Journal of Blood Group Serology and Molecular Genetics

Vo l u m e 34, N u m b e r 3, 2018 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 34, Number 3, 2018 CONTENTS

R e v i e w 85 Proceedings from the International Society of Blood Transfusion Working Party on Immunohaematology, Workshop on the Clinical Significance of Red Blood Cell Alloantibodies, September 2, 2016, Dubai Clinical significance of antibodies to antigens in the Raph, John Milton Hagen, I, Globoside, Gill, Rh-associated glycoprotein, FORS, JR, LAN, Vel, CD59, and Augustine blood group systems M. Moghaddam and A.A. Naghi

S e r o lo g i c M e t h o d R e v i e w 91 Rouleaux and saline replacement K.L. Waider

O r i g i n a l R e p o r t 93 Method-specific and unexplained reactivity in automated solid- phase testing and their association with specific antibodies M.E. Harach, J.M. Gould, R.P. Brown, T. Sanders and J.H. Herman

S e r o lo g i c M e t h o d R e v i e w 98 Utility of chloroquine diphosphate in the blood bank laboratory T. Aye and P.A. Arndt

R e v i e w 103 Proceedings from the International Society of Blood Transfusion Working Party on Immunohaematology, Workshop on the Clinical Significance of Red Blood Cell Alloantibodies, Friday, September 2, 2016, Dubai Clinical significance of antibodies to antigens in the Scianna, Dombrock, Colton, Landsteiner-Weiner, Chido/Rodgers, H, Kx, Cromer, Gerbich, Knops, Indian, and Ok blood group systems S. Lejon Crottet

C a s e R e p o r t 109 A delayed and acute hemolytic transfusion reaction mediated by an anti-c in a patient with variant RH alleles T.K. Walters and T. Lightfoot

S e r o lo g i c M e t h o d R e v i e w 113 Detecting polyagglutinable red blood cells C. Melland and C. Hintz

I n M e m o r i a m 118 Marjory Stroup Walters J. Hegarty and T.S. Casina

120 127 131 134 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 b s c r i p t i o n f o r A u t h o rs I n f o rm at 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 Pomona, California San Antonio, 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 rs 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 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 rs 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 rs 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 Margaret A. Keller, PhD Immunohematology is published quarterly (March, June, September, and December) by the Philadelphia, Pennsylvania American Red Cross, National Headquarters, Washington, DC 20006. Immunohematology is indexed and included in Index Medicus and MEDLINE on the E d i to r i a l A ss i s ta n t MEDLARS system. The contents are also cited in the EBASE/Excerpta Medica and Elsevier Linda Frazier BIOBASE/Current Awareness in Biological Sciences (CABS) databases.

P r o d u c t i o n A ss i s ta n t The subscription price is $50 for individual, $100 for institution (U.S.), and Linda Frazier $60 for individual, $100 for institution (foreign), per year. Subscriptions, Change of Address, and Extra Copies: C o p y E d i to r Frederique Courard-Houri Immunohematology, P.O. Box 40325 Philadelphia, PA 19106 P r o o f r e a d e r Or call (215) 451-4902 Wendy Martin-Shuma Web site: www.redcrossblood.org/hospitals/immunohematology E l ec t r o n i c P u b l i s h e r Copyright 2018 by The American National Red Cross Paul Duquette ISSN 0894-203X

O n O u r C o v e r Perhaps Gustav Klimt’s best-known work, The Kiss (1907), dazzlingly melds the sensual with the abstract. The painting depicts a man and woman intertwined, he standing, bowed, while she kneels on an idealized quilt-like meadow of flowers. Their proximity to the top of the painting heightens the sense of intimacy and also suggests the possibility of transcending worldly constraints. We see little of the lovers—the back of his head, her face, their hands and feet, simply rendered but wrapped in golden raiment decorated with distinct mosaics. The man’s figure is comprised of juxtaposed rectangles and the woman’s of clustered circles, a geometry that hints at both the contrast and complement of their union. Despite its shapelessness, the gilded mass of clothing serves to intensify and exalt the physical act of the kiss and thus consecrates the couple and love itself. This issue includes Waider’s review on the use of saline replacement to identify rouleaux—the stacked coin appearance of the red cells likened to “clustered circles.” David Moolten, MD

ii IMMUNOHEMATOLOGY, Volume 34, Number 3, 2018 R e v i e w Proceedings from the International Society of Blood Transfusion Working Party on Immunohaematology, Workshop on the Clinical Significance of Red Blood Cell Alloantibodies, September 2, 2016, Dubai Clinical significance of antibodies to antigens in the Raph, John Milton Hagen, I, Globoside, Gill, Rh-associated glycoprotein, FORS, JR, LAN, Vel, CD59, and Augustine blood group systems

M. Moghaddam and A.A. Naghi

This article reviews information on the clinical significance and 6 shared missense mutation c.511C>T (p.Argl71Cys) as of antibodies to antigens in the Raph, John Milton Hagen, I, well as a synonymous single-nucleotide mutation (c.579A>G) Globoside, Gill, Rh-associated glycoprotein, FORS, JR, LAN, Vel, and had no clinical features. Although the CD151 protein is CD59, and Augustine blood group systems. Antibodies to many of the antigens in these groups are rarely encountered because of the critical to cell adhesion and signaling and is implicated in high prevalence of the associated antigens in most populations. cancer progression, its significance in transfusion medicine is For many of these antibodies, the clinical significance—that is, limited to only one report of a hemolytic transfusion reaction the potential to cause reduced survival of transfused antigen- 3 positive red blood cells or a transfusion reaction (e.g., anti-P, (HTR). Least-incompatible RBC units should be selected anti-Jra, and anti-Lan), and/or hemolytic disease of the fetus and for transfusion to patients with anti-MER2.2 No information newborn (e.g., anti-RHAG4 and anti-Vel)—has been documented. on anti-MER2 causing hemolytic disease of the fetus and For other antibodies, their prevalence is so rare that information newborn (HDFN) is available.4 on the clinical significance of their antibodies is not available (e.g., anti-FORS1). Immunohematology 2018;34:85–90. John Milton Hagen Blood Group System Key Words: clinical significance, antibodies to red blood cell antigens, Raph, John Milton Hagen, I, Globoside, Gill, Rh- The John Milton Hagen (JMH) blood group system associated glycoprotein, FORS, JR, LAN, Vel, CD59, Augustine consists of six high-prevalence antigens that are recognized by the International Society of Blood Transfusion (ISBT) and are Raph Blood Group System numbered sequentially from JMH1 through JMH6. Confirmed JMH variants are named with the first letter from the antibody The Raph blood group system contains just one maker’s first name following JMH (JMH2 named JMHK, antigen, MER2 (RAPH1), located on the tetraspanin CD151 JMH3 named JMHL, JMH4 named JMHG, JMH5 named glycoprotein (TM4SF).1,2 The true MER2– phenotype, JMHM, JMH6 named JMHQ).5 These antigens are located on associated with the presence of anti-MER2, is very rare and the Sema7A protein.5,6 The chromosomal location of SEMA7A results from mutations in CD151, but there is a quantitative is 15q22.3-q23. JMH1 is the primary antigen in the system red blood cell (RBC) polymorphism in which RBCs of about 8 and is present in greater than 99 percent of all individuals. The percent of white individuals are serologically MER2–.2 JMH1– phenotype is more commonly acquired by depression of the antigen. This finding might explain the serologic Clinical Significance observation of a positive direct antiglobulin test (DAT) seen in There have been six reports of human alloantibodies to many individuals with anti-JMH.5 MER2. Three of the subjects were found to have a stop codon in the CD151 gene, which encodes a member of the tetraspanin Clinical Significance family of proteins. These three individuals had nephropathy and JMH1, commonly known as JMH, is most notable because deafness, and two of the three, who were siblings, also had skin transient depression of the antigen occurs, and (auto)anti-JMH lesions, deafness, and β-thalassemia minor. The fourth subject may develop.5 JMH– patients with anti-JMH often have no had missense mutation c.533G>A (p.Argl78His). Subjects 5 history of transfusion or pregnancy. Of seven JMH antibodies,

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five were IgG4 and two were IgG1, although IgG3 anti-JMH be hemolytic and have a thermal range up to 37°C, and some has been described. There are numerous cases where patients anti-I with a thermal range below 37°C can cause shortened with anti-JMH have been transfused with JMH+ blood survival of transfused I+ RBCs. One anti-I became potentially with no adverse effects. One such patient received 20 units clinically significant after transfusion of 6 units of I+ blood. of JMH+ blood in 10 months, with the expected hemoglobin rise. There are no reports of JMH antibodies causing HDFN, Globoside Blood Group System which is unsurprising considering that JMH antigens are expressed very weakly on cord cells. One patient with anti- The P blood group antigen of the Globoside system is a JMH is reported to have experienced an acute intravascular glycolipid structure, also known as globoside, on the RBCs HTR, but evidence that the JMH antibody was responsible is of almost all individuals worldwide. The P antigen (Gb4) is limited. Antibodies developed in the rare JMH variant types intimately related to the Pk and NOR (P1PK4) antigens. may cause reduced RBC survival.2 Today, rapid detection of The molecular genetic basis of globoside deficiency is the JMH antibodies with recombinant SEMA7A protein and the absence of functional P synthase caused by mutations at the particle gel immunoassay has been developed.7 B3GALNT1 locus. Other related glycolipid structures, the LKE and PX2 antigens, remain in the Globoside blood group I Blood Group System collection pending further evidence concerning the genes and gene products responsible for their synthesis.11 The I antigen, together with i, used to be part of the Ii blood group collection. The gene encoding the I β-1, Clinical Significance 6-N-acetylglucosamine transferase (IGNT/GCNT2) respon- Anti-P is found in the serum of all Pk individuals and sible for converting i active straight chains of carbohydrates to can be separated from serum of p individuals by adsorption 8 k k 2 I active branched chains has been cloned, and some mutations with P1 or P2 cells or by inhibition with hydatid cyst fluid. 9,10 responsible for adult i phenotype have been identified. When complement is present, anti-P will hemolyze P1 or P2 Hence, I has been promoted to the I blood group system phenotype RBCs.2 P antibodies are IgM and often also IgG, are comprising only a single antigen, the I antigen, and i remains usually reactive at 37°C, and can cause severe intravascular in the Ii collection. RBCs from adults predominantly express HTRs. Autoanti-P is associated with paroxysmal cold I antigens and only low levels of i antigens, higher levels of hemoglobinuria. P antigen is also a receptor of parvovirus B19. the latter predominate in fetal and neonatal RBCs. In a small Cytotoxic IgM and IgG3 antibodies directed against P or number of individuals, only very low levels of I can be detected, Pk antigens are associated with a higher-than-normal rate of k k and their RBCs show high levels of i (adult i phenotype). This spontaneous abortion in women with the rare p, P1 , and P2 phenotype is believed to result from lack of activity of the I phenotypes.4 branching transferase, a product of the GCNT2 gene.2 Gill Blood Group System Clinical Significance Potent cold reactive antibodies responsible for cold The Gill blood group system was added to the list of agglutinin disease are usually of I specificity. These antibodies systems already recognized by the ISBT in 2002. GIL, the only are generally monoclonal and are usually IgM, but IgG antigen of the Gill system, is an antigen of high prevalence autoanti-I can also occur. They directly agglutinate I+ RBCs at located on the water and glycerol channel aquaporin-3 4°C with varying thermal amplitude but are generally inactive (AQP3).2,12 The GIL– phenotype results from homozygosity above 30°C. One autoanti-I active at 30°C caused an acute for a splice mutation in AQP3. Anti-GIL has been identified in HTR in a small child when 2 units of blood were transfused five Gil– white women who had been pregnant at least twice.2 immediately after removal from the refrigerator. Transient, polyclonal, or oligoclonal autoanti-I may arise from infection, Clinical Significance most typically by Mycoplasma pneumoniae. Five examples of anti-GIL have been identified, all in Alloanti-I of high titer is rare and usually presents in the white women who had been pregnant at least twice. No GIL– sera of i adults. These antibodies are almost invariably IgM individual was found by screening 23,251 white American and and are active only at low temperatures. Rare examples may 2841 African American women with anti-GIL. RBCs from two

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of the babies of mothers with anti-GIL gave a positive DAT, of northern European ancestry, in Bedouin Arabs, and in one but there were no clinical symptoms of HDFN. Anti-GIL may Mexican individual. The rare Jr(a–) phenotype mostly results have been responsible for an HTR, and results of monocyte from recessive inheritance of ABCG2 null alleles caused by monolayer assay (MMA) with two GIL antibodies suggested a frameshift or nonsense changes.17,20 –22 To date, more than 25 potential to cause accelerated destruction of transfused GIL+ different mutations responsible for the absence of ABCG2 as RBCs.2 well as mutations giving rise to weakened Jra expression have been identified.16 The Rh-Associated Glycoprotein Blood Group System Clinical Significance ABCG2 expression levels in cord RBCs are higher than In 2010, the recognition that three RBC surface those in adult RBCs, and the change of ABCG2 expression antigens were located on RhAG encoded by RHAG led to in erythroid lineage cells may influence the clinical course the establishment of a new blood group system. Two high- of fetal anemia with anti-Jra.23 Anti-Jra may be stimulated prevalence antigens, Duclos (RHAG1) and DSLK (RHAG3), by transfusion or pregnancy and has been detected in two low-prevalence antigens, OI(a) and (RHAG2), and RHAG4, untransfused Jr(a–) women during their first pregnancy.24 have serologic characteristics suggestive of expression on Most anti-Jra are IgG1 and sometimes IgG3. Anti-Jra may RhAG, but RHAG4 has been shown to not exist and is under fix complement, can be a dangerous antibody in pregnancy, investigation by ISBT to be retracted.13 and has been implicated in severe and fatal HDFN; in other pregnancies with anti-Jra, however, indications of HDFN Clinical Significance have been no more than a positive DAT on cord cells or No data are available.4 mild neonatal jaundice. Many transfusions of Jr(a+) RBCs to patients have resulted in no signs of hemolysis, although FORS Blood Group System incompatible transfusion may cause a sharp rise in the titer of anti-Jra, resulting in signs of an acute HTR in subsequent This blood group system has been named FORS after its transfusions. A patient with anti-Jra developed rigors after original finder, Lund professor John Forssman. The FORS transfusion of 150 mL crossmatch-incompatible blood. Least- antigen (originally recognized as the Apae phenotype) was incompatible RBC units may be suitable for transfusion to discovered by weak reactivity of RBCs against polyclonal most patients with anti-Jra, but Jr(a–) RBCs should be selected anti-A reagents, reactivity against the lectin Helix pomatia in cases where the anti-Jra is of high titer.2

(snail anti-A), and no reactivity with the plant anti-A1 lectin, Dolichos biflorus, in two different families. Genomic analysis LAN Blood Group System of the ABO locus in both samples revealed that they were genetically group O, and the reactivity described must be due LAN (Langereis) was officially recognized by the ISBT in to other phenomena.14,15 2012 as the 33rd human blood group system. It consists of one high-prevalence antigen, Lan (LAN1).25 The ABCB6 protein is Clinical Significance the carrier of the Lan blood group antigen.16,25 The ABCB6 gene The clinical significance of anti-FORS1 is not known.4 (chromosome 2q36, 19 exons) encodes the ABCB6 polypeptide, known as a porphyrin transporter. The exceptional Lan–

JR Blood Group System individuals do not express ABCB6 (Lannull phenotype) owing to several different frameshift and missense mutations.25 To The JR blood group system (ISBT 032) consists of one date, more than 40 ABCB6 alleles that encode Lan– or Lan+w antigen, Jra, which is highly prevalent in all populations (>99%). phenotypes have been described,26 and quantitation of Lan Jra is located on the ABCG2 transporter, a multipass membrane antigen in Lan+, Lan+w, and Lan– phenotypes have been glycoprotein (also known as the breast cancer resistance protein performed.27 Despite the Lan antigen role in erythropoiesis [BCRP]), which is encoded by the ABCG2 gene on chromosome and detoxification of cells, Lan– individuals do not appear to 4q22.1.16–19 The rare Jr(a–) phenotype has been found mostly demonstrate susceptibility to any disease. in Japanese and other Asian populations, but also in people

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Clinical Significance antibody, and patients with anti-Vel should be transfused Anti-Lan has been reported in two African American with Vel– RBCs. The first anti-Vel and other examples since individuals as well as in other populations including have caused severe immediate HTRs. Anti-Vel may be missed Caucasians and Asians and may be stimulated by transfusion in compatibility testing if inappropriate methods are used.33 or pregnancy. The original anti-Lan was responsible for an Although many examples of anti-Vel have been found in immediate HTR characterized by fever and chills. There is pregnant women, anti-Vel does not usually cause HDFN, no report of naturally occurring alloanti-Lan; none of the probably because most anti-Vel are predominantly IgM, and Lan– siblings of the Lan– propositi have anti-Lan. Anti-Lan the Vel antigen is usually expressed weakly on neonatal RBCs. has been described as having variable clinical significance, Two examples of autoanti-Vel were responsible for (AIHA), either for HTRs (none to severe) or HDFN (none to mild).25 although in one case, a nine-week-old infant, the RBCs gave a Lan alloantibodies are mostly IgG1 and IgG3, although IgG2 negative DAT. 2 and IgG4 may also be present. Some anti-Lan fix complement; others do not.2 Despite challenging conditions caused by the CD59 scarcity of Lan– donors worldwide, ideally Lan– RBCs should be selected for transfusion to patients with anti-Lan, especially CD59, also known as the membrane inhibitor of reactive individuals with a high-titer antibody,2,25 although least lysis (MIRL), homologous restriction factor (HRF), and incompatible RBCs may be suitable for patients with weak membrane attack complex inhibitory factor (MACIF), is a cell examples of the antibody. The only autoanti-Lan was reported surface glycoprotein of approximately 20 kDa that limits the in a patient with mild autoimmune hemolytic anemia (AIHA) activity of the terminal complement complex C5b-9 and is with depressed Lan antigen expression.2 more effective than decay accelerating factor (DAF) or CD55 in this respect.28,34 The first demonstration of anti-CD59 was Vel Blood Group System in a patient homozygous for a CD59 deficiency, which led to the discovery of a new blood group system, CD59, and a null Vel is an RBC antigen that is expressed by more than 99.9 allele (c.146delA). percent of the population.28 The recognition of Vel dates to CD59 is attached by a glycosylphosphatidylinositol (GPI) 1952, when a patient, named Mrs. Vel, suffered a transfusion anchor not only to erythrocytes but also to various other cellular reaction due to the presence of an antibody that was found to membranes. Seven cases of an isolated CD59 deficiency due to agglutinate sera of over 10,000 individuals. The antibody was three distinct null alleles of the CD59 gene have been published named anti-Vel.29 so far.34 As well as being cell-bound through its GPI anchor, Recently, the SMIM1 protein was shown to carry the Vel soluble CD59 is also found in plasma, urine, and cerebrospinal blood group antigen. Using a high-density single nucleotide fluid. polymorphism array, Storry et al.30 identified the SMIM1 gene Absence of CD59 is associated with hemolytic anemia and residing in a 97-kb region of homozygosity on chromosome with thrombosis as well as with other autoimmune diseases 1p36 in the vicinity of the RH locus. A frameshift deletion of such as systemic lupus erythematosus.2 17 nucleotides in exon 3 of SMIM1 is responsible for the Vel– phenotype.16,31,32 Genotype screening estimated that ~1 in 17 Clinical Significance Swedish blood donors is a heterozygous deletion carrier and No data are available. ~1 in 1200 is a homozygous deletion knockout.31 Augustine Blood Group System Clinical Significance The high clinical significance of Vel is related to what Ata is a high-prevalence antigen found on the RBCs of over happens to individuals with the Vel– phenotype upon 99 percent of individuals.35 The first literature referring to the transfusion or pregnancy. Vel alloantibodies are never Ata antibody, which can be responsible for severe HTRs and naturally occurring, and most producers of anti-Vel have been mild HDFN, dates to the 1960s. Applewhaite et al.36 identified transfused, yet Vel antibodies are predominantly IgM and an antibody with a novel specificity in the serum of Mrs. fix complement. Of the two IgG anti-Vel, one was IgG1, and Augustine when the RBCs of her third child gave a positive the other contained IgG1 and IgG3. Anti-Vel is a dangerous DAT at birth. Her alloantibody, abbreviated as anti-Ata after

88 IMMUNOHEMATOLOGY, Volume 34, Number 3, 2018 ISBT conference—clinical significance of antibodies

her name, reacted with more than 6600 blood donors tested . 4 Reid ME, Lomas-Francis C, Olsson ML. The blood group at that time, indicating that Ata was a high-prevalence antigen. antigen factsbook. 3rd ed. San Diego, CA: Academic Press, 2012. All anti-Ata producers reported thus far have been of African . 5 Johnson ST. JMH blood group system: a review. ancestry, like Mrs. Augustine. Immunohematology 2014;30:18–23. Recently, it has been shown that SLC29A1 encoding the . 6 Daniels G, Flegel WA, Fletcher A, et al. International Society equilibrative nucleoside transporter 1 (ENT1) specifies a new of Blood Transfusion Committee on Terminology for Red Cell Surface Antigens: Cape Town report. Vox Sang 2007;92: candidate gene for a novel blood group system that includes the 250–3. a 37 At antigen. Daniels et al. reported that a nonsynonymous . 7 Seltsam A, Agaylan A, Grueger D, et al. Rapid detection of JMH SNP in SLC29A1 (rs45458701) is responsible for the At(a–) antibodies with recombinant Sema7A(CD108) protein and the phenotype. Although all At(a–)-reported propositi are of particle gel immunoassay. Transfusion 2008;48:1151–6. . 8 Bierhuizen MFA, Mattei MG, Fukuda M. Expression of the African ancestry with functional ENT1, they identified three developmental I antigen by a cloned human cDNA encoding siblings of European ancestry who were homozygous for a a member of a ß-1,6-N-acetylglucosaminyltransferase gene null mutation in SLC29A1 (c.589+1G>C) and thus have the family. Genes Dev 1993;7:468–78. Augustine phenotype. These individuals lacking ENT1 . 9 Yu LC, Twu YC, Chang CY, et al. Molecular basis of the adult i null phenotype and the gene responsible for the expression of the exhibit periarticular and ectopic mineralization, which human blood group I antigen. Blood 2001;98:3840–5. confirms an important role for ENT1/SLC29A1 in human 10. Inaba N, Hiruma T, Togayachi A, et al. A novel I-branching bone homeostasis,34 cardioprotection, and drug transport in ß-1,6-N-acetylglucosaminyltransferase involved in the human erythrocytes.38 blood group I antigen expression. Blood 2003;101:2870–6. 11. Hellberg A, Westman JS, Olsson ML. An update on the GLOB blood group system and collection. Immunohematology Clinical Significance 2013;29:19–24. Ata antibodies are mostly IgG, but IgM could also be 12. Roudier N, Verbavatz JM, Maurel C, et al. Evidence for the present and can directly agglutinate At(a+) RBCs. Of two IgG presence of aquaporin-3 in human red blood cells. J Biol Chem 1998;273:8407–12. anti-Ata, one was IgG1, and the other consisted of IgG1, IgG3, 13. Tilley L, Gren C, Poole J, et al. A new blood group system, a 51 and IgG4. At antibodies facilitate rapid destruction of Cr- RHAG: three antigens resulting from amino acid substitutions labeled At(a+) RBCs in vivo and give positive results in the in in the Rh-associated glycoprotein. Vox Sang 2010;8:151–9. vitro functional assay, the MMA. Ideally, At(a–) RBCs should 14. Barr K, Korchagina E, Popova I, et al. Monoclonal anti-A a activity against the FORS1 (Forssman) antigen.Transfusion be selected for transfusion to patients with anti-At , although 2014;55:129–36. least incompatible RBC units may be suitable for patients with 15. Svensson L, Hult AK, Stamps R, et al. Forssman expression weak examples of the antibody.2 on human erythrocytes: biochemical and genetic evidence of a One anti-Ata caused an immediate HTR with chills and new histo-blood group system. Blood 2013;121:1459–68. nausea during an RBC survival study, and another caused 16. Storry J. Five new blood group systems: what next? ISBT Sci Ser 2014;9:136–40. a severe delayed HTR after transfusion of multiple units of 17. Castilho L, Reid ME. A review of JR blood group system. At(a+) RBCs. Despite numerous pregnancies involving anti- Immunohematology 2013;29:63–8. Ata, only one of the infants had moderately severe HDFN 18. Saison C, Helia V, Ballif BA, et al. Null alleles of ABCG2 requiring phototherapy.2 In three At(a–)-reported patients encoding the breast cancer resistance protein define the new blood group system Junior. Nat Genet 2012;44:174–7. from the southern United States, the anti-Ata was concomitant 19. Zelinski T, Coghlan G, Liu XQ, et al. ABCG2 null alleles define 5 with autoimmune disease. the Jr(a–) blood group phenotype. Nat Genet 2012;44:131–2. 20. Ogasawara K, Osabe T, Susuki Y, et al. A new ABCG2 null allele References with a 27kb deletion including the promoter region causing the Jr(a–) phenotype. Transfusion 2015;55:1467–71. . 1 Crew VK, Burton N, Kagan A, et al. CD151, the first member of 21. Coghlan G. The JR blood group system: genetic and molecular the tetraspanin (TM4) super family detected on erythrocytes investigations. ISBT Sci Ser 2012;7:260–3. is essential for the correct assembly of human basement 22. Hue-Roye K, Lomas-Francis C, Coghlan G, et al. The JR blood membranes in kidney and skin. Blood 2003;102:4a. group system (ISBT 032): molecular characterization of the . 2 Daniels G. Human blood groups. 3rd ed. London, UK: Wiley- three new null alleles. Transfusion 2013;53:1575–9. Blackwell, 2013. 23. Fujita S, Kashiwagi H, Tomimatsu T, et al. Expression levels . 3 Hayes M. Raph blood group system. Immunohematology of ABCG2 on cord red blood cells and study of fetal anemia 2014;30:6–10. associated with anti-Jr(a). Transfusion 2016;56:1171–81.

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24. Endo Y, Ito S, Ogiyama Y. Suspected anemia caused by maternal 34. Anliker M, Zabera I, Hochsmann B, et al. A new blood group anti-Jr(a) antibodies: a case report. Biomark Res 2015;3:23. antigen is defined by anti-CD59 detected in a CD59-deficient 25. Peyrard T. The LAN blood group system: a review. patient. Transfusion 2014;54:1817–22. Immunohematology 2013;29:131–5. 35. McBean R, Liew Y, Wilson B, et al. Genotyping confirms 26. Reid ME, Hue-Roye K, Huamg A, et al. Alleles of the LAN inheritance of the rare At(a–) type in a case of hemolytic blood group system: molecular and serologic investigation. disease of the newborn. J Path Clin Res 2016;2:53–5. Transfusion 2014;54:398–404. 36. Applewhaite F, Ginsberg V, Gerena J, et al. A very frequent red a 27. McBean R, Wilson B, Liew Y, et al. Quantitation of Lan antigen cell antigen At . Vox Sang 1967;13:444–5. in Lan+, Lan+w, and Lan– phenotypes. Blood Transfus 2015; 37. Daniels G, Ballif B, Helias V, et al. Lack of the nucleoside 13:662–5. transporter ENT1 results in the Augustine-null blood type. 28. Hayer-Wigman I, deHaas M, van der Schoot CE. The immune Blood 2015;125:3651–4. response to the VEL antigen is HLA class II DRB1*11 restricted. 38. Rose JB, Naydenova Z, Bang A, et al. Equilibrative nucleoside Vox Sang 2013;105(suppl 1):29. transporter 1 plays an essential role in cardioprotection. Am J 29. Race RR, Sanger R. Blood groups in man. 6th ed. Philadelphia, Physiol Heart Circ Physiol 2010;298:H771–7. PA: Blackwell Science Ltd., 1975:413. 30. Storry JR, Joud M, Christophersen MK, et al. Homozygosity Mostafa Moghaddam, MA, CLS(ASCP)BB, Head of for a null allele of SMIM1 defines the Vel-negative blood group Immunohematology Reference Laboratory, (corresponding author), phenotype. Nat Gen 2013;45:537–41. Department of Immunohematology, Blood Transfusion Research 31. Vejic A, Haer-Wigman L, Stephens JC, et al. SMIM1 underlines Center, High Institute for Research and Education in Transfusion the Vel blood group and influences red blood cell traits. Nat Medicine, Iranian Blood Transfusion Organization, IBTO Tower, Genet 2013;45:542–5. Hemat Expressway, Tehran, Iran, [email protected]; 32. Ballif BA, Helia V, Peyrard T, et al. Disruption of SMIM1 causes the Vel– blood type. EMBO Mol Med 2013;5:751–61. and Amir Ali Naghi, DMT, Senior Technologist, Department of Immunohematology, Blood Transfusion Research Center, High 33. Storry JR, Mallory D. Misidentification of anti-Vel due to inappropriate use of techniques. Immunohematology 1994;10: Institute for Research and Education in Transfusion Medicine, 83–6. Iranian Blood Transfusion Organization, Tehran, Iran.

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90 IMMUNOHEMATOLOGY, Volume 34, Number 3, 2018 Rouleaux and saline replacement

K.L. Waider

Rouleaux is a phenomenon that commonly occurs in patients who have an increased number of circulating protein macromolecules. Reagents/Supplies It is a benign, in vitro reaction that appears microscopically as red blood cells (RBCs) line up against each other; many liken the RBC Reagents Supplies aggregation to “stacked coins.” This unexpected reactivity may • 0.9% saline or PBS (pH • Transfer pipettes cause confusion in direct agglutination testing such as reverse 6.5–7.5) • 10 × 75 or 12 × 75 mm tubes blood typing and crossmatching. Saline replacement is the established method to resolve rouleaux. True agglutination will • Calibrated serologic centrifuge remain when plasma is replaced with saline for resuspension of • Microscope the RBC button. Rouleaux will no longer be seen when the plasma PBS = phosphate-buffered saline. proteins are removed. Immunohematology 2018;34:91–92. SEROLOGIC METHOD REVIEW METHOD SEROLOGIC Procedural Steps Key Words: rouleaux, stacked coins, saline replacement, macromolecule • Confirm the presence of rouleaux microscopically following initial RBC resuspension. Principle • Re-centrifuge the test tube. • Remove patient’s plasma/serum using transfer pipettes. • Replace with saline. Rouleaux is a type of red blood cell (RBC) aggregation • Resuspend. Confirm. with a distinct “stacked coins” appearance when observed RBC = red blood cell. microscopically. These aggregates frequently have a more refractive appearance than classic RBC agglutination. There are typically many “stacks” in the sample, and their size can vary greatly. The rouleaux aggregates disperse differently Indications from the main RBC button during resuspension, such that experienced technologists can sometimes recognize this Rouleaux may be present in any direct agglutination phenomenon even before confirming it microscopically. test containing plasma or serum. Examples are reverse ABO It is most typically seen in patients with disease states that typing, immediate spin crossmatch, and immediate spin and/ cause an increase in plasma protein macromolecules, such as or 37°C reading of antibody detection and/or identification multiple myeloma, diabetes mellitus, and many infections.1 testing. If unexpected positive results present after routine The macromolecules are thought to cause rouleaux by two incubation and centrifugation, rouleaux should be considered. unrelated mechanisms: the bridging model or the depletion Once confirmed, the saline replacement method3 should model. One or both may occur in an individual. In the bridging be performed using the tube method. It is important to model, the macromolecules partially adsorb into the RBC and remember that rouleaux can only be found in the presence of bridge to the RBC closest to it. In the depletion model, the patient plasma or serum, since this is where the interfering macromolecules move away from the RBCs and change the macromolecules are found. Rouleaux is not seen in the osmotic pressure between RBCs causing them to aggregate antiglobulin phase of testing because the wash step rids the (Fig. 1). 2 Once recognized, rouleaux can be resolved using the test system of the patient’s plasma/serum. saline replacement method. By replacing the patient’s plasma/ serum with saline, the macromolecules are removed, and the Procedure RBCs dissociate. Examine suspected reactivity microscopically. Rouleaux will appear as stacked coins, whereas true agglutinates are

IMMUNOHEMATOLOGY, Volume 34, Number 3, 2018 91 K.L. Waider

Once saline replacement has been performed, only direct agglutination testing may be interpreted. Serum/plasma may not be reintroduced to the test system. If test reading at additional phases are desired, the test must be repeated using a separate tube for each phase where a reading will occur.

(A) Limitations

Rouleaux does not typically cause interference in column testing, although high protein levels can lead to false positives or hazy reactions in this method.4 Confirmation of rouleaux and resolution must be performed with the tube method.

Acknowledgments

The author would like to thank Jan Hamilton for her guidance while working on this manuscript.

(B) References

. 1 Samsel RW, Perelson AS. Kinetics of rouleau formation. I. A mass action approach with geometric features. Biophys J 1982;37:493–514. . 2 Bäumler H, Neu B, Donath E, et al. Basic phenomena of red blood cell rouleaux formation. Biorheology 1999;36:439–42. . 3 Fung MK, Ed. Method 3-7: Detecting antibodies in the presence of rouleaux-saline replacement. In: Technical manual. Fig. 1 (A) Schematic drawing of the bridging model. The 19th ed. Bethesda, MD: AABB, 2017. adsorption of dextran into single cells and the formation of bridges during the absence of shear flow lead to rouleaux formation of . 4 Blood grouping reagent MTS A/B/D monoclonal and reverse cells. (B) Schematic drawing of the depletion model. Depletion grouping card [package insert]. Pompano Beach, FL: Micro of macromolecules from the interface (with or without a weak Typing Systems Inc., 2008. adsorption) leads to rouleaux formation if the cells come into close proximity due to the osmotic pressure difference between the CM pressure in the bulk phase and in the gap. (Reprinted from Bäumler Kayla L. Waider, BS, MLS(ASCP) , Immunohematology Reference et al.2 with permission from Biorheology.) Laboratory Technologist III, American Red Cross Biomedical Services, Southeastern Michigan Region, 100 Mack Avenue, Detroit, MI 48201, [email protected]. amorphous clumps. After confirming the presence of rouleaux, re-centrifuge the plasma (or serum) and cell mixture. Carefully remove the plasma using transfer pipettes, leaving the RBC button undisturbed. This effect can be achieved by tilting the tube so that the plasma is away from the cell button, allowing the pipette to aspirate it without touching the RBCs. Gently replace the plasma with an equal volume of saline without disturbing the RBC button. Resuspend the RBC button, and observe for agglutination. Rouleaux will disperse when suspended in saline, but true agglutination is stable in the presence of saline and will remain.

92 IMMUNOHEMATOLOGY, Volume 34, Number 3, 2018 O r i g i n a l R e p or t Method-specific and unexplained reactivity in automated solid-phase testing and their association with specific antibodies

M.E. Harach, J.M. Gould, R.P. Brown, T. Sanders, and J.H. Herman

The inherent tradeoff between sensitivity and specificity in the as many clinically significant antibodies as possible while detection of unexplained antibodies has been the objective of avoiding as many clinically insignificant antibodies.1 many studies, editorials, and journal articles. Many publications This inherent tradeoff between sensitivity and specificity note that no method is capable of detecting all clinically significant antibodies while avoiding all clinically insignificant in the detection of unexplained antibodies has been the antibodies. This study describes the frequency of nonspecific objective of many studies, editorials, and journal articles. reactivity and unexplained reactivity in solid-phase testing, along Although all these publications note that no method is with the subsequent development of specific antibodies (Abs). In this study, nonspecific reactivity (NS) is defined as method- capable of detecting all clinically significant antibodies specific panreactivity detected by solid-phase testing only, with while avoiding all clinically insignificant antibodies, some of no reactivity in other methods. Unexplained reactivity (UR) is these studies reported decreased detection of insignificant defined as reactivity present and detectable in all test methods antibodies by gel,2 and others noted the superior detection after all clinically significant antibodies were ruled out following 3 a standard antibody identification algorithm using selected cell of clinically significant antibodies by solid-phase testing. In panels. This retrospective study evaluated antibody detection their study, Liu and Grossman4 noted that in gel an “antibody tests of patients at a single center for 2 years using two automated of undetermined specificity (AUS) was reported 1442 times solid-phase instruments that used the same three-cell antibody (18%) and was the single most reported event” of the 8121 detection test. Antibody identification was performed with solid- phase panels supplemented with a polyethylene glycol tube antibodies evaluated. In the same study, they document that method as needed. Of the 1934 (5%) samples with a positive in patients presenting with AUS for the first time, the majority antibody detection test, 29 had unavailable work-up data, of AUS persist in subsequent workups and that some AUSs leaving 1905 (98.5%) samples eligible for inclusion in the study. 4 The data revealed the following: Ab only 999 (52.4%); UR only even develop into clinically significant antibodies. The 2017 429 (22.5%); Ab and UR 227 (11.9%); NS only 206 (10.8%); Ab study by Miller et al.5 noted the increased sensitivity of general and NS 24 (1.3%); UR and NS 14 (0.7%); and Ab, UR, and NS nonspecific reactivity in solid-phase testing when compared 6 (0.3%). Patients with a positive follow-up antibody detection with tube method testing using polyethylene glycol (PEG) test had UR and NS replaced with a specific Ab in 23 of 656 UR (3%) and 8 of 230 NS (3%) cases, respectively. Additionally, six and recommends this nonspecific reactivity to not be ignored patients with UR developed a specific Ab along with persistent because of the noteworthy proportion of these antibodies that UR, and no patients with persistent NS developed a specific Ab. develop into specific and clinically significant antibodies. The The study concluded that both UR and NS can be encountered in Miller et al. study, however, did not focus on the frequency with solid-phase testing, and both UR and NS can persist in follow-up testing. Specific Ab was observed to replace UR in a few patients. which nonspecific reactivity might progress to the level of true Immunohematology 2018;34:93–97. antigen specificity, as did the Liu and Grossman study of the gel method. In our current study, nonspecific reactivity (NS) Key Words: immunohematology, red blood cell serology, is defined as method-specific panreactivity detected by solid- blood groups, nonspecific reactivity, unexpected reactivity phase testing only, with no reactivity in other methods. This reactivity is in contrast to unexplained reactivity (UR), defined Transfusion medicine professionals have the responsibility as reactivity present and detectable in all test methods after to identify and provide compatible blood components for all clinically significant antibodies were ruled out following transfusion to their patients. This responsibility includes our standard antibody identification algorithm using selected the detection and identification of unexplained antibodies. cell panels.6 Our data analysis attempts to enumerate the According to the 18th edition of the AABB Technical Manual, frequency of UR and NS as well as evaluate their association the goals of antibody detection are to detect in a timely manner with development of specific antibodies (Abs) over time.

IMMUNOHEMATOLOGY, Volume 34, Number 3, 2018 93 M.E. Harach et al.

Materials and Methods Results of antibody identification testing revealed UR was present in 917 of 1905 (48%) patients, and NS was present in This retrospective study evaluated the antibody detection 250 of 1905 (13%) patients. The various combinations of Ab, test results of patients between 1 January 2012 and 31 UR, and NS were as follows: Ab only 999 (52.4%); UR only December 2013. In this single facility, for the time period under 429 (22.5%); Ab and UR 227 (11.9%); NS only 206 (10.8%); evaluation, all antibody detection tests were performed on two Ab and NS 24 (1.3%); UR and NS 14 (0.7%); and Ab, UR, and different automated instruments (Echo and Galileo; Immucor, NS 6 (0.3%). For the samples in which UR and NS were both Norcross, GA). Both instruments use the same three-cell noted, UR was detected in a different antibody identification antibody detection test plates (Capture-R Ready-Screen, method (PEG tube) after NS was detected in the solid-phase Immucor) to reduce ambiguity between donor cells. Antibody method. The results are summarized in Table 1. There was a detection test results were read and graded by the automated history of a specific Ab in 141 of 656 (21%) patients with UR instruments or by a medical laboratory scientist who read and and in 42 of 230 (18%) patients with NS. graded (negative, weak+, 1+, 2+, 3+, or 4+) results according to method 1–9 in the AABB Technical Manual.1 Table 1. Solid-phase antibody evaluation summary In the instance of a positive antibody detection test result, Antibody work-up results (N = 1905) the patient’s historical antibody records were reviewed in n %* addition to the ensuing antibody identification performed on Antibody only 999 52.4 the current sample. Antibody identification was performed Unexplained reactivity only 429 22.5 primarily with solid-phase panels (Capture-R Ready-ID, Antibody and unexplained reactivity 227 11.9 Capture-R Ready-ID Extend I and Extend II; Immcor). If Nonspecific reactivity only 206 10.8 needed, this testing was supplemented with a PEG additive Antibody and nonspecific reactivity 24 1.3 (Gamma PeG, Immucor) tube method using a 10-minute Unexplained reactivity and nonspecific 14 0.7 incubation. Antibody reactions were read and graded by the reactivity same methods as previously described. Antibody, unexplained reactivity, and 6 0.3 An observation of UR or NS resulted in reviewing the past nonspecific reactivity records of the associated patient and evaluating subsequent *Percent of total, rounded to nearest 0.1%. samples for the presence of UR and/or NS and for the development of specific Abs. Based on the results of the follow- For samples in which UR or NS was observed, follow-up up testing of these subsequent samples, patients were assigned testing of subsequent samples was evaluated, and the patients to one of the following categories: were assigned to one of the following categories: • Category 1: No testing performed • Category 1: No testing performed in 326 of 656 UR • Category 2: Positive antibody detection test; antibody (50%) and 129 of 230 NS (56%) identification unresolved • Category 2: Positive antibody detection test; antibody • Category 3: Negative antibody detection test identification unresolved in 89 of 656 UR (14%) and 51 • Category 4: Positive antibody detection test; UR or NS of 230 NS (22%) replaced by specific Ab • Category 3: Negative antibody detection test in 178 of • Category 5: Positive antibody detection test; UR or NS 656 UR (27%) and 23 of 230 NS (10%) only remained • Category 4: Positive antibody detection test; UR or NS replaced by specific Ab in 23 of 656 UR (3%) and 8 of Results 230 NS (3%) • Category 5: Positive antibody detection test; UR only In the 24-month study period, antibody detection testing remained in 40 of 656 UR (new specific antibody was performed on a total of 38,178 patients. Of these, 1934 developed in 6 of 40 of these) or NS only in 19 of 230 NS (5%) had a positive antibody detection test. The antibody Category 4 and 5 results are graphically depicted in identification workup was unavailable for 29 of these patients, Figure 1. leaving 1905 (98.5%) eligible for inclusion in this study.

94 IMMUNOHEMATOLOGY, Volume 34, Number 3, 2018 Method-specific and unexplained reactivity

A

Solid-phase nonspecific reactivity (NS)

HLA antibody

Cold antibody

Anti-Lea

Anti-K

Anti-Jkb

Anti-D

Anti-c

Anti-E

Anti-Jka

0 1 2 3 4 5 6 7 8

Number of patients

B

UR

Warm autoantibody and UR

Warm autoantibody

Cold antibody

Anti-Jkb and UR 0 1 2 3 4

Number of patients

Fig. 1 (A) Unexplained reactivity (UR) replaced with antibodies and reactivity, as shown. (B) Nonspecific reactivity (NS) replaced with antibodies and reactivity, as shown. HLA = human leukocyte antigen.

Discussion 40 of 241 (17%), and NS was undetectable in 31 of 50 (62%) and persisted in 19 of 50 (38%). Specific Ab development in Our data also shed light on the constant balance between patients in categories 3, 4, and 5 occurred in 29 of 241 (12%) sensitivity and specificity. During follow-up testing, we of previous UR and 8 of 50 (16%) of NS. Figure 1 depicts the determined the development from UR and/or NS to true Ab specific Abs identified in place of the UR/NS. specificity. Those samples in categories 1 and 2 can be omitted Of interest is that during the study period, the because of either a lack of follow-up or no resolution of specific manufacturer (Immucor) alerted users to an increase in UR antibody identification. Of those samples with evaluable follow- reported to them by their customers. Steps were taken during up antibody detection and identification in categories 3, 4, and reagent manufacturing, and resolution of the increase in UR 5, UR was undetectable in 201 of 241 (83%) and persisted in was communicated to users based on customer reports. To

IMMUNOHEMATOLOGY, Volume 34, Number 3, 2018 95 M.E. Harach et al.

1.4

1.2

1.0 Echo UR 0.8 Echo NS

Percent 0.6 Galileo UR Galileo NS 0.4

0.2

0 Pre-alert Alert Post-alert

Fig. 2 The frequency of unexplained reactivity (UR)/nonspecific reactivity (NS) changed during the study period, which overlapped with the manufacturer’s alert regarding UR/NS. The pre-alert period is defined as 1 January 2012 to 30 September 2012, the alert period is 1 October 2012 to 30 April 2013, and the post-alert period is 1 May 2013 to 31 December 2013. determine whether the UR alert affected UR and NS rates patients, it can be concluded that UR and NS antibodies are at our facility, the study periods before, during, and after the encountered in solid-phase testing and may be transient. In alert period were compared. Based on the issuance date of the these patients, the UR was undetectable in 201 of 241 (83%) alert (22 March 2013), the pre-alert period was defined as 1 and persisted in 40 of 241 (17%) of the patients on follow-up January 2012 through 30 September 2012, the alert period testing. Also, the NS was undetectable in 31 of 50 (62%) and was defined as 1 October 2012 through 30 April 30 2013, and persisted in 19 of 50 (38%) of the patients upon follow-up the post-alert period was defined as 1 May 2013 through 31 testing. December 2013. UR and NS antibodies detected in solid-phase testing may A decrease in the rate of UR and NS after the alert period develop into true antibody specificity. Of those patients with was noted on the smaller instrument (Echo, Immucor) (Fig. 2). evaluable follow-up antibody detection tests (categories 3, 4, There was a 26 percent overlap of patients with antibody and 5), a new antibody developed in 29 of 241 patients with detection performed on either instrument (Galileo or Echo, a previous UR (12%) and in 8 of 50 patients with a previous Immucor) on different dates, but because of the retrospective NS (16%). Based on a high regard for patient safety, which nature of the study and the relatively small numbers, no includes the maximum detection of clinically significant further trend was apparent in distinguishing UR and NS on antibodies, this facility has selected the automated solid-phase the two solid-phase devices. Further studies are in progress at method—knowing the rate of UR and NS—because of the well- this facility to fully evaluate the impact of the improvements documented superior sensitivity of Capture-R3,7 compared communicated on the UR and NS rates. Additionally, these with other testing methods such as tube testing. subsequent studies use a newer instrument (Galileo NEO, Immucor) and will generate data as a second phase to this References original study with the updated automated instrument. . 1 Fung MK, Grossman BJ, Hillyer CD, Westhoff CM. Technical manual. 18th ed. Bethesda, MD: AABB, 2014. Conclusions . 2 Judd WJ, Steiner EA, Knafl PC. The gel test: sensitivity and specificity for unexplained antibodies to blood group antigens. Immunohematology 1997;13:132–5. The patients who have evaluable follow-up antibody . 3 Weisbach V, Kohnauser T, Zimmermann R, et al. Comparison detection testing are described in categories 3, 4, and 5. Based of performance of microtube column systems and solid- on analysis of the testing performed on samples from these phase systems and the tube low-ionic-strength solution

96 IMMUNOHEMATOLOGY, Volume 34, Number 3, 2018 Method-specific and unexplained reactivity

additive indirect antiglobulin test in the detection of red cell alloantibodies. Transfus Med 2006;16:276–84. Mary E. Harach, BS, MT(ASCP), Quality Assurance Coordinator (corresponding author), Thomas Jefferson University Hospital, . 4 Liu C, Grossman BJ. Antibody of undetermined specificity: frequency, laboratory features, and natural history. Transfusion Department of Transfusion Medicine, 111 South 11th Street, Suite 2013;53:931–7. 8250 Gibbon, Philadelphia, PA 19107, [email protected]; . 5 Miller NM, Johnson ST, Carpenter E, Naczek CA, Karafin MS. Joy M. Gould, BB(ASCP)C, SBB, Blood Bank Supervisor, Thomas Patient factors associated with unidentified reactivity in solid- Jefferson University Hospital, Department of Transfusion Medicine, phase and polyethylene glycol antibody detection methods. Philadelphia, PA; Rosemary P. Brown, MT(ASCP), Senior Medical Transfusion 2017;57:1288–93. Technologist–Blood Bank, Thomas Jefferson University Hospital, . 6 US Food and Drug Administration. Subpart D, Reagent Red Department of Transfusion Medicine, Philadelphia, PA; Tricia Blood Cells. Testing of source material. 21 CFR Sect. 660.33 Sanders, MBA, MLS(ASCP)CMSBBCM, Global Education and Technical (2017). eCFR Code of Federal Regulations. Available from Marketing Program Manager, Immucor Diagnostics, Norcross, https://www.ecfr.gov/cgi-bin/text-idx?SID=26cb4151fc77996 GA; and Jay H. Herman, MD, Thomas Jefferson University 051b7706a77bcb400&mc=true&node=pt21.7.660&rgn=div5 #se21.7.660 _133. Hospital, Department of Pathology, Anatomy and Cell Biology, . 7 Kay B, Poisson JL, Tuma CW, Shulman IA. Anti-Jka that are Philadelphia, PA. detected by solid-phase red blood cell adherence but missed by gel testing can cause hemolytic transfusion reactions. Transfusion 2016;56:2973–9.

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].

IMMUNOHEMATOLOGY, Volume 34, Number 3, 2018 97 Utility of chloroquine diphosphate in the blood bank laboratory

T. Aye and P.A. Arndt

Chloroquine diphosphate (CDP) is a helpful tool in the blood bank Reagents/Supplies — Room Temperature for two main applications. The most common application is to render direct antiglobulin test–positive red blood cells (RBCs) Reagents Supplies free from membrane-bound IgG; these treated RBCs can then be used for autologous adsorption and/or to determine the patient’s • Anti-IgG • Pipettes RBC phenotype. Another common use of CDP is to remove • IgG-coated RBCs • Test tubes: 10 × 75 or 12 × 75 mm human leukocyte antigens (HLAs) from RBCs to help identify or • 16–20% CDP solution at • Calibrated centrifuge exclude the presence of antibodies to HLAs expressed on RBCs, pH 5.0 ± 1 for example, Bennett-Goodspeed (Bg) antigens. In this review, • Isotonic saline the principles, applications, and limitations of using CDP are RBCs = red blood cells; CDP = chloroquine diphosphate. discussed. Immunohematology 2018;34:98–102.

Procedural Steps — Room Temperature Key Words: chloroquine diphosphate, direct antiglobulin test, human leukocyte antigen • Mix 1 volume of washed, packed RBCs with 4 volumes of 16–20% CDP. • Incubate mixture at room temperature. Antigen typing of red blood cells (RBCs) using an indirect • At 30-minute intervals, wash a small aliquot of RBCs at least three antiglobulin test (IAT) method can be challenging when RBCs times, and perform DAT using anti-IgG to monitor the process of IgG dissociation. The mixture can be incubated for up to 2 hours. are coated with IgG antibodies and thus have a positive direct • When the DAT is negative, wash the remaining CDP-treated RBCs antiglobulin test (DAT). Commercially available monoclonal at least three times with isotonic saline. antisera that can be used at the direct agglutination phase RBCs = red blood cells; CDP = chloroquine diphosphate; without an IAT are not available for typing of all blood group DAT = direct antiglobulin test. antigens. For those monoclonal antisera that are available, false-positive and false-negative results can occur when Reagents/Supplies — 37°C testing untreated RBCs if they are strongly coated with IgG.1–3

SEROLOGIC METHOD REVIEW The majority of “in-house” or “rare” antiserum is collected Reagents Supplies from patients and is usually reactive only by the IAT. When • Anti-IgG • Pipettes there are no commercially available antisera that can be used • IgG-coated RBCs • Test tubes: 10 × 75 or 12 × 75 mm by direct agglutination tests and/or the patient’s sample has • 16–20% CDP solution at • Calibrated centrifuge pH 5.0 ± 1 a strongly positive DAT, there is a need for a reliable method • 37°C water bath or heat block • Isotonic saline to dissociate membrane-bound IgG to then obtain an accurate RBCs = red blood cells; CDP = chloroquine diphosphate. RBC phenotype using an IAT method. Although chloroquine diphosphate (CDP) was reported Procedural Steps — 37°C in the 1970s to remove warm autoantibodies from DAT+ 4,5 • Mix 1 volume of washed, packed RBCs with 4 volumes of 16–20% RBCs, its use was not applied to antigen-typing of RBCs with CDP. a positive DAT until the 1980s (by Edwards et al.6) There were • Incubate mixture at 37°C. only two other procedures available at that time—gentle heat • At 5-minute intervals, wash a small aliquot of RBCs at least three 7 times and perform a DAT using anti-IgG to monitor the process of elution (e.g., 45°C for 10–30 minutes or 56°C for 3 minutes) IgG dissociation. The mixture can be incubated for up 30 minutes. or treatment with ZZAP, a combination of dithiothreitol (DTT) • When DAT is negative, wash the remaining CDP-treated RBCs at least three times with isotonic saline. and papain.8 Heating RBCs above 37°C weakens antigens relative to the temperature and time of incubation9 and ZZAP RBCs = red blood cells; CDP = chloroquine diphosphate; DAT = direct antiglobulin test. destroys antigens sensitive to DTT and papain (Kell, Duffy,

98 IMMUNOHEMATOLOGY, Volume 34, Number 3, 2018 Use of CDP

MNS, etc.). Gentle heat elution and ZZAP are good methods Principle for preparing RBCs for autoadsorption; CDP has also been described for this use. CDP diluted to 16–20 percent (wt/vol) at a final pH of Treatment of DAT+ RBCs with glycine-HCl/EDTA (also 5.0 ± 0.1 is used to dissociate membrane-bound IgG from known as EDTA glycine acid [EDTA acid]) was described RBCs.6,15 Issitt and Anstee16 suggested that the most likely by Louie et al.10 and reviewed by Kosanke.11 It is quick and mechanism for this dissociation involves neutralization of reliable but destroys Kell system antigens and Era. Microwave charged groups on amino acids that influence the shape irradiation was also described to remove IgG from RBCs and of the antibody molecules that bind to antigens. Edwards allow phenotyping.12 All these methods have their limitations. et al.6 found that CDP was effective on 84 percent of in vivo Most significant is the potential for weakening or destroying sensitized DAT+ samples tested; results of other studies are RBC antigens when the treatment is done to prepare RBCs summarized in Table 1. Factors that influence the ability of for phenotyping. Because of these limitations, there is a need CDP to dissociate antibodies from DAT+ RBCs may be the IgG to use more than one method in an immunohematology subclass of the antibody, the antibody affinity, the accessibility reference laboratory. Depending on the antigens of interest, an of the antigen-antibody complex, and the quantity of antibody appropriate method can be selected to dissociate membrane- bound to the RBCs.6 The temperature of CDP treatment (e.g., bound IgG from DAT+ RBCs. room temperature versus 37°C) and the sensitivity of the DAT Bennett-Goodspeed (Bg) antigens are human leukocyte method (e.g., tube versus gel) can also affect the outcome of antigen (HLA) class I (HLA-A and HLA-B) weakly expressed CDP treatment. on RBCs, including the reagent RBCs used in blood bank CDP can remove HLAs expressed on RBCs (e.g., Bg testing. Despite their clinical insignificance, Bg antibodies antigens).13,14 This fact can be useful in antibody detection may interfere in routine antibody detection and identification and identification tests where the antibodies may show weak tests by showing weak unexpected reactions without a distinct reactivity with no apparent specificity. By testing a patient’s pattern. CDP and EDTA acid destroy Bg antigens on RBCs; plasma with CDP-treated RBCs in parallel with untreated thus, these reagents can be useful in identifying or excluding RBCs, the presence of Bg antibodies may be identified reactions caused by Bg antibodies.13,14 presumptively. The Bg antibodies may then be confirmed

Table 1. Studies on the efficiency of CDP for clearing IgG versus other methods

IgG-coated RBC samples Reference Number Type* Strength Results using RT or 37°C CDP method Results by other methods 6 56 In vivo 1–4+ RT = 47 (84%) neg NA 40 In vitro 3–4+ RT = 22 (55%) neg NA 10 30 In vivo Strong+ RT = 26 (87%) neg EDTA acid = 30 (100%) neg; ZZAP = 28 (93%) neg 12 15 In vivo 1–3+ RT = 4 (27%) neg Microwave = 7 (47%) neg 17 20 In vitro Variable RT = 10 (50%) neg ZZAP = 18 (90%) neg 37°C = 20 (100%) neg 18 8 In vivo 1–4+ RT = 26 (52%) neg by tube, EDTA acid = 48 (96%) neg by tube, 22 (44%) neg by gel 42 (84%) neg by gel 42 In vitro 56°C heat = 46 (92%) neg by tube, 25 (50%) neg by gel 19 26 In vivo ±–4+ RT = 25 (96%) neg EDTA acid = 25 (96%) neg 20 93 In vivo w+–4+ RT = 29 (31%) â by gel EDTA acid = 59 (63%) â by gel 56°C heat = 36 (39%) â by gel 17 In vitro No data available RT = 5 (29%) neg by gel EDTA acid = 12 (71%) neg by gel 56°C heat = 9 (53%) neg by gel *In vivo = direct antiglobulin test (DAT)+ red blood cells (RBCs); In vitro = RBCs coated with antibody in the laboratory. CDP = chloroquine diphosphate; RT = room temperature; neg = negative; NA = not applicable; â = reduction in strength.

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or excluded by further testing with routine HLA laboratory used, the manufacturer’s recommendations supersede what is methods or by testing with RBCs known to show strong described here. expression of Bg antigens. Along with Bg, CDP may destroy The most common method involves CDP treatment at or weaken other important RBC antigens (see Limitations). room temperature with an incubation time that varies from Because of the possible effect that CDP can have on these other 30 to 120 minutes.6,15 Briefly, 4 volumes of 16–20 percent antigens, caution must be taken in antibody exclusions when chloroquine, pH 5.0 ± 0.1, are added to 1 volume of washed, identifying interfering reactions due to Bg antibody using packed DAT+ RBCs. After 30 minutes, a small subsample is CDP-treated RBCs. Other blood group antigens reported to be removed and washed, and a DAT is performed using anti- affected by CDP are summarized in Table 2. IgG. If the DAT is negative, then the rest of the sample can be washed and used for phenotyping and/or autoadsorption. If Procedural Steps the DAT is still positive, then the incubation can continue for another 30 minutes and the process is repeated (to a maximum The supplies and procedural steps for CDP treatment incubation time of 2 hours). An alternative method involves are provided. If commercially manufactured CDP reagent is CDP treatment at 37°C.17,24,29 Beaumont et al.24 studied RBC

Table 2. Blood group antigens reported to be significantly affected or not affected by CDP

Blood group system/ antigen Significantly affected by CDP Not affected by CDP ABH NDA (NI)6,13 Rh (D, C, e; reduced and saline antisera)21; (D; direct testing, few by (NI)6,13,20; (C)23; (D, C, E, c, e at 37°C)24; (C, c, E, e)18 IAT)22; (D, C, E, c, e, Cw; weakened at RT and 37°C)17 MNS (Mta)25 (NI)6,13,20; (S, s)18,19,26; (S)27; (Mg, Mta, He)13; (M, N, S, s at RT and 37°C)17 P1PK (P1 weakened at 37°C)17 (NI)6; (P1)13; (P1 at RT)17 Lewis (Lea, Leb, weakened at 37°C)17 (NI)6,13; (Lea, Leb at RT)17 Kell NDA (NI)6,13,20; (K, k)26; (K, k, Kpa, Kpb at RT and 37°C)17; (K, k at 37C)24; (K, k, Kpb)18; (K, Kpa)2 Duffy (Fyb)26; (Fyb, sig â after 2 hours)27; (NI, weakened)20 (NI)6,13; (Fya, Fyb)18,19,26; (Fya)2,27; (Fya, Fyb at RT and 37°C)17; (Fya, Fyb at 37°C)24 Kidd (Jkb weakened after 2 hours at RT, Jka weakened at 37°C)28; (NI)6,13,20; (Jka, Jkb)18,19,26–28; (Jka, Jkb at RT and 37°C)17; (Jkb at 37°C)24 (Jka at 37°C)24 Knops (NI)25 (Kna)27; (Kna, McCa, Yka)13; (Kna, McCa, Sla, Yka)23 Lutheran (Lub)26; (Lub, slight â)27 (Lub)26 JMH (JMH)26; (JMH, slight â)27 (JMH)13 Vel (Vel, sig â after 120 minutes)27 NDA Yt (Yta)26; (Yta, sig â after 2 hours)27 (Yta)13,26 Chido/Rodgers NDA (Ch, Rg)13 Lan NDA (Lan)13 Cromer NDA (Cra)13 Colton NDA (Coa)13 Dombrock (NI)25 (Gya)13 Diego NDA (Bpa, Rba, Wda, Wra, Vga)13 Indian NDA (Ina)13 Cost NDA (Csa)13,23 700 Series NDA (Bya)13 Miscellaneous NDA (Cad)13 CDP = chloroquine diphosphate; NDA = no data available; NI = specificities not indicated; IAT = indirect antiglobulin test; RT = room temperature; sig = significant;â = decrease.

100 IMMUNOHEMATOLOGY, Volume 34, Number 3, 2018 Use of CDP

treatment with CDP at 18°C, 25°C, 30°C, and 37°C. Incubation Limitations at 37°C is known to facilitate IgG elution; when incubating at 37°C, a subsample is removed and washed, and a DAT is Weakening or denaturation of some RBC antigens was performed using anti-IgG every 5 minutes (to a maximum observed in some instances (Table 2). Per the manufacturer’s incubation time of 30 minutes).24 In both methods of treatment instructions for Gamma-Quin,15 CDP-treated RBCs can be (room temperature or 37°C), there is a threshold for incubation typed with high-protein reagents or those reactive by IAT but time where further incubation will not facilitate dissociation should not be typed with saline-reactive or monoclonal blood of antibody from RBCs, and the RBCs start to deteriorate. typing reagents because of weaker-than-expected reactions. An alternative treatment method to dissociate IgG should be Mallory and Reid26 reported false-negative antigen typing of considered. some CDP-treated RBCs when tested with some antiglobulin For removal of Bg antigens, Swanson and Sastamoinen13 reacting antisera. McShane and Cornwall29 tested 90 and Champagne et al.14 treated RBCs with commercially antibodies (specificities not given) and found that 63 showed available chloroquine (Gamma-Quin; now available from reduced titration scores, and 25 were nonreactive when testing Immucor, Norcross, GA) at room temperature for 90 minutes13 RBCs that had been treated with CDP for 30 minutes at 37°C. or up to 2 hours.14 Most of these 25 antibodies were low titer or low avidity. They also treated RBCs with CDP at room temperature and saw Indications a loss of reactivity, although less than that at 37°C.29 These investigators thus cautioned the use of rare single donor CDP treatment can be used when a patient’s RBCs antibodies that are “low titer” or “low avidity” to obtain a are coated with IgG and there is a need to perform antigen phenotype on CDP-treated RBCs because of possible antigen typing using antisera that are reactive only at the IAT phase weakening.29 Factors affecting the strength of antigens on and/or to prove the presence of an autoantibody by testing a CDP-treated RBCs include time and temperature of treatment; patient’s plasma, serum, or eluate against DAT– autologous concentration, volume, and pH of CDP solution; and potency RBCs. Treatment with CDP dissociates membrane-bound of antisera. Weaker results with treated RBCs will be more IgG, allowing the treated RBCs to be tested at the IAT phase apparent if titration scores are used for comparison rather without interference. CDP treatment can also be used to than results with undiluted antisera. avoid false-positive or false-negative results with monoclonal CDP does not always completely remove IgG from RBCs. antisera when testing a patient’s RBCs that are strongly coated Different publications show a wide variation (27–96%) in with IgG. For institutions that advocate the use of specific the effectiveness of CDP (Table 1). The variations may be antigen–matched blood for transfusion in the presence of a due to differences in the antibody-coated RBCs and the CDP warm autoantibody, an RBC phenotype from DAT+ RBCs treatment methods used. RBCs with only partial removal of can be obtained by treating the RBCs with a chemical such as IgG may still be useful for antigen typing or autoadsorption CDP, as long as the patient has not been recently transfused. purposes. Most studies comparing CDP with other methods CDP treatment can also be used to remove or reduce RBC- found more effective IgG removal using EDTA acid (Table 1). bound IgG so the patient’s RBCs can be used in autologous Another disadvantage of CDP, compared with EDTA adsorptions (again, as long as the patient has not been recently acid, is that CDP treatment requires longer incubation time. transfused). At room temperature, CDP treatment requires a minimum When Bg antibodies are suspected to cause weak incubation of 30 minutes (maximum, 2 hours) whereas EDTA interfering reactions in antibody detection or identification acid treatment requires no more than a 2-minute incubation. testing and a laboratory does not have the capability to identify In urgent situations where antigen typing must be performed HLA class I antibodies in a patient’s plasma, CDP-treated cells on DAT+ RBCs—for example, to confirm the specificity of can be tested in parallel with untreated RBCs to presumptively alloantibodies—it may be more practical to use EDTA acid than exclude or identify Bg antibodies and to allow the identification CDP. CDP, like EDTA acid, does not dissociate membrane- of other RBC alloantibodies. The abrogation of reactivity with bound complement, so it is important to use anti-IgG when CDP-treated RBCs suggests that the weak interfering reactions testing CDP-treated or EDTA acid-treated RBCs. Note that, may be caused by Bg antibodies. unlike CDP or EDTA acid, ZZAP can be used to remove RBC- bound complement.30

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When using commercially manufactured CDP for 14. Champagne K, Spruell P, Chen J, et al. EDTA/glycine-acid clinical testing, it is necessary to follow the manufacturer’s versus chloroquine diphosphate treatment for stripping Bg antigens from red blood cells. Immunohematology 1999; recommendations. Any steps taken outside the manufacturer’s 15:66–8. recommendation should be validated in-house before 15. Gamma-Quin. Manufacturer’s instructions. Norcross, GA: implementing their use. If there is a concern about weakening Immucor, 2013. of antigen by CDP treatment, an aliquot of RBCs known to carry 16. Issitt PD, Anstee DJ. Applied blood group serology. 4th ed. Durham, NC: Montgomery Scientific, 1998. a single dose expression of the antigen(s) under investigation 17. Burich MA, AuBuchon JP, Anderson HJ. Evaluation of should be treated and tested in parallel. The CDP-treated rapid antibody dissociation techniques. Immunohematology RBCs should be tested with a protein medium (e.g., 6–10% 1986;2:76–80. albumin) in parallel with the blood grouping reagents. If the 18. Burin des Roziers N, Squalli S. Removing IgG antibodies from intact red cells: comparison of acid and EDTA, heat and results with the albumin control are positive, then the antigen chloroquine elution methods. Transfusion 1997;37:497–501. typing results are invalid. 19. Sererat TS, Veidt D, Dutched A. A quick and simple method for phenotyping IgG-sensitized red blood cells. References Immunohematology 2000;16:154–6. 20. Katharia R, Chaudhary RK. Removal of antibodies from red . 1 Rodberg K, Tsuneta R, Garratty G. Discrepant Rh phenotyping cells: comparison of three elution methods. Asian J Transfus results when testing IgG-sensitized RBCs with monoclonal Rh Sci 2013;7:29–32. reagents (abstract). Transfusion 1995;35(Suppl):67S. 21. Sassetti R, Nichols D. Decreased antigenic reactivity caused . 2 Lee E, Hart K, Burgess G, et al. Efficacy of murine monoclonal by chloroquine (letter). Transfusion 1982;22:537. Moulds antibodies in RBC phenotyping of DAT-positive samples. JJ, Edwards JM, Judd WJ (response). Transfusion 1982;22: Immunohematology 2006;22:161–5. 537–8. . 3 DePalma H, Lomas-Francis C, Wilson-Sandberg K, et al. 22. Prewitt PL, Steane EA, Steane SM. Decreased agglutinability Unexpected antigen typing discrepancies in testing of DAT+ with anti-D of chloroquine-treated red cells (abstract). RBCs with monoclonal reagents (abstract). Transfusion Transfusion 1983;23:434. 2017;57(Suppl S3):37A. 23. Chen J, Spruell P, Moulds M. Studies using chloroquine . 4 Holtz G, Mantel W, Buck W. The inhibition of antigen-antibody diphosphate to strip HLA A and B antigens from red blood cells reactions by chloroquine and its mechanism of action. Z (RBCs). ISBT/AABB Book of Abstracts 1990;152. Immunitatsforsch Exp Klin Immunol 1973;146:145–57. 24. Beaumont AE, Stamps R, Booker DJ, Sokol RJ. An improved . 5 Mantel W, Holtz G. Characterisation of autoantibodies method for removal of red cell-bound immunoglobulin using to erythrocytes in autoimmune haemolytic anaemia by chloroquine solution. Immunohematology 1994;10:22–4. chloroquine. Vox Sang 1976;30:453–63. 25. Reid ME, Lomas-Francis C, Olsson ML. The blood group . 6 Edwards JM, Moulds JJ, Judd WJ. Chloroquine dissociation antigen factsbook. San Diego, CA; Academic Press, 2012. of antigen-antibody complexes: a new technique for typing red 26. Mallory D, Reid M. Misleading effects of chloroquine (letter). cells with a positive antiglobulin test. Transfusion 1982;22: Transfusion 1984;24:412. 59–61. 27. Shin C, Mallory D, Reid M. False negative red cell antigen . 7 Widmann FK, Ed. AABB technical manual. 8th ed. typing following chloroquine treatment (abstract). Transfusion Philadelphia, PA: JB Lippincott, 1981. 1984;24:416. . 8 Branch DR, Petz LD. A new reagent (ZZAP) having multiple 28. Brazell J. Use of monoclonal Jk(a) and Jk(b) reagents in applications in immunohematology. Am J Clin Pathol 1982; phenotyping red cells with a positive direct antiglobulin test. 78:161–7. Immunohematology 1994;10:16–8. . 9 Petz LD, Garratty G. Acquired immune hemolytic anemia. 29. McShane K, Cornwall S. Chloroquine reduces antigen strength New York: Churchill Livingstone, 1980. (letter). Transfusion 1985;25:83. 10. Louie JE, Jiang AF, Zaroulis CG. Preparation of intact 30. Leger RM, Garratty G. A reminder that ZZAP reagent antibody-free red blood cells in autoimmune hemolytic anemia removes complement in addition to IgG from coated RBCs. (abstract). Transfusion 1986;26:550. Immunohematology 2006;22:205–6. 11. Kosanke J. EDTA glycine acid treatment of red blood cells. Immunohematology 2012;28:95–6. Thandar Aye, MLS(ASCP)SBB, Senior Technologist (corresponding 12. McCullough JS, Torloni AS, Brecher ME, et al. Microwave dissociation of antigen-antibody complexes: a new elution author), Immunohematology Reference Laboratory, Bloodworks technique to permit phenotyping of antibody-coated red cells. NW, 921 Terry Avenue, Seattle, WA 98104, taye@bloodworksnw. Transfusion 1993;33:725–9. org; and Patricia A. Arndt, MS, MT(ASCP)SBB, Lead Technologist, 13. Swanson JL, Sastamoinen R. Chloroquine stripping of HLA Special Immunohematology Laboratory, American Red Cross Blood A,B antigens from red cells (letter). Transfusion 1985;25: Services, Southern California Region, Pomona, CA. 439–40.

102 IMMUNOHEMATOLOGY, Volume 34, Number 3, 2018 R e v i e w Proceedings from the International Society of Blood Transfusion Working Party on Immunohaematology, Workshop on the Clinical Significance of Red Blood Cell Alloantibodies, September 2, 2016, Dubai Clinical significance of antibodies to antigens in the Scianna, Dombrock, Colton, Landsteiner- Weiner, Chido/Rodgers, H, Kx, Cromer, Gerbich, Knops, Indian, and Ok blood group systems

S. Lejon Crottet

This article reviews information regarding the clinical Table 1. Antigen and antibody characteristics of the Scianna significance of antibodies to antigens in the Scianna, Dombrock, blood group system Colton, Landsteiner-Wiener, Chido/Rodgers, H, Kx, Cromer, Antigen Antibody Gerbich, Knops, Indian, and Ok blood group systems. Like most blood group systems, antibodies to many of the antigens in these ISBT name Trivial name Prevalence HTR HDFN groups are rarely encountered because of the high prevalence SC1 Sc1 High No report No report (DAT+) of the associated antigens in most populations. For many, the SC2 Sc2 Low No No to Mild clinical significance—that is, the potential to cause reduced (1 case) (DAT+) survival of transfused antigen-positive red blood cells or a SC3 Sc3 High No to mild/ Mild transfusion reaction (e.g., anti-Ge2, anti-H) and/or hemolytic delayed disease of the fetus and newborn (e.g., anti-Coa, anti-Ge3)— has been documented. Some of these antibodies are not always SC4 Rd Low No Mild to severe clinically significant, and because of the high prevalence of the SC5 STAR High No report No report antigen, antigen-negative blood may be extremely difficult to find SC6 SCER High No report No report (e.g., anti-LW, anti-Inb). The use of a monocyte monolayer assay may be helpful when making transfusion decisions for patients SC7 SCAN High Yes, delayed No report with these antibodies. For others, their prevalence is so rare (1 case) that information on the clinical significance of their antibodies ISBT = International Society of Blood Transfusion; HTR = hemolytic is not available (e.g., anti-Co4, anti-Ok). Immunohematology transfusion reaction; HDFN = hemolytic disease of the fetus and newborn; 2018;34:103–108. DAT = direct antiglobulin test.

Key Words: clinical significance, antibodies to red blood Clinical Significance cell (RBC) antigens, Scianna, Dombrock, Colton, Landsteiner- SC antibodies are usually IgG and are detected by the Wiener, Chido/Rodgers, H, Kx, Cromer, Gerbich, Knops, indirect antiglobulin test (IAT). There are rare cases of Indian, Ok hemolytic disease with enough information to convincingly attribute clinical relevance to SC antibodies (Table 1).3 If Scianna Blood Group System possible, antigen-negative blood should be selected. SC:–1,2,3 blood may be available but is extremely rare. Donors being The Scianna (SC) blood group system, named in 1974, SC:–1,–2,–3 are not available.4 contains seven antigens: five high-prevalence antigens (Sc1, Antibodies to the two low-prevalence antigens, Sc2 and Sc3, Sc5, Sc6, and Sc7) and two low-prevalence antigens (Sc2 Sc4, have not been reported to cause hemolytic transfusion and Sc4) (Table 1).1 These antigens result from variants in the reactions (HTRs). However, they have been implicated in erythroid membrane-associated protein (ERMAP).2 The Sc hemolytic disease of the fetus and newborn (HDFN). In the protein is expressed weakly on leukocytes and other tissues case of transfusion, IAT-compatible blood (most donors) such as fetal liver, thymus, lymph nodes, spleen, and bone should be selected.2 marrow in adults.1

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Dombrock Blood Group System (Table 2).5 Serologically least-incompatible RBC units are recommended for patients with weak examples of the antibody, Besides the two antithetical antigens, Doa and Dob, the but antigen-negative RBCs should be provided for patients Dombrock blood group system (DO) contains eight antigens of who demonstrate strong examples of the antibody.4 high prevalence: Gya, Hy, Joa, DOYA, DOMR, DOLG, DOLC, and DODE (Table 2).1,5,6 Doa and Dob are polymorphic with Colton Blood Group System a prevalence of 67 and 82 percent, respectively, in Caucasian individuals. DO is a glycoprotein attached to the red blood cell The Colton blood group system (CO) consists of three high- (RBC) membrane by a glycosylphosphatidylinositol anchor.5 prevalence antigens (Coa, Co3, and Co4) and one polymorphic antigen (Cob) (Table 3). The water channel-forming protein, Clinical Significance aquaporin-1, is the carrier of the CO blood group antigens.1 Anti-Doa and -Dob have caused immediate and delayed 1 Table 3. Antigen and antibody characteristics of the Colton blood HTRs, but no clinical HDFN (Table 2). They are mostly group system found in sera containing multiple RBC antibodies and often Antigen Antibody disappear in vivo. Anti-Doa and -Dob are usually IgG-reacting, ISBT name Trivial name Prevalence HTR HDFN optimally by the IAT using papain-treated RBCs. CO1 Coa High No to moderate/ Mild to severe For blood transfusion, antigen-negative blood should be D or I/H selected.4 Because of the lack of reliable anti-Do antisera, it used CO2 Cob Polymorphic No to moderate/ Mild to be difficult to find compatible blood. With today’s approach D/H of molecular testing of donors, compatible donors are more CO3 Co3 High Mild; H Severe easily found. Note, though, that the presence of other antibody CO4 Co4 High No report No report specificities may complicate the finding of compatible blood. ISBT = International Society of Blood Transfusion; HTR = hemolytic Anti-Gya, -Hy, -Joa, and other DO antibodies are rare but transfusion reaction; HDFN = hemolytic disease of the fetus and newborn; D = delayed; I = immediate; H = hemolytic. have been reported to cause HTRs. Because of the rarity of these antibodies, evidence of clinical significance is limited Clinical Significance Anti-Coa has caused delayed HTRs and severe HDFN, Table 2. Antigen and antibody characteristics of the Dombrock a blood group system and patients with anti-Co should be transfused with Co(a–) blood.4 Antigen Antibody Whereas anti-Coa is often seen as a single specificity, ISBT name Trivial name Prevalence HTR HDFN anti-Cob is not. This rare antibody, detecting an antigen with DO1 Doa Polymorphic Yes; I/D No report (DAT+) a prevalence of about 10 percent,1 has been reported to cause b DO2 Do Polymorphic Yes; I/D No report (DAT+) both acute HTR and a mild delayed HTR (Table 3).7 About 90 a DO3 Gy High No to moderate; No report (DAT+) percent of the donors are IAT compatible; these donor RBC D units should be selected in case of a transfusion. There are DO4 Hy High No to moderate; No report (DAT+) D no reports of anti-Cob being implicated in a serious HDFN, 1 DO5 Joa High No to moderate; No report although mild HDFN has been reported. D Anti-Co3 and anti-Co4 are rare antibodies detecting DO6 DOYA High No report No report antigens of very high prevalence (Table 3). Anti-Co3 has caused (one case, pretransfusion a mild HTR and serious HDFN. Only four Co4– probands medication) have been identified, and because of the rarity of anti-Co4, no DO7 DOMR High No report No report data are available on its clinical significance.1 Co(a–b–) blood DO8 DOLG High No report No report should be selected for compatibility testing, but because it is DO9 DOLC High No report No report extremely rare serologically, least incompatible blood may be DO10 DODE High No report No report given with extra caution.4 ISBT = International Society of Blood Transfusion; HTR = hemolytic transfusion reaction; HDFN = hemolytic disease of the fetus and newborn; I = immediate; D = delayed; DAT = direct antiglobulin test.

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Landsteiner-Wiener Blood Group System Table 5. Antigen and antibody characteristics of the CH/RG blood group system

Currently, there are three antigens assigned to the Antigen Antibody Landsteiner-Wiener blood group system (LW): LWa, LWab, and ISBT name Trivial name Prevalence HTR HDFN LWb (Table 4).1 The LW antigens are carried on an intracellular CH/RG1 Ch1 High No hemolytic No report adhesion molecule, glycoprotein ICAM-4.7 LWa and LWb are CH/RG2 Ch2 High No report No report a antithetical, with LW being of high prevalence (100%) and CH/RG11 Rg1 High No hemolytic No report b LW being of low prevalence (<1%) in most populations. The CH/RG12 Rg2 High No report No report b prevalence of LW is greatest in the Baltic region (e.g., 8% in Five additional antigens within the CH/RG blood group system have been Estonians, 6% in Finns), and thus the likelihood of finding identified but with no information.1 LW(a−) donors is greatest in these areas.1 ISBT = International Society of Blood Transfusion; HTR = hemolytic transfusion reaction; HDFN = hemolytic disease of the fetus and newborn. Autoantibodies developed after antigen suppression are not uncommon, which makes the differentiation between allo- Clinical Significance and autoantibodies difficult. Anti-Ch and anti-Rg have not been found to cause an HTR or HDFN (Table 5).2 Antigen-negative blood is not required for Table 4. Antigen and antibody characteristics of the Landsteiner- Wiener blood group system transfusion. Severe anaphylactic reactions have been reported in a few patients with antibodies to Ch or Rg antigens after Antigen Antibody infusion of plasma products and platelet concentrates since ISBT name Trivial name Prevalence HTR HDFN these contain soluble Ch/Rg antigen; however; these events LW5 LWa High No to mild; D No to mild are exceptional.8 LW6 LWab High No report Mild LW7 LWb Low No to mild No to mild H Blood Group System ISBT = International Society of Blood Transfusion; HTR = hemolytic transfusion reaction; HDFN = hemolytic disease of the fetus and newborn; D = delayed. The H blood group system contains only one antigen, H. The H antigen is present in all individuals except those with the

Clinical Significance Bombay (Oh) phenotype. Individuals with the para-Bombay 1 LW antibodies are usually IgG and are detected by the (A h or Bh) phenotype have very low levels of H (Table 6). IAT. Mild cases of HTR and HDFN with anti-LWa, anti-LWab, and anti-LWb have been reported (Table 4).1 For transfusion, Table 6. Antigen and antibody characteristics of the H blood group system antigen-negative blood is not required, but D– blood should be selected if possible, since the antigen expression is lower than Antigen Antibody that of D+ blood. In the presence of anti-LWb, IAT-compatible ISBT name Trivial name Prevalence HTR HDFN blood should be selected.4 H H High No to severe; No report I/D/H ISBT = International Society of Blood Transfusion; HTR = hemolytic Chido/Rodgers Blood Group System transfusion reaction; HDFN = hemolytic disease of the fetus and newborn; I = immediate; D = delayed; H = hemolytic. The antigens of the Chido/Rodgers blood group system (CH/RG) are carried on the C4d fragment of complement Clinical Significance component C4; Ch antigens are carried on the C4B allotype Individuals with the Oh phenotype (RBC H-deficient, and Rg antigens are carried on the C4A allotype.1 These non-secretor) always have anti-H in their serum/plasma. Like antigens adsorb onto the RBC surface in vivo.1,8 Currently, anti-A and -B, anti-H is likely to cause a severe immediate nine antigens have been identified in the CH/RG system: six of HTR. HDFN may occur, but there are no reports (Table 6).1 high prevalence and one that is polymorphic for CH and two In case of transfusion, blood of the Oh phenotype must be of high prevalence for RG (Table 5). selected.

Individuals with the non-secretor Ah or Bh phenotype usually have anti-H in their serum/plasma, although often of lower titer. There is little information on the clinical

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significance of anti-H in Ah or Bh individuals. Preferably, blood Gerbich Blood Group System of the Oh phenotype should be transfused, but if not available, then RBCs of the appropriate ABO group (A for Ah, B for Bh) The Gerbich blood group system is expressed on may be transfused with extra caution.4 glycophorin C and D and contains 11 antigens: 6 of high 10 Individuals with the secretor Ah or Bh phenotype may prevalence and 5 of low prevalence (Table 8). have anti-HI present in their serum/plasma. Typically, anti-HI is not reactive at 37°C, and ABO-identical blood, compatible at Table 8. Antigen and antibody characteristics of the Gerbich blood group system 37°C, can be used for transfusion.4 Antigen Antibody Anti-HI may also be found in group A1, A1B, or B individuals. If the antibody is reactive at 37°C, blood of the ISBT name Trivial name Prevalence HTR HDFN ABO group of the patient should be used for transfusion; GE2 Ge2 High No to moderate; No report I/D (DAT+) blood of group O and A2 should not be used. If the antibody is GE3 Ge3 High No to moderate; Positive DAT reactive only at lower temperatures, blood compatible at 37°C I/D to severe 4 can be used for transfusion, regardless of ABO group. GE4 Ge4 High No report No report GE5 Wb Low No report No report Kx Blood Group System GE6 Lsa Low No report No report GE7 Ana Low No report No report Kx was assigned blood group system status in 1990 GE8 Dha Low No report 1 report, and contains one antigen of very high prevalence (100%), very severe Kx (Table 7). The Kx antigen is covalently linked to the GE9 GEIS Low No report No report Kell molecule on RBCs and is controlled by a gene on the GE10 GEPL High No report No report X chromosome. The presence of the Kx protein is critical GE11 GEAT High No report No report to normal RBC morphology, and silencing mutations are GE12 GETI High No report No report associated with McLeod syndrome, neuroacanthocytosis, and 9 ISBT = International Society of Blood Transfusion; HTR = hemolytic neuromuscular disorders in later life. transfusion reaction; HDFN = hemolytic disease of the fetus and newborn; I = immediate; D = delayed; DAT = direct antiglobulin test. Clinical Significance Anti-Kx is often found together with anti-Km (anti- Clinical Significance KEL20) in men with the McLeod phenotype and X-linked Anti-Ge2, made by Yus, Gerbich, or Leach phenotypes, chronic granulomatous disease. These two antibodies (also and anti-Ge3, made by Gerbich or Leach phenotypes, are called anti-KL) can cause severe HTRs; thus, antigen-negative usually IgG antibodies and can cause moderate immediate or (McLeod phenotype) blood should be selected for transfusion delayed HTRs. Anti-Ge4 (made by Leach phenotypes), -GEPL, (Table 7).4 -GEAT, and -GETI are very rare, and no data are available on their clinical significance (Table 8).10 Antigen-negative blood is not usually required for transfusion but should be considered Table 7. Antigen and antibody characteristics of the Kx blood group system for strong examples of these antibodies.4 Although Anti-Ge2 has not been implicated in clinical Antigen Antibody HDFN, newborns have shown positive direct antiglobulin ISBT name Trivial name Prevalence HTR HDFN tests (DATs).1 Anti-Ge3 has caused severe HDFN due to XK1 Kx High Mild; D Not applicable* suppression of the erythroid progenitor cell growth in the *Anti-Kx only made by male individuals with McLeod phenotype infant. Initial treatment of the infant at birth with subsequent ISBT = International Society of Blood Transfusion; HTR = hemolytic transfusion reaction; HDFN = hemolytic disease of the fetus and newborn; follow-up for several weeks after birth may be indicated.1 D = delayed. There is no information on the clinical significance with regard to HDFN for anti-Ge4, -GEPL, -GEAT, and -GETI.1 The antibodies to the low-prevalence antigens of the Gerbich system can be naturally occurring and may be IgM

106 IMMUNOHEMATOLOGY, Volume 34, Number 3, 2018 ISBT conference—clinical significance of antibodies

or IgG. There are no reports that these antibodies have caused Knops Blood Group System HTRs or HDFN.1 The antigens of the Knops blood group system are carried Cromer Blood Group System on complement receptor 1 (CR1). The major functions of CR1 are the binding of C4b/C3b opsonized complexes as well The antigens of the Cromer blood group system are carried as complement regulation. The Knops blood group system on the decay accelerating factor (DAF), a protein belonging contains nine antigens that are polymorphic, of low prevalence to the regulators of the complement activation family. The or of high prevalence depending on the origin of the individual system contains 16 high-prevalence antigens and three low- (Table 10).1,12 prevalence antigens (Table 9).11 Table 10. Antigen and antibody characteristics of the Knops blood Table 9. Antigen and antibody characteristics of the Cromer blood group system group system Antigen Antibody Antigen Antibody ISBT name Trivial name Prevalence HTR HDFN ISBT name Trivial name Prevalence HTR HDFN* KN1 Kna High No No CROM1 Cra High No to moderate No KN2 Knb Low No report No report a CROM2 Tc High No to severe No KN3 McCa High No No CROM3 Tcb Low No report No report KN4 Sla High (non- No to mild No CROM4 Tcc Low No to mild No black) CROM5 Dra High No to mild No Polymorphic (black) CROM6 Esa High Mild No KN5 Yka High No No CROM7 IFC High No to mild No b a KN6 McC Low (non- No report, No report CROM8 WES Low No to mild No black) unlikely CROM9 WESb High No report No, DAT+ Polymorphic (black) CROM10 UMC High No report No CROM11 GUTI High No report No KN7 Vil Low (non- No report, No report black) unlikely CROM12 SERF High No data No Polymorphic CROM13 ZENA High No data No (black) CROM14 CROV High No data No KN8 Sl3 High No report, No report unlikely CROM15 CRAM High No data No KN9 KCAM High (non- No report, No report CROM16 CROZ High No data No black) unlikely *Decay accelerating factor adsorbs maternal antibody Polymorphic ISBT = International Society of Blood Transfusion; HTR = hemolytic (black) transfusion reaction; HDFN = hemolytic disease of the fetus and newborn; ISBT = International Society of Blood Transfusion; HTR = hemolytic DAT = direct antiglobulin test. transfusion reaction; HDFN = hemolytic disease of the fetus and newborn.

Clinical Significance Clinical Significance Antibodies to Cromer antigens are rare (Table 9). Most of the Knops antibodies are not considered clinically There is evidence that the antibodies may cause accelerated significant since they do not cause apparent HTRs or HDFN, destruction of transfused RBCs, although functional assays thus antigen-negative units are not indicated in the case of on clinical significance are ambiguous.11 Because of the high transfusion (Table 10).4 concentration of maternal DAF on the apical surface of the trophoblasts in the placenta, which is thought to adsorb the Indian Blood Group System maternal antibodies, there is no risk of HDFN associated with Cromer system antibodies. Antigen-negative blood is not The Indian blood group system contains four antigens usually required for transfusion, but should be considered for that are carried by CD44. Ina and Inb are antithetical antigens strong-reacting antibodies.4 of low prevalence and high prevalence, respectively. INFI and INJA are both antigens of high prevalence (Table 11).2

IMMUNOHEMATOLOGY, Volume 34, Number 3, 2018 107 S. Lejon Crottet

Table 11. Antigen and antibody characteristics of the Indian blood Clinical Significance group system Ok antibodies have not been implicated in HDFN.1 There Antigen Antibody is almost no information on the clinical significance of Ok ISBT name Trivial name Prevalence HTR HDFN antibodies, but in vivo survival tests and cellular functional IN1 Ina Low Decreased cell No, DAT+ assays suggest that anti-Oka is clinically significant (Table survival 12).1 Because Ok(a–) blood is extremely rare, serologically b IN2 In High No to severe D No, DAT+ least-incompatible blood may be given with extra caution.4 IN3 INFI High No report Mild IN4 INJA High No report No report References ISBT = International Society of Blood Transfusion; HTR = hemolytic transfusion reaction; HDFN = hemolytic disease of the fetus and newborn; . 1 Reid ME, Lomas-Francis C, Olsson ML. Blood group antigen DAT = direct antiglobulin test; D = delayed. factsbook. 3rd ed. San Diego, CA: Academic Press, 2012. . 2 Wagner FF, Poole J, Flegel WA. Scianna antigens including Rd Clinical Significance are expressed by ERMAP. Blood 2003;101:752–7. Indian antibodies are mostly IgG, reacting preferably by . 3 Bunker PAR, Flegel WA. Scianna: the lucky 13th blood group system. Immunohematology 2011;27:41–57. the IAT (Table 11). These antibodies do not bind complement . 4 Thornton N. The clinical significance of blood group and do not cause in vitro hemolysis.6 Ina is rare in populations alloantibodies and the supply of blood for transfusion, 2016. of European origin, but has a prevalence of about 3 percent in SPECIFICATION SPN214/4. Available from http://hospital. blood.co.uk/media/29271/spn2144-the-clinical-significance- Indian individuals and 10 percent in Arab individuals. Anti- of-blood-group-alloantibodies-and-the-supply-of-blood-for- Ina is not reported to be clinically significant, although limited transfusion.pdf. data are available. Blood compatible by IAT can be selected for . 5 Lomas-Francis C, Reid ME. The Dombrock blood group transfusion.4 system: a review. Immunohematology 2010;26:71–8. Anti-Inb may cause none to severe/delayed HTRs.1 In(b–) . 6 Storry JR, Castilho L, Chen Q, et al. International society of blood transfusion working party on red cell immunogenetics blood is not usually required for transfusion but should be and terminology: report of the Seoul and London meetings. considered for strong-reacting antibodies. ISBT Sci Ser 2016;11:118–22. No clinical data are available on anti-INFI and anti- . 7 Bryne KM, Bryne PC. Review: other blood group systems— Diego, Yt, Xg, Scianna, Dombrock, Colton, Landsteiner- INJA; INFI– and INJA– blood types are extremely rare. For Wiener, and Indian. Immunohematology 2004;20:50–8. transfusion, serologically least-incompatible blood should be . 8 Daniels G. Human blood groups. 3rd ed. Oxford, U.K.: used with extra caution.4 Blackwell Science, 2013. Anti-Ina, -Inb, and -INJA have not been implicated in . 9 Denomme GA. Kell and Kx blood group systems. HDFN.1 Anti-INFI was implicated in one case of mild HDFN.13 Immunohematology 2015;31:14–9. 10. Walker PS, Reid ME. The Gerbich blood group system: a review. Immunohematology 2010;26:60–5. Ok Blood Group System 11. Storry JR, Reid ME, Yaser MH. The Cromer blood group system: a review. Immunohematology 2010;26:109–18. The Ok blood group system contains three antigens 12. Moulds JM. The Knops blood group system: a review. carried on CD147 (Table 12).14 The Ok blood group system was Immunohematology 2010;26:2–7. 13. Poole J, Tilley L, Warke N, et al. Two missense mutations in the established in 1999. To date, the null phenotype, Ok(a–) has CD44 gene encode two new antigens of the Indian blood group been found in eight Japanese families only. system. Transfusion 2007;47:1306–11. 14. Smart EA, Storry JR. The OK blood group system: a review. Table 12. Antigen and antibody characteristics of the Ok blood Immunohematology 2010;26:124–6. group system

Antigen Antibody Sofia Lejon Crottet, PhD, Head of Immunohematology, Interregional ISBT name Trivial name Prevalence HTR HDFN Blood Transfusion Service, Swiss Red Cross, Bern, Switzerland, OK1 Oka High 51Cr cell No [email protected]. survival reduced OK2 OKGV High No report No report OK3 OKVM High No report No report ISBT = International Society of Blood Transfusion; HTR = hemolytic transfusion reaction; HDFN = hemolytic disease of the fetus and newborn.

108 IMMUNOHEMATOLOGY, Volume 34, Number 3, 2018 Ca s e R e p or t A delayed and acute hemolytic transfusion reaction mediated by anti-c in a patient with variant RH alleles

T.K. Walters and T. Lightfoot

The Rh system is the most complex of the human blood groups. locus consists of a pair of adjacent homologous genes, RHD Of the 55 antigens that have been characterized, the system’s and RHCE, that respectively encode for D and for C or c and principal antigens D, C, E, c, and e are responsible for the E or e antithetical pairs. The antigenic diversity present in majority of clinically significant Rh antibodies. In the last few years, advancements in molecular testing have provided a wealth this blood group system is attributed to a variety of molecular of information on the genetic diversity of the Rh locus. This case mechanisms including nucleotide substitutions and gene report describes a patient with variant RHD*DAR alleles inherited conversion.4 These molecular mechanisms lead to the creation in conjunction with two compound heterozygote RHCE*ceEK/ RHCE*ceAR alleles. The patient was previously alloimmunized to of unique protein sequences with resultant unique antigens D, C, and E and possibly hrS. Further transfusion of D–C–E–K– and an increase in the number of antigens in the Rh system RBCs resulted in a suspected acute hemolytic transfusion reaction relative to other blood group systems. Further adding to and the subsequent identification of anti-c. Monocyte monolayer the complexity, variant RHD and RHCE alleles can encode assay testing suggests clinical significance with a range of 29.5–38.5 percent reactive monocytes. Immunohematology qualitative and/or quantitative changes in the Rh antigen 2018;34:109-112. expression.5 Individuals with such variants, when exposed to the conventional antigen during transfusion, will recognize Key Words: RH variant alleles, anti-hrS, RH allele matching, those epitopes that they lack as foreign, and an immune partial antigens, Rh antigens, delayed hemolytic transfusion response may be elicited. Routine serologic RBC phenotyping reaction, monocyte monolayer assay, hrS does not differentiate between conventional and variant Rh antigens. Within the Rh blood group system, partial D, C, and The Rh system is the most complex of the human blood e variants and their corresponding alloantibodies have been groups. Of the 55 antigens that have been characterized, the well described. Alloanti-c in a c+ individual was first reported system’s principal antigens D, C, E, c, and e are responsible by Moulds et al.6 in 1982, with a number of additional cases for the majority of clinically significant Rh antibodies. reported since that time.7–9 Antibodies to antigens in the Rh system have been implicated Peyrard et al.10 reported the first case of alloanti-c/ce in in hemolytic disease of the fetus and newborn (HDFN) as well a patient with the RHCE*ceAR allele that encodes a partial as acute and delayed hemolytic transfusion reactions (HTRs). c antigen. Hipsky et al.11 also described the identification of In the last few years, advancements in molecular testing have alloanti-c in the plasma of an African American patient with provided a wealth of information on the genetic diversity sickle cell disease carrying the RHCE*ceAR allele. of the Rh locus. The Rh system is a very polymorphic and We report a case of a 72-year-old multi-transfused immunogenic blood group system that is second only to the African American woman with altered RHD and RHCE alleles ABO system in its clinical significance for blood transfusion. predicting partial D, partial c, and partial e antigens who Nucleotide changes can result in qualitative and quantitative developed a clinically significant alloanti-c. changes in Rh antigen expression.1 Individuals who express a partial D on their red blood cells (RBCs) can be immunized Case Report to make anti-D. Similarly, people who are homozygous or hemizygous for alleles encoding partial c and/or partial e The patient is a 72-year-old African American woman can also produce alloantibodies.2 In addition to the common with a past medical history of multiple sclerosis, congestive antigens of the Rh system (D, C, c, E, e) there are 50 additional heart failure, and recurrent gastrointestinal (GI) bleeding antigens that constitute the Rh blood group system.3 The Rh secondary to diverticulosis. The patient presented to the

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emergency department with acute GI bleeding and received The facility was contacted to report the anti-e–like fluid support and 2 units of packed red blood cells (PRBCs). reactivity, and molecular genotype testing was recommended She was admitted to the hospital for further evaluation and for further characterization. No compatible units negative for treatment. A colonoscopy was performed that revealed a D, C, E, e, and K were available for immediate transfusion. diverticular bleed in the ascending colon; this bleed was Therefore, after consultation with the facility, units negative successfully cauterized. During this time, her hemoglobin for D, C, E, and K were recommended in the event emergent (Hgb) was observed to fall to 4.9 g/dL with a hematocrit transfusion was needed. (Hct) of 16 percent (normal 12.0–15.5 g/dL and 37–48%, Unfortunately, there was insufficient time to perform respectively); she was emergently transfused 4 units of PRBCs. a monocyte monolayer assay (MMA) before transfusion of Initial serologic results indicated the patient to be group A, incompatible blood.12 An MMA can be performed to help weak D positive with a positive antibody detection test. The assess the clinical significance of an antibody when antigen- patient’s previous serologic history included identification of negative units are rare and not likely to be readily available. anti-D, -C, -E, warm autoantibody, and cold autoantibody. Given the patient’s clinical situation (GI bleed with a low Hgb The initial antibody screening was performed by the hospital, level), the facility transfused 2 crossmatch-incompatible units and the presence of additional RBC antibodies was suspected. of PRBCs negative for D, C, E, and K over a 48-hour time The patient’s sample was referred to the Immunohematology period. No adverse reactions were noted after transfusion of Reference Laboratory (IRL) (American Red Cross, e+ units in the presence of the anti-e–like antibody. Charlotte, NC) for further serologic evaluation and antibody Molecular testing was performed by the American Red identification. Cross National Molecular Laboratory. Results indicated the patient carried a RHD*DAR variant allele inherited with Results compound heterozygote RHCE*ceEK/RHCE*ceAR alleles, predicting partial D, partial c, partial e, and the lack of the Initial serologic testing performed by the IRL indicated high-prevalence antigens, hrS and Hr (Table 1). that the patient was group A, weak D positive with a negative direct antiglobulin test (DAT). Serologic phenotype testing Table 1. Molecular testing results was performed and the results were C–E–c+e+; K–; Fy(a–b–); Allele name Antigens encoded by allele Jk(a+b+); S+s–. The patient’s plasma exhibited panreactivity Probable RHD RHD*DAR (hemizygous Partial D with D– phenotypically similar RBCs in the presence of a genotype or homozygous) negative autocontrol. This reactivity suggested the presence Probable RHCE RHCE*ceEK/ Partial c, partial e, Hr–, S of an additional antibody (ies) unrelated to the previously genotype hr – identified alloanti-D, -C, and -E. Plasma studies showed 1+ RHCE*ceAR Partial c, partial e, Hr–, hrS–, V+w, VS– reactivity with D– phenotypically similar cells using low- Predicted Partial D+C–E– partial c+ ionic-strength saline solution in the indirect antiglobulin phenotype partial e+ Hr– V+w VS– hrS– test (IAT) and 3+ reactivity with polyethylene glycol (PEG) in the IAT. The reactivity was resistant to ficin treatment as well as to 0.2 M dithiothreitol. Given the patient’s history The molecular results confirmed suspected serologic of warm and cold autoantibodies, adsorption studies were reactivity. RHD*DAR allele is associated with D typing performed at 37°C using ZZAP-treated autologous cells. No discrepancies and the production of alloanti-D. In addition, reduction in strength was observed with the autoadsorbed the patient had two altered RHCE alleles predicting the hrS– plasma. Allogeneic adsorptions were performed using papain- phenotype and the production of alloanti-e and/or anti-hrS. treated R0 RBCs. Clinically significant antibodies to common The facility was contacted to report the additional probable RBC blood group antigens were excluded in the alloadsorbed anti-hrS reactivity in the presence of previously identified plasma. An acid eluate was prepared from the adsorbing cells anti-D, -C, and -E. Consultation was made with the American to ascertain the identity of the panreactivity. The acid eluate Rare Donor Program, and no RH allele–matched units were demonstrated an anti-e–like antibody. The eluate was reactive available for further transfusion needs. Despite the presence with three sources of D– e+ RBCs and negative with three of the probable anti-hrS, the patient tolerated the previous 2 sources of D– hrB– RBCs. units of PRBCs well, with no adverse effects. The patient’s Hgb

110 IMMUNOHEMATOLOGY, Volume 34, Number 3, 2018 HTR due to anti-c

increased to 7.7 g/dL with an Hct of 23 percent, and she was autocontrol remained negative. Adsorption studies were discharged. performed using aliquots of R1R1 cells. The adsorbed plasma Five days after the initial transfusion, the patient was demonstrated the presence of anti-c. To further confirm the readmitted to the hospital for a suspected delayed HTR. anti-c specificity, an additional aliquot of the plasma was

The patient presented with dark urine, Hgb of 6.3 g/dL, adsorbed with R2R2 cells. An acid eluate was then prepared total bilirubin of 2.6 mg/dL (normal 0.1–1.2 mg/dL), lactate from the adsorption cells. This eluate reacted with D–c+E– dehydrogenase 1034 IU (normal 100–190 IU), and haptoglobin RBCs and was nonreactive with Rhnull RBCs. Clinically less than 15 mg/dL (normal 30–200 mg/dL). The patient then significant antibodies to other major blood group antigens received 2 additional units of D–C–E–K– PRBCs. were excluded in the allogeneic adsorbed eluate and plasma. On completion of transfusion of the second unit, the The presence of anti-c in conjunction with anti-D, -C, -E, patient began to display symptoms of a possible acute HTR. and probable anti-hrS presented significant challenges in the The patient’s temperature increased from 98.0 to 99.5°F, with possibility of locating any compatible blood for this patient. accompanying symptoms of rigors, dyspnea, and severe back An MMA was performed by the American Red Cross National pain. Blood pressure increased from 149/68 mmHg to 166/94 Reference Laboratory for Blood Group Serology in an attempt mmHg, with an increase in pulse from 85 bpm to 93 bpm. to assess the clinical significance of the alloantibody to the c Oxygen saturation remained unchanged at 99 percent. antigen. Two random sources of group O, D–C–E–K– RBCs The facility began an initial transfusion reaction and autogeneic cells were used. Results are listed in Table 2. investigation. The post-transfusion sample was severely Because the patient had antibodies to both c and e as well as hemolyzed, but the DAT remained negative. Gram stain to D, C, and E, phenotypically similar cells (D–C–E–) were was performed and was negative for any microorganisms. A chosen for the MMA. Cells that could further distinguish the current blood sample was drawn and sent to the local IRL for significance of the reactivity with regard to the partial anti-c additional serologic testing. The DAT on both the pre- and or probable anti-hrS were not available. MMA testing using post-transfusion samples was negative. genotypically similar RBCs would have been ideal, but none Because of the recent transfusion and suspected are currently available in the United States. transfusion reaction, an eluate was prepared despite the Table 2. Monocyte monolayer assay results negative DAT. Expecting the eluate to contain the previously identified probable anti-hrS, the eluate was tested against a IgG DAT* % Reactive monocytes† panel of e+ and e– cells. All cells were reactive 4+ at PEG-IAT, Random #1 4+ 30.3 with fresh complement and the patient’s EDTA glycine acid–treated autologous cells D–C–E–K–Fy(a–b–)s– 4+ 34.0 without fresh complement were negative, as was the last wash. All cells tested with the Random #2 4+ 38.5 with fresh complement eluate were positive except for Rhnull RBCs. Two Rhnull RBCs D–C–E–K– 4+ 29.5 without fresh complement were tested and found to be negative with the patient’s neat Autologous cells Negative 0.3 without fresh complement eluate. Additionally, one RBC with the Rh:–46 phenotype was *Results of coated test red blood cells after 37°C incubation. available for testing and was significantly weaker in reactivity †The normal range for the monocyte monolayer assay is 0–3 percent reactive monocytes. Values above 3 percent suggest that the antibody may and displayed only a 1+ reaction. This cell was negative for c cause accelerated clearance of antigen-positive red blood cells.18 and had a weakened expression of e. Given that the patient’s DAT = direct antiglobulin test. molecular results indicated an altered expression of c, the possibility of anti-c or anti-Hr was suspected. The eluate was Discussion tested against a panel of hrS– cells to investigate the presence of anti-Hr, but all cells were 3+ reactive with the neat eluate. The We report the serologic and molecular findings on a eluate was adsorbed using R1R1 cells. Anti-c was demonstrated 72-year-old multi-transfused African American female in the adsorbed eluate at PEG-IAT. Additionally, the previously patient. The presence of anti-c in conjunction with anti-D, identified anti-hrS was also shown to be demonstrating in the -C, -E, and probable anti-hrS posed significant challenges in neat eluate when tested with a c–e+ RBC. the identification and provision of compatible blood for this Plasma studies were also performed on the post- patient. The MMA results, the patient’s clinical status, and transfusion sample. The plasma was reactive 4+ with two post-transfusion laboratory and serologic results indicate that phenotypically similar cells [D–C–E–s–K–Fy(a–b–)]. The the alloanti-c is clinically significant.

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Variants of c are much less frequent than variant forms of . 5 Avent ND, Reid ME. The Rh blood group system: a review. the other major Rh antigens.12 Westhoff et al.13 postulate that Blood 2000;95:375–87. the less frequent occurrence of c variants in comparison with . 6 Moulds JJ, Case J, Anderson TD, Cooper ES. The first example of allo-anti-c produced by a c-positive individual. In: Recent other Rh antigens is because the two proline residues involved advances in haematology, immunology and blood transfusion: in c expression form a stable structure that is resistant to the proceedings of the plenary sessions of the joint meeting of the changes that often occur with RHCE. Individuals of African 19th congress of the International Society of Haematology and the 17th Congress of the International Society of Blood descent have a much higher incidence of variant Rh antigens Transfusion, Budapest, August 1982. Wiley, 1983. as compared with individuals of European descent, with the . 7 Ong J, Walker PS, Schmulbach E, et al. Alloanti-c in a latter constituting the largest percentage of blood donors c-positive, JAL-positive patient. Vox Sang 2008;96:240–3. by ethnicity.14,15 The high prevalence of variant antigens . 8 Pham BN, Peyrard T, Juszczak G, et al. Alloanti-c (RH4) revealing that the (C)ces haplotype encodes a partial c antigen. in African American patients thereby increases the risk of Transfusion 2009;49:1320–4. alloimmunization when these patients are exposed to the . 9 Hipsky CH, Lomas-Francis C, Fuchisawa A, et al. RHCE*ceCF conventional antigen during transfusion.16,17 encodes partial c and partial e not CELO, an antigen antithetical to Crawford. Transfusion 2011;51:25–31. The detection of the presence of a variant antigen, 10. Peyrard T, Pham BN, Poupal S, et al. Alloanti-c/ce in a c+ ceAR/ identification of the corresponding alloantibody, determination Ce patient suggests that the rare RHCE*ceAR allele (ceAR) of its clinical significance, and the logistics of finding encodes a partial c antigen. Transfusion 2009;49:2406–11. compatible blood can be quite challenging. Rhnull RBCs were 11. Hipsky CH, Lomas-Francis C, Fuchisawa A, Reid recommended for any future transfusions for this patient, until M. RHCE*ceAR encodes a partial c (RH4) antigen. Immunohematology 2010;26:57–9. such time that a possible RH allele–matched donor unit could 12. Simon TL, McCullough J, Snyder EL, Solheim BG, Strauss RG. be identified for further compatibility testing and evaluation. Rossi’s principles of transfusion medicine. 5th ed. West Sussex, This case demonstrates the increasingly important role of UK: Blackwell Publishing, 2016. molecular testing in the overall clinical/laboratory assessment 13. Westhoff CM, Silberstein LE, Wylie DE. Evidence supporting the requirement for two proline residues for expression of c. of alloimmunized patients with partial RBC antigens and for Transfusion 1997;37:1123–30. subsequent transfusion recommendations and guidance. 14. Wang D, Lane C, Quillen K. Prevalence of RhD variants, confirmed by molecular genotyping, in a multiethnic prenatal Acknowledgments population. Am J Clin Pathol 2010;134:438–42. 15. Mezokh N, Song Y, Moulds JM, et al. Reliable identification of diverse RHD and RHCE variants in African American donors The authors would like to thank Dr. Carol Weida (Atrium by Beadchip analysis (abstract). Transfusion 2009;49:120A. Health, Charlotte, NC) for assistance with the management 16. Westhoff CM, Vege S, Horn T, et al. RHCE*ceMO is frequently of this case and the procurement of the clinical data for this in cis to RHD*DAU0 and encodes a hr(S)-, hr (B)-, RH:-61 phenotype in black persons: clinical significance. Transfusion manuscript. The authors would also like to thank the staff at 2013;53:2983–9. the American Red Cross National Molecular Laboratory and 17. Noizat-Pirenne F, Lee K, Pennec PY, et al. Rare RHCE National Reference Laboratory for Blood Group Serology for phenotypes in black individuals of Afro-Caribbean origin: their assistance with molecular and MMA testing. Identification and transfusion safety. Blood 2002;100: 4223–31. 18. Nance SJ, Arndt P, Garratty G. Predicting the clinical References significance of red cell alloantibodies using a monocyte monolayer assay. Transfusion 1987;27:449–52. . 1 Fung MK, Eder AF, Spitalnik SL, Westhoff CM. Technical manual. 19th ed. Bethesda, MD: American Association of Blood Banks, 2017. Tiffany K. Walters MT(ASCP)SBBCM, Director, (corresponding . 2 Reid ME, Lomas-Francis C, Olsson ML. The blood group author), Immunohematology Reference Laboratory, American antigens factsbook. 3rd ed. San Diego, CA: Elsevier, 2012. Red Cross, Carolinas Charlotte, Carolinas Durham, River Valley, . 3 International Society of Blood Transfusion. Table of blood Tennessee Valley, and South Carolinas Regions, 2425 Park Road, group antigens v.8_180620. Available at www.isbtweb. Charlotte, NC, 28203, [email protected]; and Thomas org/fileadmin/user_upload/Red_Cell_Terminology_and_ Lightfoot, MD, Medical Director, American Red Cross, Charlotte, NC, Immunogenetics/Table_of_blood_group_antigens_within_ 28203. systems_v8_180620.pdf. Accessed August 2018. . 4 Klein HC, Anstee DJ. Mollison’s blood transfusion in clinical medicine. 11th edition. Malden, MA: Blackwell Publishing, 2005.

112 IMMUNOHEMATOLOGY, Volume 34, Number 3, 2018 Detecting polyagglutinable red blood cells

C. Melland and C. Hintz

Polyagglutination is a condition in which red blood cells (RBCs) are agglutinated by normal adult human sera but not by autologous Reagents/Supplies or newborn sera. Polyagglutination is caused by changes in the Reagents Supplies RBC membrane that enable patient RBCs to agglutinate with normal human sera; this agglutination can interfere with blood • Adult group O RBCs • Centrifuge or cell washer bank testing. Depending on the cause, polyagglutination may or • Phosphate-buffered saline • Alsever’s solution (7.0–7.5 pH) may not be the cause of RBC hemolysis. Lectins and human sera • 0.9% saline can be used to detect polyagglutinable RBCs. Identification of this • Lectins (if available): Arachis • 6% albumin phenomenon can be helpful in providing not only transfusion hypogea, Glycine soja, Salvia recommendation information for physicians but also information sclarea, Salvia horminum (or • 10 × 75 mm tubes Dolichos biflores if the patient is associated with pathogens (i.e., Streptococcus pneumoniae) and non–group A) severity of illness. Testing with ABO group–compatible adult REVIEW METHOD SEROLOGIC human sera can determine if a patient’s RBCs are polyagglutinable. • Polyagglutinable RBCs (frozen or prepared) Further testing with a variey of lectins may identify the kind of polyagglutination. Immunohematology 2018;34:113–117. • Donor pooled group AB plasma or serum • Pooled cord serum Key Words: polyagglutination, lectin, acquired B, T, Tk, Th, • Neuraminidase, 1 IU/mL from Vibrio Tx, Tr, HEMPAS, Cad, Tn cholera RBCs = red blood cells. Principle and Background Procedural Steps Polyagglutination was first described in 1988 by Peter Preparing controls Hermann Stillmark in his doctoral thesis. The agglutinin • Treat adult group O RBCs with neuraminidase. used was isolated from seeds of the castor tree (i.e., ricin) • Pool the serum from three to five cord blood samples. and was highly toxic. Ricin became commercially available, • Grind up lectin seeds, incubate in 0.9% saline, and harvest the supernatant. which prompted Paul Ehrlich to use it for studies in the 1890s Process to establish fundamental principles of immunology. Ehrlich • Mix lectin and/or human sera with a 3–5% suspension of patient found that mice could become tolerant to a lethal dose of ricin RBCs. • Incubate at room temperature. by repeated small injections of the lectin, creating protective • Centrifuge, and read macroscopically for agglutination. antibodies to the ricin.1 RBCs = red blood cells. Karl Landsteiner detected hemagglutinating activity in some nontoxic plant extracts. In 1909, he observed that the hemagglutinating activity of certain seed extracts or “lectins” was inhibited by either heat-treated serum or by mucin.2 consanguinity; and (3) a disease state such as lymphoma During the 1940s and 1950s, there was increased research or leukemia. This RBC alteration causes panagglutination in the area of lectin extracts from plant seeds. Some lectins when these RBCs are combined with normal adult human showed hemagglutinating activity with all RBCs, and others sera which contain naturally occurring antibodies to these showed blood group–specific activity, such as A or H. altered antigens but not when they are combined with cord There are two categories of polyagglutination: acquired (T, sera which do not.3 Understanding the mechanisms by Tk, Th, Tx, acquired B) and inherited (Tr, HEMPAS, Cad, Tn). which polyagglutinable RBCs occur can help in recognizing In both categories, the surface of human RBCs can become these conditions when working on patient samples and in altered by (1) microbial enzyme modification, usually through determining when polyagglutination screening is indicated. sepsis; (2) homozygosity of rare altered alleles, often due to

IMMUNOHEMATOLOGY, Volume 34, Number 3, 2018 113 C. Melland and C. Hintz

Microbial Enzyme Modification have been associated with bacterial infections of the intestinal Polyagglutination caused by microbial enzyme tract and pneumococcal infections, respectively.12,13 modification occurs when enzymes excreted from bacteria cleave portions of the RBC membrane, exposing hidden Inheritance of Rare Altered Alleles antigens termed “cryptantigens” on the RBC surface. There Polyagglutination caused by the inheritance of rare, altered are many types of polyagglutination due to bacterial action alleles is associated with increased or altered glycosylation or including but not limited to T, acquired B, Tk, Th, and Tx. changes in the antigen expression on the RBC surface, which Identifying and understanding the type of polyagglutination causes the RBC to react with naturally occurring antibodies in often linked to pathogens can be helpful for the clinician.4,5 human sera. Examples of inherited polyagglutination include but are not limited to Tr, HEMPAS, and Cad. NOR has now T-activation been assigned to the P1PK blood group system (ISBT 003.004 During T-activation, the neuraminidase enzyme cleaves or 3.4) and is no longer considered a polyagglutinable state.14,15 terminal N-acetylneuraminic acid (NeuAc) from the RBC membrane, exposing a hidden crypt or T antigen that can Tr agglutinate with naturally occurring IgM anti-T present in Tr polyagglutination has been shown to be caused by the most adult sera. This phenomenon was first described in 1938 lack of glycosylation of membrane proteins.16 In this case, when the RBCs of a 4-year-old child with bronchopneumonia the parents were consanguineous, and the propositus was were agglutinated by 15 percent of ABO-compatible human homozygous for the altered allele. sera.6 Anti-T IgM antibodies are absent in newborns and form over time in response to T-like antigenic stimulation of HEP M AS bacteria from intestinal exposure. This process is analogous HEMPAS is an acronym for hereditary erythroblastic to the formation of anti-A and anti-B from exposure to A- and multinuclearity with a positive acidified serum lysis test and B-like antigens in the environment.7 Unlike anti-A and anti-B, is caused by congenital dyserythropoietic anemia type II. The however, the binding of anti-T has not been proven to cause RBCs of these individuals react with anti-I and anti-i.17 These hemolysis during the acute disease state. T-activated RBCs abnormal RBC membranes result in increased susceptibility are agglutinated by human sera and by Arachis hypogea and to lysis and a compensating anemia.18 HEMPAS RBCs react Glycine soja.5,8 with Glycine soja.

Acquired B, Tk, Th, Tx Cad Acquired B, Tk, Th, and Tx polyagglutination are Individuals with Cad polyagglutination do not have a caused by similar processes. Acquired B is associated with compensating anemia but have a very strong expression of a gastrointestinal (GI) disease and occurs when A1 RBCs Sd on their RBCs which reacts with weak naturally occurring (N-acetylgalactosamine) are degraded by bacterial enzymes to anti-Sda.19–21 Cad+ RBCs react with Glycine soja and Salvia galactosamine and therefore look similar to B RBCs but react horminum. with human anti-B.9 Before the advent of monoclonal reagents, human Polyagglutination and Disease polyclonal reagents were used for routine ABO blood group Polyagglutination has been associated with leukemia in testing. These reagents were prepared from human sera some individuals, and Tn polyagglutination characterized which contained naturally occurring antibodies to such by persistent mixed-field agglutination has been linked to “cryptantigens”. Acquired B polyagglutination caused blood myelocytic leukemia.22 The RBC membrane change is through group discrepancies in the forward typing results when somatic mutation resulting in one cell population lacking polyclonal reagents were used.10 Tk polyagglutination is also NeuAc.23 associated with GI infection. Bacteria that produce endo- beta-galactosidase remove galactose residues, exposing Tn activation N-acetylglucosamine in the paragloboside system and causing Tn activation has been described in healthy individuals Tk polyagglutination.11 Th and Tx polyagglutination are not as and some infants. It may be caused by incomplete expression well understood as the other types of microbial infection but of the gene responsible for converting the Tn into T expressed

114 IMMUNOHEMATOLOGY, Volume 34, Number 3, 2018 Detecting polyagglutinable RBCs

on RBCs.24,25 Incomplete synthesis of T on RBCs in this type to ensure potency and specificity. T-activated RBCs can be of polyagglutination causes agglutination with Salvia sclarea, prepared by treating normal RBCs with neuraminidase. Salvia horminum, and Glycine soja.26 These RBCs can then be used as a positive control for Arachis hypogea, for Glycine soja, and for the pooled human plasma. Indications To prepare T-activated RBCs, wash adult group O RBCs from a single donor source with 0.9 percent saline Indications for polyagglutination can come from three to four times. Dilute one part neuraminidase with discrepant results obtained during ABO testing caused by the nine parts phosphate-buffered saline (PBS) (i.e., one drop of naturally occurring antibody (e.g., anti-T) in human polyclonal neuraminidase with nine drops of PBS). In one tube, mix one antisera.27 Since the advent of monoclonal reagents, however, volume (e.g., three drops) of the packed RBCs with an equal most examples of polyagglutination are only suspected after volume of diluted neuraminidase. In a second tube, mix one reviewing the clinical findings of the patient who has suspected volume (e.g., three drops) of the packed RBCs with an equal hemolysis or anemia of unknown origin. Lectins and normal volume of 6 percent albumin to serve as a negative control adult human sera can be used to detect the surface changes on when testing to ensure T-activation has occurred. Incubate polyagglutinable RBCs. By observing the patterns of reactivity both tubes at 37°C for approximately 15 minutes, then wash of the RBCs in question with human sera and with the various three to four times with 0.9 percent saline, and create a 3–5 lectins, the specific polyagglutinable state of the patient’s RBCs percent concentration in 0.9 percent saline. can be identified. Test the treated and untreated RBCs with Glycine soja to ensure activation of the treated cells and no activation with Materials the untreated cells. T-activated RBCs can be stored in liquid nitrogen if not used immediately for testing. Prepare human sera for testing against patient RBCs by pooling the plasma or serum from four to six group AB donors Quality Control drawn within 48 hours. This plasma pool can be stored frozen for later use.26 To prepare the positive control, add one drop of T-activated Pooled serum from two to three cord blood samples can RBCs to one drop of each lectin tested. To prepare the negative be used as a negative control for the polyagglutination screen. control, add one drop of cord sera to a 3–5 percent suspension

Mix one drop each of group A1 and group B RBCs with two of the patient’s RBCs. Mix, and incubate all tubes using the drops of pooled cord sera. Incubate at room temperature for same time and temperature as specified in the manufacturer’s approximately 5 minutes. Centrifuge, and read macroscopically instructions or as determined in the in-house standardization for agglutination. The cord sera should not agglutinate the of the lectin in use. Centrifuge, and read macroscopically for group A1 or group B RBCs. If there is reactivity, cord sera are agglutination. contaminated with maternal antibody and should not be used.28 The T-activated RBCs will react with Arachis hypogea, If lectins are not available commercially, they can be Glycine soja, and the pooled plasma/sera control but will not extracted from a variety of seeds.29 Salvia horminum lectin react with Salvia sclarea, Salvia horminum/Dolichos biflores, is also known as annual sage, Salvia sclarea is clary sage, or pooled cord sera. See Table 1.27,30 Arachis hypogea is from raw peanuts, and Glycine soja (also known as Glycine max) is from wild soybean. To prepare the Procedure lectins, measure 1 g of seeds. Wash, and grind up the seeds and then incubate in 5 mL of 0.9 percent saline. Let the Mix one drop of a 3–5 percent suspension of the patient’s mixture sit overnight. Centrifuge the mixture, and harvest the RBCs with two drops of pooled donor plasma or sera. If lectins supernatant.30 To obtain a clear supernatant, run the lectin are commercially available, mix one drop of the patient’s RBC through a filter. suspension with each lectin following the manufacturer’s instructions for amount, time, and temperature indicated. If Quality Control for In-House Prepared Lectins in-house prepared lectins are used, the amount of lectin would Although it is relatively easy to create lectins, it is difficult be dependent upon in-house standardization. Centrifuge, and to find polyagglutinable RBCs to use as positive controls read macroscopically for agglutination.

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Table 1. Examples of lectin reactivity with examples of References polyagglutinable RBCs . 1 Sharon N, Lis H. History of lectins: from hemagglutinins Polyagglutinable RBCs Normal to biological recognition molecules. Glycobiology 2004;14: Lectin RBCs Cad+ Tn+ T+ Tk+ NOR HEMPAS 53R–62R. Arachis hypogea 0 0 0 + + 0 0 . 2 Gorakshakar AC, Ghosh K. Use of lectins in immunohematology. Asian J Transfus Sci 2016;10:12–21. Salvia sclarea 0 0 + 0 0 0 0 . 3 Hubener G. Investigation of isoagglutination, with special Salvia horminum/ 0 + + 0 0 0 0 consideration of apparent deviations from the blood grouping Dolichos biflores scheme. [In German] Z Immunitatsforsch 1926;45:223–48. Glycine soja 0 + + + 0 0 + . 4 Poulsen PE. Experimentally produced “polyagglutinability” Pooled plasma/sera 0 + + + + + + (T-transformation of erythrocytes in vivo) in guinea pigs control infected with pneumococci. Nature 1954;173:82–3. Pooled cord sera 0 0 0 0 0 0 0 . 5 Crookston KP, Reiner AP, Cooper LJN, Sacher RA, Blajchman MA, Heddle NM. RBC T activation and hemolysis: implications RBCs = red blood cells. for pediatric transfusion management. Transfusion 2000;40: 801–12. . 6 Levine P, Katzin EM. Temporary agglutinability of red blood Interpretation cells. Proc Soc Exp Biol NY 1938;39:167–9. . 7 Ramasethu J, Luban N. T Activation. Br J Haematol 2001;112: If adult sera are reactive with the RBCs tested and the cord 259–63. sera are nonreactive, the RBCs are polyagglutinable. If the . 8 Moh-Klaren J, Bodivit G, Jugie M, et al. Severe hemolysis after plasma transfusion in a neonate with necrotizing adult sera are nonreactive and the cord sera are nonreactive, enterocolitis, Clostridium perfringens infection and red blood then the RBCs are not polyagglutinable. If the cord sera are cell T-polyagglutination. Transfusion 2017;57:2571–7. reactive, then consider spontaneous agglutination due to . 9 Gerbal A, Maslet C, Salmon C. Immunological aspects of the antibodies present in the plasma. acquired-B antigen. Vox Sang 1975;28:398. 10. Judd WJ, McGuire-Mallory D, Anderson KM, et al. Note that additional lectins are available to help determine Concomitant T- and Tk-activation association with acquired-B the specific type of polyagglutination present. Table 1 shows antigens. Transfusion 1979;19:293–8. examples of lectin reactivity with examples of polyagglutinable 11. Doinel C, Andreu G, Cartron JP, Salmon C, Fukuda MV. Tk RBCs.15,31 polyagglutination produced by in vitro by an endo–beta– galacto–sidase. Vox Sang 1980;38:94–8. 12. Bird GW, Wingham J, Beck ML, Pierce SR, Oates GD, Pollock Limitations A. Th, a “new” form of erythrocyte polyagglutinability. Lancet 1978;1:1215-6. Lectins kits are no longer available from commercial 13. Bird GW, Windham J, Seger R, Kenny AB. Tx, a “new” red cell cryptantigen exposed by pneumococcal enzymes. Blood manufacturers. Noncommercial lectins should be used for Transf Immunohaemat 1982;25215–16. testing with the appropriate controls tested the day of use. 14. Suchanowska A, Kaczmarek R, Duk M, et al. A single point Cad and Tn polyagglutinable RBCs are needed to create mutation in the gene encoding Gb3/CD77 synthase causes positive controls for Salvia horminum and Salvia sclarea. The a rare inherited polyagglutination syndrome. J Biol Chem 2012;287:38220–30. unavailability of these polyagglutinable RBCs is problematic 15. Reid ME, Lomas-Francis C, Olsson M. The blood group antigen for using these lectins. If lectins are not made with the proper factsbook. 3rd ed. San Diego, CA: Academic Press, 2012. solutes, they cannot be stored frozen. 16. Halverson GR, Lee AH, Oyen R, Reiss RF, Hurlet-Jensen A, Patient RBCs that are not completely polyagglutinated Reid ME. Altered glycosylation leads to Tr polyagglutination. Transfusion 2004;44:1588–92. or are transiently polyagglutinable may not react by testing 17. Mawby WJ, Tanner MJA, Anstee DJ, Clamp JR. Incomplete with lectins or human sera. Conversely, some lectins have glycosylation of erythrocyte to membrane protein in congenital antibacterial activity and may agglutinate RBCs coated with dyserythropoietic anemia Type II. (CDA II) Br Haematol bacterial polysaccharides. This reactivity is not the same 1983;55:357–68. 18. Fukuda MN. HEMPAS: Hereditary erythroblastic reactivity caused by the state of polyagglutination and justifies multiniclearity with positive acidified serum lysis test. Biochim the use of pooled human sera to support the polyagglutination Biophys Acta 1999;1455:231–9. test.32 19. Sanger R, Gavin J, Tippett P, Teesdale P, Eldon K. Plant agglutinin for another human blood-group. Lancet 1971;i:1130.

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20. Pickles MM, Morton JA. The Sda blood group antigen. In: 28. Judd, WJ. The use of purified lectins in immunohematology. Human blood groups. 5th International Convocation on Transfusion 1979;19:769–9. Immunology, Buffalo, NY, 1977:277–86. 29. King MJ, Liew YW, Moores PP, Bird GWG. Enhanced reaction 21. Sringarm S, Chiewsilp P, Tubrod J. Cad receptor in Thai blood with Vicia graminea lectin and exposed terminal N-acetyl-D- donors. Vox Sang 1974;26:462–6. glucosaminyl residues on a sample of human red cells with Hb 22. Bird GW, Wingham J, Pippard MJ, Hoult JG, Melikian V. M-Hyde Park. Transfusion 1988;28:549–55. Erythrocyte membrane modification in malignant diseases of 30. Mollison PL, Engelfriet CP, Contreras M. Blood transfusion myeloid and lymphoreticular tissues. I. Tn–polyagglutination in clinical medicine. 10th ed. Oxford, UK: Blackwell Science, in acute myelocytic leukaemia. Br J Haematol 1976;33: 1997. 289–94. 31. Issitt PD. Applied blood group serology. 3rd ed. Miami, FL: 23. Cartron JP, Cartron J, Andreu G, et al. Selective deficiency Montgomery Scientific iPublications, 1985:457. of 3-B-galactosyl-transferase (T-transferase) in Tn- 32. Hemo bioscience. Lectin package insert. Version 2.1 Revised polyagglutinable erythrocytes. Lancet 1978;i:856–7. 03-2015. Morrisville, NC: Hemo bioscience. 24. Beck ML. Tn: a nonmicrobial form of polyagglutination. In:

Beck ML, Judd WJ, Eds. Polyagglutination. Washington, DC: CM AABB, 1980:55–70. Cami Melland, MLS(ASCP)SBB , Clinical Laboratory Scientist, Manager (corresponding author), Immunohematology Reference 25. Schulz M, Fortes P, Brewer L, et al. “In Utero” exposure of Tn and Th cryptantigens (abstract). Transfusion 1983;23:422. Laboratory, Bonfils Blood Center, 717 Yosemite Street, Denver, 26. Judd WJ. Judd’s methods in immunohematology. 3rd ed. CO 80230, [email protected]; and Connie Hintz, Durham, NC: Montgomery Scientific Publications, 2008. MT(ASCP)SBB, Clinical Laboratory Scientist, Immunohematology 27. Judd WJ. Review: polyagglutination. Immunohematology Reference Laboratory, Bonfils Blood Center, Denver, CO. 1992;8:58–69.

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

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E-mail information to [email protected] or fax to (215) 451-2538

IMMUNOHEMATOLOGY, Volume 34, Number 3, 2018 117 I n M e mor i a m

Marjory Stroup Walters 1924–2018

Marjory was born in 1924 in Iola, Kansas. She was the sixth D+ child of a D– mother, but fortunately was unaffected by Rh antibodies. She received her medical technology degree in 1947 and worked as a medical technologist at the University of Kansas Hospital. Marjory joined Ortho Pharmaceutical Corporation (Raritan, NJ) in 1951, working first in Quality Assurance and manufacturing blood bank antisera before moving to the Blood Consultation Service. The Blood Consultation Service/ Philip Levine Laboratory had been established after the discovery of Rh and its role in erythroblastosis fetalis. The Blood Consultation Service rapidly became a world-recognized center for immunohematology research, investigating approximately 12,000 immunohematology problems per year, as well as becoming a center for immunohematology education. A generation of blood bank technologists learned immunohematology at the Ortho Immunohematology Schools under Marjory’s leadership. She was a mentor and friend to many of the leading lights in blood banking in the late 20th and early 21st centuries and inspired many technologists to become “blood bankers.” Marjory was the author and co-author with Margaret Treacy of numerous publications including the Ortho “Red Books” on The ABO & Rh Systems, Compatibility Testing, and Hemolytic Disease of the Newborn. In 1982, Blood Group Antigens and Antibodies by Marjory and Margaret continued immunohematology education. She was a sought-after lecturer and educator and traveled the world speaking about blood banking topics and conducting workshops. “Have knit dress, will travel,” said Marjory. Marjory and other leading immunohematologists found that they were always the ones giving presentations but never had the opportunity to learn much themselves, so Marjory and Shirley Busch decided that a special meeting of reference-lab level immunohematologists was needed. Thus was born the first sessions of the Reference Lab meetings, sponsored by the AABB, which later separated from AABB and became the Invitational Conference of Investigative Immunohematologists (ICII). It continues today as a leading gathering of blood bank specialists. Marjory’s red blood cells (RBCs) were positive for many important antigens, so they were used in the laboratory as a screening cell. At first it had been thought that Marjory was D–, even when she had donated blood. But further attention showed she was D+, having a D suppressed by her C. As screening cells, Marjory’s RBCs were useful, especially being Le(a+) and very strongly P1+. Every now and then, however, her RBCs would be reactive when no other cells were. Samples were sent to Robert Race and Ruth Sanger in London, who found them also to be Cw+. During her 30+ years in blood banking, Marjory made innumerable discoveries and important observations. She showed that Jsa was part of the Kell system. She described the first examples of anti-Cra, -Jra, -Lu8, -Lu13, -K12, -K13, and -K19. She was involved in extensive work on several systems including Diego, Kell, and Lutheran.

118 IMMUNOHEMATOLOGY, Volume 34, Number 3, 2018 Marjory was an active member of AABB for more than 50 years. Among her numerous awards were two AABB honors: the Ivor Dunsford award in 1973 and the Emily Cooley award in 1983. Marjory was a pioneer in the field, and her work contributed to the modern practice of transfusion medicine. She will be remembered by scientists, business colleagues, and friends, not only for her significant contributions, but also for her commitment to quality, leadership, and education delivered with an irrepressible spirit and enthusiasm.

Acknowledgments The authors thank Steve Pierce whose extensive knowledge and work on the history of blood banking contributed to this piece.

About the Authors JoAnn Hegarty, MT(ASCP)SBB, Ortho Clinical Diagnostics (Formerly); and Tony S. Casina, MT(ASCP)SBB, (corresponding author), Ortho Clinical Diagnostics, 1001 U.S. Highway 202 South, Raritan, NJ 08869, [email protected].

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 For more information, send an e-mail to [email protected]. [email protected].

IMMUNOHEMATOLOGY, Volume 34, Number 3, 2018 119 A n n o u n c e m e n t s

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

120 IMMUNOHEMATOLOGY, Volume 34, Number 3, 2018 Announcements, cont.

IMMUNOHEMATOLOGY, Volume 34, Number 3, 2018 121 Announcements, cont.

122 IMMUNOHEMATOLOGY, Volume 34, Number 3, 2018 Announcements, cont.

IMMUNOHEMATOLOGY, Volume 34, Number 3, 2018 123 Announcements, cont.

124 IMMUNOHEMATOLOGY, Volume 34, Number 3, 2018 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 2018 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 34, Number 3, 2018 125 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 2018. 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

126 IMMUNOHEMATOLOGY, Volume 34, Number 3, 2018 Announcements, cont.

A dv e r t i s e m e n t s

IMMUNOHEMATOLOGY, Volume 34, Number 3, 2018 127 Advertisements, cont.

Becoming a Specialist in Blood Banking (SBB)

What is a certified Specialist in Blood Banking (SBB)? • Someone with educational and work experience qualifications who successfully passes the American Society for Clinical Pathology (ASCP) board of registry (BOR) examination for the Specialist in Blood Banking. • This person will have advanced knowledge, skills, and abilities in the field of transfusion medicine and blood banking. Individuals who have an SBB certification serve in many areas of transfusion medicine: • Serve as regulatory, technical, procedural, and research advisors • Perform and direct administrative functions • Develop, validate, implement, and perform laboratory procedures • Analyze quality issues preparing and implementing corrective actions to prevent and document issues • Design and present educational programs • Provide technical and scientific training in 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 BloodCenter of Wisconsin Milwaukee, WI

Revised October 2017

128 IMMUNOHEMATOLOGY, Volume 34, Number 3, 2018 Advertisements, cont.

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IMMUNOHEMATOLOGY, Volume 34, Number 3, 2018 129 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

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130 IMMUNOHEMATOLOGY, Volume 34, Number 3, 2018 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 34, Number 3, 2018 131 132 IMMUNOHEMATOLOGY, Volume 34, Number 3, 2018 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 34, Number 3, 2018 133 Journal of Blood Group Serology and Molecular Genetics

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