmatches. This article will review each of the hemolytic anemia in non–group O patients, a finding potential transfusion service challenges associated with IVIg reported to be about 34 percent of patients receiving IVIg administration. Immunohematology 2019;35:13–15. in one prospective study.12 The hemolysis can occasionally be severe, even life-threatening.12–16 Some manufacturers Key Words: IVIg, ABO discrepancies, DAT, isoagglutinins, have removed the majority of ABO isoagglutinins from IVIg alloantibodies by a chromatographic step in their processing, which may limit the risk of hemolytic anemia.13,17 Thrombosis can also Introduction result from IVIg infusion, but this is quite rare compared with IVIg-associated hemolysis.18 In addition to adverse Preparations of intravenous immune globulin (intravenous events, administration of IVIg can create challenges for the gamma globulin; IVIg) are produced from large pools of donor transfusion service including ABO discrepancies, positive plasma. One lot of IVIg is produced by pooling the plasma of direct antiglobulin tests (DATs), positive antibody detection up to 60,000 donors.1,2 IVIg was first produced in the 1970s tests, and incompatible crossmatches. after the development of the cold alcohol fractionation process (Cohn fractionation) of antibodies from pooled plasma; the Reverse ABO Grouping final product contains mostly IgG.3–6 The original use for IVIg was as antibody replacement for patients, mostly children, Because up to 4 g/kg of IVIg may be given to an individual with primary immunodeficiency.3,5,6 In 1981,7 while treating with blood group A, B, or AB, these patients will often have children with primary immunodeficiency and concomitant circulating anti-A and anti-B due to passive transfer. In ABO thrombocytopenia, Paul Imbach observed that platelet testing, these antibodies can interfere with an accurate reverse counts increased after the administration of IVIg. Imbach plasma/serum grouping. Because these passively transfused suggested that IVIg might be useful for the treatment of antibodies are IgG, they may not directly agglutinate the immune thrombocytopenia (ITP); this hypothesis was quickly reverse typing cells. However, because of the high antigen site supported by a number of studies.8 The efficacy of high- density of ABO antigens,13 it is possible that they could cause dose (1 to 2 g/kg) IVIg use in ITP led other investigators to some direct agglutination. show that IVIg can effectively ameliorate a number of other

IMMUNOHEMATOLOGY, Volume 35, Number 1, 2019 13 D.R. Branch

When trying to resolve an ABO discrepancy between these antibodies may suggest the presence of an alloantibody forward and reverse typing where unexpected antibodies when the patient’s RBCs are antigen negative. Passive transfer are detected, it should be determined whether the patient has of antibodies should be suspected if the patient has received recently received high-dose IVIg. If so, the discrepancy is most high-dose IVIg and has no history of sensitization, transfusion, likely due to the passive transfer of isoagglutinins. Repeating or pregnancies. the reverse typing several days after the last IVIg dose should provide accurate reverse grouping results. Additionally, if a Summary group B individual receiving high-dose IVIg has an ABO cell/ plasma discrepancy and anti-A cannot be demonstrated by the IVIg given at high doses is an effective therapy for assorted manufacturers’ recommended direct agglutination method autoimmune/inflammatory conditions, and its clinical use (where the result may suggest a subtype), then do not test the will continue to increase. Unfortunately, serologic problems in plasma against group A1 or B red blood cells (RBCs) using the the blood bank can arise due to passive transfer of antibodies indirect antiglobulin test (IAT). Positive reactions could be contained in IVIg. This review has addressed these potential seen with both cells due to the circulating IVIg instead of the problems and proposed ways to resolve them. true ABO antibody. A similar caveat must be heeded with a group A individual. References

1. Barahona Afonso AF, Joao CM. The production processes and Direct Antiglobulin Test biological effects of intravenous immunoglobulin. Biomolecules 2016;6:15. When group A, B, or AB patients receive high-dose IVIg, 2. Buchacher A, Iberer G. Purification of intravenous immunoglobulin G from human plasma—aspects of yield and the IgG isoagglutinins can bind to the patients’ RBCs in vivo virus safety. Biotechnol J 2006;1:148–63. 15 and result in a positive DAT due to IgG sensitization. The DAT 3. Eibl MM. History of immunoglobulin replacement. Immunol strength of reactivity can be as strong as 3+.19 An eluate would Allergy Clin North Am 2008;28:737–64. be nonreactive with group O screening cells but positive with 4. Greenbaum BH. Differences in immunoglobulin preparations for intravenous use: a comparison of six products. Am J Pediatr A or B reverse typing cells by IAT depending on the patient’s 1 Hematol Oncol 1990;12:490–6. ABO type. When a group A, B, or AB patient is found to have 5. Branch DR. Unraveling the IVIG mystique. Transfusion a positive DAT with an eluate that demonstrates isoagglutinin 2013;53:242–4. reactivity, determine whether the patient has received high- 6. Schroeder HW Jr, Dougherty CJ. Review of intravenous dose IVIg therapy within the last 3–5 days. The clinical team immunoglobulin replacement therapy trials for primary humoral immunodeficiency patients. Infection 2012;40: of such a patient should be alerted to closely watch the patient 601–11. for development of a hemolytic event.15,16,19 7. Imbach P, Barandun S, d’Apuzzo V, et al. High dose intravenous Rarely, IVIg may contain RBC alloantibodies to antigens immunoglobulin for idiopathic thrombocytopenic purpura in childhood. Lancet 1981;1:1228–31. other than A or B.20,21 If IVIg is infused in a patient having the 8. Imbach P, Morell A. Idiopathic thrombocytopenic purpura corresponding antigen, this may result in a positive IgG DAT. (ITP): immunomodulation by intravenous immunoglobulin An eluate may reveal an antibody that would resemble an (IVIg). Int Rev Immunol 1989;5:181–8. autoantibody, but with a common specificity, such as anti-D 9. Schwartz SA. Intravenous immunoglobulin (IVIG) for the or anti-C.22–35 Rarely, passive antibodies to antigens other therapy of autoimmune disorders. J Clin Immunol 1990;10: 81–9. 14,35 than A or B can cause hemolytic anemia. Reports of IVIg 10. Chipps E, Skinner C. Intravenous immunoglobulin: containing antibodies to Rh or other RBC antigens are now less implications for use in the neurological patient. J Neurosci common due to more stringent manufacturing standards.23 Nurs 1994;26:8–17. 11. Perez EE, Orange JS, Bonilla F, et al. Update on the use of immunoglobulin in human disease: a review of evidence. J Unexpected Alloantibodies in Antibody Screening Allergy Clin Immunol 2017;139:S1–S46. 12. Pendergrast J, Binnington B, Tong TN, et al. Incidence and risk As mentioned earlier, some preparations of IVIg may factors for IVIG-mediated hemolysis. Blood 2017;130:2398. contain antibodies to RBC antigens such as D, C, or K.22–35 Such 13. Branch DR. Anti-A and anti-B: what are they and where do they come from? Transfusion 2015;55(Suppl 2):S74–9. antibodies could be detected in antibody detection tests and/or result in incompatible crossmatches. Indeed, the detection of

14 IMMUNOHEMATOLOGY, Volume 35, Number 1, 2019 Challenges associated with IVIg

14. Quinti I, Pulvirenti F, Milito C, et al. Hemolysis in patients 26. Turner CE, Thorpe SJ, Brasher MD, Thorpe R. Anti-Rh with antibody deficiencies on immunoglobulin replacement D activity of commercial intravenous immunoglobulin treatment. Transfusion 2015;55:1067–74. preparations. Vox Sang 1999;76:55–8. 15. Michelis FV, Branch DR, Scovell I, et al. Acute hemolysis 27. Garratty G. Problems associated with passively transfused after intravenous immunoglobulin amid host factors of ABO- blood group alloantibodies. Am J Clin Pathol 1998;109: mismatched bone marrow transplantation, inflammation, 769 –77. and activated mononuclear phagocytes. Transfusion 2014;54: 28. Lucas GS, Jobbins K, Bloom AL. Intravenous immunoglobulin 681–90. and blood-group antibodies. Lancet 1987;2:742. 16. Branch DR, Hellberg A, Bruggeman CA, et al. ABO zygosity, 29. Romer J, Morgenthaler J-J, Scherz R, Skvaril F. but not secretor or Fc status, is a significant risk factor for Characterization of various immunoglobulin preparations for IVIG-associated hemolysis. Blood 2018;131:830–5. intravenous application I: protein composition and antibody 17. Hoefferer L, Glauser I, Gaida A, et al. Isoagglutinin reduction content. Vox Sang 1982;42:62–73. by dedicated immunoaffinity chromatography step in the 30. Kluge A, Dopfer R, Pfeiffer-Wolf I, Roelcke D. Immunoglobulin manufacturing process of human immunoglobulin products. high-dose therapy: RBC-alloantibodies in commercial Transfusion 2015;55(Suppl 2):S117–21. preparations and haemolytic anaemia: a case report. Beitr 18. Funk MB, Gross N, Gross S, et al. Thromboembolic events Infusionsther Transfusionsmed 1994;32:474–5. associated with immunoglobulin treatment. Vox Sang 2013; 31. Lichtiger B, Rogge K. Spurious serologic test restuls in patients 105:54–64. receiving infusions of intravenous immune gammaglobulin. 19. Puga Yung G, Seebach JD, Baerenzung N, Pendergrast J, Arch Pathol Lab Med 1991;115:467–9. Cserti-Gazdewich C, Branch DR. IgG subclasses determined 32. Garcia L, Huh YO, Fischer HE, Lichtiger B. Postivie from eluates of DAT positive patients after high-dose IVIg immunohematologic and serologic test results due to high dose therapy do not predict hemolysis and are primarily of the IgG2 intravenous immune globulin administration. Transfusion subclass. Transfusion (in press). 1987;27:503. 20. Moscow JA, Casper AJ, Kodis C, Fricke WA. Positive direct 33. Steiner EA, Butch SH, Carey JL, Oberman HA. Passive antiglobulin test results after intravenous immune globulin anti-D from intravenous immune serum globulin. Transfusion administration. Transfusion 1987;27:248–9. 1983;23:363. 21. Robertson VM, Dickson LG, Romond EH, Ash RC. Positive 34. Hoppe I. Antibody screening of commercially available antiglobulin tests due to intravenous immunoglobulin in immunoglobulins. erythrocyte-, HLA- and autoantibodies. patients who received bone marrow transplant. Transfusion Blut 1979;39:9–16. 1987;27:28–31. 35. Lang GE, Veldhuis B. Immune serum globulin—a cause for anti- 22. Whitsett CF, Pierce JA, Daffin LE. Positive direct antiglobulin Rh(D) passive sensitization. Am J Clin Pathol 1973;60:205–7. tests associated with intravenous gamma globulin use in bone marrow transplant recipients. Transplantation 1986;41: 663–4. Donald R. Branch, PhD, Professor, Departments of Medicine and 23. Thorpe SJ, Fox B, Heath A, Behr-Gross M-E, Virata ML, Yu Laboratory Medicine and Pathobiology, University of Toronto, M-Y. International collaborative study to assess candidate Division of Experimental Therapeutics, Toronto General Hospital reference preparations to control the level of anti-D in IVIG Research Institute, University Health Network, and the Centre for for use in Europe and the United States. Biologicals 2006;34: Innovation, Canadian Blood Services, 67 College Street, Toronto, 209–12. Ontario M5G 2M1, Canada, [email protected]. 24. Sakem B, Matozan K, Nydegger UE, Weigel G, Griesmacher A, Risch L. Anti-red blood cell antibodies, free light chains, and antiphospholipid antibodies in intravenous immunoglobulin preparations. Isr Med Assoc J 2013;15:617–21. 25. Thorpe SJ, Fox BJ, Dolman CD, Lawrence J, Thorpe R. Batches of intravenous immunoglobulin associated with adverse reactions in recipients contain atypically high anti-Rh D activity. Vox Sang 2003;85:80–4.

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IMMUNOHEMATOLOGY, Volume 35, Number 1, 2019 15 B l o o d G r o u p R e v i e w An update on the Knops blood group system

J.M. Moulds

This update of the Knops (KN) blood group system (Moulds (PfRhs). Serologists are well aware that the proteins carrying JM. The Knops blood group system. Immunohematology MNS and Ge are involved in parasite invasion. Therefore, it 2010;26:2–7) adds no new antigens to this system (International should not come as a surprise that the Knops protein (i.e., CR1) Society of Blood Transfusion system 22), which currently has nine antigens. However, the molecular basis of York, KN5, or is also an invasion ligand, but for the sialic acid–independent Yka has been identified as c.4223C>T and designatedKN*01.- pathway.4,5 The binding site has been localized to SCR1, and 05. Although not considered clinically significant in the field of the Knops antigens in SCR25 do not appear to have any effect transfusion medicine, there has been great interest in the Knops 6,7 polymorphism by investigators working on malaria documented on binding. What does appear to be important is erythrocyte by numerous studies over the past 8 years. Immunohematology CR1 copy number (E-CR1). 2019;35:16–18. Low E-CR1 (Helgeson Phenotype) Key Words: Knops, blood group antigens, malaria, PfRh4 The serological null in the Knops system is known as the Helgeson phenotype and most often results from low York Antigen expression of E-CR1. Pham et al.8 suggested that the Helgeson phenotype may also be due to the lack of a high-prevalence York (Yk a) was assigned when the Knops (KN) blood group KN antigen. Patients with anti-KCAM often have the Helgeson system (system 22) was established.1 The number given for the phenotype (J.M.M., unpublished data). There is a genetically Yk(a–) phenotype was KN:–5, and the allele was designated controlled expression of E-CR1 that can be detected by as KN*01.-05. Although the molecular mechanism for the Southern blot or polymerase chain reaction–restriction more well-known high-prevalence antigens (e.g., Kna, McCa, fragment-length polymorphism methods. In individuals of and Sl1) were identified fairly rapidly, that for Yka was elusive. European descent, there is good correlation with a HindIII In 2011, Veldhuisen et al.2 identified a mutation at c.4223C>T polymorphism (Q981H), but this correlation has not been well that resulted in the change of threonine to methionine at investigated in people of African descent. amino acid position 1408 in Yk(a–) samples. It is interesting In studies of parasite rosetting, erythrocytes with low to note that the Yka mutation is found in exon 26, which is at E-CR1 formed fewer and less stable rosettes. The clinical role the beginning of a long homologous repeat (LHR-D), while all for low E-CR1 in malaria was first observed in Papua New the other known Knops polymorphisms are located in exon Guinea when Cockburn et al.9 found that reduced rosetting 29. Recently, Kretzschmar et al.3 found that Yk(a–) examples protected children from severe malaria. In addition to the (p.1408Met) resulted in a twofold increase in susceptibility association with rosetting, the Helgeson phenotype appears to leprosy. They postulated that this change altered the CR1 to also result in reduced merozoite invasion thru PfRh4.6,7 conformation, which adversely affected a terminal cleavage site Numerous anti-CR1 monoclonal antibodies exist as well as leaving more CR1 on the cell for attachment to the invading recombinant soluble CR1 (sCR1).10 Both have been shown to Mycobacterium. inhibit invasion and may be considered for therapeutic use in the future. Because only the terminal portion of CR1 (SCR1) Knops and Malaria is needed for inhibition, transfusion of this product would not cause alloimmunization of patients to Knops, since these CR1 as an Invasion Receptor antigens are in SCR25. It has been known for some time that Plasmodium species use multiple red blood cell (RBC) ligands to gain entry into Sl, McCoy, and KCAM the erythrocyte. P. falciparum merozoites have two major Serologists knew for years that Sl2, namely Sl(a–), groups of proteins used in invasion: erythrocyte-binding– and McC(b+) were more common among individuals of like antigens and reticulocyte-binding–like homolog proteins African descent. Because of these known differences, it was

16 IMMUNOHEMATOLOGY, Volume 35, Number 1, 2019 Update on KN

hypothesized that the Knops blood group polymorphisms References might be important in malaria. Numerous studies have now 1. Moulds JM. The Knops blood group system. been performed throughout Africa as well as South America Immunohematology 2010;26:2–7. and Asia.11–13 All have shown that Sl2 and McC(b+) are found 2. Veldhuisen B, Legthart PC, Vidarsson G, et al. Molecular with the highest prevalence among people of African descent; analysis of the York antigen of the Knops blood group system. Transfusion 2011;51:1389–96. however, their exact role in malaria remains controversial. 3. Kretzschmar GC, Oliveira LC, Nisihara RN, et al. Complement Of note, KCAM-negative examples occur in approximately receptor 1 (CR1, CD35) association with susceptibility to 80 percent of people of African descent, but there have been leprosy. PLoS Negl Trop Dis 2018;8:e0006705. few studies of its impact on malaria susceptibility or disease 4. Spadafora C, Awandare GA, Kopydlowski KM, et al. Complement receptor 1 is a sialic acid-independent progression.14 erythrocyte receptor of Plasmodium falciparum. PLoS Pathog Two conflicting reports have been published from Ghana. 2010;6:e1000968. The first study of 150 children found that McC(a–b+) was 5. Wai-Hong T, Wilson DW, Lopaticki S, et al. Complement associated with a reduced risk of severe malaria.14 A slightly receptor 1 is the host erythrocyte receptor for Plasmodium falciparum PfRh4 invasion ligand. PNAS 2010;107:17327–32. larger study of 267 patients found no association with Knops 6. Tham W-H, Schmidt CQ, Hauhart RE, et al. Plasmodium 15 antigens, however. A very large study of over 4000 case and falciparum uses a key functional site in complement control subjects in Kenya16 found Sl2 associated with decreased receptor type-1 for invasion of human erythrocytes. Blood odds of cerebral malaria and death when α-thalassemia genes 2011;118:1923–33. 7. Park HJ, Guariento M, Maciejewski M, et al. Using were present, although McCb was associated with increased mutagenesis and structural biology to map the binding site for odds. Because of the variability in performing research using the Plasmodium falciparum merozoite protein PfRh4 on the human samples, some investigators have used recombinant human immune adherence receptor. J Biol Chem 2014;289: 45–63. soluble CR1 fragments for invasion and rosetting studies and b 17 8. Pham BN, Kisserli A, Donvito B, et al. Analysis of complement found no association with Sl2 or McC . receptor type 1 expression on red blood cells in negative phenotype of the Knops blood group system, according to CR1 Conclusions gene allotype polymorphisms. Transfusion 2010;50:1435–43. 9. Cockburn IA, Mackinnon MJ, O’Donnell A, et al. A human complement receptor one polymorphism that reduces The role of CR1 and the Knops blood group in malaria Plasmodium falciparum rosetting confers protection against remains unanswered in this author’s opinion. A major problem severe malaria. PNAS 2004;101:272–7. is that many of the studies have used only molecular methods to 10. Lim NT, Harder MJ, Kennedy AT, et al. Characterization a/b of inhibitors and monoclonal antibodies that modulate the assess the role of Sl1/2 and McC . Without RBC phenotyping, interaction between Plasmodium falciparum adhesin PfRh4 it cannot be assured that these genes are expressed and, with its erythrocyte receptor complement receptor 1. J Biol furthermore, even when expressed, they may be present in low Chem 2015;290:25307–21. E-CR1 copy numbers. There is good evidence that low E-CR1 11. Covas DT, De Oliveira FS, Rodriques ES, et al. Knops blood group haplotypes among distinct Brazilian populations. (i.e., the Helgeson phenotype) is protective, since it results in Transfusion 2006;47:147–53. lower rates of parasite invasion as well as a lower number of 12. Sandri LT, Adukpo S, Giang DP, Nguetse CN, Antunes Andrade rosettes. The role of the CR1 blood group polymorphism is just F. Geographical distribution of complement receptor 1 variants now being investigated in other diseases such as tuberculosis, and their associated disease risk. PLoS One 2017;12:e175973. 3,18 13. Fontes AN, Kashima S, Bonin-Silva R, et al. Association leprosy, and Alzheimer’s disease. Clearly, the Knops system between Knops blood group polymorphisms and susceptibility will remain a point of interest for many more years. to malaria in an endemic area of the Brazilian Amazon. Genet Mol Biol 2011;34:539–45.

IMMUNOHEMATOLOGY, Volume 35, Number 1, 2019 17 J.M. Moulds

14. Tettey R, Ayeh-KUmi P, Tettey P, et al. Severity of malaria in 18. Mahmoudi R, Feldman S, Kisserli A, et al. Inherited and relation to a complement receptor 1 polymorphism: a case- acquired decreases in complement receptor one (CR1) density control study. Pathog Glob Health 2015;109:247–52. on red blood cells associated with high levels of soluble CR1 in 15. Hansson HH, Kurtzhals JA, Goka BQ, et al. Human genetic Alzheimer’s disease. Int J Mol Sci 2018;8:2175. polymorphisms in the Knops blood group are not associated with a protective advantage against Plasmodium falciparum Joann M. Moulds, PhD, MT(ASCP)SBB, Immunohematology malaria in Southern Ghana. Malaria J 2013;12:400. Consultant, 127 Wynnpage Drive, Dripping Springs, TX 78620, 16. Opi DH, Swann O Macharia A, et al. Two complement receptor [email protected]. one alleles have opposing associations with cerebral malaria and interact with α+ thalassemia. eLife 2018;7:e31579. 17. Tetteh-Quarcoo, Schmidt Q, Tham WH, et al. Lack of evidence from studies with soluble protein fragments that the Knops blood group polymorphism in complement receptor-type one are driven by malaria. PLoS One 2012;7:e34820.

For information concerning the National Reference Immunohematology is on the Web! Laboratory for Blood Group Serology, including the American Rare Donor Program, contact Sandra Nance, by phone at www.redcrossblood.org/hospitals/immunohematology (215) 451-4362, by fax at (215) 451-2538, or by e-mail at [email protected]. For more information, send an e-mail to [email protected].

18 IMMUNOHEMATOLOGY, Volume 35, Number 1, 2019 Inhibition of blood group antibodies by soluble substances

K.M. Byrne, C.M.C. Mercado, T.N. Nnabue, T.D. Paige, and W.A. Flegel

The presence of multiple alloantibodies or an antibody to a high- Reagents/Supplies prevalance antigen in a patient sample can pose challenges in antibody identification. The pattern of reactivity seen on an Reagents Supplies antibody panel may show various strengths of reactivity by • Reactive blood sample (plasma or • Test tubes (gel cards different methods of testing or same strength of reactivity at serum) if that method is used) one or more phases of testing. To ensure proper identification, • Soluble substance (antigen, natural, or multiple investigative tools may be used. We review one of synthetic) • Pipettes these methods—inhibition by soluble substances—which has • Inert substance, such as albumin or • 37°C incubator become an expansion of our toolbox within the past 10 years. saline • Centrifuge REVIEW METHOD SEROLOGIC Alloantibodies can be inhibited using specific soluble substances. • Other reagents as appropriate, such as These soluble substances occur naturally in various fluids or anti-IgG, IgG-coated RBCs can be manufactured. When a patient sample contains multiple RBCs = red blood cells. antibodies, clinically significant or not, inhibition of one may help determine specificities of others. Specific inhibition of a particular antibody will also help to confirm its presence. Procedural Steps Immunohematology 2019;35:19–22. • Prepare soluble substance for use. • Mix soluble substance with test sample. Key Words: soluble blood group substance, dilution control, • Prepare a dilution control containing equal volumes of test sample neutralization, soluble peptide, inhibition, recombinant blood and an inert substance, such as albumin or saline. • Incubate at optimal temperature and time. group proteins • Test the sample plus the substance and the sample plus the inert substance (dilution control) against RBCs that previously gave a Background positive reaction. RBCs = red blood cells. When performing pretransfusion testing, serologic results may indicate the presence of one or more alloantibodies. There are many methods that can be used to identify and to the detection of underlying alloantibodies.5–8 Unlike tradi- separate specificities.1 One such method is based on the tional soluble substances that are found naturally in human principle of inhibition. The ability to specifically inhibit one and other animal sources, rBGPs are manufactured.5–7 The antibody may help identify that antibody and allow other result of the manufacturing is a very specific rBGP that could antibody specificities to also be identified. Inhibition can aid aid in antibody identification.7 Gone are the days when the only in the identification of an antibody to an antigen that shows tools available to the investigational immunohematologist were variable expression among individuals, such as anti-P1. Some RBCs, polyclonal antibodies, lectins, and natural inhibitory antibodies can be inhibited by soluble substances such as substances.9 Recombinant peptides and proteins represent the sugars, proteins, and peptides; examples include ABH, Lewis, latest addition to our growing toolbox. P1, Sda, Chido/Rodgers, and ID. Human saliva, hydatid cyst fluid, pigeon egg white, human or guinea pig urine, human Principle serum, and human milk have been used as soluble substances to inhibit red blood cell (RBC) antibodies before the 1990s.1–4 Inhibition takes place when plasma or serum containing Since then, recombinant blood group proteins (rBGPs) an antibody is incubated with a soluble substance (natural or have also been shown to be effective in the identification of synthentic) of corresponding specificity. Subsequent testing antibodies to high-prevelance antigens that are single pass (hemagglutination) reveals the lack of reactivity with RBCs and glycosylphosphatidylinositol-linked proteins, thus leading that tested positive before inhibition. Other antibodies, if

IMMUNOHEMATOLOGY, Volume 35, Number 1, 2019 19 K.M. Byrne et al.

present, in treated plasma or serum should remain unaffected treated with monoclonal antibodies. Besides the surface and can be identified. The principle of this reaction is based proteins on the target cells, some of this novel drug binds on the inhibition of the antibody by the corresponding soluble to RBCs. Examples are anti-CD3814,15 and anti-CD4716 protein. monoclonal antibody therapy. Both drugs can be inhibited by recombinant soluble proteins, CD3817 and CD47 (unpublished Indications results), although their high titers in patients may preclude the effective inhibition in neat plasma samples. A recent Inhibition of blood group antibodies by soluble substances article by Velliquette et al.18 evaluated anti-CD47 (Hu5F9-G4) can aid in the identification of specific antibodies. Antibody interference in pretransfusion testing and offered mitigation activity of known specificity can be selectively “removed” strategies. Covering the target blood group antigens on the by using the inhibition method, thus leaving behind other RBC surface is another approach. This novel and attractive antibodies to be identified. Other indications for inhibition are alternative will add to our toolbox and should eventually to determine ABH secretor status and immunoglobulin class become the topic of another review. of anti-A and/or anti-B. Inhibition using rBGP has also helped classify a new blood group antigen, CD59.6 One may consider Procedure using rBGPs based on serological clues of the specimen and the availability and specificity of the rBGP. Obtain plasma or serum sample for testing. If necessary, process the substance (Table 2). Once ready for testing, the Soluble Substances most common procedure starts with labeling two test tubes: one for the sample and one for the dilution control. Combine One must determine the appropriate soluble substance test sample and soluble substance into the tube labeled to use. Today, the choices are many (Table 1). Blood group “sample.” To the tube labeled “dilution control,” combine test substances in water-soluble form in tissue fluids and secretions sample and inert substance. Incubate both tubes for a specific of the body have been known since the 1930s.10 Agglutination time and temperature determined by the known “ideal” for inhibition tests using A and B substances and boiled saliva were the target specificity. After incubation, test samples against being used as early as 1940.11,12 In 1996, soluble CR1 produced previously reactive RBCs selected by phenotype. Inhibition by recombinant DNA techniques was used to identify Knops has occurred when the “sample” is nonreactive and the system antibodies.13 “dilution control” is still reactive. These results confirm The latest development is the use of soluble proteins to that inhibition of antibody has taken place and the lack of inhibit drugs in the plasma and serum of patients who are reactivity was not caused by dilution. If the “dilution control”

Table 1. Known soluble substances and their use

Blood group system ISBT number Antibody inhibited Immmunoglobulin class Soluble substance ABO 001 Anti-A; anti-B IgM; IgG Human saliva (secretor) H 018 Anti-H IgM Human saliva (secretor) Lewis 007 Anti-Lea; anti-Leb IgM Human saliva (secretor) P1PK 003 Anti-P1 IgM Hydatid cyst fluid, pigeon egg albumin 901 series 901 Anti-Sda IgM>IgG Human urine or saliva, guinea pig urine Chido/Rodgers 017 Anti-Ch; anti-Rg IgG Pooled plasma Cromer 021 Most Cromer antibodies IgG Human serum, plasma, or concentrated urine Scianna 013 Anti-Sc1 (Seltsam et al.5) IgG Recombinant protein CD59, Lutheran, Yt, Dombrock, Chido/ 035, 005, 011, 014, See Anliker et al.6 and Usually IgG Soluble recombinant Rodgers, Cromer, Knops, JMH 017, 021, 022, 026 Seltsam et al.7 proteins from eukaryotic expression systems ISBT = International Society of Blood Transfusion.

20 IMMUNOHEMATOLOGY, Volume 35, Number 1, 2019 Inhibition of blood group antibodies

Table 2. Preparation of soluble substances Quality Control

Source material Preparation Guinea pig urine After collection of urine, boil for 10 minutes. The dilution control containing the sample plus inert Dialyze for 48 h at 4°C against multiple changes substance should result in a positive reaction when tested of pH 7.4 PBS. Centrifuge, aliquot, and freeze.19 against an RBC positive for the corresponding antigen to the Human milk Collect milk from lactating women, centrifuge at 1000g (serofuge at 3400 rpm) for 10 minutes, antibody under investigation. The lack of reactivity in the remove and discard the cream layer, incubate dilution control indicates dilution of weakly reactive low-titer milk in boiling water for 10 minutes, mix 1 volume of milk with 1 volume of PBS. Aliquot and antibody and invalidates the test. freeze.2 Human saliva Collect 2 mL saliva, boil for 10 minutes, Precautions centrifuge at 1000g (serofuge at 3400 rpm) for 10 minutes, harvest supernate, aliquot, and freeze.2 Use caution when using any of these soluble substances. Human urine Collect urine from three individuals, pool, Follow the procedure as written and test using the centrifuge, dilute with equal volume of distilled water, check pH (dialysis with PBS may be recommended test system, such as tube or gel method. For needed to obtain pH between 6 and 8.5), example, if one chooses to change from testing in tube to gel aliquot, and store frozen until needed.2 method, the nonstandard test method should be validated Hydatid cyst fluid Incubate HCF (with scolices) from animal or human sources at 56°C for 1 hour. Dilute 1 before use. Immunoglobulin class (IgG versus IgM) should be volume of hydatid cyst fluid with 9 volumes of considered when evaluating an unexpected result, such as a PBS. Aliquot and freeze.2 false positive. Pigeon egg albumin Separate egg white from the yolk. Prepare dilutions of 1:100 to 1:1000 in PBS. Test the dilutions using a potent anti-P1 to determine the Summary best dilution for inhibition studies. Make aliquots of appropriate dilution and freeze.2 Pooled plasma Purchase commercial pooled plasma or prepare Inhibition has proven to be useful in separating, pooled plasma from six or more donors. Aliquot identifying, and detecting alloantibodies that may be present and freeze.3 in a patient’s sample. Traditional sources (saliva, plasma, and Recombinant protein May be commercially available.5–7,15 urine) of soluble substances and anti-drug proteins can be PBS = phosphate-buffered saline. used to inhibit antibodies, allowing for their detection and identification. The use of rBGPs has expanded our ability to is nonreactive, the test is invalid. The hemagglutination test inhibit a greater number of antibody specificities and can can be done in tubes as described here or by other methods be used in different assays to detect and identify distinct such as column agglutination or solid phase if recommended antibodies. by the manufacturer or validated in-house. Acknowledgments Limitations We thank Debrean A. Loy, Marina U. Bueno, and Kshitij A positive reaction when testing the sample plus soluble Srivastava for providing the inhibition of anti-CD47 by soluble substance may indicate that additional alloantibodies are protein (unpublished results). present in the sample. Testing additional RBCs using the This article was authored as a group writing assignment sample plus soluble substance may be indicated to determine as part of the Specialist in Blood Bank (SBB) training program possible additional antibody specificities. It may not be at the National Institutes of Health (NIH) Clinical Center, possible to test low-titer antibodies because of the required Department of Transfusion Medicine (www.cc.nih.gov/dtm/ dilution that cannot be achieved. If one wants to use specific education.html).21 rBGPs, they are commercially available if the molecular and The authors are federal employees. This work was genetic basis are known.20 It may not always be possible to supported by the Intramural Research Program (project ID inhibit high-titer antibodies, particularly monoclonal antibody Z99 CL999999) of the NIH Clinical Center. There were no drug formulations, because the required high concentration of nonfederal sources of support. the soluble substance cannot be achieved.

IMMUNOHEMATOLOGY, Volume 35, Number 1, 2019 21 K.M. Byrne et al.

References 15. Flegel WA, Chen Q, Castilho L, et al. Molecular immuno- haematology round table discussions at the AABB annual 1. Fung MK, Eder AF, Spitalnik SL, Westhoff CM, Eds. Technical meeting, Orlando 2016. Blood Transfus 2018;16:447–56. manual. 19th ed. Bethesda, MD: AABB Press, 2017. 16. Tong B, Wang M. CD47 is a novel potent immunotherapy target 2. Beattie K, Crawford M, Mallory D, Mougey R. Inhibition of in human malignancies: current studies and future promises. blood group antibodies by soluble antigens. In: Mallory D, Ed. Future Oncol 2018;14:2179–88. Immunohematology methods procedures. Washington, DC: 17. Oostendorp M, Lammerts van Bueren JJ, et al. When blood American Red Cross, 1993, 31:1. transfusion medicine becomes complicated due to interference 3. Judd WJ, Johnson ST, Storry JR. Judd’s methods in by monoclonal antibody therapy. Transfusion 2015;55; Immunohematology. 3rd ed. Bethesda, MD: AABB Press, 1555–62. 2008. 18. Velliquette RW, Aeshlimann J, Kirkegaard J, Shakarian G, 4. Reid M, Lomas-Francis C, Olsson M. The blood group antigen Lomas-Francis C, Westhoff CM. Monoclonal anti-CD47 factsbook. 3rd ed. San Diego, CA: Academic Press, 2012. interference in red cell and platelet testing. Transfusion 5. Seltsam A, Grueger D, Blasczyk R, Flegel WA. Easy 2019;59:730–7. identification of antibodies to high-prevalence Scianna 19. Judd WJ. Inhibition tests with soluble blood group substances. antigens and detection of admixed alloantibodies using soluble In: Judd WJ, Ed. Methods in immunohematology. 2nd ed. recombinant Scianna protein. Transfusion 2009;49:2090–6. Durham, NC: Montgomery Scientific Publications, 1994;240. 6. Anliker M, von Zabern I, Höchsmann B, et al. A new blood 20. Seltsam A, Blasczyk R. Recombinant blood group proteins group antigen is defined by anti-CD59, detected in a CD59- in clinical practice: from puzzling to binary antibody testing. deficient patient. Transfusion 2014;54:1817–22. ISBT Sci Ser 2016;11(Suppl 1):243–9. 7. Seltsam A, Wagner F, Lambert M, et al. Recombinant blood 21. Byrne KM, Sheldon SL, Flegel WA. Organization and group proteins facilitate the detection of alloantibodies to high- management of an accredited specialist in blood bank (SBB) prevalence antigens and reveal underlying antibodies: results technology program. Transfusion 2010;50:1612–7. of an international study. Transfusion 2014;54:1823–30. 8. Rojewski MT, Schrezenmeier H, Flegel WA. Tissue distribution Karen M. Byrne, MDE, MT(ASCP)SBB (corresponding author), of blood group membrane proteins beyond red cells: evidence from cDNA libraries. Transfus Apher Sci 2006,35:71–82. SBB Program Education Coordinator, National Institutes of Health (NIH) Clinical Center, Department of Transfusion Medicine, 10 9. Flegel WA, Henry SM. Can anti-A1 cause hemolysis? Tra nsf usion 2018;58:3036 –7. Center Drive, Bethesda, MD 20892-1184, [email protected]; Chloe CM 10. Morgan WTJ. The human ABO blood group substances. Marie C. Mercado, MS, MLS(ASCP)SBB , Reference Technologist, Experientia 1947;III:257–300. American Red Cross, Pomona, CA; Thaddeus N. Nnabue, MSc, CM 11. Witebsky E, Klendshoj NC. The isolation of the blood group MLS(ASCP) , Training Technologist, Transfusion Medicine, Walter specific B substance. J Exp Med 1940;72:663–7. Reed National Military Medical Center, Bethesda, MD; Traci D. 12. Grubb R. Correlation between Lewis blood group and secretor Paige, MT(ASCP)SBBCM, SBB Program Director and Supervisor of character in man. Nature 1948;162:933. Transfusion Services, National Institutes of Health (NIH) Clinical 13. Moulds JM, Rowe KE. Neutralization of Knops system Center, Department of Transfusion Medicine, Bethesda, MD; antibodies using soluble complement receptor 1. Transfusion and Willy A. Flegel, MD, SBB Program Medical Director, Chief of 1996;36:517–20. Laboratory Services Section, National Institutes of Health (NIH) 14. Wong SW. CD38 Monoclonal antibody therapies for multiple Clinical Center, Department of Transfusion Medicine, Bethesda, MD. myeloma. Clin Lymphoma Myeloma Leuk 2015;15:635–45.

Attention: SBB and BB Students

You are eligible for a free 1-year subscription to Immunohematology.

Ask your education supervisor to submit the name and Important Notice About Manuscripts for Immunohematology complete address for each student and the inclusive dates Please e-mail all manuscripts to [email protected]. of the training period to [email protected].

22 IMMUNOHEMATOLOGY, Volume 35, Number 1, 2019 B l o o d G r o u p R e v i e w An update on the Lutheran blood group system

G. Daniels

This update of the Lutheran (LU) blood group system (Daniels propositus had a mutation in the X-linked gene for the erythroid G. Lutheran. Immunohematology 2009;25:152–9) describes transcription factor GATA1. The c.1240T>C mutation converts six new antigens of the Lutheran system (International Society the translation termination codon to an arginine codon, of Blood Transfusion system 5). These antigens are numbered LU22 to LU27, resulting in a total of 25 antigens in the system. predicting a GATA1 protein with an extraneous 41 amino

The molecular background of LU7 is also described. New KLF1 acids at the carboxy terminus. In addition to the Lumod RBC mutations responsible for In(Lu) have been identified, and the phenotype, the propositus had a hemoglobin count slightly gene responsible for the X-linked form of Lumod has been identified. Immunohematology 2019;35:23–24. below normal, a low platelet count, and a history of bruising.

Key Words: Lutheran, BCAM, CD239, KLF1, GATA1 New Nomenclature

New Antigens of the Lutheran System and The Lutheran gene is now named BCAM, and the Lutheran Molecular Basis of LU7 protein is now named the basal cell adhesion molecule or CD239. It is located on chromosome 19 at 19q13.32 (19:44.81- Since publication of the original review in 2009,1 the 44.82 Mb). molecular basis for LU7 has been resolved,2 and six new high-prevalence antigens have been added to the Lutheran References (LU) blood group system (Table 1). The antigen-negative 1. Daniels G. Lutheran. Immunohematology 2009;25:152–9. phenotype of each of these new antigens, except LU22, results 2. Hue-Roye K, Reid ME. The molecular basis of the LU:7 and from homozygosity for one or two nucleotide changes in LU:−7 phenotypes. Immunohematology 2012;28:130–1. the Lutheran gene. LU22 is more complex, however. LU22 3. Karamatic Crew V, Thornton N, Burton N, Poole J, Search S, expression requires the presence of both Arg77 (Lub) and Daniels G. Two heterozygous mutations in an individual result in the loss of a novel high incidence Lutheran antigen LURC Arg75 on the same molecule. The LU:–22 propositus, who (abstract). Transfus Med 2009;19(Suppl 1):10. made anti-LU22, was Lu(a+b+w) and heterozygous for codons 4. Hustinx H, Lejon-Crottet S, Henny C, et al. LUIT: a novel 75 and 77. Cys75 with Arg77 results in weak Lub and no LU22; high incidence antigen in the Lutheran blood group system (abstract). Vox Sang 2014;107(Suppl 1):172. Arg75 with His77 results in Lua, no Lub, and no LU22; Arg75 b 3 5. Brennan S, Shakarian G, Vege S, Hue-Roye K, Lomas-Francis with Arg77 results in expression of Lu and LU22. C, Westhoff CM. A new antibody in the Lutheran blood group system against a novel high-prevalence antigen named LUGA (abstract). Transfusion 2015;55(Suppl 3):36A. Lumod 6. Karamatic Crew V, Laundy R, Bahashwan A, et al. Two novel high incidence antigens in the Lutheran blood group system Since the discovery that the Lumod In(Lu) phenotype (LUAC and LUBI) (abstract). Vox Sang 2016;111(Suppl 1):63. resulted from heterozygosity for mutations in the KLF1 gene, 7. Vrignaud C, Ramelet S, Amiranoff D, et al. Characterization of many more mutations responsible for that phenotype have a novel high prevalence antigen in the Lutheran blood group system (abstract). Transfusion 2018;58:42A. been identified and are listed in Daniels,8 Singleton et al.,9 10 8. Daniels G. Human blood groups. 3rd ed. Oxford, U.K.: Wiley- and Fraser et al. Flow cytometric analyses revealed reduced Blackwell, 2013. expression of OK (CD147) and LW (ICAM4) blood group 9. Singleton BK, Frayne J, Anstee DJ. Blood group phenotypes antigens, in addition to those previously reported, in red blood resulting from mutations in erythroid transcription factors. cells (RBCs) of the In(Lu) phenotype.10 Curr Opin Hematol 2012;19:486–93. 10. Fraser SN, Knauth CM, Schoeman, EM, et al. Investigation The molecular background of a previously described Lumod of the variable In(Lu) phenotype caused by KLF1 variants. phenotype with an X-linked mode of inheritance in a single Transfusion 2018;58:2414–20. 11 Australian family was described by Singleton et al. The Lumod

IMMUNOHEMATOLOGY, Volume 35, Number 1, 2019 23 G. Daniels

Table 1. New antigens of the Lutheran system and molecular basis of LU7

Antigen Molecular basis of antigen-negative phenotype Protein Number Name Nucleotides Exon Amino acids IgSF domain Reference LU1 Lua c.230A>G 3 p.His77Arg 1 1 LU2 Lub c.230G>A 3 p.Arg77His 1 1 LU3 Lu3 Various 1 LU4 Lu4 1. c.524G>A 5 1. p.Arg175Gln 2 1 2. c.524G>T 5 2. p.Arg175Leu LU5 Lu5 c.326G>A 3 p.Arg109His 1 1 LU6 Lu6 c.824C>T 7 p.Ser275Phe 3 1 LU7 Lu7 c.1274A>C 10 p.Glu425Ala 4 2 LU8 Lu8 c.611T>A 6 p.Met204Lys 2 1 LU9 Lu9 c.824T>C 7 p.Phe275Ser 3 1 LU11 Lu11 Not known 1 LU12 Lu12 1. c.99-104del 2 1. p.delArg34+Leu35 1 1 2. c.419G>A 3 2. p.Arg140Gln LU13 Lu13 c.1340C>T 11 p.Ser447Leu 5 1 1742A>T 13 Gln581Leu LU14 Lu14 c.611A>T 6 p.Lys204Met 2 1 LU16 Lu16 c.679C>T 6 p.Arg227Cys 2 1 LU17 Lu17 c.340G>A 3 p.Glu114Lys 1 1 LU18 Aua c.1615A>G 12 p.Thr529Ala 5 1 LU19 Aub c.1615G>A 12 p.Ala529Thr 5 1 LU20 Lu20 c.905C>T 7 p.Thr302Met 3 1 LU21 Lu21 c.282C>G 3 p.Asp94Glu 1 1 LU22 LURC c.223C>T 3 p.Arg75Cys* 1 3 LU23 LUIT c.469G>A 4 p.Gly157Arg 2 4 1289C>T 10 Thr430Ile 4 LU24 LUGA c.212G>A 3 p.Arg71His 1 5 LU25 LUAC c.662C>T 6 Thr221Ile 2 6 LU26 LUBI c.1495C>T 12 Arg449Trp 5 6 LU27 LUYA c.1184G>A 9 p.Arg395His 4 7 *LU22 expression requires the presence of both Arg77 (Lub) and Arg75 on the same molecule. IgSF = immunoglobulin superfamily.

11. Singleton BK, Roxby DJ, Stirling JW, et al. A novel GATA1 mutation (Stop414Arg) in a family with the rare X-linked Geoffrey Daniels, PhD, retired, previously Head of Diagnostics, blood group Lu(a−b−) phenotype and mild macrothombocytic International Blood Group Reference Laboratory, Bristol, UK, geoff. thrombocytopenia. Br J Haematol 2013;161:139–42. [email protected].

24 IMMUNOHEMATOLOGY, Volume 35, Number 1, 2019 A nnouncements

September 19, 2019

37th Annual Immunohematology and Blood Transfusion Symposium The Department of Transfusion Medicine, Clinical Center, National Institutes of Health (NIH), and the American Red Cross are co-hosting this symposium on the NIH campus in Bethesda, MD. There is no registration fee, but advance registration is encouraged. Contact Karen Byrne, NIH/CC/DTM, Bldg. 10/Rm. 1C711, 10 Center Drive MSC 1184, Bethesda, MD 20892‑1184, e-mail: [email protected], or visit the Web site: http://www.cc.nih.gov/dtm/research/symposium.html

September 20, 2019

9th Annual Red Cell Genotyping Symposium 2019: Patients First The Department of Transfusion Medicine, National Institutes of Health (NIH) Clinical Center, and Versiti are co-hosting this symposium on the NIH campus in Bethesda, MD. For information, registration fee, and advance registration, contact Natasha Leon, Versiti, P.O. Box 2178, Milwaukee, WI 53021-2178, e-mail: [email protected], or visit the Web site: https://Versiti. org/rcg2019

IMMUNOHEMATOLOGY, Volume 35, Number 1, 2019 25 Announcements, cont.

26 IMMUNOHEMATOLOGY, Volume 35, Number 1, 2019 Announcements, cont.

The Johns Hopkins Hospital Specialist in Blood Bank Technology Program

The Johns Hopkins Hospital was founded in 1889. It is located in Baltimore, MD, on the original founding site, just 45 minutes from Washington, DC. There are approximately 1,000 inpatient beds and another 1,200 outpatient visits daily; nearly 600,000 patients are treated each year.

The Johns Hopkins Hospital Transfusion Medicine Division is one of the busiest in the country and can provide opportunities to perform tasks that represent the entire spectrum of Immunohematology and Transfusion Medicine practice. It provides comprehensive support to all routine and specialized areas of care for surgery, oncology, cardiac, obstetrics, neonatal and pediatric, solid organ and bone marrow transplant, therapeutic apheresis, and patients with hematological disorders to name a few. Our intradepartment Immunohematology Reference Laboratory provides resolution of complex serologic problems, transfusion management, platelet antibody, and molecular genotype testing.

The Johns Hopkins Hospital Specialist in Blood Bank Technology Program is an onsite work-study, graduate-level training program for certified Medical Technologists, Medical Laboratory Scientists, and Technologists in Blood Banking with at least 2 years of full-time Blood Bank experience.

The variety of patients, the size, and the general intellectual environment of the hospital provide excellent opportunities for training in Blood Banking. It is a challenging program that will prepare competent and knowledgeable graduates who will be able to effectively apply practical and theoretical skills in a variety of employment settings. The Johns Hopkins Hospital Specialist in Blood Bank Technology Program is accredited by the Commission on Accreditation of Allied Health Education Programs (CAAHEP). Please visit our Web site at http://pathology.jhu.edu/department/divisions/ transfusion/sbb.cfm for additional information.

Contact: Lorraine N. Blagg, MA, MLS(ASCP)CMSBB Program Director E-mail: [email protected] Phone: (410) 502-9584

The Johns Hopkins Hospital Department of Pathology Division of Transfusion Medicine Sheikh Zayed Tower, Room 3100 1800 Orleans Street Baltimore, MD 21287

Phone (410) 955-6580 Fax (410) 955-0618 Web site: http://pathology.jhu.edu/department/divisions/transfusion/index.cfm

IMMUNOHEMATOLOGY, Volume 35, Number 1, 2019 27 Announcements, cont.

28 IMMUNOHEMATOLOGY, Volume 35, Number 1, 2019 Announcements, cont.

Masters of Science (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 2019 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 a Diploma in Transfusion and Transplantation Sciences or a Certificate in Transfusion and Transplantation Sciences.

The course is accredited by the Institute of Biomedical Sciences.

Further information can be obtained from the Web site: http://ibgrl.blood.co.uk/MSc/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: [email protected]

IMMUNOHEMATOLOGY, Volume 35, Number 1, 2019 29 Announcements, cont.

30 IMMUNOHEMATOLOGY, Volume 35, Number 1, 2019 Announcements, cont.

IMMUNOHEMATOLOGY, Volume 35, Number 1, 2019 31 Announcements, cont.

Online Specialist in Blood Bank (SBB) Certificate and Masters in Clinical Laboratory Management Program Rush University | College of Health Sciences

Continue to work and earn graduate credit while the Rush University SBB/MS program prepares you for the SBB exam and the Diplomat in Laboratory Management (DLM) exam given by ASCP Board of Certification! (Please note acceptable clinical experience is required for these exams.)

Rush University offers online graduate level courses to help you achieve your career goals. Several curricular options are available. The SBB/MS program at Rush University is currently accepting applications for Fall 2019. For additional information and requirements, please visit our Web site at: www.rushu.rush.edu/cls/

Rush University is fully accredited by the Higher Learning Commission (HLC) of the North Central Association of Colleges and Schools, and the SBB Certificate Program is accredited by the Commission on Accreditation of Allied Health Education Programs (CAAHEP).

Applications for the SBB/MS Program can be submitted online at the following Web site: http://www.rushu.rush.edu/admiss/hlthadm.html

Contact: Laurie Gillard, MS, MLS(ASCP)SBB Director of the Specialist in Blood Banking Program Assistant Professor, Department of Medical Laboratory Science, Rush University 312-942-2402 (o) | 312-942-6464 (f) | [email protected] Denise Harmening, PhD, MT(ASCP) Director of Curriculum, [email protected]. 600 S. Paulina Street | Suite 1021 AAC | Chicago, IL 60612

32 IMMUNOHEMATOLOGY, Volume 35, Number 1, 2019 A dvertisements

IMMUNOHEMATOLOGY, Volume 35, Number 1, 2019 33 Becoming a Specialist in Blood Banking (SBB)

What is a certified Specialist in Blood Banking (SBB)? • Someone with educational and work experience qualifications who successfully passes the American Society for Clinical Pathology (ASCP) board of registry (BOR) examination for the Specialist in Blood Banking. • This person will have advanced knowledge, skills, and abilities in the field of transfusion medicine and blood banking. Individuals who have an SBB certification serve in many areas of transfusion medicine: • Serve as regulatory, technical, procedural, and research advisors • Perform and direct administrative functions • Develop, validate, implement, and perform laboratory procedures • Analyze quality issues preparing and implementing corrective actions to prevent and document issues • Design and present educational programs • Provide technical and scientific training in transfusion medicine • Conduct research in transfusion medicine Who are SBBs? Supervisors of Transfusion Services Managers of Blood Centers LIS Coordinators Educators Supervisors of Reference Laboratories Research Scientists Consumer Safety Officers Quality Assurance Officers Technical Representatives Reference Lab Specialists Why become an SBB? Professional growth Job placement Job satisfaction Career advancement How does one become an SBB? • Attend a CAAHEP-accredited SBB Technology program OR • Sit forthe examination based on criteria established by ASCP for education and experience. However: In recent years, a greater percentage of individuals who graduate from CAAHEP-accredited programs pass the SBB exam. Conclusion: The BEST route for obtaining an SBB certification is . . . to attend a CAAHEP-accredited Specialist in Blood Bank Technology Program. Facilities with CAAHEP-accredited programs, onsite or online, are listed below. Additional information can be found by visiting the following Web sites: www.ascp.org, www.caahep.org, and www.aabb.org.

California American Red Cross Blood Services Pomona, CA Florida Academic Center at OneBlood St. Petersburg, FL Illinois Rush University Chicago, IL Indiana Indiana Blood Center Indianapolis, IN Louisiana LifeShare Blood Center Shreveport, LA University Medical Center New Orleans New Orleans, LA Maryland National Institutes of Health Clinical Center Bethesda, MD The Johns Hopkins Hospital Baltimore, MD Walter Reed National Military Medical Center Bethesda, MD Texas University Health System and Affiliates School of Blood Bank Technology San Antonio, TX University of Texas Medical Branch Galveston, TX Wisconsin Versiti Wisconsin, Inc. Milwaukee, WI

Revised March 2019

34 IMMUNOHEMATOLOGY, Volume 35, Number 1, 2019 Reference and Consultation Services IgA Testing

Antibody identification and problem resolution IgA testing is available to do the following:

HLA-A, B, C, and DR typing • Identify IgA-deficient patients

HLA-disease association typing • Investigate anaphylactic reactions

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For information, contact: For additional information contact: Mehdizadeh Kashi Sandra Nance (215) 451-4362 at (503) 280-0210 or e-mail: or write to: [email protected]

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National Reference Laboratory Donor IgA Screening for Blood Group Serology • Effective tool for screening large volumes of donors Immunohematology Reference Laboratory • Gel diffusion test that has a 15-year proven track record: AABB, ARC, New York State, and CLIA licensed Approximately 90 percent of all donors identified as 24-hour phone number: IgA deficient by this method are confirmed by the more (215) 451-4901 Fax: (215) 451-2538 sensitive testing methods

American Rare Donor Program For additional information: 24-hour phone number: (215) 451-4900 Kathy Kaherl Fax: (215) 451-2538 at (860) 678-2764 [email protected] e-mail: Immunohematology [email protected] Phone, business hours: (215) 451-4902 or write to: Fax: (215) 451-2538 Reference Laboratory [email protected] American Red Cross Biomedical Services Quality Control of Cryoprecipitated–AHF Connecticut Region Phone, business hours: 209 Farmington Avenue (215) 451-4903 Farmington, CT 06032 Fax: (215) 451-2538

IMMUNOHEMATOLOGY, Volume 35, Number 1, 2019 35 Advertisements, cont.

National Reference Laboratory National Neutrophil Serology Reference Laboratory for Specialized Testing Our laboratory specializes in granulocyte antibody detection Diagnostic testing for: and granulocyte antigen typing. • Neonatal alloimmune thrombocytopenia (NAIT) Indications for granulocyte serology testing • Post-transfusion purpura (PTP) include: • Refractoriness to platelet transfusion • Alloimmune neonatal neutropenia (ANN) • Heparin-induced thrombocytopenia (HIT) • 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) • Solid-phase red cell adherence (SPRCA) assay TRALI investigations also include: • Molecular analysis for HPA-1a/1b • HLA (PRA) Class I and Class II antibody detection

For further information, contact: For further information, contact: Platelet Serology Laboratory (215) 451-4205 Neutrophil Serology Laboratory (651) 291-6797 Sandra Nance (215) 451-4362 Randy Schuller (651) 291-6758 [email protected] [email protected]

American Red Cross Biomedical Services American Red Cross Biomedical Services Musser Blood Center Neutrophil Serology Laboratory 700 Spring Garden Street 100 South Robert Street Philadelphia, PA 19123-3594 CLIA licensed St. Paul, MN 55107 CLIA licensed

36 IMMUNOHEMATOLOGY, Volume 35, Number 1, 2019 Advertisements, cont.

Immunohematology Instructions for Authors

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

IMMUNOHEMATOLOGY, Volume 35, Number 1, 2019 37 38 IMMUNOHEMATOLOGY, Volume 35, Number 1, 2019 Immunohematology Instructions for Authors | New Blood Group Allele Reports

A. For describing an allele that has not been described in a peer-reviewed publication and for which an allele name or provisional allele name has been assigned by the ISBT Working Party on Blood Group Allele Terminology (http://www.isbtweb.org/working-parties/red-cell-immunogenetics-and-blood-group-terminology/blood-group- terminology/blood-group-allele-terminology/)

B. Preparation 1. Title: Allele Name (Allele Detail) ex. RHCE*01.01 (RHCE*ce48C) 2. Author Names (initials and last name of each [no degrees, ALL CAPS])

C. Text 1. Case Report i. Clinical and immunohematologic data ii. Race/ethnicity and country of origin of proband, if known 2. Materials and Methods Description of appropriate controls, procedures, methods, equipment, reagents, etc. Equipment and reagents should be identified in parentheses by model or lot and manufacturer’s name, city, and state. Do not use patient names or hospital numbers. 3. Results Complete the Table Below: Phenotype Allele Name Nucleotide(s) Exon(s) Amino Acid(s) Allele Detail References e weak RHCE*01.01 48G>C 1 Trp16Cys RHCE*ce48C 1

Column 1: Describe the immunohematologic phenotype (ex. weak or negative for an antigen). Column 2: List the allele name or provisional allele name. Column 3: List the nucleotide number and the change, using the reference sequence (see ISBT Blood Group Allele Terminology Pages for reference sequence ID). Column 4: List the exons where changes in nucleotide sequence were detected. Column 5: List the amino acids that are predicted to be changed, using the three-letter amino acid code. Column 6: List the non-consensus nucleotides after the gene name and asterisk. Column 7: If this allele was described in a meeting abstract, please assign a reference number and list in the References section. 4. Additional Information i. Indicate whether the variant is listed in the dbSNP database (http://www.ncbi.nlm.nih.gov/snp/); if so, provide rs number and any population frequency information, if available. ii. Indicate whether the authors performed any population screening and, if so, what the allele and genotype frequencies were. iii. Indicate whether the authors developed a genotyping assay to screen for this variant and, if so, describe in detail here. iv. Indicate whether this variant was found associated with other variants already reported (ex. RHCE*ce48C,1025T is often linked to RHD*DIVa-2).

D. Acknowledgments

E. References

F. Author Information List first name, middle initial, last name, highest degree, position held, institution and department, and complete address (including ZIP code) for all authors. List country when applicable.

IMMUNOHEMATOLOGY, Volume 35, Number 1, 2019 39 40 IMMUNOHEMATOLOGY, Volume 35, Number 1, 2019 Journal of Blood Group Serology and Molecular Genetics

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