Journal of Blood Group Serology and Molecular Genetics

Vo l u m e 30, N u m b e r 1, 2014

Immunohematology Journal of Blood Group Serology and Molecular Genetics Volume 30, Number 1, 2014 CONTENTS

R e p o r t 1 Indirect antiglobulin test-crossmatch using low-ionic-strength saline–albumin enhancement medium and reduced incubation time: effectiveness in the detection of most clinically significant antibodies and impact on blood utilization C.L. Dinardo, S.L. Bonifácio, and A. Mendrone, Jr.

R e v i e w 6 Raph blood group system M. Hayes

R e p o r t 11 I-int phenotype among three individuals of a Parsi community from Mumbai, India S.R. Joshi

C a s e R e p o r t 14 Evans syndrome in a pediatric liver transplant recipient with an autoantibody with apparent specificity for the KEL4 (Kpb) antigen S.A. Koepsell, K. Burright-Hittner, and J.D. Landmark

R e v i e w 18 JMH blood group system: a review S.T. Johnson

R e p o r t 24 Demonstration of IgG subclass (IgG1 and IgG3) in patients with positive direct antiglobulin tests A. Singh, A. Solanki, and R. Chaudhary

I n M e m o r ia m 28 George Garratty, 1935–2014 Patricia A. Arndt and Regina M. Leger 30 A nnouncements 34 A dv e r t i s e m e n t s 39 I n s t r u c t i o n s f o r A u t h o r s E d i to r - i n -C h i e f E d i to r ia l B oa r d Sandra Nance, MS, MT(ASCP)SBB Philadelphia, Pennsylvania Patricia Arndt, MT(ASCP)SBB Paul M. Ness, MD Pomona, California Baltimore, Maryland M a n ag i n g E d i to r James P. AuBuchon, MD Thierry Peyrard, PharmD, PhD Cynthia Flickinger, MT(ASCP)SBB Seattle, Washington Paris, France Philadelphia, Pennsylvania Barbara J. Bryant, MD Mark Popovsky, MD Tec h n i c a l E d i to r s Milwaukee, Wisconsin Braintree, Massachusetts Christine Lomas-Francis, MSc Lilian Castilho, PhD S. Gerald Sandler, MD New York City, New York Campinas, Brazil Washington, District of Columbia Joyce Poole, FIBMS Martha R. Combs, MT(ASCP)SBB Jill R. Storry, PhD Bristol, United Kingdom Durham, North Carolina Lund, Sweden Dawn M. Rumsey, ART(CSMLT) Geoffrey Daniels, PhD David F. Stroncek, MD Norcross, Georgia Bristol, United Kingdom Bethesda, Maryland Anne F. Eder, MD Nicole Thornton S e n i o r M e d i c a l E d i to r Washington, District of Columbia Bristol, United Kingdom Ralph R. Vassallo, MD Philadelphia, Pennsylvania Brenda J. Grossman, MD St. Louis, Missouri E m e r i t u s E d i to r s Delores Mallory, MT(ASCP) SBB A s s o c iat e M e d i c a l E d i to r s Christine Lomas-Francis, MSc P. Dayand Borge, MD New York City, New York Supply, North Carolina Baltimore, Maryland Geralyn M. Meny, MD Marion E. Reid, PhD, FIBMS New York City, New York David Moolten, MD San Antonio, Texas Philadelphia, Pennsylvania

M o l ec u l a r E d i to r Margaret A. Keller Philadelphia, Pennsylvania

E d i to r ia l A s s i s ta n t Immunohematology is published quarterly (March, June, September, and December) by the Sheetal Patel American Red Cross, National Headquarters, Washington, DC 20006. Immunohematology is indexed and included in Index Medicus and MEDLINE on the P r o d u c t i o n A s s i s ta n t MEDLARS system. The contents are also cited in the EBASE/Excerpta Medica and Elsevier Marge Manigly BIOBASE/Current Awareness in Biological Sciences (CABS) databases. The subscription price is $50 for individual, $100 for institution (U.S.) and C o p y E d i to r $60 for individual, $100 for institution (foreign) per year. Mary L. Tod Subscriptions, Change of Address, and Extra Copies: Immunohematology, P.O. Box 40325 P r o o f r e a d e r Philadelphia, PA 19106 Lucy Oppenheim Wendy Martin-Shuma Or call (215) 451-4902 Web site: www.redcross.org/about-us/publications/immunohematology E l ec t r o n i c P u b l i s h e r Copyright 2014 by The American National Red Cross Paul Duquette ISSN 0894-203X

O n O u r C o v e r

Henri Rousseau, or Le Douanier (the customs officer), as he was nicknamed for his primary occupation as a toll collector, failed to impress either the art world or the public in his lifetime (1844–1910). Self- taught, he adhered to a simple, childlike style at odds with his contemporaries. Although he received little recognition from and was even ridiculed by critics, he became the darling of artists and writers such as Picasso, Brancusi, and Apollinaire and is now regarded as a genius. The Tabby, or Le Chat Tigre, exemplifies both the primitive and yet subtly dreamlike character of his work. The JMH blood group discussed by S.T. Johnson in this issue includes “The Cat” among its nicknames. David Moolten, MD R e p o r t Indirect antiglobulin test-crossmatch using low-ionic-strength saline–albumin enhancement medium and reduced incubation time: effectiveness in the detection of most clinically significant antibodies and impact on blood utilization

C.L. Dinardo, S.L. Bonifácio, and A. Mendrone, Jr.

Indirect antiglobulin test-crossmatch (IAT-XM) using Crossmatching of donor red blood cells (RBCs) and enhancement media such as low-ionic-strength saline (LISS) recipient’s serum is an important step required to complete and polyethylene glycol (PEG) usually requires 15 minutes of pretransfusion tests. Crossmatch can be performed incubation. These methods are necessary when testing samples from blood recipients who have a higher risk of alloimmunization. electronically or by an immediate-spin method, in which In emergency situations, IAT-XM can be time-consuming and no incubation or anti-human globulin (AHG) steps are can influence presurgery routine, resulting in more red blood cell performed. Those modalities of crossmatch are applied to (RBC) units being tested and stored to avoid the transfusion of uncrossmatched ones. The objective of this study was to evaluate nonalloimmunized recipients, with the detection of ABO the performance of a LISS-albumin enhancer to intensify mismatches their main goal. Whenever there is a positive antigen-antibody reaction after 5 minutes of 37°C incubation and antibody screen (current or past) or a history of pregnancy, compare this performance with that of other enhancers, gel, and crossmatch demands 37°C incubation and AHG steps conventional tube testing. Second, the study evaluated the impact of this method’s implementation in the C:T ratio (crossmatched (indirect antiglobulin test-crossmatch; IAT-XM), increasing to transfused RBC units) of a transfusion laboratory. Ninety the time needed for completion of pretransfusion tests, which serum samples containing alloantibodies of potential clinical can become critical in emergency situations. significance were tested against phenotyped RBCs using four In Brazil, legislation requires IAT-XM for all recipients different methods: (1) tube with LISS-albumin enhancer (5 minutes of incubation), (2) tube with LISS-albumin and PEG who have ever been transfused, pregnant, or alloimmunized, (15 minutes of incubation), (3) gel, and (4) conventional tube and as a consequence, the routine of performing a type and method (60 minutes of incubation). In parallel, the study compared screen on samples from surgical patients is frequently not the C:T ratio of a tertiary-hospital transfusion laboratory in two different periods: 3 months before and 3 months after the applied, as most transfusion laboratories perform IAT-XM implementation of the 5-minute IAT-XM protocol. The use of before the surgery and segregate all the units that may be used LISS-albumin with 5 minutes of incubation exhibited the same during the surgical procedure until its end. This obviously performance as LISS-albumin, PEG, and gel with 15 minutes increases the number of blood units in store and the costs of incubation. Conventional tube method results were equally comparable, but reactions were significantly less intense, except of the pretransfusion tests. Also, the implementation of any for anti-c (p = 0.406). Accuracy was 100 percent for all selected automation may be compromised. methods. After the implementation of the 5-minute IAT-XM Classic saline indirect antibody test (SIAT) demands 60 protocol, the C:T ratio fell from 2.74 to 1.29 (p < 0.001). IAT-XM minutes of incubation (37°C) and three wash steps before can have its incubation time reduced to 5 minutes with the use of LISS-albumin enhancement. We suggest this strategy should the addition of AHG and interpretation of the results. This be used to quickly prepare RBC units for surgical patients, method allows 99 percent of antibody uptake onto RBCs for keeping transfusion safety without compromising blood supplies. the detection of an immunoglobulin G (IgG) antibody present Immunohematology 2014;30:1–5. in the recipient’s serum.1 This length of incubation can be shortened with the help of enhancement media such as low- Key Words: crossmatch, alloantibody, antibody screening, ionic-strength saline (LISS), albumin, polyethylene glycol LISS, PEG, gel testing (PEG), and hexadimethrine bromide (Polybrene), whose

IMMUNOHEMATOLOGY, Volume 30, Number 1, 2014 1 C.L. Dinardo et al.

major functions are to speed up the rate of antigen-antibody Gel Microcolumn Assay Method association, promoting a higher rate of antibody uptake by RBCs were washed once in 0.9 percent saline and RBCs in a shorter time. suspended in LISS (ID-Diluent 2, DiaMed) to achieve a final In emergency situations, even when data referring to 0.8 to 1 percent concentration. Then 50 µL of 0.8 to 1 percent recipients’ previous alloimmunization is not known, saline RBC suspension and 25 µL of sera were added to LISS-Coombs immediate-spin crossmatch (IS-XM) is frequently chosen by gel cards (IgG/C3d, DiaMed), incubated for 15 minutes at laboratory analysts, as it allows fast availability of RBC units. For 37°C, and centrifuged (85g) for 10 minutes according to recipients who present a higher risk of alloimmunization, such manufacturer’s instructions. as those with sickle cell anemia, myelodysplastic syndrome, or multiparity, this approach may be risky. Enhancement Tube Testing medium containing a mixture of LISS and albumin typically RBCs were washed three times in 0.9 percent saline requires 15 minutes of incubation before the AHG phase. Our and then adjusted to a final concentration of 3 percent. After hypothesis is that this time can be shortened to 5 minutes this step, 50 µL of each RBC suspension and 100 µL of each without losing sensitivity, making this method suitable for specific serum were added to each tube. For the conventional RBC and serum crossmatch in emergency situations. method, tubes were incubated for 1 hour at 37°C. For the The main objective of this study is to evaluate the methods involving enhancers 2 and 3, tubes were incubated performance of a LISS-albumin enhancer to intensify the for 15 minutes at 37°C. After addition of enhancer 1, the tubes antigen-antibody reaction after 5 minutes of 37°C incubation, were incubated for 5 minutes at 37°C. After the incubation allowing the detection of clinically significant alloantibodies step, all tubes were washed three times with 0.9 percent saline present in recipients’ serum. Second, the impact of this method’s before AHG was added and final reading of the results was implementation in the routine of a transfusion laboratory will performed. All reactions were read macroscopically. Positive also be evaluated. To meet this second objective, we chose to reactions were graded from 1+ to 4+ and expressed in scores.2 evaluate the C:T ratio (ratio of units crossmatched to units transfused) before and after the implementation of the type Statistical Analysis and screen associated with the 5-minute IAT-XM protocol in We compared the scores obtained from the five studied our presurgery routine. In most countries, a C:T ratio less than methods (median value) for each specific antibody using 2:1 is an indicator of efficient preparation of blood units for the Friedman statistical test (nonparametric statistical test elective surgery. suitable for multiple comparisons of different methods or treatments, SPSS version 17, SPSS Inc., Chicago, IL). We Materials and Methods considered as statistically significant a probability value less than 0.05. Scores were calculated based on AABB standards. Ninety serum samples containing alloantibodies of potential clinical significance (anti-D, -c, -K, -Jka, -Jkb, -Fya) Analysis of Crossmatched-to-Transfused Ratio from our inventory were tested against phenotyped RBCs From June 2011 to September 2011, after the implemen- using two different methods: tube and column agglutination tation of our new legislation, only an ABO/Rh type and antibody gel (DiaMed Latino América, Lagoa Santa, Brazil). Tube testing screen was performed for all preoperative transfusion requests was performed conventionally (without enhancers) and with (type and screen strategy), except in cases of alloimmunization, two different enhancement media: LISS-albumin (Dialiss, in which phenotyped units were prepared. If during the surgery DiaMed, enhancer 1, and LISS ADD, Lorne Laboratories, any RBC unit was requested, our laboratory analysts performed Lower Earley, UK, enhancer 2) and PEG (BioPeg, Fresenius the IAT-XM using the LISS-albumin enhancer and 5 minutes HemoCare, São Paulo, Brazil, enhancer 3). One of the LISS- of 37°C incubation. We calculated the C:T ratio at the end of albumin enhancers (enhancer 1) was chosen to be tested with this 3-month period and compared it with our retrospective the proposed protocol of 5 minutes of incubation. All tests C:T ratio (March 2011 to May 2011). were performed in parallel by the same analyst, according to the specific method described in a later section. We chose Results RBCs from donors who were heterozygous for the allele encoding the corresponding antigen against the alloantibody Enhancer 1 (LISS-albumin, 5 minutes of incubation), being tested. enhancer 2 (LISS-albumin, 15 minutes of incubation),

2 IMMUNOHEMATOLOGY, Volume 30, Number 1, 2014 Rapid technique for IAT-crossmatch

enhancer 3 (PEG, 15 minutes of incubation), and gel enhancement medium, without loss of sensitivity or specificity. microcolumn agglutination methods exhibited similar This strategy improved our C:T ratio, an important quality performance, expressed in terms of score, for all selected indicator of blood utilization, as unexpected needs for urgent antibodies. Conventional tube method results were equally transfusions directed to surgical patients could be met in less comparable, but reactions were significantly less intense than than 10 minutes. those presented by the other methods, except in the case of Reducing time without losing sensitivity in RBC antigen- anti-c (p = 0.406). Sensitivity and specificity were 100 percent antibody reactions was always a subject of discussion in for all selected methods. Enhancer 1 presented the highest the literature before the emergence of the gel microcolumn scores in the presence of anti-Jkb, -c, -K, and -Fya. In the case agglutination method. PEG and LISS, either isolated or of anti-D and -Jka, gel microcolumn agglutination method combined with albumin, are the most widely used enhancement exhibited higher scores, but they were not statistically different media and intensify RBC sensitization while decreasing the from those presented by all other enhancers. Table 1 shows required incubation time of 60 to 90 minutes (conventional mean scores for the different antibodies using each selected tube method) to 15 to 20 minutes.3 Even though extending method. Figures 1 and 2 summarize the data. the incubation time in the presence of enhancers may increase After the implementation of the type and screen routine the detection of antibodies directed against antigens of clinical associated with the 5-minute IAT-XM protocol using a LISS- significance,4 extending this time to more than 40 minutes albumin enhancer, we had 2080 elective surgeries: 360 RBC may result in a paradoxical loss of sensitivity.3 units were crossmatched and only 81 were not used during Performance of RBC crossmatch between donor and the surgery. Three months before the implementation of this recipient before transfusion is suggested either when the protocol, we had a C:T ratio of 2.74 (2040 elective surgeries, antibody screen is positive or when the risk of previous 795 units crossmatched and 290 units transfused). Our C:T alloimmunization is higher than usual (multiply transfused ratio fell from 2.74 to 1.29 (p < 0.001) in 3 months. patients and multiparous). There are a considerable number After the implementation of the 5-minute IAT-XM of reports of hemolytic transfusion reactions caused by protocol, no transfusion reactions suggestive of hemolysis were antibodies that were not detected by antibody screen or by reported to the blood bank. In one case, antibody screening IS-XM, but that would have been detected by IAT-XM.5–8 was negative (gel microcolumn agglutination method) and Even though those antibodies mainly are cold-reactive or IAT-XM was positive. The antibody identified after the are against low-incidence antigens, the odds of encountering performance of 16°C antibody screening (tube method) was them increase in patients who are immunologic responders an anti-M without activity at 37°C. In all other cases, IAT-XM and consequently prone to RBC alloimmunization.9 Indeed, was negative. the decision to eliminate IAT-XM is associated with a risk of hemolytic transfusion reactions of 1:2000 transfused units,10 Discussion which is a significant value, especially in large transfusion laboratories. Our results demonstrate that the required 37°C The IS-XM approach is suitable for detecting donor- incubation time for detection of significant RBC alloantibodies patient ABO incompatibilities and may be used in situations can be decreased to 5 minutes when using a LISS-albumin when antibody screening is negative. However, there is the

Table 1. Performance of different IAT methods in detecting clinically significant antibodies*

Antibody Enhancer 1 (LISS-albumin): Enhancer 2 (LISS-albumin): Enhancer 3 (PEG): (number of samples) 5-min incubation 15-min incubation 15-min incubation Gel Tube: 60-min incubation Anti-D (50) 9.32 ± 0.79 9.3 ± 1.18 9.18 ± 0.92 9.52 ± 0.93 5.64 ± 1.27 Anti-Jka (12) 3.34 ± 2.23 4 ± 2.89 3.92 ± 1.88 4.25 ± 3.17 1.67 ± 1.37 Anti-Fya (11) 6.82 ± 2.75 6 ± 2.24 6 ± 2.28 6 ± 2.24 2.91 ± 1.87 Anti-Jkb (4) 8.5 ± 1 8.5 ± 1 6.5 ± 1.73 8 ± 0 4 ± 0 Anti-c (11) 12 ± 0 12 ± 0 11.81 ± 0.6 12 ± 0 11.81 ± 0.6 Anti-K (48) 9.38 ± 1.61 9.15 ± 1.51 8.75 ± 1.64 7.48 ± 1.89 5.27 ± 1.97 IAT = indirect antiglobulin test; LISS = low-ionic-strength saline; PEG = polyethylene glycol. *Mean ± standard deviation.

IMMUNOHEMATOLOGY, Volume 30, Number 1, 2014 3 C.L. Dinardo et al.

anti-D anti-Jka anti-Fya 10

8 x x x x 6 x x

4 Mean Score

2 x

0 Enhancer 1 Enhancer 2 Enhancer 3 GEL Conventional Enhancer 1 Enhancer 2 Enhancer 3 GEL Conventional Enhancer 1 Enhancer 2 Enhancer 3 GEL Conventional Error bars: 95% Cl

Fig. 1 Mean scores ± 95 percent confidence interval (CI) of antigen-antibody reactions using different indirect antiglobulin test methods for the detection of anti-D, -Jka, and -Fya. Enhancers 1, 2, and 3 had a performance similar to that of gel testing. Conventional tube testing expressed significantly lower reaction intensity. *p < 0.05; **p < 0.01, ***p < 0.001.

anti-Jkb anti-c anti-K 12

10

8

x x x 6

x x 4 Mean Score

2

0 Enhancer 1 Enhancer 2 Enhancer 3 GEL Conventional Enhancer 1 Enhancer 2 Enhancer 3 GEL Conventional Enhancer 1 Enhancer 2 Enhancer 3 GEL Conventional Error bars: 95% Cl

Fig. 2 Mean scores ± 95 percent confidence interval (CI) of antigen-antibody reactions using different indirect antiglobulin test methods for the detection of anti-Jkb, -c, and -K. Enhancers 1, 2, and 3 had a performance similar to that of gel testing. Conventional tube testing expressed significantly lower reaction intensity, except for anti-c. *p < 0.05; **p < 0.01, ***p < 0.001.

substantial risk of not detecting an A2B (donor)-B (recipient) requests, we were able to reduce significantly the number of mismatch or the presence of an alloantibody against a low- units that were prepared and not transfused. In our country incidence antigen.11 In countries where IAT-XM is required for this strategy is of great importance, as our legislation requires all patients with a previous history of pregnancy or transfusion, IAT-XM for all recipients who have ever been transfused, as in ours (Brazil), or in areas where the incidence of some low- pregnant, or alloimmunized. As a referral hospital, we have incidence antigens is higher owing to ethnic characteristics, a plenty of previously transfused or pregnant patients, and 5-minute IAT-XM may be a less time-consuming and more preparing and reserving RBC units for all of them compromised effective strategy than a 15-minute IAT-XM. our blood supplies. Our present results indicate that the LISS-albumin In conclusion, crossmatch incubation time can be reduced enhancement medium detects clinically significant antibodies to 5 minutes with the use of LISS-albumin enhancement after 5 minutes of incubation and that the intensity of reactions medium. We suggest this strategy be used to quickly prepare did not differ from that presented by this same enhancer or RBC units for surgical patients, maintaining transfusion safety by PEG after 15 minutes of incubation. After implementation without compromising blood supplies. of routine type and screen testing on samples from surgical patients and of preparing RBC units only after intraoperation

4 IMMUNOHEMATOLOGY, Volume 30, Number 1, 2014 Rapid technique for IAT-crossmatch

References 8. Koshy R, Patel B, Harrison JS. Anti-Kpa-induced severe delayed hemolytic transfusion reaction. Immunohematology 1. Rumsey DH, Ciesielski DJ. New protocols in serologic 20 09;25:4 4 –7. testing: a review of techniques to meet today’s challenges. 9. Higgins JM, Sloan SR. Stochastic modeling of human RBC Immunohematology 2000;16:131–7. alloimmunization: evidence for a distinct population of 2. Roback JD, Grossman BJ, Harris T, Hillyer CD. Technical immunologic responders. Blood 2008;112:2546–53. manual. 17th ed. Bethesda, MD: American Association of 10. Judd WJ, Fullen DR, Steiner EA, Davenport RD, Knafl PC. Blood Banks, 2011. Revisiting the issue: can the reading for serologic reactivity 3. Jørgensen J, Nielsen M, Nielsen CB, Nørmark J. The influence following 37 degrees C incubation be omitted? Transfusion of ionic strength, albumin and incubation time on the sensitivity 1999;39:295–9. of the indirect Coombs’ test. Vox Sang 1979;36:186–91. 11. Chapman JF, Milkins C, Voak D. The computer crossmatch: 4. Skaik YA. Detection of Rh antibodies using two low ionic a safe alternative to the serological crossmatch. Transfus Med diluents: extension of the incubation time and the number of 2000;10:251–6. Rh antibodies detected. J Postgrad Med 2008;54:4–6. 5. Reznicek MJ, Cordle DG, Strauss RG. A hemolytic reaction Carla Luana Dinardo, MD, PhD, student (corresponding author), implicating Sda antibody missed by immediate spin crossmatch. Vox Sang 1992;62:173–5. Chief of Immunohematology Division, Sílvia Leão Bonifácio, MSc 6. Judd WJ, Steiner EA, Abruzzo LV, et al. Anti-i causing acute student, Chief of Immunohematology Quality Control Department, hemolysis following a negative immediate-spin crossmatch. and Alfredo Mendrone Júnior, MD, PhD, Director of Fundação Pró- Transfusion 1992;32:572–5. Sangue/Hemocentro de São Paulo, Avenida Dr. Enéas de Carvalho 7. Wise SC, Larson PJ, Cook LO. Antibodies to low-incidence Aguiar, 155—1st floor, Cerqueira César, São Paulo—SP, Brazil antigens and elimination of the antihuman globulin phase of 05403-000. the crossmatch-case report: anti-Wra. Immunohematology 1997;13:20–2.

IMMUNOHEMATOLOGY, Volume 30, Number 1, 2014 5 R e v i e w Raph blood group system

M. Hayes

This review describes the current state of knowledge of the Raph The first report of human alloantibodies with MER2 blood group system, which consists of a single antigen, MER2. specificity was in 1988 and involved three Jews originating MER2 was initially classified as a high-incidence antigen in the in India and living in Israel (Table 1).3 Two were siblings 901 series of blood groups, formerly known as 901011, but was reclassified as an antigen in the Raph blood group system in (Subjects 1 and 2) and the third was unrelated (Subject 3); 2004. There have been six reports of human alloantibodies to all possessed the Raph-null phenotype.3,4 A fourth example of MER2. Three of the subjects were found to have a stop codon an antibody with MER2 specificity was described in 2002 in in the CD151 gene, which encodes a member of the tetraspanin 4 family of proteins. These three individuals had nephropathy a healthy Turkish blood donor (Subject 4). In 2008, two more and deafness, and two of the three, who are siblings, also had examples of anti-MER2 were reported in pregnant women, skin lesions and β-thalassemia minor. The fourth subject had one of Pakistani and the other of Turkish origin (Subjects 5 missense mutation c.533G>A (p.Arg178His). Subjects 5 and 6 and 6, respectively).5 shared missense mutation c.511C>T (p.Arg171Cys) as well as a synonymous single-nucleotide mutation (c.579A>G) and had no clinical features. Although the CD151 protein is critical to cell- Biochemistry to-cell interactions and cell signaling and is implicated in cancer progression, the significance in transfusion medicine is limited In 2004, Crew et al.4 demonstrated that MER2 is carried to one report of a hemolytic transfusion reaction in Subject 5. Immunohematology 2014;30:6–10. on CD151, a member of the cluster of differentiation (CD) family. CD proteins are found mostly on leukocytes and are Key Words: MER2, CD 151, MER2 red blood cell often used as laboratory tools to determine developmental polymorphism and functional characteristics of T and B cells.7 Tetraspanins (TSPs) span the cell membrane, with two extracellular and two History intracellular loops and short intracellular, cytoplasmic N- and C-termini. The first extracellular loop (EC1) is small, whereas MER2 , the single antigen in the Raph blood group system, the second (EC2) is large. EC2, sometimes characterized as a was first described in 1985.1 The authors described a red blood “mushroom head,”8 contains cysteines that form at least two cell (RBC) polymorphism called DEN that subsequently was conserved disulfide bonds (Fig. 1). CD151 is a TSP with six named MER2. MER2 was the first RBC surface antigen to be cysteines in EC2 including those in CCG, PXXCC, and GC defined by monoclonal antibodies, reacting with monoclonal cysteine patterns found in other TSP EC2 domains (Fig. 1).8 antibodies 2F7 and 1D12.2 The incidence of MER2 in a Tetraspanins interact with each other as well as a variety of random white population was estimated at 92 percent, with other transmembrane proteins, including integrins. They are approximately 8 percent of individuals showing the Raph-null thought to be involved in cell membrane stability, cell-to-cell phenotype.2 communication, cell migration, and maintenance of cell-to- cell contacts.4

Table 1. Molecular studies of CD151

Subject number Ethnicity Antigen/ antibody status Nucleotide changes Amino acid changes Allele name Reference 1, 2, 3 Israeli MER2–Anti-MER2 present c.383insG p.Lys127fs + Glu140X RAPH*01N.01 4 4 Turkish MER2–Anti-MER2 present c.533G>A p.Arg178His RAPH*-01.02 4 5 Pakistani MER2–Anti-MER2 present c.511C>T p.Arg171Cys RAPH*-01.01 5 c.579A>G 6 Turkish MER2–Anti-MER2 present c.511C>T p.Arg171Cys RAPH*-01.01 5 c.579A>G 7 White MER2–Anti-MER2 present c.494G>A p.Arg165Gln N/A 6 N/A = not available.

6 IMMUNOHEMATOLOGY, Volume 30, Number 1, 2014 Raph blood group system review

Genetics and Inheritance

The CD151 gene is 4.3 kilobases in length and located on chromosome 11p15.5.2,19,20 The CD151 gene is made up of 8 exons (Fig. 2) and encodes a 253–amino acid protein product.21 MER2 is inherited as a Mendelian dominant trait. As a result, individuals who express MER2 via one CD151 allele will type positive for MER2.7 The reference allele is called RAPH*01 and encodes a RAPH:1 or MER2+ phenotype.

Fig. 2 CD151 gene structure shows the 4.3 kilobases of the gene with its 8 exons (boxes). Exonic regions in gray depict the untranslated N-terminus C-terminus regions whereas those in black depict the coding regions.

Fig. 1 CD151 protein structure shows the protein with its four transmembrane domains spanning the cell membrane, including After the CD151 gene was found to encode the blood group the small first extracellular domain (EC1) and the large second antigen MER2, it was reclassified a blood group system by extracellular domain (EC2). EC2 contains six cysteines (C, black circles) in patterns of amino acids found in other TSP family the International Society of Blood Transfusion Committee on members. The gray circles represent noncysteine residues including Terminology for Red Cell Surface Antigens19 as described in proline (P) and glycine (G). X represents any amino acid. Table 2.22

Table 2. Raph blood group system terminology21

CD151 is the first member of the TSPs to be found on Phenotype: Phenotype: RBCs, being present on immature RBCs in the bone marrow. System System Gene numerical alternative System name symbol name Antigen terminology terminology As the RBC matures, the amount of CD151 on the cell 025 Raph RAPH CD151 RAPH1 MER2 RAPH:1 MER2+ surface decreases.4 MER2 is present on cord cells.7 CD151 also has been found on epithelium, endothelium, muscle, renal glomeruli and proximal and distal tubules, Schwann The CD151 genes of Subjects 1, 2, and 3, all with the cells, and dendritic cells.9 High levels of CD151 have been Raph-null phenotype and anti-MER2 , were subjected to DNA found on both platelets and megakaryocytes4,10 as well as on sequence analysis.4 Subjects 1 and 2 were homozygous for a other cell types.11 CD151 is co-located with integrins α3β1 single-nucleotide insertion in exon 5 (c.383insG). The insertion and α6β4 in hemi-desmosomes at the basolateral surface of causes a frameshift mutation that leads to a premature stop basal keratinocytes12 and with α6β4 on endothelial cells.13 codon at amino acid 140. The resulting truncated protein Given the impaired bone marrow responsiveness of CD151- would be predicted to lack a significant part of the second deficient individuals, this molecule may play an important large extracellular loop (EC2) and would be unlikely to fold role in erythroid progenitor membrane assembly such that properly or reach the plasma membrane.4 Subject 4 was found the cells are responsive to signals including those via the to be homozygous for a nonsynonymous single-nucleotide erythropoietin receptor.4 polymorphism (SNP) at nucleotide 533 in exon 6. The CD151 plays a role in signal transduction. Overexpression c.533G>A nucleotide change results in an arginine to histidine of CD151 leads to activation of phosphoinositide 3-kinase at amino acid 178 (p.Arg178His) in the EC2 domain. Protein (PI3K), hepatocyte growth factor (HGF), and the c-Met modeling did not predict that the amino acid change would signaling pathway. In turn, PI3K upregulates matrix have a functional effect on the protein.5 Subjects 5 and 6 were metallopeptidase 9 (MMP-9).14 CD151 also regulates RhoA homozygous for a nonsynonymous SNP in exon 6 at c.511.5 and cell-to-cell contacts, and, in doing so, maintains vascular The nucleotide 511C>T change results in an amino acid change stability.15–17 Regulation of cell-to-cell contact through CD151 of arginine to cysteine at amino acid 171 (p.Arg171Cys). In involves palmitoylation.18 addition, both individuals were homozygous for a synonymous

IMMUNOHEMATOLOGY, Volume 30, Number 1, 2014 7 M. Hayes

SNP (c.579A>G) in exon 6. Protein modeling of this variant MER2 may be associated with other disease states. CD151 suggested that the p.Arg171Cys variant protein would retain has been linked to modulation of platelet function.26 CD151- its integrin-binding capacity but would not have the MER2 null mice show increased bleeding time and decreased clotting epitope.5 Subject 7 is a white woman negative for MER2 ability. 27 Intravital microscopy in mice with and without with anti-MER2 reported to have a CD151 c.494G>A change CD151 expression demonstrated that the protein is required resulting in an amino acid change of arginine to glutamine at for regulating thrombus formation in vivo.9 In addition, there amino acid 165 (p.Arg165Gln).6 is a growing body of evidence suggesting that increased expression of CD151 is associated with poor prognosis in a Antibodies variety of cancers.28–32 Weakened or reduced expression of MER2 on RBCs has Anti-MER2 is reactive in the antiglobulin phase of been associated with the inheritance of In(Lu).7 The In(Lu) testing. MER2 is resistant to treatment with sialidase, phenotype is caused by a mutation in the promoter of EKLF papain, and neuraminidase.7,23 Dithiothreitol (DTT), trypsin, (KLF1), 33 a transcription factor that is required for expression chymotrypsin, pronase, and 2-aminoethylisothiouronium of Lutheran and other blood group antigens. Although bromide (AET) denature MER2 by breaking the disulfide regulation of CD151 is not well understood,34 if EKLF plays bonds within EC2.7,24 Blocking studies found that human a role in its expression, this is consistent with the finding of alloanti-MER2 blocked binding of both murine anti-CD151 weakened MER2 expression on RBCs of the In(Lu) phenotype. and murine anti-MER2 with MER2+ RBCs.4 Mice lacking CD151 show abnormal basement membrane Alloanti-MER2 has been reported in individuals who do biosynthesis and maturation, maintenance, and function of not have the antigen expressed on any cell types.4 Most people the kidney filter.35 Thus, animal modeling and the clinical whose RBCs type as MER2– express MER2 on other cell presentation of Subjects 1, 2, and 3 suggest a link between types. The presence of CD151 on multiple cell types supports Raph and the basement membrane of the kidney, skin, inner early speculation that although 8 percent of the population ear, and other tissues. Mice lacking CD151 also demonstrate phenotypically types as MER2– on RBCs, they are not at risk abnormal wound healing.36 of alloimmunization to MER2 because of the presence of the antigen on other cells. Conclusions

Clinical Significance The Raph blood group system is currently made up of a sole antigen, MER2, encoded by CD151. MER2 is expressed Subjects 1 and 2, who were siblings, as well as Subject 3 on RBCs as well as other cell types. CD151 has been associated had kidney disease.5 All three subjects with the null mutation with kidney function, cell-to-cell interactions, platelet function, had nephritic syndrome, which progressed to end-stage renal and cancer progression. Evidence suggests that MER2– failure requiring dialysis. Subjects 1 and 2 also showed pretibial individuals express MER2 on other cell types.4 This would be bullous skin lesions, neurosensory deafness, bilateral lacrimal similar to Fyb in the Duffy system, in which theFYB allele can duct stenosis, nail dystrophy, and β-thalassemia minor.25 The be silenced in RBCs based on the presence of an SNP in the male sibling had a single right kidney and defective teeth, promoter region, while being expressed in other tissues.37 and the female sibling had agenesis of the distal vagina and The clinical significance of anti-MER2 in RBC transfusion bilateral cervical ribs.25 is not clear. Additional examples of the antibody will need to The fourth subject with anti-MER2 was a Turkish blood be studied to determine the impact on RBC survival. Given donor (Subject 4).4 This individual had no apparent clinical that approximately 8 percent of whites are MER2–, it should symptoms or abnormalities. be possible to crossmatch such individuals. Subjects 5 and 6 were pregnant women; neither was reported to have symptoms associated with renal failure.5 Subject 5 experienced a hemolytic transfusion reaction after transfusion of three units of RBCs. A monocyte monolayer assay suggested the antibody could be clinically significant, and the patient’s serum contained no other alloantibodies.5

8 IMMUNOHEMATOLOGY, Volume 30, Number 1, 2014 Raph blood group system review

References 15. Zhang F, Michaelson JE, Moshiach S, et al. Tetraspanin CD151 maintains vascular stability by balancing the forces of cell 1. Daniels GL, Tippett P, Palmer DK, Miller YE, Geyer D, adhesion and cytoskeletal tension. Blood 2011;118:4274–84. Jones C; MRC Blood Group Unit. DEN, a new human red 16. Johnson JL, Winterwood N, DeMali KA, Stipp CS. Tetraspanin cell polymorphism defined by monoclonal antibodies and CD151 regulates RhoA activation and the dynamic stability of controlled by a locus on chromosome 11. (Abstract) Transfusion carcinoma cell-cell contacts. J Cell Sci 2009;122:2263–73. 1985;25:482. 17. Yamada M, Sumida Y, Fujibayashi A, et al. The tetraspanin 2. Daniels GL, Tippett P, Palmer DK, Miller YE, Geyer D, Jones CD151 regulates cell morphology and intracellular signaling C. MER2: a red cell polymorphism defined by monoclonal on laminin-511. FEBS J 2008;275:3335–51. antibodies. Vox Sang 1987;52:107–10. 18. Sharma C, Yang XH, Helmer ME. DHHC2 affects 3. Daniels GL, Levene C, Berrebi A, et al. Human alloantibodies palmitoylation, stability, and functions of tetraspanins CD9 detecting a red cell antigen apparently identical to MER2. Vox and CD151. Mol Biol Cell 2008;19:3415–25. Sang 1988;55:161–4. 19. Daniels GL, Fletcher A, Garratty G, et al; International Society 4. Karamatic Crew V, Burton N, Kagan A, et al. CD151, the of Blood Transfusion. Blood group terminology 2004: from first member of the tetraspanin (TM4) superfamily detected the International Society of Blood Transfusion committee on erythrocytes, is essential for the correct assembly of on terminology for red cell surface antigens. Vox Sang human basement membranes in kidney and skin. Blood 2004;87:304–16. 2004;104:2217–23. 20. Lögdberg L, Reid ME, Lamont RE, Zelinski T. Human blood 5. Karamatic Crew V, Poole J, Long S, et al. Two MER2- group genes 2004: chromosomal locations and cloning negative individuals with the same novel CD151 mutation and strategies. Transfus Med Rev 2005;19:45–57. evidence for clinical significance of anti-MER2. Transfusion 21. Whittock NV, McLean WHI. Genomic organization, 2008;48:1912–16. amplification, fine mapping, and intragenic polymorphisms 6. Karamatic Crew V, Poole J, Bullock T, Burton N, Muniz-Diaz of the human hemidesmosomal tetraspanin CD151 gene. E, Daniels G. A new case and a novel molecular background Biochem Biophys Res Comm 2001;281:425–30. in a MER2-negative (RAPH:−1) individual with anti-MER2 22. International Society of Blood Transfusion. Blood Group (abstract). Vox Sang 2012;103(Suppl 1):210–11. Allele Terminology. Available at: http://www.isbtweb.org/ 7. Issitt PD, Anstee DJ. Applied blood group serology. 4th ed. working-parties/red-cell-immunogenetics-and-blood-group- Durham, NC: Montgomery Scientific Publications, 1998. terminology/blood-group-terminology/blood-group-allele- 8. Huang S, Yuan S, Dong M, et al. The phylogenetic analysis terminology/. Accessed January 21, 2014. of tetraspanins projects the evolution of cell-cell interactions 23. Daniels G. Human blood groups. 3rd ed. Oxford: Wiley- from unicellular to multicellular organisms. Genomics 2005; Blackwell, 2013. 86:674–84. 24. Roback JD, Combs MR, Grossman BJ, Hillyer CD. Technical 9. Sincock PM, Mayrhofer G, Ashman LK. Localization of the manual. 16th ed. Bethesda, MD: AABB, 2008:432. transmembrane 4 superfamily (TM4SF) member PETA-3 25. Kagan A, Feld S, Chemke J, Bar-Khayim Y. Occurrence of (CD151) in normal human tissues: comparison with CD9, hereditary nephritis, pretibial epidermolysis bullosa and CD63, and α5β1 integrin. J Histochem Cytochem 1997; beta-thalassemia minor in two siblings with end-stage renal 45:515–25. disease. Nephron 1988;49:331–2. 10. Orlowski E, Chand R, Yip J, et al. A platelet tetraspanin 26. Lau L, Wee JL, Wright MD, et al. The tetraspanin superfamily superfamily member, CD151, is required for regulation of member CD151 regulates outside-in integrin α β signaling thrombus growth and stability in vivo. J Thromb Haemost IIb 3 and platelet function. Blood 2004;104:2368–75. 2009;7:2074–84. 27. Wright MD, Geary SM, Fitter S, et al. Characterization of mice 11. Rojewski MT, Schrezenmeier H, Flegel WA. Tissue distribution lacking the tetraspanin superfamily member CD151. Mol Cell of blood group membrane proteins beyond red cells: evidence Biol 2004;24:5978–88. from cDNA libraries. Transfus Apher Sci 2006;35:71–82. 28. Ang J, Lijovic M, Ashman LK, Kan K, Frauman AG. CD151 12. Sterk LMT, Geuijen CA, Oomen LC, Calafat J, Janssen protein expression predicts the clinical outcome of low-grade H, Sonnenberg A. The tetraspanin molecular CD151, a primary prostate cancer better than histologic grading: a novel constituent of hemidesmosomes, associates with the new prognostic indicator? [Erratum in Cancer Epidemiol integrin α6β4 and may regulate the spatial organization of Biomarkers Prev 2005;14:553]. Cancer Epidemiol Biomarkers hemidesmosomes. J Cell Biol 2000;149:969–82. Prev 2004;13:1717–21. 13. Sincock PM, Fitter S, Parton RG, Berndt MC, Gamble JR, 29. Hashida H, Takabayashi A, Tokuhara T, et al. Clinical Ashman LK. PETA-3/CD151, a member of the transmembrane significance of transmembrane 4 superfamily in colon cancer. 4 superfamily, is localised to the plasma membrane and Br J Cancer 2003;89:158–67. endocytic system of endothelial cells, associates with multiple integrins and modulates cell function. J Cell Sci 1999;112: 30. Ke AW, Shi GM, Zhou J, et al. Role of overexpression of 833–44. CD151 and/or c-Met in predicting prognosis of hepatocellular carcinoma. Hepatology 2009;49:491–503. 14. Yañez-Mó N, Barreiro O, Gonzalo P, et al. MT1-MMP collagenolytic activity is regulated through association 31. Tokuhara T, Hasegawa H, Hattori N, et al. Clinical significance with tetraspanin CD151 in primary endothelial cells. Blood of CD151 gene expression in non-small cell lung cancer. Clin 2008;112:3217–26. Cancer Res 2001;7:4109–14.

IMMUNOHEMATOLOGY, Volume 30, Number 1, 2014 9 M. Hayes

32. Sadej R, Romanska H, Baldwin G, et al. CD151 regulates 36. Cowin AJ, Adams D, Geary SM, Wright MD, Jones JC, Ashman tumorigenesis by modulating the communication between LK. Wound healing is defective in mice lacking tetraspanin tumor cells and endothelium. Mol Cancer Res 2009;7:787–98. CD151. J Invest Dermatol 2006;126:680–9. 33. Singleton BK, Burton NM, Green C, Brady RL, Anstee DJ. 37. Chaudhuri A, Polyakova J, Zbrzezna V, Pogo AO. The coding Mutations in EKLF/KLF1 form the molecular basis of the rare sequence of Duffy blood group gene in humans and simians: blood group In(Lu) phenotype. Blood 2008;112:2081–8. restriction fragment length polymorphism, antibody and 34. Wang J, Liu X, Ni P, Gu Z, Fan Q. SP1 is required for basal malarial parasite specificities, and expression in nonerythroid activation and chromatin accessibility of CD151 promoter tissues in Duffy-negative individuals. Blood 1995;85:615–21. in liver cancer cells. Biochem Biophys Res Commun 2010;393:291–6. Michele Hayes, MT(ASCP)SBB, MS HHSA, Director, IRL, Greater 35. Baleato RM, Guthrie PL, Gubler M, Ashman LK, Roselli S. Alleghenies American Red Cross, 250 Jari Drive, Johnstown, PA Deletion of Cd151 results in a strain-dependent glomerular 15904. disease due to severe alterations of the glomerular basement membrane. Am J Pathol 2008;173:927–37.

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 not necessarily reflect the official policy of the American of Immunohematology for distribution, please send request, Red Cross. 4 months in advance, to [email protected]

For information concerning the National Reference Immunohematology is on the Web! Laboratory for Blood Group Serology, including the American www.redcross.org/about-us/publications/ Rare Donor Program, contact Sandra Nance, by phone at 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]

10 IMMUNOHEMATOLOGY, Volume 30, Number 1, 2014 R e p o r t I-int phenotype among three individuals of a Parsi community from Mumbai, India

S.R. Joshi

The red blood cells (RBCs) of most adult individuals display an It is interesting to note that all three subjects belonged to the I+i– phenotype, whereas those of newborns and some rare adult Parsi community, an ethnic population group that migrated individuals are typed as I–i+. The phenotype in the latter category, centuries ago from Persia, i.e., present-day Iran. designated as adult i, is under genetic influence as the RBCs of I+i+ individuals display strengths of I and i antigen expression intermediate to that of ordinary adults and ii-adults. As there Materials and Methods was no information on the occurrence of adult i phenotype in the Indian population, the present study was undertaken. The RBCs of randomly selected subjects were screened with anti-I and Blood samples used in screening for I and i were obtained anti-i reagents by a saline tube technique at 22°C. Individuals from the local blood center in Mumbai. The RBCs of 5864 with unusual I and i antigen reactivity patterns were further randomly selected donors were screened with anti-I and anti-i tested by a semi-quantitative method with a battery of anti-I and reagents by a saline tube technique at 22°C. Individuals with anti-i reagents, followed by family studies. Three of the 5864 donors tested showed an elevated strength of i antigen. Further unusual I and i antigen reactivity patterns were further tested study revealed an intermediate strength of both I and i antigens by a semiquantitative method with a battery of anti-I and anti-i compared with those on RBCs from adult and cord blood samples. reagents, followed by family studies. The control rare ii-adult All three probands came from an ethnic Parsi community. The RBC specimen was made available through the Serum, Cells, phenotype (referred to as I-int) was shown to be inherited, being passed through two generations, but none of the members of the and Rare Fluids Exchange (SCARF; Houston, TX). Reagent families had displayed an adult i phenotype. The I-int phenotype antisera used including anti-I (Ste) and anti-i (Mac) were a gift detected showed an ethnic association because all three subjects from the late M.C. Crookston, Toronto, Ontario, Canada; anti-i belonged to an endogamous Parsi community that has migrated to India some centuries ago from Persia, the present-day Iran. (Ziag) was from Peter Issitt, Cincinnati, OH; and anti-i (Mort) Immunohematology 2014;30:11–13. was from Carolyn Giles, Chelsea, London, U.K. The other two anti-I sera, Gov and Gan, were from locally diagnosed patients Key Words: I-int phenotype, Parsi community, India with cold agglutinin disease. The methods used were standard serologic techniques recommended by Bhatia.3 Antigen I and i blood group antigens are considered developmental strength on RBCs was obtained by titration of antisera and antigens. Antigenic strength gradually alters during the first expressed as score values calculated as per Marsh.4 18 months of life. The I antigen, weakly expressed at birth, becomes stronger, whereas the i antigen, strongly expressed Results at birth, becomes weaker to undetectable through this period.1 Almost all adult red blood cells (RBCs) are observed as I+i–. Three (RD, RV, and ND) of the 5864 donors tested The rare adult I–i+ RBC phenotype is comparable to that of showed an elevated strength of i antigen. The RBC I and i newborns, is under genetic influence, and is designated as the antigen strengths were expressed as score values obtained adult i phenotype. The I+i+ phenotype with levels of I and i by titration of different antisera using RBCs from the three antigen strength intermediate between ordinary adults and ii- donors alongside appropriate control samples. All three anti-I adults or newborns is referred to as the I-int phenotype.1 I-int sera gave a lower antigen score on these donors as compared phenotype is often detected in the parents or the offspring with the control RBCs from adults. Anti-I (Ste) showed score of ii-adults. Earlier, Joshi and Bhatia2 described a unique values of 20, 22, and 23 for RD, RV, and ND, respectively, as phenotype in adults with traces of I antigen comparable to compared with the control value of 36; anti-I (Gov) showed that on RBCs of newborn infants but without any reciprocal i respective score values of 14, 17, and 20 in contrast to a control antigen. This phenotype was designated as I–i–.2 The present value of 44; similarly, anti-I (Gan) showed respective scores report describes the I-int phenotype in three individuals, and of 39, 31, and 21 versus a control adult RBC score of 55 (see their families, encountered while screening donors for I and i. Table 1).

IMMUNOHEMATOLOGY, Volume 30, Number 1, 2014 11 S.R. Joshi

Table 1. Comparison of the I and i antigen scores of the proband and their family members Although the phenotype showed having I-int phenotype with control RBCs vertical inheritance, none of the Anti-I Anti-i members in these families have the Red blood cells ABO groups Ste Gov Gan Ziag McD Mort ii-adult phenotype. RD (Proband) O 20 14 39 22 56 21 RD Family Father O 23 18 34 26 56 25 Discussion ND (Proband) B 23 20 21 26 61 12 Father B 21 25 25 26 64 19 ND Family I and i antigens are considered Sister B 22 16 19 23 53 19 to be developmental antigens as Niece B 23 25 25 18 59 21 they show a remarkable reciprocal RV Family RV (Proband) A1B 22 17 31 23 50 10 relationship of expression on RBCs Adults B; O 36 44 55 0 26 2 of individuals in early childhood and Controls Cord B; O 4 0 3 34 64 36 subsequent adult life.1 Most adult ii-Adult O 2 0 1 38 70 NT RBCs are characterized by a strong NT = not tested. expression of I antigen and a weak expression of i antigen, whereas the On the other hand, the three anti-i reagents showed RBCs of newborns have weak I and strong i antigens. The higher antigen scores on RBCs from these donors as compared rare individuals defined as ii-adult have an I/i antigen profile with the control RBCs from adults. Anti-i (Ziag) showed score similar to that found in newborn infants. Additionally, these values of 22, 23, and 26 for RD, RV, and ND, respectively, individuals have naturally occurring alloanti-I. whereas control RBCs from adults showed a score value of Joshi and Bhatia2 earlier found weak expression of I 0; anti-i (McD) gave respective score values of 56, 50, and 61 antigen without concurrent increase in i antigen on RBCs of as compared with control RBCs from adults showing score certain individuals and defined the phenotype as I–i–. Family values of 26; and anti-i (Mort) showed respective scores of 21, study showed that this I–i– phenotype demonstrated vertical 10, and 12 as compared with control adult RBCs showing a inheritance with some members showing an apparently partial score value of 2. The i antigen strengths on these donors’ RBCs expression of the I–i– character. However, the phenotype were lower than those found on control RBCs of the newborns appeared to be passing through generations without showing or ii-adults. The results are displayed in Table 1. any pattern of Mendelian inheritance.5 Joshi and Bhatia5 In the family of donor ND, the I-int phenotype was reported an association with group A1 or A1B phenotype that found in three generations in the father (II-1), sister (III-4), was characterized by a remarkable increase in A1 antigen and niece (IV-2) of the proband (III-1; Fig. 1), whereas in the expression on RBCs, thereby suggesting an influence of family of donor RD, the phenotype was detected through two A1 blood group on expression of I in this phenotype. In the generations in the father (I-1) and the proband (II-1; Fig. 2). present cases, the I-int phenotype showed no bearing on the

I I

1 2 1 2

II II

1 2 3 1 2

Fig. 2 I-int phenotype in members of the RD family. III

1 2 3 4 LEGEND

IV

1 2 Deceased Normal I-I Phenotype I-int phenotype Proband Fig. 1 I-int phenotype in members of the ND family.

12 IMMUNOHEMATOLOGY, Volume 30, Number 1, 2014 I-int phenotype among Parsi community

A1 blood group, as two of the three probands were group B References and O. The reduced I antigen in the present I-int phenotype 1. Jenkins WJ, Marsh WL, Noades J, Tippett P, Sanger R, Race appeared with an increase in i as is seen among the obligate RR. The I antigen and antibody. Vox Sang 1960;5:97–106. heterozygotes (e.g., parents or offspring) in the families of the 2. Joshi SR, Bhatia HM. A new red cell phenotype I–i–: red cells ii-adult probands.6 Although there was no ii-adult phenotype lacking both l and i antigens. Vox Sang 1979;36:34–8. found in the present families studied, such a rare phenotype 3. Bhatia HM, ed. Procedures in blood banking and immunohaematology. Bombay: Blood Group Reference Centre 7 was investigated by Joshi et al. in a blood donor from Iran (ICMR), 1977. with depressed RBC ABH antigen expression. However, the 4. Marsh WL. Scoring of hemagglutination reactions. Transfusion three donors in the present study had normal features of ABH 1972;12:352–3. antigens. The Parsi community has its ancestral origin in Iran, 5. Joshi SR, Bhatia HM. I–i– phenotype in a large kindred Indian family. Vox Sang 1984;46:157–60. so it is conceivable that the I-int phenotype in the present cases 6. Daniels G. I and I antigens, and cold agglutination. In: Human bears some ethnic relation to the ii-adult phenotype detected in blood groups. 3rd ed. Chichester, UK: John Wiley & Sons, the Irani donor. However, the level of I and i antigen strength 2013:469–84. found among those donors with an I-int phenotype as well 7. Joshi SR, Pourazar A, Clarke VA, Ala FA. Para-Bombay phenotype with altered I-i blood group antigens in an Iranian as those reported among the family members of the ii-adult donor. Transfusion Today 2000:3–6. probands may potentially reflect a dosage effect shown by the I and i antibodies, a concept that, as per the author’s knowledge, Sanmukh R. Joshi, PhD, Associate Professor, Allianze University has not been proposed to date. College of Medical Sciences, Waziria Medical Square, Jalan Bertam 2, Mukim 6, Kepala Batas 13200, Penang, Malaysia.

Attention: SBB and BB Students

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

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

For information concerning Immunohematology or the Notice to Readers Immunohematology Methods and Procedures manual, Immunohematology is printed on acid-free paper. contact us by e-mail at [email protected]

IMMUNOHEMATOLOGY, Volume 30, Number 1, 2014 13 Ca s e R e p o r t Evans syndrome in a pediatric liver transplant recipient with an autoantibody with apparent specificity for the KEL4 (Kpb) antigen

S.A. Koepsell, K. Burright-Hittner, and J.D. Landmark

Although most warm red blood cell (RBC) autoantibodies react In transplant recipients, the presence of an apparent broadly with panel cells in addition to the patient’s own RBCs, autoantibody also raises the possibility that the pathologic occasionally an autoantibody with specificity for a specific blood antibody may not be a true autoantibody, but rather an group antigen is encountered. Rare cases of warm autoantibodies with specificity for the Kpb antigen of the Kell blood group system alloantibody arising from lymphocytes of donor origin from have been described. We report a pediatric transplant recipient the transplanted organ, a term called passenger lymphocyte with anemia, immune-mediated hemolysis, thrombocytopenia, syndrome (PLS).9 PLS often occurs within weeks of transplant and a warm autoantibody with apparent anti-Kpb specificity. The patient’s autoimmune anemia and thrombocytopenia responded and usually involves an antibody with defined specificity well to discontinuing the immunosuppressant tacrolimus, trans- that is mismatched between the donor and the recipient. An fusions with Kp(b–) RBCs, and intravenous immunoglobulin additional consideration in transplant recipients who develop therapy, with disappearance of the pathologic antibody. During red blood cell (RBC) autoantibodies are immunosuppressant the autoimmune hemolysis, the patient’s RBCs did not react with antisera specific for Kpb. However, repeat testing of the patient’s drugs, which may have unknown consequences on the ability RBCs with Kpb-specific antisera 15 months after the resolution of of the immune system to regulate autoimmunity. Tacrolimus hemolysis showed reactivity, indicating that the RBC autoantibody in particular is an immunosuppressant that has been reported was associated with a transient disappearance of the Kpb antigen. to be associated with AIHA by an unknown mechanism in Immunohematology 2014;30:14–17. transplant recipients based on the observation that pathologic Key Words: autoimmune hemolytic anemia, Kell blood warm autoantibodies seem to resolve after discontinuation of group, Evans syndrome the drug.10,11 We report here an unusual case of a liver transplant Warm autoantibodies are a broad category of immuno- recipient who developed an AIHA and thrombocytopenia globulins that react with a patient’s own antigens optimally (Evans syndrome), with the autoantibody having apparent at 37°C. Their incidence is 1 in 50,000 to 80,000 patients.1 specificity for the Kpb antigen. The patient responded well to Depending on clinical background, antibody specificity, discontinuing her tacrolimus, transfusion of Kp(b–) RBCs, antibody titer, immunoglobulin subclass, and other less and intravenous immunoglobulin (IVIG) therapy, with the defined variables, warm autoantibodies may be associated autoantibody becoming undetectable. with an autoimmune hemolytic anemia (AIHA), especially if the direct antiglobulin test (DAT) is reactive for complement Case Report C3 in addition to immunoglobulin G (IgG).2 Although most warm autoantibodies display broad The patient was a 2-year-old group O, D+ girl who specificity, many examples have been reported of autoanti- received a cadaveric liver transplant at 7 months of age to bodies with well-defined specificity, most commonly against treat congenital biliary atresia. Her postoperative course antigens of the Rh and LW blood group systems.2,3 Although was significant for pneumonia, one episode of liver rejection, extremely rare, warm autoantibodies with anti-Kpb specificity and ascites. Eight months after transplantation, the patient have been reported.4–8 The most common of three antithetical presented with difficulty breathing and fatigue. She was found antigens, Kpb (KEL4) is an antigen that is formed by the to have a hemoglobin (Hb) concentration of 6.2 g/dL, down presence of an arginine at amino acid 281 of the Kell protein. from 10.0 g/dL 2 weeks earlier. Her immunosuppression Substitution of the arginine with tryptophan generates the regimen was tacrolimus at 1.5 mg, twice daily. Pretransfusion Kpa antigen (present in 2.3% of whites), and substitution with testing at this time showed a negative antibody screen. glutamine generates the Kpc antigen, which is extremely rare. The patient subsequently received 15 mL/kg of irradiated,

14 IMMUNOHEMATOLOGY, Volume 30, Number 1, 2014 Autoantibody with Kpb specificity

leukocyte-reduced, cytomegalovirus (CMV)-negative group O, treated with an ethylenediaminetetraacetic acid–glycine acid D+ RBCs. Her Hb level increased to 9.4 g/dL after transfusion. (EGA) solution and were DAT-negative. The patient’s plasma Infectious disease testing at this time showed positive nucleic and eluate both reacted with the EGA-treated cells, indicating acid amplification results for Epstein-Barr virus (28,000 a true autoantibody. copies/mL), human herpes virus 6, and rhinovirus. The The patient’s plasma was then tested against a panel patient recovered and was discharged 1 day after admission. of antigen-negative rare RBCs that included cells negative Five days after being discharged, the patient was referred for the Rh, Joa, Hy, PP1Pk, and Kpb antigens in low-ionic- to a pediatric hematologist after being seen in the clinic with strength saline (LISS) at 37°C. Reactivity was observed with an Hb concentration of 8.4 g/dL. Eleven days after discharge, all the cells tested, with the exception of the Kp(b–) cells. The the patient had an Hb level of 6.8 g/dL, an absolute reticulocyte patient’s plasma was nonreactive with four different reagent count of 70,000/µL, and a platelet count of 45,000/µL, and her cells that were Kp(a+b–). Antisera with Kpb specificity did RBCs were negative in the DAT with polyspecific antiglobulin not agglutinate the patient’s RBCs. The American Red Cross reagent. Nucleic acid amplification testing for parvovirus B19 reference laboratory also performed molecular genotyping was negative. Physical examination was significant for an of the patient’s peripheral blood leukocytes using HEA enlarged spleen. The patient again had a negative antibody Beadchip™. This technology identified that patient as screen and received 90 mL of irradiated, leukocyte-reduced, demonstrating the Kpb genotype in her peripheral blood CMV-negative group O, D+ RBCs. A bone marrow biopsy was leukocyte DNA, despite the nonreactivity with the Kpb-specific performed, which showed trilineage hematopoiesis with a antisera. decreased number of megakaryocytes. During this time, the patient underwent both a lower and Ten days after the bone marrow biopsy and transfusion, upper endoscopy, which ruled out significant gastrointestinal the patient’s Hb concentration was 8.7 g/dL and her platelet bleeding or pathology. Her immunosuppressant regimen was count was 15,000/µL. The patient then received two units of changed from tacrolimus to cyclosporin, 50 mg twice per day. single-donor, leukocyte-reduced, irradiated, CMV-negative A lactate dehydrogenase (LDH) level at this time was elevated platelets, and her platelet count increased to 228,000/µL. at 392 units (normal, 140–304 units). Sixteen days after the Again, the patient’s Hb level continued to decline to 7.8 g/dL, first positive DAT, the patient was provided with Kp(b–) and and the patient was given RBCs, which brought her Hb K– RBCs, which were nonreactive with the patient’s serum concentration up to 10.8 g/dL. On the following day, the patient without enhancement but did have weak reactivity with received another unit of platelets. Six days after the last unit LISS and anti-IgG reagent. Her Hb concentration increased of RBCs and 5 days after the last unit of platelets, the patient from 6.0 g/dL to 10.8 g/dL. Four days after her Kp(b–) RBC was again anemic with an Hb concentration of 7.4 g/dL. An transfusion, the patient was started on IVIG, 12 g per day for antibody screen performed at this time was positive, with all 4 days. During the third day of IVIG treatment, an antibody three cells in the panel reacting with 1+ reactivity at the anti- screen was negative and a subsequent polyspecific DAT was human globulin phase. A polyspecific DAT was 2+ positive, also negative. All antibody screens and DATs performed since with similar reactivity observed with anti-IgG monospecific this episode have been negative. Fifteen months later, the reagent. A peripheral blood smear showed RBCs with crenated patient’s RBCs were reactive with Kpb antisera. The patient’s and teardrop morphology. clinical course is summarized in Table 1. The patient’s specimen was forwarded to the American Red Cross regional reference laboratory, where the DAT Discussion was confirmed to be positive (1+) with anti-IgG reagent, but negative with anti-complement reagent. An antibody screen We report a liver transplant recipient who developed including an auto-control was reactive with all cells at room AIHA. The etiology of the autoantibody is unknown, but may temperature, showing 1+ to 2+ reactivity. As part of the protocol have been associated with her antecedent viral illnesses that for initial testing of a suspected autoantibody, the sample was occurred near the time of her first bout of anemia and before prewarmed, which did not resolve the reactivity. An eluate her first positive antibody screen. Although the possibility was prepared and tested, which subsequently reacted with all exists that the anti-Kpb was actually an alloantibody as a cells tested. Its reactivity with the ficin-treated reagent cells result of PLS, this is unlikely, as the Kpb antigen is highly increased to 4+ at 37°C, and no reactivity was observed with prevalent in the population. In addition, the timing of the dithiothreitol-treated reagent cells. The patient’s cells were patient’s hemolysis occurred 8 months after transplantation,

IMMUNOHEMATOLOGY, Volume 30, Number 1, 2014 15 S.A. Koepsell et al.

Table 1. Clinical and laboratory data obtained from a pediatric liver transplant recipient who developed autoimmune hemolytic anemia*

Hemoglobin Platelets Date Before transfusion After transfusion Before transfusion After transfusion DAT Ab Screen Transfusion 10/29/2009 10 288 11/13/2009 6.2 9.4 212 Neg 135 mL PRBC 11/25/2009 6.8 8.7 45 Neg Neg 90 mL PRBC 12/7/2009 15 133 50 mL A+ platelets 12/8/2009 54 228 45 mL A+ platelets 12/15/2009 7.8 64 Neg 12/16/5009 10.8 64 135 mL PRBC 12/17/2009 29 92 51 mL A+ platelets 12/22/2009 7.4 54 Pos Pos 12/23/2009 Tacrolimus discontinued 1/7/2010 6.0 7.8 121 100 mL Kp(b–) PRBC 1/8/2010 7.8 10.8 100 mL Kp(b–) PRBC 1/12/2010 Initiation of IVIG therapy 1/14/2010 9.0 179 Neg 1/22/2010 9.9 231 Neg 3/14/2010 12.6 13.3 343 Neg 100 mL PRBC 3/24/2010 11.9 12.3 278 Neg 4/6/2010 10.9 364 Neg DAT = direct antiglobulin test; Ab = antibody; Neg = negative; PRBC = packed red blood cells; Pos = positive; IVIG = intravenous immunoglobulin G. *Timeline of the patient’s hemoglobin (g/dL), platelet count (cells/μL), DAT, Ab screen, and quantity and type of transfused product. All cellular blood products were irradiated. whereas PLS usually occurs within a couple of weeks after this period, she was clinically diagnosed as having AIHA transplantation. Further, genetic analysis performed on the because of the lack of clinical evidence of bleeding, the increased formalin-fixed paraffin-embedded liver biopsy obtained from LDH level, and the increased level of circulating reticulocytes the transplanted liver while the organ was being prepared for along with the warm autoantibody that was identified. The transplant was homozygous for the Kpb allele, essentially ruling patient also had persistent thrombocytopenia during this out the possibility that donor lymphocytes could recognize the time, which may indicate that this case of AIHA may actually Kpb antigen as foreign.12 be part of an Evans syndrome. Interestingly, a case of Evans Serologic workup found a warm autoantibody in her syndrome in a pediatric liver transplant recipient has been plasma that had specificity for the Kpb antigen. However, reported in which the cytopenias were successfully treated by antisera specific for the Kpb antigen did not agglutinate the switching the patient from tacrolimus to cyclosporine.13 patient’s RBCs at the time of the anemia. To classify the Previous case reports have identified that patients with pathologic antibody as an autoantibody or alloantibody, Kpb-specific autoantibodies have a depression in the Kell molecular genotyping was performed that identified the patient antigens on their RBCs that rebounds after resolution of the as being homozygous for the gene encoding Kpb. The patient autoantibody.8,14 A similar observation was made in this case, was successfully treated with discontinuing her tacrolimus, as the patient’s RBCs failed to agglutinate with Kpb-specific transfusion of Kp(b–) blood, and administration of IVIG, with antisera during the peak of her AIHA. Appropriate controls complete resolution of the pathologic antibody. showed that the Kpb-specific antisera agglutinated reagent Table 1 shows that from mid-November 2009 through cells. Fifteen months after the resolution of the patient’s mid-January 2010, the child developed repeatedly low anemia, her cells did agglutinate with Kpb-specific antisera. hemoglobin levels requiring transfusions. Clinically, there Thus, the apparent negative reaction with anti-Kpb may be a was no evidence of bleeding. During this period, the child’s result of a similar mechanism of Kell antigen downregulation hemoglobin decreased at an initial rate of 0.24 g/dL/day, which in response to an autoantibody as previously reported,15 or increased to over 1 g/dL/day before starting IVIG therapy. that our patient simply had a serologically “blocked” antigen Although the patient did not have elevated bilirubin during owing to her specific autoantibody. The process of how an

16 IMMUNOHEMATOLOGY, Volume 30, Number 1, 2014 Autoantibody with Kpb specificity

autoantibody to the Kpb antigen results in downregulation of 8. Beck ML, Marsh WL, Pierce SR, DiNapoli J, Oyen R, Nichols b the antigen is not known. ME. Auto anti-Kp associated with weakened antigenicity in the Kell blood group system: a second example. Transfusion A common theme among the handful of case reports 1979;19:197–202. b describing warm autoantibodies with Kp specificity is resolu- 9. Audet M, Panaro F, Piardi T, et al. Passenger lymphocyte tion of the autoantibody with either time or treatment.8,14,16 syndrome and liver transplantation. Clin Dev Immunol 2008; Our case report affirms this trend, with our patient’s bKp 2008:715769. 10. Valentini RP, Imam A, Warrier I, et al. Sirolimus rescue for autoantibody completely disappearing 23 days after first tacrolimus-associated post-transplant autoimmune hemolytic being discovered. The patient’s hemoglobin responded well anemia. Pediatr Transplant 2006;10:358–61. to Kp(b–) blood transfusions. Discontinuing tacrolimus and 11. Botija G, Ybarra M, Ramos E, et al. Autoimmune cytopaenia administering IVIG may have played a role as well, as the after paediatric intestinal transplantation: a case series. Transpl Int 2010;23:1033–7. antibody became undetectable shortly after IVIG therapy was 12. Koepsell SA, Landmark JD. Passenger lymphocyte syndrome: administered. In summary, we report a pediatric solid-organ use of archived donor organ biopsy obtained at the time of transplant recipient who developed a warm autoantibody with transplantation for diagnosis. Am J Transplant 2013;13:2227. apparent Kpb specificity that responded well to treatment and 13. Domenech C, Mialou V, Galambrun C, et al. Successful b treatment of Evans syndrome post liver transplant with was associated with transient depression of the Kp antigen on splenectomy and switch from tacrolimus to cyclosporine. the patient’s RBC surface. Transpl Int 2008;21:397–9. 14. Seyfried H, Gorska B, Maj S, Sylwestrowicz T, Giles CM, References Goldsmith KL. Apparent depression of antigens of the Kell blood group system associated with autoimmune acquired 1. Issitt PD, Anstee DJ. Applied blood group serology. 4th ed. haemolytic anaemia. Vox Sang 1972;23:528–36. Durham, NC: Montgomery Scientific Publications, 1998. 15. Williamson LM, Poole J, Redman C, et al. Transient loss of 2. Wheeler CA, Calhoun L, Blackall DP. Warm reactive proteins carrying Kell and Lutheran red cell antigens during autoantibodies: clinical and serologic correlations. Am J Clin consecutive relapses of autoimmune thrombocytopenia. Br J Pathol 2004;122:680–5. Haematol 1994;87:805–12. 3. Garratty G. Specificity of autoantibodies reacting optimally at 16. Win N, Kaye T, Mir N, Damain-Willems C, Chatfield C. b 37 degrees C. Immunohematology 1999;15:24–40. Autoimmune haemolytic anaemia in infancy with anti-Kp specificity. Vox Sang 1996;71:187–8. 4. Villa MA, Coluccio E, Revelli N, Drago F, Morelati F, Rebulla P. Successful transfusion of Kp (a–b+) red cells incompatible for auto anti-Kpb. Haematologica 2005;90:ECR07. Scott A. Koepsell, MD, PhD (corresponding author), Assistant 5. Lee E, Burgess G, Win N. Autoimmune hemolytic anemia Professor of Pathology and Microbiology, University of Nebraska b and a further example of autoanti-Kp . Immunohematology Medical Center; Kerry Burright-Hittner, MT(ASCP)SBB, Director, 2005;21:119–21. Immunohematology Reference Laboratory, American Red Cross, 6. Puig N, Carbonell F, Marty ML. Another example of mimicking Omaha; and James D. Landmark, MD, Associate Professor of anti-Kpb in a Kp(a+b–) patient. Vox Sang 1986;51:57–9. Pathology and Microbiology, University of Nebraska Medical Center, 7. Manny N, Levene C, Sela R, Johnson CL, Mueller KA, Marsh 983135 Nebraska Medical Center, Omaha, NE 68198-3135. WL. Autoimmunity and the Kell blood groups: auto-anti-Kpb in a Kp(a+b–) patient. Vox Sang 1983;45:252–6.

IMMUNOHEMATOLOGY, Volume 30, Number 1, 2014 17 R e v i e w JMH blood group system: a review

S.T. Johnson

Dedication This review is dedicated to John J. Moulds, MT(ASCP)SBB, as he was instrumental in bringing a group of antibodies with interesting serologic characteristics to the blood group it is today. In the classic “Moulds” way throughout his life and career, he always challenged young SBBs and laboratory scientists to question unusual serologic results. He would say, “They are telling you something.” The JMH blood group system is a testament to his beliefs. He is coauthor on many papers in this review. John called me to provide additional information he found important to the JMH story. I am honored and privileged to have known John, to have been challenged by John, and to write this review as a tribute to his work.

The JMH blood group system consists of six high-prevalence Not to leave the ladies out of describing these antibodies, the antigens. These antigens are located on the Sema7A protein. The nickname “The Cat” came from one of the first women who molecular basis of the JMH1– phenotype is not known; however, produced anti-JMH. She is said to have claimed the anti-JMH single nucleotide changes in the SEMA7A gene on chromosome 15 account for the other JMH antigens. JMH1, commonly known occurred when her cat died (Marilyn Moulds, March 2012, as JMH, is most notable because transient depression of the personal communication). antigen occurs and anti-JMH may develop. These antibodies are The significance of antibodies to JMH antigens in trans- most commonly observed and are not significant in transfusion. Antibodies developed in the rare JMH variant types may cause fusion and pregnancy are minimal and will be discussed. reduced red cell survival. This review provides a general overview of the JMH blood group system, including the serologic and History molecular characteristics as well as proposed functions of the Sema7A protein. Immunohematology 2014;30:18–23. In March 1973, a male patient in his 60s was to have Key Words: John Milton Hagen group, semaphorin 7A elective orthopedic surgery. He had no history of transfusions. (CDw108 glycoprotein), SEMA7A gene, high-prevalence red A weakly reactive antibody detected by the indirect antiglobulin cell blood group antigen test (IAT) was not reactive with papain- or ficin-pretreated cells. No compatible blood was available, and units collected for Introduction autologous transfusions were never given. It was reported as an “antibody to high-incidence unknown factor,” and samples The JMH blood group system has the distinction of being were sent to other laboratories for investigation. This result led the blood group with the most “nicknames.” The “over 60 to a group of antibodies being collected in the 1970s that were group,” “John Milton Hagen group,” “The Boys,” “The Cat,” compatible with this individual’s red blood cells (RBCs) and and the “Old Boys’ Club” have all been used in reference to the had similar reactivity. They were first mentioned in print by antibody and antibody makers.1 The earliest accounts in the Issitt in 1975 as the John Milton Hagen group of antibodies, 1970s were of antibody reactivity, followed by description of as he termed them “belonging to a group of high-incidence the protein carrying the antigens (semaphorin 7A or CD108), antigens of which little is known about.” His remarks were and finally with identification of the gene SEMA7A, JMH based on personal communication with John J. Moulds.3 The earned its rightful status of blood group system 026 in 2001. first antibody was reported to be recognized in 1970 per Sabo Antibodies to JMH antigens are encountered infrequently et al.,4 who further characterized 49 sera with similar reactivity but have unique characteristics landing them their nicknames. and proposed giving this antibody the symbol JMH, naming it The “over 60 group,” “The Boys,” and the “Old Boys’ Club” after one of the original antibody makers, and adding it to the all are derived from early observations that many of the list of high-titer, low-avidity antibodies. These antibodies were antibodies were found in older gentleman. Older has been of high titer and weakly reactive in saline IAT with all RBCs defined as >50 or 60 years of age. Like Issitt,2 I concur that tested, except autologous cells and other JMH– RBCs. old is recognized as mature, wiser, and experienced patients.

18 IMMUNOHEMATOLOGY, Volume 30, Number 1, 2014 JMH blood group system review

In the 1980s, work was done to further define the serologic Nomenclature characteristics of anti-JMH and its reactivity with chemically modified RBCs, namely those treated with proteases, Nomenclature resulting from serologic observations and sulfhydryl-reducing agents, and neuraminidase. In addition, biologic and molecular studies has remained very simple for attempts were made to predict the clinical significance of anti- the blood group with so many nicknames. The antigen name JMH in transfusion using chromium-51 RBC survival studies commonly known by serologists remains JMH and is officially and subclassing.5 In 1982, a monoclonal antibody named H8 recognized by the International Society for Blood Transfusion was described with JMH specificity.6,7 This antibody was (ISBT) as JMH1. Confirmed JMH variants are sequentially important to further work in characterizing the JMH protein. numbered for example, JMH2 and named with the first letter In addition, J.J. Moulds reported evidence that there was from the antibody maker’s first name (JMHK) following JMH. heterogeneity in reactivity of different anti-JMH.8 Telen et al.9 reported in 1990 that several high-incidence JMH Glycoprotein antigens including the JMH antigen were absent on RBCs of individuals with paroxysmal nocturnal hemoglobinuria Antigens in the JMH blood group system are carried (PNH). PNHIII RBCs were previously shown to lack on the protein semaphorin 7A, also known as Sema7A and glycosylphosphatidylinositol (GPI)-linked proteins, and CD108. Mature Sema7A consists of 525 amino acids. Changes when these RBCs were tested with human anti-JMH and in amino acids 207 and 460/461 were noted by Seltsam in monoclonal H8, no reactivity was seen. This result suggested JMH-variant individuals. A three-dimensional model of the that JMH must reside on a GPI-linked membrane protein. crystal structure of Sema7A was proposed (Fig. 1A and B).14 Immunoprecipitation and immunoblotting experiments on Position 207 is located at the top face of the sema domain, human anti-JMH and H8 showed JMH resides on a 76-kD whereas positions 460 and 461 are on the bottom. phosphatidylinositol-linked protein.10 Finally, the location of As mentioned previously, Sema7A protein binds to the cell JMH was determined to be on the CDw108 glycoprotein, now membrane by a GPI linkage. Not only is it present on RBCs, known as semaphorin 7A.11 it is present on lymphoid and myeloid cells as well as bone Having knowledge of the location of JMH, the cDNA clone cells, neurons of the brain and spinal cord, thymus, spleen, containing the CDw108 gene was identified in 1999.12 The gut, kidney, heart, and placenta. Sema7A is found primarily in gene resides on the middle of the long arm of chromosome 15. activated T cells and thymocytes.18 This CDw108 gene is now known as SEMA7A and is located on 15q23–24. The genetic information earned JMH its own blood group system in 2001 named John Milton Hagen, 026, and its symbol JMH.13 JMH1 is the antigen detected by most individuals making anti-JMH. Four additional variant JMH genotypes were added in 2007.14 JMHQ was proposed in 2011, reported in four Native Americans whose RBCs were JMH1–, with most examples of anti-JMH, and officially recognized later that year.15 Today, there are six recognized antigens in the system summarized in Table 1.16,17 Each antigen is defined by antibodies nonreactive with JMH1– RBCs.

Table 1. The John Milton Hagen blood group system

Number Name Prevalence Molecular basis of antigen-negative phenotype JMH1 JMH High Not known JMH2 JMHK High 619C>T R207W JMH3 JMHL High 620G>A R207Q Fig. 1 Three-dimensional model of Sema7A (reprinted with permission from AABB). Image courtesy of A. Setsam. (A) A cartoon JMH4 JMHG High 1379G>A R460H of the sema domain when looking down on the protein. The numbers JMH5 JMHM High 1381C>T R461C represent propellers. (B) A side view of the sema domain. The JMH6 JMHQ High 1040G>T R347L polymorphic amino acid positions 207, 460, and 461 are circled.

IMMUNOHEMATOLOGY, Volume 30, Number 1, 2014 19 S.T. Johnson

Numerous roles for Sema7A have been proposed. It is variable reactivity is seen when the specificity of each antibody clear it is involved in cytokine expression, chemotaxis, and is cross-tested with other JMH variants (Table 2).28 JMH axon guidance signaling.19 In effector T cells, it likely promotes antigens are destroyed by various chemicals, as shown in macrophage recruitment to sites of inflammation.18,20,21 A Table 3, characteristic of other blood group antigens on GPI- recent report shows the Sema7A_R461C variant (JMH5) linked proteins. PNHIII cells can be used as a source of JMH– causes different T-cell responses than the wild-type Sema7A.22 RBCs as well.29 It is able to strongly activate CD4+ T cells and change their Table 2. Anti-JMH and JMH-related antibodies tested with JMH– phenotype to a more cytotoxic one, and evidence is provided and variant JMH red blood cells that Sema7A_R461C delivers costimulatory signals to T cells, Anti- causing them to increase their release of cytokines such as Phenotype JMH1 JMH2 JMH3 JMH4 JMH5 JMH6 interleukin (IL)-1β, IL-6, and IL-8. JMH:-1 0 0 0 0 0 0 Sema7A has also been reported to be an important JMH:-2 + 0 0 + + NA regulator of tissue remodeling. In genetic studies in Korean JMH:-3 + 0 0 + + NA women with decreased bone mineral density, mutations in JMH:-4 + + + 0 + NA 23 SEMA7A were found. Sema7A also contributes to regulation JMH:-5 + + + 0 0 NA 24 of tissue fibrosis and remodeling. JMH:-6 + NA NA NA NA NA The exact function of semaphorin 7A on RBCs is not NA = not available. known, but it likely plays a role in cell migration as an adhesion Adapted from Daniels.27 molecule.25 It may also play a role as a receptor for Plasmodium falciparum. It was identified as a receptor for theP. falciparum Table 3. Characteristics of antigens in the John Milton Hagen blood group system merozoite-specific thrombospondin-related anonymous pro- tein (TRAP) homolog in laboratory experiments using Sensitive to ficin or papain treatment recombinant protein.26 Sensitive to trypsin and alpha chymotrypsin treatment Sensitive to 2-aminoethyl-isothiouronium bromide and 200 mmol/L/ 50 mmol/L dithiothreitol treatment JMH Antigen Characteristics Sensitive to sialidase treatment

JMH1 is the primary antigen in the system and is Resistant to chloroquine treatment present in greater than 99 percent of all individuals. The Absent from paroxysmal nocturnal hemoglobinuria-III red blood cells JMH1-negative phenotype occurs as either inherited or more Weakly expressed on cord cells commonly acquired depression of the antigen. The JMH antigen is most commonly depressed in a transient manner. In these individuals, the antigen is often below the level of The SEMA7A Gene: Genetics and Inheritance detection by standard serologic methods and they make anti- JMH. This result likely explains the serologic observation SEMA7A is located on 15q22.3-q23, the middle of the of a positive direct antiglobulin test (DAT) seen in many long arm of chromosome 15 (GenBank accession number individuals with anti-JMH. There has been no genetic change BC101647), and is organized in 14 exons. To date, no nucleotide identified in theSEMA7A gene of these individuals.14 The changes have been identified in individuals with JMH– RBCs. transient phenomenon may be triggered by an autoimmune JMH variants result from changes in nucleotides in exons 6 process like that seen in other blood group systems. and 11 (Table 1). There is only one family described with the inherited Rare individuals who lack Sema7A on their RBCs possess negative phenotype identified when randomly screening Sema7A on other cells. Given the information provided from donors. In this family, it was shown that three generations of molecular analysis performed to date, this is likely because of individuals possessed JMH– RBCs, consistent with autosomal a posttranscriptional mechanism.14 dominant inheritance.27 None had made anti-JMH, and all had a negative DAT. JMH Antibodies Rare JMH variants have been described with reduced or variable expression of JMH antigen (Table 1).14 All possess an Anti-JMH is found most often as the acquired form and antibody that is not reactive with JMH1– RBCs. However, as such can be found in individuals with no prior transfusions

20 IMMUNOHEMATOLOGY, Volume 30, Number 1, 2014 JMH blood group system review

or pregnancies. JMH antibodies are weakly reactive in a saline useful in confirming anti-JMH specificity as well as in ruling IAT. All RBCs tested will show positive reactivity. Using a out underlying alloantibodies. scale of 0 to 4+, weak macroscopic to 2+ reactivity is common. A novel approach to detecting anti-JMH is through the More consistent, stronger positive reactivity may be seen when use of particles coated with purified, recombinant Sema7A testing donor RBCs using column agglutination or solid-phase protein in a gel card format.35 If recombinant proteins could methods. All anti-JMH, whether found in JMH– or JMH- be manufactured for all common blood group antigens, they variant individuals, are negative with JMH:-1 RBCs. could replace the need for donor RBCs. This is an ongoing area Some JMH antibodies made by JMH-variant individuals of investigation. are nonreactive with other JMH variants (Table 2). These individuals will not have a positive DAT or positive autocontrol, Clinical Importance as seen in the majority of individuals with depressed JMH antigen. The acquired type JMH-negative individuals producing As mentioned previously, PNHIII cells may also be used anti-JMH have not been associated with adverse transfusion as a source of JMH– selected cells, as they lack all GPI-linked episodes.36 It is routine transfusion practice today to give proteins.29 The characteristic finding is the autocontrol being crossmatch-incompatible blood to these patients. weakly positive and weaker than the antibody reactivity with Rare, inherited-variant JMH-negative individuals who panel cells. The DAT is also weakly positive with polyspecific have made anti-JMH have been associated with decreased anti-human globulin (AHG) and anti-IgG. Interestingly, the RBC survival.37 One additional patient, later confirmed to be eluate is usually negative, but there are rare reports of anti- a JMH variant by molecular analysis, was also reported to JMH in the eluate.30 experience an acute hemolytic transfusion reaction.14,38 JMH antibodies are predominantly IgG4 subclass.31–33 A Another report of an IgG3 antibody and a positive rare example of a presumed significant IgG3 anti-JMH has monocyte monolayer assay suggested the antibody was also been described.34 The IgG subclass is important to keep in significant, but the patient did not require transfusion.34 mind when identifying anti-JMH. Some AHG reagents on the Molecular studies were not performed to determine whether market lack anti-IgG4. If using this AHG reagent, no reactivity this was a JMH-variant individual. will be observed in any IAT regardless of methodology. Very little is known about anti-JMH in pregnancy Additional characteristics of JMH antibodies are summarized because most individuals with anti-JMH are older women or in Table 4. men. In addition, JMH is very weakly expressed on cord cells. Soluble recombinant JMH proteins have been produced One example of a 32-year-old pregnant woman with a JMH- that will inhibit anti-JMH.14 These soluble proteins are not weak phenotype and anti-JMH gave birth to a baby with no available commercially at this time. However, they could be evidence of hemolytic disease of the fetus and newborn.14

Table 4. Characteristics of antibodies to John Milton Hagen Conclusions antigens Immunoglobulin G (IgG) The John Milton Hagen blood group system is fairly Usually IgG4-negative with anti-human globulin lacking IgG4 anti-IgG straightforward, but it took more than 30 years to organize Usually weakly reactive (<2+) in test tube methods serologic observations of the original group of antibody Nonreactive with papain or ficin-treated RBCs makers to determine its biochemistry and molecular genetics, Nonreactive with trypsin or alpha chymotrypsin and to it finally being named its own blood group system, 026. Reactive with chloroquine-treated RBCs Investigation of this system, led by the work of John J. Moulds, Nonreactive with 200 mmol/L/50 mmol/L dithiothreitol treatment or taught us the value of sharing rare RBCs and fluids to further 2-aminoethyl-isothiouronium bromide-treated RBCs identify unusual antibodies and to fully evaluate antibody Not inhibited with pooled normal serum identification results that do not make sense. To build on his Do not bind complement legacy, readers are encouraged to continue to question unusual Antibodies may be transient (detectable when individuals RBCs have serologic results and use the molecular tools available to find reduced expression of antigen) new, interesting, and exciting findings to add to this system Do not cause hemolytic disease of the fetus and newborn and other blood group systems, or perhaps to discover a new RBC = red blood cell. system.

IMMUNOHEMATOLOGY, Volume 30, Number 1, 2014 21 S.T. Johnson

References and blood group terminology: Berlin report. Vox Sang 2011; 101:77–82. 1. Rolih SD. High-titer, low-avidity (HTLA) antibodies and 18. Suzuki K, Okuno T, Yamamoto M, et al. Semaphorin 7A initiates antigens: a review. Transfus Med Rev 1989;3:128–39. T-cell-mediated inflammatory responses through alpha1beta1 2. Issitt PD. Applied blood group serology. 4th ed. Durham, NC: integrin. Nature 2007;446:680–4. Montgomery Scientific Publications, 1998. 19. Kikutani H, Kumanogh A. Semaphorins in interactions 3. Issitt PD. Applied blood group serology. 2nd ed. Oxnard, CA: between T cells and antigen-presenting cells. Nat Rev Immunol Spectra Biologicals, 1975. 2003;3:159–67. 4. Sabo B, Moulds JJ, McCreary J. Anti-JMH: another high 20. Yazdani U, Terman JR. The semaphorins. Genome Biol 2006; titer-low avidity antibody against a high frequency antigen 7:211. (abstract). Transfusion 1978;18:387. 21. Holmes S, Downs AM, Fosberry A, et al. Sema7A is a potent 5. Baldwin ML, Ness PM, Barrasso C, et al. In vivo studies of the monocyte stimulator. Scand J Immunol 2002;56:270–5. long-term 51Cr red cell survival of serologically incompatible 22. Gras C, Eiz-Vesper B, Seltsam A, Immenschuh S, Blasczyk red cell units. Transfusion 1985;25:34–8. R, Figueiredo. Semaphorin 7A protein variants differentially 6. Daniels GL, Green C, Lomas C, Tippet P. Monoclonal antibodies regulate T-cell activity. Transfusion 2013;53:270–83. recognizing high-frequency RBC antigens, including type 2H 23. Koh J-M, Oh B, Lee JY, et al. Association study of semaphorin and JMH (abstract). Transfusion 1981:21;612. 7a (sema7a) polymorphisms with bone mineral density and 7. Daniels GL, Knowles RW. A monoclonal antibody to the high fracture risk in postmenopausal Korean women. J Hum Genet frequency red cell antigen JMH. J Immunogenet 1982;9:57–9. 2006;51:112–17. 8. Moulds JJ, Levene C, Zimmernam S. Serological e vidence 24. Kang H-R, Lee CG, Homer RJ, Elias JA. Semaphorin 7A plays for heterogeneity among antibodies compatible with JMH- a critical role in TGF-beta1-induced pulmonary fibrosis. J Exp negative red cells (abstract). Abstracts of the Joint Meeting of Med 2007;204:1083–93. the 19th Congress of the International Society of Haematology 25. Anstee DJ. The functional importance of blood group-active and the 17th Congress of the International Society of Blood molecules in human red blood cells. Vox Sang 2011;100:140–9. Transfusion. 1982:287. 26. Bartholdson SJ, Bustamente LY, Crosnier C, et al. Semaphorin- 9. Telen MJ, Rosse WF, Parker CJ, Moulds MK, Moulds JJ. 7A is an erythrocyte receptor for P. falciparum merozoite- Evidence that several high-frequency human blood group specific TRAP homolog, MTRAP. PLoS Pathog 2012; antigens reside on phosphatidylinositol-linked erythrocyte 8:e1003031. membrane proteins. Blood 1990;75:1404–7. 27. Kollmar M, South SF, Tregellas WM. Evidence of a genetic 10. Bobolis KA, Moulds JJ, Telen MJ. Isolation of the JMH antigen mechanism for the production of the JMH negative phenotype on novel phosphatidylinositol-linked human membrane (abstract). Transfusion 1981;21:612. protein. Blood 1992;79:1574–81. 28. Daniels G. Human blood groups. 3rd ed. Wiley-Blackwell, 11. Mudud R, Rao N, Angelisova P, Horejsi V, Telen MJ. Evidence 2013:467. that CDw108 membrane protein bears the JMH blood group 29. Stroka-Lee AH, Halverson GR. Collecting GPI-negative RBCs antigen. Transfusion 1995;35:566–70. from patients with paroxysmal nocturnal hemoglobinuria 12. Yamada A, Kubo K, Takeshita T, et al. Molecular cloning of a for use in serological investigations (abstract). Transfusion glycosylphosphatidylinositol-anchored molecule CDw108. J 2010;50(Suppl):167A. Immunol 1999;162:4094–100. 30. Whitsett CF, Moulds M, Pierce JA, Hare V. Anti-JMH identified 13. Daniels GL, Anstee DJ, Cartron JP, et al. International Society in serum and eluate from red cells of a JMH-negative man. of Blood Transfusion Working Party on Terminology for Red Transfusion 1983;23:344–5. Cell Surface Antigens. Vox Sang 2001;80:193–7. 31. Tregellas WM, Pierce SR, Harding JT, Beck ML. Anti-JMH: 14. Seltsam A, Strigens S, Levene C, et al. The molecular diversity IgG subclass composition and clinical significance (abstract). of Sema7A, the semaphorin that carries the JMH blood group Transfusion 1980;20:628. antigens. Transfusion 2007;47:133–46. 32. Garratty G, Arndt P, Nance S. IgG subclass of blood 15. Richard M, St-Laurent J, Perreault J, Long A, St-Louis M. group alloantibodies to high frequency antigens (abstract). A new SEMA7A variant found in Native Americans with Transfusion 1996;36(Suppl 9S):50S. alloantibody. Vox Sang 2011;100:322–6. 33. Pope J, Lubenko A, Lai WYY. A survey of the IgG subclasses of 16. Daniels G, Flegel WA, Fletcher A, et al. International Society antibodies to high frequency red cell antigens (abstract). Transf of Blood Transfusion Committee on Terminology for Red Med 1991;1(Suppl 2):58. Cell Surface Antigens: Cape Town report. Vox Sang 2007;92: 34. Geisland J, Corgan M, Hillard B. An example of anti-JMH 250–3. with characteristics of a clinically significant antibody. 17. Storry JR, Castilho L, Daniels G, et al. International Society of Immunohematology 1990;6:9–11. Blood Transfusion Working Party on red cell immunogenetics

22 IMMUNOHEMATOLOGY, Volume 30, Number 1, 2014 JMH blood group system review

35. Seltsam A, Agaylan A, Grueger D, Meyer O, Blasczyk R, Salama 38. Hoppe B, Pastucha L, Seltsam A, Greinacher A, Salama A. A. Rapid detection of JMH antibodies with recombinant Acute haemolytic transfusion reactions due to weak antibodies Sema7A (CD108) protein and the particle gel immunoassay. that in vitro did not seem to be clinically significant. Vox Sang Transfusion 2008;48:1151–5. 2002;82:207–10. 36. Baldwin ML, Ness PM, Barrasso C, et al. In vivo studies of the long-term 51Cr red cell survival of serologically incompatible Susan T. Johnson, MSTM, MT(ASCP) SBB, Director, Transfusion red cell units. Transfusion 1985;25:34–8. Medicine Program, Marquette University, Milwaukee, WI; Director 37. Mudad R, Rao N, Issitt PD, Roy RB, Combs MR, Telen MJ. of the SBB Program, Director, Clinical Education, Blood Center of JMH variants: serologic, clinical, and biochemical analyses in two cases. Transfusion 1995;35:925–30. Wisconsin, 638 N. 18th Street, Milwaukee, WI.

IMMUNOHEMATOLOGY, Volume 30, Number 1, 2014 23 R e p o r t Demonstration of IgG subclass (IgG1 and IgG3) in patients with positive direct antiglobulin tests

A. Singh, A. Solanki, and R. Chaudhary

Serologic characterization of autoantibodies helps in the electrophoresis.5 The tube agglutination assay has been the management and monitoring of the course of autoimmune standard antibody detection method for RBC serology tests hemolytic anemia (AIHA). The purpose of this study was to for decades; however, use of the gel microcolumn antibody evaluate gel centrifugation test (GCT) cards for immunoglobulin G (IgG) titer and determination of IgG subclasses IgG1 and IgG3 detection system is growing and can be used in place of the tube and their influence on hemolysis. Eighty direct antiglobulin test agglutination assay. Gel technology, which was introduced by (DAT)-positive patients were examined with the help of GCT Lapierre et al. in 1990,6 is used to determine ABO group and D cards for IgG titer and IgG subclasses. The results were correlated 6,7 8–14 with the presence and absence of hemolysis. A statistically type, detect unexpected serum antibodies, and perform significant (p < 0.005) association of hemolysis with increasing the direct antiglobulin test (DAT).15 Compared with the tube anti-IgG titer was observed. When IgG titer was 30 or less, 28 agglutination assay, the gel microcolumn assay requires less (50.91%) patients had no hemolysis, whereas 15 (93.75%) patients test sample and reagent, is easier to read, needs no washing, had features of hemolysis when titer was at least 300. Statistically 15 significant (p < 0.005) association of subclass of IgG (IgG1, IgG3) and is more readily automated. Although FC is a very coating the red blood cells with intravascular hemolysis was also sensitive method and can detect very small amounts of Ig seen. Twenty-nine (80.56%) patients had evidence of hemolysis molecules coating the RBCs, it has the disadvantage of being when IgG1 or IgG1-IgG3 both were present. Gel technology is costly and not easily available in many of the hospital-based helpful to demonstrate red blood cell–bound autoantibodies and their characterization with regard to class, subclass, and blood transfusion services in developing countries. titer. This information is useful to identify patients with AIHA Here we discuss our experience of gel technology to who are at risk of severe hemolysis with adverse prognosis. demonstrate IgG subclasses (IgG1 and IgG3) in DAT+ cases Immunohematology 2014;30:24–27. and their correlation with hemolysis.

Key Words: autoimmune hemolytic anemia, gel centrifuga- Materials and Methods tion test, direct antiglobulin test, hemolysis The study was conducted in the immunohematology Autoimmune hemolytic anemia (AIHA) is characterized laboratory of the Department of Transfusion Medicine, by increased red blood cell (RBC) destruction or decreased Sanjay Gandhi Postgraduate Institute of Medical Sciences, RBC survival as a result of autoantibodies directed against Lucknow, India, from January 2009 to June 2012. self-antigen on RBCs.1 The degree of hemolysis depends on Ethylenediaminetetraacetic acid (EDTA) whole blood samples characteristics of the bound antibody as well as the target from 80 DAT+ patients were tested for IgG subclasses using antigen. Immunoglobulin M (IgM) antibodies readily activate gel technology (Diamed, Cressier, Switzerland). Normal blood the classical complement pathway and produce cytolysis. donors were not included in this retrospective study, which IgG antibodies are relatively poor activators of the classical was a limitation of this study. complement pathway but are easily recognized by phagocytic Hemolysis in a patient was documented when at least cells.2–4 IgG subclasses have a varying affinity for Fc receptors, three of the following laboratory parameters were abnormal: with IgG3 having a higher affinity for mononuclear phagocytes. (1) hemoglobin (Hb; <9 g/dL), (2) percentage of reticulocytes Therefore, monocyte reactions with IgG3 are much more rapid, (>2%), (3) total serum bilirubin (>2 mg/dL), and (4) lactate requiring few molecules for initiation of erythrophagocytosis dehydrogenase (LDH; >500 IU/mL).16 and thus having greater potential for RBC destruction. RBC-bound Ig can be detected using conventional tube technique, gel technique, flow cytometry (FC), or capillary

24 IMMUNOHEMATOLOGY, Volume 30, Number 1, 2014 IgG subclass in DAT positive patients

Gel Technology 28 30 27 50.91% 49.09% For quantification and IgG subclass determination, three Hemolysis types of gel centrifugation test (GCT) dilution cards were used. 25 No hemolysis The first type (IgG dilution cards) contained dilutions of rabbit 20 anti-human polyclonal anti–IgG-g-chain serum of 1 in 10, 1 15 p value <0.05 93.75% in 30, 1 in 100, 1 in 300, and 1 in 1000 and a negative diluent 15 control. The second type contained monospecific AHG (ID- Card DC Screening I, Diamed). This card consisted of five 10 7 77.78% different monospecific AHG reagents to anti-IgG, anti-IgA, 5 2 22.22% 1 anti-IgM, anti-C3c (all rabbit), and anti-C3d (monoclonal cell 6.25% line C139-9). If antibody was IgG in nature, then the subclass 0 30 = 100 30 was determined by a third type of monospecific anti-IgG1 and ≤ ≥ anti-IgG3 gel card (ID-Card DAT IgG1/IgG3, Diamed). This Fig. 1 Correlation of IgG titer with in vivo hemolysis. card consisted of monoclonal anti-IgG1 (cell line M345/795) in two different dilutions (1:1 and 1:100), anti-IgG3 (cell line had evidence of hemolysis. Similarly, 77.78 percent and 93.75 M346/805) in two different dilutions (1:1 and 1:100), anti-IgG percent of the patients experienced hemolysis when anti-IgG 1:10 (rabbit), and a negative control. The tests were performed titer was 100 and at least 300, respectively. following the department’s standard operating procedure There was a statistically significant (p < 0.005) manual as described previously.17 association of subclass of IgG (IgG1, IgG3) coating the RBCs with intravascular hemolysis. Of 80 DAT+ samples, 25 had Elution IgG1 and 11 had both IgG1 and IgG3, of which 20 (80.0%) To confirm that the positive DAT result is caused by an and 9 (81.82%) had evidence of hemolysis, respectively. In 44 autoantibody, cold acid elution was performed.18 The eluate patients, IgG1 or both IgG1 and IgG3 were not detected; of was tested with a three-cell screening panel, ID DiaCell I-II- these, 24 (54.55%) had no hemolysis (Table 1). III (Diamed), by indirect antiglobulin test using gel cards. Table 1. Correlation of IgG subclass with hemolysis

Statistical Analysis Number Data were entered in Microsoft Office Excel (Microsoft IgG subclass of patients Hemolysis No hemolysis Corporation, Redmond, WA) and analyzed with Statistical IgG1 25 20 (80.00%) 5 (20.00%) Package for the Social Sciences (SPSS) version 16.0 (SPSS, Inc., IgG1 and IgG3 11 9 (81.82%) 2 (18.18%) Chicago, IL). Pearson’s χ2 test was used to analyze the relations IgG3 only or neither IgG1 44 20 (45.45%) 24 (54.55%) between IgG titer with hemolysis and IgG subclass with nor IgG3 detected hemolysis. Probability values less than 0.05 were considered Total 80 49 (61.25%) 31 (38.75%) significant. p value <0.005.

Results Whenever DAT was more than 2+ in strength, cold acid elution was performed. Eluate was tested with reagent RBC A total of 80 DAT+ samples were evaluated. The median panels (Diamed). It was found to be pan-reactive in all the age of patients was 32.5 years, and the female-to-male ratio samples, confirming the presence of autoantibodies. was 2.33. Of the 80 patients, 49 had features of hemolysis as shown in Fig. 1. The median levels of Hb, reticulocyte count, Discussion total serum bilirubin, and LDH were 6.9 g/dL (range, 3–14.3 g/dL), 4.7 percent (range, 0.4–41%), 1.35 mg/dL (range, 0.3– Introduction of specialized gel cards, such as monospecific 22.7 mg/dL), and 847.5 IU/mL (range, 125–9620 IU/mL), Ig cards, IgG subclass cards, and IgG dilution cards, have respectively. made it possible to characterize RBC-bound antibodies in a A statistically significant (p < 0.005) association of simplified and rapid way. These detailed investigations help hemolysis with increasing anti-IgG titer was observed (Fig. predict the severity of hemolysis and disease outcome and 1). When anti-IgG titer was 30 or less, 27 (49.09%) patients permit the planning of therapy in autoimmune disorders.17

IMMUNOHEMATOLOGY, Volume 30, Number 1, 2014 25 A. Singh et al.

In this study, we demonstrated IgG subclasses (IgG1 and A 14 IgG3) in DAT+ patients using specialized gel cards (Diamed). IgG2 and IgG4 were not studied, as these are rare antibodies 12 involved in AIHA; moreover, Diamed does not provide gel 10 cards for detection of these subclasses. IgG1 or both IgG1 and

IgG3 were observed with hemolysis in 29 (59.18%) patients. 8 Hb This finding was almost the same as that of Das et al.16 They reported IgG subclasses, IgG1 or IgG3 or both, coating the 6 RBC membrane in 46.5 percent of their DAT+ patients with AIHA. We have observed a statistically significant association 4 of IgG1 and IgG3 with intravascular hemolysis (Table 1, 2 19,20 Fig. 2). This result is in agreement with previous studies. G1 G1, G3, Both No G1/G3 Garratty21 found that most of the RBC-bound IgG was of G1 or G3 the IgG1 subclass similar to our study, but did not find any B 50 correlation with the quantity of RBC-bound IgG and the rate of in vivo RBC destruction. 40 One factor that possibly contributes to the degree of hemolysis is the IgG subclass. Four human subclasses of 30 the IgG molecule can be differentiated (IgG1, IgG2, IgG3, Retic and IgG4) of which IgG1 is predominant. Although all four 20 subclasses are able to induce hemolysis, types 1 and 3 bind to the Fc receptor of phagocytic cells with greater affinity than 10 do types 2 and 4. Hemolysis is therefore expected to occur to a greater extent in association with the former.22 0 The extent of RBC destruction depends not only on the G1 G1, G3, Both No G1/G3 density of the cell-bound antibodies and the IgG subclass, but G1 or G3 also on the type of effector cells, the Fc receptors of the latter, Fig. 2 Correlation of IgG subclass (G1 or G3) with various hemolytic and complement activation. Immune hemolysis also depends parameters: (A) hemoglobin (Hb), (B) reticulocyte count (Retic), (C) total bilirubin (TB), and (D) lactate dehydrogenase (LDH). on other factors influencing the activity of the monocyte/ macrophage system, on the kind and stage of underlying disease, or on therapeutic actions.23 reagent, is easier to read, needs no washing, and achieves a We found that increasing the titer of IgG was significantly stable agglutination that allows reading the result over a long associated with hemolysis. When titer was at least 300, 15 of 16 period, makes it very useful in practice. (93.75%) patients had features of hemolysis. Lai et al.24 found We have used gel technology for demonstration of IgG in their study that in 81 percent of AIHA patients, the IgG titer subclasses on DAT+ RBCs. Flow cytometry has been used was at least 300; however, they reported a strong association by some workers20 to demonstrate IgG subclasses on RBC of DAT strength with hemolysis compared with anti-IgG titer. membranes, as it is more precise and reproducible than Lynen et al.20 reported that immune hemolysis is unlikely if the standard techniques. In addition, FC can actually quantitate titer is 30 or less, whereas immune mechanisms are probably the number of IgG subclass molecules on RBC membranes.20 the cause of hemolysis if the titer is at least 300. Although However, FC may not actually be useful in predicting there is a correlation between the amount of immunoglobulin hemolysis. molecules coating the RBCs and in vivo hemolysis, the actual Although blood samples from donors were not included in threshold required to induce hemolysis is not exactly known. this study, the testing performed on referred patient samples There are several methods of quantitative determination illustrated that gel technology is helpful to demonstrate RBC- of IgG subclasses of antibodies.25 In general, they are very bound autoantibodies and their characterization with regard useful, especially for a better understanding of immune RBC to class, subclass, and titer. This information is useful to destruction, but for routine use, they are not very practical. identify patients of AIHA who are at risk of severe hemolysis The simplicity of GCT, as it requires less test sample and with adverse prognosis.

26 IMMUNOHEMATOLOGY, Volume 30, Number 1, 2014 IgG subclass in DAT positive patients

C 25.0 laboratory. Arch Pathol Lab Med 1999;123:693–7. 10. Titlestad K, Georgsen J, Andersen H, Kristensen T. Detection of irregular red cell antibodies: more than 3 years of experience 20.0 with a gel technique and pooled screening cells. Vox Sang 1997;73:246–51.

15.0 11. Rumsey DH, Ciesielski DJ. New protocols in serologic testing: a review of techniques to meet today’s challenges.

TB Immunohematology 2000;16:131–7. 10.0 12. Eichler H, Kretschmer V. The gel test: investigation into the aetiology and the significance of a positive auto-control. Infusionsther Transfusionsmed 1995;22:232–6. 5.0 13. Novaretti MCZ, Jens Silveira E, Filho EC, Dorlhiac-Llacer PE, Chamone DA. Comparison of tube and gel techniques for antibody identification. Immunohematology 2000;16:138–41. 0 G1 G1, G3, Both No G1/G3 14. Weisbach V, Ziener A, Zimmerman R, Glaser A, Zingsen J, G1 or G3 Eckstein R. Comparison of the performance of four microtube 10000 column agglutination systems in the detection of red cell D alloantibodies. Transfusion 1999;39:1045–50. 15. Greco VA, Byrne KM, Procter JL, Stroncek DF. Detection of 8000 antibodies in acid eluates with the gel microcolumn assay. Transfusion 2002;42:698–701. 16. Das SS, Nityanand S, Chaudhary R. Clinical and serological 6000 characterization of autoimmune hemolytic anemia in a tertiary care hospital in north India. Ann Hematol 2009;88:727–32. LDH 4000 17. Das SS, Chaudhary RK. Application of gel technology in the serologic characterization of autoantibody in DAT-positive autoimmune disease. Immunohematology 2007;23:59–62. 2000 18. Rekvig OP, Hannestad K. Acid elution of blood group antibodies from intact erythrocytes. Vox Sang 1977;33:280–5. 19. Li Z, Shao Z, Xu Y, et al. Subclasses of warm autoantibody 0 G1 G1, G3, Both No G1/G3 IgG in patients with autoimmune hemolytic anemia and their clinical implications. Chin Med J (Engl) 1999;112:805–8. G1 or G3 20. Lynen R, Krone O, Legelar TJ, Köhler M, Mayer WR. A newly developed gel centrifugation test for quantification of RBC- References bound IgG antibodies and their subclasses IgG1 and IgG3: comparison with flow cytometry. Transfusion 2002;42: 1. Gehrs BC, Friedberg RC. Autoimmune hemolytic anemia. Am 612–18. J Hematol 2002;69:258–71. 21. Garratty G. Effect of cell-bound proteins on the in vivo survival 2. Abramson N, Gelfand EW, Jandl JH, Rosen FS. The interaction circulating blood cells. Gerontology 1991;37:68–94. between human monocytes and red cells: specificity for IgG subclasses and IgG fragments. J Exp Med 1970;132:1207–15. 22. Mackenzie SB. Hemolytic anemia due to extrinsic factors. In: Textbook of hematology. Baltimore, MD: Williams & Wilkins, 3. Anderson CL, Looney RJ. Human leukocyte IgG Fc receptors. 1996:245–74. Immunol Today 1986;7:264–6. 23. Engelfriet C, Overbeeke M, Dooren M, Ouwehand WH, 4. Ravetch J, Kinet JP. Fc receptors. Annu Rev Immunol 1991; von dem Borne AE. Bioassays to determine the clinical 9:457–92. significance of red cell alloantibodies based on Fc receptor- 5. Katsougraki P, Heliopoulos J, Lamari F, et al. Identification induced destruction of red cells sensitized by IgG. Transfusion of kappa and lambda chains of the major immunoglobulin 1994;34:617–26. G subclasses by capillary zone electrophoresis. Biomed 24. Lai M, Leone G, Landolf R. Autoimmune hemolytic anemia Chromatogr 2002;16:234–6. with gel-based immunohematology tests. Am J Clin Pathol 6. Lapierre Y, Rigal D, Adam J, et al. The gel test: a new way to 2013;139:457–63. detect red cell antigen-antibody reactions. Transfusion 1990; 25. Fabijańska-Mitek J, Lopieńska H, Zupańska B. Gel test 30:109–13. application for IgG subclass detection in auto-immune 7. Langston MM, Procter JL, Cipolone KM, Stroncek DF. haemolytic anaemia. Vox Sang 1997;72:233–7. Evaluation of the gel system for ABO grouping and D antigen typing. Transfusion 1999;39:300–5. 8. Reis KJ, Chachowski R, Cupido A, Davies D, Jakway J, Ashutosh Singh, MD, Senior Resident, Archana Solanki, MD, Senior Setcavage TM. Column agglutination technology: the Resident, and Rajendra Chaudhary, MD (corresponding author), antiglobulin test. Transfusion 1993;33:639–43. Professor and Head, Department of Transfusion Medicine, Sanjay 9. Cate JC 4th, Reilly N. Evaluation and implementation of the gel Gandhi Postgraduate Institute of Medical Sciences (SGPGIMS), test for indirect antiglobulin testing in a community hospital Lucknow 226014, India.

IMMUNOHEMATOLOGY, Volume 30, Number 1, 2014 27 In Memoriam

George Garratty 1935–2014

Dr. George Garratty was born in 1935 in England. His original career plan was to attend the Royal Veterinary College in London and become a veterinarian. While waiting to go into the Army Veterinary Corps, he applied for a summer job at the local hospital (knowing that hospitals kept animal houses). The hospital was Hammersmith Hospital in London, and Dr. Dacie (later Professor Sir John Dacie) had some openings in his laboratory in the Hematology Department. Professor Patrick L. Mollison, the Director of Blood Transfusion, worked just down the hall. George became excited about the fields of hematology and transfusion medicine, and his career plans changed. At the time, he did not realize exactly how famous Dr. Dacie and Prof. Mollison were and imposed on them, asking many questions and discussing interesting cases. Hematologists from all over the world came to study with Dr. Dacie at the Royal Postgraduate Medical School of London; one of them was Dr. Lawrence Petz from the United States. George’s friend Peter Issitt also worked at Hammersmith in Dr. Dacie’s department and with Prof. Mollison before moving to the New York Blood Center in 1964. Peter wrote letters to George, persuading him to come to the United States. In 1968, George joined Dr. Petz in San Francisco, California, to carry out research on complement for 2 years. He and Dr. Petz ended up working together for 10 years, studying autoimmune hemolytic anemia (AIHA) and drug-induced immune hemolytic anemia (DIIHA). The culmination of their work was the first edition ofAcquired Immune Hemolytic Anemias published in 1980. In 1978, the American Red Cross in Los Angeles, California, offered George a job as Scientific Director to start a research program. The Research Department began with two technologists and expanded over the years to eventually include both Cellular Immunology and Transfusion Transmitted Diseases in addition to the Immunohematology Research Laboratory. Much of the latter group’s work was applied research on immune cell destruction. Some major projects included development of assays to help diagnose AIHA, functional assays to predict the clinical significance of antibodies (e.g., monocyte monolayer assay), applications of flow cytometry to the study of blood group antigens and antibodies, research on “stealth” cells (e.g., red blood cells treated with polyethylene glycol), and continuing studies on DIIHA. George received his PhD in immunology in 1985 and his Fellow of Royal College of Pathologists

28 IMMUNOHEMATOLOGY, Volume 30, Number 1, 2014 (FRCPath) in 1990. He published more than 300 Dr. Garratty was very involved over the years scientific papers and was coauthor of three and editor with committees at the state level (e.g., CBBS of six textbooks. The second edition of Petz and President 1985–86), national level (e.g., the AABB’s Garratty’s Immune Hemolytic Anemias was published Standards, Annual Meeting, and Nominating and in 2004. Awards committees), and the international level In addition to his continuing research at the (e.g., the International Society of Blood Transfusion’s American Red Cross Blood Services, Southern Working Party on Red Cell Immunogenetics and California Region, Dr. Garratty became responsible Blood Group Terminology). He was an Associate for the Immunohematology Reference Laboratory (red Editor of Transfusion for 31 years (1982–2013), cell and HLA/platelet immunology) and Community and on the boards of other journals, including Education department (including a Specialist in Immunohematology. Blood Banking program). He was also Clinical Dr. Garratty will be remembered by many for his Professor of Pathology and Laboratory Medicine at great sense of humor and ability to connect to anyone the University of California, Los Angeles. Education he met. He and his wife Eileen shared their home was important to Dr. Garratty. He was always eager with two to three Great Danes (12 over the years). His to share information via face-to-face discussions presence among us will be missed, and probably most at meetings, phone and e-mail consultations, and significantly, his passing marks a defining moment at lectures, publications, and textbooks. Dr. Garratty the end of an era. He lived the history that we must was very supportive of new people in the field and teach to those who follow. was a personal inspiration to many. He was a highly sought lecturer and traveled the world giving invited Patricia A. (Pat) Arndt lectures. In addition, visitors from around the world Regina M. (Gina) Leger came to Southern California to learn from him. Immunohematology Research Laboratory Dr. Garratty was honored during his 50+ years American Red Cross Blood Services, Southern in the field with numerous awards at the state, California Region national, and international levels as well as through 100 Red Cross Circle lectureships. Two honors most special to him were Pomona, CA 91768 the James Blundell Award from the British Blood (909) 859-7407 phone Transfusion Society in 2007 and the AABB’s Bernard (909) 859-7718 fax Fantus Lifetime Achievement Award with Dr. Petz in [email protected] 2010. The latter award is given only every 5 or more [email protected] years. In 2014, he posthumously received the Herb Perkins Lectureship Award at the California Blood Bank Society (CBBS) meeting; this award will now be known as the Perkins-Garratty Memorial Award to honor Dr. Garratty and his achievements.

IMMUNOHEMATOLOGY, Volume 30, Number 1, 2014 29 A n n o u n c e m e n t s

Annual Symposium Announcements

September 17, 2014 September 18, 2014 Red Cell Genotyping 2014: Clinical Benefits. The department National Institutes of Health, Clinical Center, Department of Transfusion Medicine, Clinical Center, National Institutes of Transfusion Medicine, 33rd Annual Symposium: of Health and the BloodCenter of Wisconsin are co-hosting Immunohematology and Blood Transfusion. The symposium the 4th Annual Symposium on Red Cell Genotyping. For is co-hosted by the American Red Cross and is free of charge, information and registration fee, contact Phyllis Kirchner, but advance registration is encouraged. Contact Karen Byrne, BloodCenter of Wisconsin, P.O. Box 2178, Milwaukee, WI NIH/CC/DTM, Bldg. 10/Rm. 1C711, 10 Center Drive MSC 53021-2178, e-mail: [email protected] or visit our 1184, Bethesda, MD 20892-1184, e-mail: [email protected] Web site: www.bcw.edu/rcg2014 or visit our Web site: www.cc.nih.gov/dtm/education.html.

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

Free Classified Ads and Announcements Immunohematology will publish classified ads and announcements (SBB schools, meetings, symposia, etc.)without charge.

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

30 IMMUNOHEMATOLOGY, Volume 30, Number 1, 2014 Announcements, cont.

The Johns Hopkins Hospital Specialist in Blood Bank Technology Program

The Johns Hopkins Hospital was founded in 1889. It is located in Baltimore, Maryland, 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 two 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. The program is a challenging one 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, Maryland 21287

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

IMMUNOHEMATOLOGY, Volume 30, Number 1, 2014 31 Announcements, cont.

32 IMMUNOHEMATOLOGY, Volume 30, Number 1, 2014 Announcements, cont.

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

Applications are invited from medical or science graduates for the Master of Science (MSc) degree in Transfusion and Transplantation Sciences at the University of Bristol. The course starts in October 2014 and will last for 1 year. A part-time option lasting 2 or 3 years is also available. There may also be opportunities to continue studies for PhD or MD following the MSc. The syllabus is organized jointly by The Bristol Institute for Transfusion Sciences and the University of Bristol, Department of Pathology and Microbiology. It includes: • Scientific principles of transfusion and transplantation • Clinical applications of these principles • Practical techniques in transfusion and transplantation • Principles of study design and biostatistics • An original research project

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

The course is accredited by the Institute of Biomedical Sciences.

Further information can be obtained from the Web site: http://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 30, Number 1, 2014 33 A dv e r t i s e m e n t s

Reference and Consultation Services IgA/Anti-IgA Testing

Antibody identification and problem resolution IgA and anti-IgA testing are available to do the

HLA-A, B, C, and DR typing following:

HLA-disease association typing • Identify IgA-deficient patients • Investigate anaphylactic reactions Paternity testing/DNA • Confirm IgA-deficient donors Our ELISA for IgA detects protein to 0.05 mg/dL. For information, contact:

Mehdizadeh Kashi For additional information contact:

at (503) 280-0210 Cynthia Flickinger at (215) 451-4909

or e-mail: or write to: [email protected]

Tissue Typing Laboratory or write to: American Red Cross Biomedical Services American Red Cross Biomedical Services

Pacific Northwest Region Musser Blood Center 700 Spring Garden Street 3131 North Vancouver Philadelphia, PA 19123-3594 Portland, OR 97227 ATTN: Cynthia Flickinger CLIA licensed, ASHI accredited CLIA licensed

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

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

CLIA licensed

34 IMMUNOHEMATOLOGY, Volume 30, Number 1, 2014 Advertisements, cont.

IMMUNOHEMATOLOGY, Volume 30, Number 1, 2014 35 Advertisements, cont.

National Platelet Serology Reference Laboratory National Neutrophil Serology Reference Laboratory

Diagnostic testing for: Our laboratory specializes in granulocyte antibody detection • Neonatal alloimmune thrombocytopenia (NAIT) and granulocyte antigen typing. • Posttransfusion purpura (PTP) • Refractoriness to platelet transfusion Indications for granulocyte serology testing • Heparin-induced thrombocytopenia (HIT) include: • Alloimmune idiopathic thrombocytopenia purpura (AITP) • Alloimmune neonatal neutropenia (ANN) Medical consultation available • Autoimmune neutropenia (AIN) • Transfusion-related acute lung injury (TRALI) Test methods: • GTI systems tests Methodologies employed: — detection of glycoprotein-specific platelet antibodies • Granulocyte agglutination (GA) — detection of heparin-induced antibodies (PF4 ELISA) • Platelet suspension immunofluorescence test (PSIFT) • Granulocyte immunofluorescence by flow cytometry (GIF) • Solid phase red cell adherence (SPRCA) assay • Monoclonal antibody immobilization of neutrophil antigens • Monoclonal immobilization of platelet antigens (MAIPA) (MAINA) • Molecular analysis for HPA-1a/1b TRALI investigations also include: For further information, contact: • HLA (PRA) Class I and Class II antibody detection Platelet Serology Laboratory (215) 451-4205 For further information, contact: Cynthia Flickinger (215) 451-4909 Neutrophil Serology Laboratory (651) 291-6797 [email protected] Sandra Nance (215) 451-4362 Randy Schuller (651) 291-6758 [email protected] [email protected]

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

36 IMMUNOHEMATOLOGY, Volume 30, Number 1, 2014 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 Certification (BOC) 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 nonconformances • Design and present educational programs • Provide technical and scientific training in transfusion medicine • Conduct research in transfusion medicine Who are SBBs? Supervisors of Transfusion Services Executives and Managers of Blood Centers LIS Coordinators 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? CAAHEP-accredited SBB Technology program or grandfather the exam based on ASCP education and experience criteria. Fact: In recent years, a greater percentage of individuals who graduate from CAAHEP-accredited programs pass the SBB exam compared to individuals who grandfather the exam. The BEST route for obtaining an SBB certification is to attend a CAAHEP-accredited Specialist in Blood Bank Technology Program. Which approach are you more compatible with? Contact the following programs for more information: Additional information can be found by visiting the following Web sites: www.ascp.org, www.caahep.org, and www.aabb.org

 Onsite or : Online Program Contact Name Phone Contact E-mail Contact Web Site Program Blood Systems Laboratories Marie P. Holub 602-996-2396 [email protected] www.bloodsystemslaboratories.org : Walter Reed Army Medical Center William Turcan 301-295-8605 [email protected] www.militaryblood.dod.mil/Fellow/default.aspx  [email protected] American Red Cross, Southern California Region Catherine Hernandez 909-859-7496 [email protected] www.redcrossblood.org/socal/communityeducation  ARC-Central OH Region Joanne Kosanke 614-253-2740 ext. 2270 [email protected] none  Blood Center of Wisconsin Phyllis Kirchner 414-937-6271 [email protected] www.bcw.edu  Community Blood Center/CTS Dayton, Ohio Nancy Lang 937-461-3293 [email protected] www.cbccts.org/education/sbb.htm : Gulf Coast Regional Blood Center Clare Wong 713-791-6201 [email protected] www.giveblood.org/services/education/sbb-distance-program : Hoxworth Blood Center, University of Cincinnati Pamela Inglish 513-558-1275 [email protected] www.grad.uc.edu  Medical Center Indiana Blood Center Jayanna Slayten 317-916-5186 [email protected] www.indianablood.org : Johns Hopkins Hospital Lorraine N. Blagg 410-502-9584 [email protected] http://pathology.jhu.edu/department/divisions/transfusion/sbb.cfm  Medical Center of Louisiana Karen Kirkley 504-903-3954 [email protected] www.mclno.org/webresources/index.html  NIH Clinical Center Blood Bank Karen Byrne 301-496-8335 [email protected] www.cc.nih.gov/dtm  Rush University Yolanda Sanchez 312-942-2402 [email protected] www.rushu.rush.edu/cls : Transfusion Medicine Center at Florida Blood Services Marjorie Doty 727-568-5433 ext. 1514 [email protected] www.fbsblood.org : Univ. of Texas Health Science Center at San Antonio Linda Myers 210-731-5526 [email protected] www.sbbofsa.org  University of Texas Medical Branch at Galveston Janet Vincent 409-772-3055 [email protected] www.utmb.edu/sbb : University of Texas SW Medical Center Lesley Lee 214-648-1785 [email protected] www.utsouthwestern.edu/education/school-of-health-professions/ : programs/certificate-programs/medical-laboratory-sciences/index.html Revised May 2012

IMMUNOHEMATOLOGY, Volume 30, Number 1, 2014 37 38 IMMUNOHEMATOLOGY, Volume 30, Number 1, 2014 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…), ending A. Each component of the manuscript must start on a new page in the following with a period. If figure is submitted as a glossy, place first author’s name and order: 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. JONES, skills 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 patient’s 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…) use no punctuation at the end of the title. Send all manuscripts by e-mail to [email protected]

IMMUNOHEMATOLOGY, Volume 30, Number 1, 2014 39 40 IMMUNOHEMATOLOGY, Volume 30, Number 1, 2014 Immunohematology Instructions for Authors | New Blood Group Allele Reports

A. For describing an allele which 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 Reference 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 30, Number 1, 2014 41

NON PROFIT U.S. POSTAGE PAID Musser Blood Center AMERICAN 700 Spring Garden Street RED CROSS Philadelphia, PA 19123-3594

(Place Label Here)