Vo l u m e 28, N u m b e r 3, 2012

Immunohematology Volume 28, Number 3, 2012

CONTENTS

C a s e R e p o r t 85 Blood group genotyping in a multitrauma patient: a case report J. Curvers, V. Scharnhorst, M. de Haas, L. Warnier-Wandel, and D. van de Kerkhof

R e v i e w 88 Warm autoadsorption with enzyme-treated red blood cells F. Tsimba-Chitsva, S. Bishop, and K. Kezeor

R e p o r t 91 Implications of the Kidd blood group system in renal transplantation A. Rourk and J.E. Squires

R e v i e w 95 EDTA glycine acid treatment of red blood cells J. Kosanke

R e p o r t 97 Molecular background of RH in Bastiaan, the RH:–31,–34 index case, and two novel RHD alleles M.E. Reid, C.H. Hipsky, R.W. Velliquette, C. Lomas-Francis, K. Larimore, and C. Olsen

C o m m u n i c at i o n 104 Letter from the Editor Serologic method reviews—a new feature

105 A nnouncements

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

112 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 Cynthia Flickinger, MT(ASCP)SBB James P. AuBuchon, MD Thierry Peyrard, PharmD, PhD Philadelphia, Pennsylvania Seattle, Washington Paris, France Lilian Castilho, PhD Joyce Poole, FIBMS S e n i o r M e d i c a l E d i to r Campinas, Brazil Bristol, United Kingdom Ralph R. Vassallo, MD Martha R. Combs, MT(ASCP)SBB Mark Popovsky, MD Philadelphia, Pennsylvania Durham, North Carolina Braintree, Massachusetts

Tec h n i c a l E d i to r s Geoffrey Daniels, PhD Marion E. Reid, PhD, FIBMS Christine Lomas-Francis, MSc Bristol, United Kingdom New York City, New York New York City, New York Anne F. Eder, MD S. Gerald Sandler, MD Dawn M. Rumsey, ART(CSMLT) Washington, District of Columbia Washington, District of Columbia Norcross, Georgia George Garratty, PhD, FRCPath Jill R. Storry, PhD Pomona, California Lund, Sweden A s s o c iat e M e d i c a l E d i to r s Brenda J. Grossman, MD David F. Stroncek, MD David Moolten, MD St. Louis, Missouri Bethesda, Maryland Philadelphia, Pennsylvania Christine Lomas-Francis, MSc Joy Fridey, MD New York City, New York E m e r i t u s E d i to r Pomona, California Geralyn M. Meny, MD Delores Mallory, MT(ASCP) SBB Supply, North Carolina M o l ec u l a r E d i to r San Antonio, Texas 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 $40.00 (U.S.) and $50.00 (foreign) per year. C o p y E d i to r 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 Or call (215) 451-4902

E l ec t r o n i c P u b l i s h e r Web site: www.redcross.org/about-us/publications/immunohematology Paul Duquette Copyright 2012 by The American National Red Cross ISSN 0894-203X

O n O u r C o v e r

Subjects of the San rock paintings in the Drakensberg mountains of South Africa range from depictions of daily life to those of sacred events and of animals native to the region, such as the eland and reedbuck. Caves and overhangs are numerous in the mountains due to the ease with which the terrain’s abundant sandstone erodes. These caves shelter the paintings, some of which date back millennia. Tens of thousands of rock paintings have been documented in hundreds of locations. The artists made use of natural pigments such as red and yellow ocher, hematite, manganese oxide, and charcoal. Anthropological evidence suggests the San people may have been present in the region as far back as 100,000 years ago. As Reid et al. remind us in this issue of Immunohematology, anti-hrB (-RH31) and anti-HrB (-RH34) were discovered 40 years ago in the serum of a South African woman. Their report characterizes Rh in Bastiaan using molecular techniques. David Moolten, MD Ca s e R e p o r t Blood group genotyping in a multitrauma patient: a case report

J. Curvers, V. Scharnhorst, M. de Haas, L. Warnier-Wandel, and D. van de Kerkhof

Currently DNA-based analysis of blood groups is mainly used to at that time the transfusion laboratory received the first sample improve transfusion safety by reducing alloantibody formation for crossmatching. Serologically it was no longer possible to in multiply transfused patients and by monitoring pregnancies at determine a blood group owing to the number of transfused risk for hemolytic disease of the fetus and newborn. We present a case in which genotyping was performed after massive transfusion RBC units. Even a mixed-field reaction was hardly visible with unmatched group O, D– blood in a trauma setting. Our (Fig. 1). Also, a reverse blood group typing was not possible as patient was genotyped as O1A1 and predicted to be D–, and we a result of the number of FFP units she had received. After the therefore transfused group A, D– red blood cell concentrates. This case demonstrates how the use of blood group genotyping surgical procedure, the patient was stable and was transferred in an acute setting can lead to a decrease in the unnecessary to the intensive care unit with a hemoglobin concentration of use of group O, D– blood products. Immunohematology 8.8 g/dL (5.5 mmol/L). Three additional FFP units and two 2012;28:85–7. five-donor platelet concentrates were transfused because of low levels of fibrinogen (0.87 g/L) and platelets (76/nL), Key Words: genotyping, serology, massive transfusion, respectively. Altogether, our patient received 18 units of group unmatched transfusion O, D– leukocyte-reduced packed cells, 12 units of FFP (blood

Case Report

Our transfusion laboratory received an emergency request for six unmatched group O, D– red blood cell (RBC) concentrates for a patient. These units were to be transfused not inside the hospital but at the scene of a car accident (at a peripheral highway), where the patient was bleeding heavily while trapped in a car wreck. Again, another four unmatched RBC units were ordered together with five group AB, D– fresh-frozen plasma (FFP) units and one randomly selected unit of platelets obtained from five donors. All products were leukocyte reduced, with less than 106/L white blood cells per unit. We had not received blood for crossmatching. When released from the car wreck, the 62-year-old white female patient arrived at the hospital emergency department and was admitted to the operating room immediately. The patient suffered from complex high-impact trauma. Her car had slid underneath a truck, and the patient had been trapped for at least 1.5 hours. She had multiple fractures, in the nose, Case XXXXXXX left wrist, left humerus, bilateral femur, and ramus superior and inferior, and she had a pneumothorax. Also, the patient exhibited various superficial wounds and a head injury that Fig. 1. Blood group typing cards (Ortho Clinical Diagnostics, Inc., was closed using staples. Besides damage-control surgery, Raritan, NJ) from our patient’s blood upon arrival at the hospital, showing a mixed-field reaction with anti-A (left lane). The mixed- additional acute surgery was necessary to implant a stent field reaction was only visible after addition of 10x the erythrocyte because of dissection of the descending aorta. During this volume used in the usual procedure. No reaction with the A1 cells procedure the patient was in need of more blood products, and and B cells (Ortho Clinical Diagnostics, Inc.) is visible.

IMMUNOHEMATOLOGY, Volume 28, Number 3, 2012 85 J. Curvers et al.

group AB), and 6 units of leukocyte-reduced five-donor platelet We were not able to confirm the blood group by reverse concentrates (group O, D–) during her intensive care stay. blood typing because of the amount of FFP received. However, Unfortunately, we were unable to obtain information after several days, serum anti-B was found but no anti-A; this regarding the blood group of our patient (even after contacting is consistent with the ABO genotype we found. the patient’s general practitioner and the local blood bank). The multiplex PCR for genotyping the Rh blood group Therefore, we chose to determine her blood group by means system is based on single-nucleotide polymorphisms (SNPs) of DNA analysis to avoid further (unnecessary) use of group using six different primer sets that cover exons 3, 4, 5, 6, 7, and O, D– RBC products. We collected venous blood samples 9 of RHD and is able to identify most RHD variants.2 Based on

(anticoagulated in K3-EDTA) and sent them to the laboratories the genotyping results, our patient was predicted to be D– , of the Sanquin Blood Supply Foundation. ABO genotyping and for our white patient, the frequency of RHDψ or a variant was performed using a sequence-specific primer polymerase D is very low. We did not confirm genotyping with buccal cells chain reaction (SSP-PCR).1 RHD genotyping was performed because the clinical consequences of a false-negative result are using a multiplex reverse transcriptase (RT) PCR method.2 far less severe than those of a false-positive result. Serologically, Our patient’s genotype was determined to be O1A1, and she on increasing volumes of RBCs to observe mixed-field was predicted to be D–. reaction in our column technique, the D phenotype remained Fortunately, the patient appeared to no longer need blood negative (Fig. 1). When blood group serology was repeated products. She stayed in the intensive care unit for another 20 before transfusion 2 weeks after the accident, no mixed-field days, during which time only one unit of RBCs was transfused agglutination was observed, although transfused RBCs were (blood group A, D–). At that time the patient’s blood sample likely to have been present. This was consistent with a D– was serologically typed as group A, D– (without mixed-field phenotype and showed concordance between genotype and reactions), and the screening for irregular antibodies was phenotype results. Again, using a three-cell screening panel, negative (using a three-cell screening panel, Ortho Clinical we found no irregular erythrocyte antibodies. Diagnostics, Inc.). She was then moved to the surgery Of course, interpretation of genotyping results should be department for necrotomy of wounds and was transferred to a performed carefully because ethnic differences (e.g., presence nursing home 2 months after the car accident. of RHDψ, or the DAR variant) and silencing alleles (e.g., GATA box mutations) exist that could lead to discrepancies between Results genotype and phenotype.3,4 When the patient is of a certain ethnicity with a higher risk of ABO variants, sequencing may The chosen SSP-PCR is based on the presence of O1, be an option, although costly and time-consuming. O2, B, or A2 genes (the presence of glucosyltransferases on chromosome 9q34-2) in DNA isolated from peripheral Discussion blood samples.1 A PCR product is produced only when the specific chosen primer binds to and amplifies the sequence Recently, excellent reviews5,6 have been published of interest. Eight mixtures of four allele-specific primer sets regarding genotyping of the blood donor population and are used. The PCR products are transferred to an ethidium- its possible drawbacks. However, in transfusion practice, bromide gel, and patterns are read and interpreted manually. genotyping is not commonly performed on patient samples. Extrapolating results of eight different incubations (O1, O2, B, The Dutch national guideline currently states that a blood A2, non-O1, non-O2, non-B, and non-A2) led to the genotype. group can only be confirmed when it has been established Therefore, this assay cannot be used for ABO variant analysis. by testing two different samples, irrespective of the method In our case, blood group A1 was predicted as determined by used.7 One method may be genotyping. exclusion of the other genotypes present. This phenotype has a From 2001, leukocytes are depleted from all blood products frequency of 33 percent in the white population. The presence within the Netherlands by means of filtration, resulting in the 6 of an A2 blood group was excluded, as was the presence of a presence of less than 1 × 10 white blood cells per unit. In case

B or Bombay (Oh) phenotype. The frequency of other type A of massive transfusion of RBCs and platelet or plasma units, (or B) blood groups is very rare in the white population, and the load of donor white blood cells in the patient remains low would not have led to a mixed-field reaction of the patient’s (undetectable when compared with the patient’s white blood RBCs with the anti-A serum (Fig. 1). cell count, on average 5 × 109/L), hence genotyping on DNA from the patient’s white blood cells is achievable. Our patient

86 IMMUNOHEMATOLOGY, Volume 28, Number 3, 2012 Genotyping in massive transfusion

had a white blood cell count of 4.5 leukocytes/nL at the time of References DNA analysis. Of these, a maximum of 4 percent (0.16/nL: 16 . 1 Gassner C, Schmarda A, Nussbaumer W, Schönitzer D. ABO 6 blood products times 10 white blood cells) could potentially glycosyltransferase genotyping by polymerase chain reaction originate from the received RBC units. To circumvent these using sequence-specific primers. Blood 1996;88:1852–6. limitations or when white blood cell counts in peripheral blood . 2 Maaskant-van Wijk PA, Faas BH, de Ruijter JA, et al. Genotyping of RHD by multiplex polymerase chain reaction analysis of six are too low to perform genotyping, other material can be used, RHD-specific exons. Transfusion 1998;38:1015–21. such as nails or buccal epithelial cells (with potential errors . 3 Castilho L, Rios M, Bianco C, et al. DNA-based typing of blood in bone marrow transplant patients).3,4,8 Therefore, leukocyte groups for the management of multiply-transfused sickle cell counting for the patient is essential before performing these disease patients. Transfusion 2002;42:232–8. . 4 Tax MGHM, van der Schoot CE, van Doorn R, Douglas- analyses, and care should be taken in case of neutropenic or Berger L, van Rhenen DJ, Maaskant-vanWijk PA. RHC and lymphopenic patients. RHc genotyping in different ethnic groups. Transfusion 2002; Ideally, all transfusions should be closely monitored, 42:634–44. especially transfusions of unmatched RBC units, to detect acute . 5 Veldhuisen B, van der Schoot CE, de Haas M. Blood group genotyping: from patient to high-throughput donor screening. transfusion reactions in case of transfusion of incompatible Vox Sang 2009;97:198–206. blood group(s). Unfortunately the clinical practice shows that . 6 Avent NO. Large-scale blood group genotyping: clinical unmatched units are transfused in emergency situations, in implications. Br J Haematol 2009;144:3–13. which close monitoring of transfusion can be difficult. Because . 7 CBO (Centraal Begeleidings Orgaan) consensus. Richtlijn bloedtransfusie (Blood transfusion directive) 2004. transfused RBCs remain in circulation for several weeks and Kwaliteitsintituut voor de Gezondheidszorg (Dutch Institute can cause mixed-field agglutination when antigen typing for Healthcare Improvement). Utrecht, The Netherlands. is performed, genotyping can determine the blood group of . 8 Rozman P, Dovc T, Gassner C. Differentiation of autologous trauma patients who have received repeated unmatched RBC ABO, RHD, RHCE, KEL, JK, and FY blood group genotypes by analysis of peripheral blood samples of patients who units. have recently received multiple transfusions. Transfusion 2000;40:936–42. Conclusions Joyce Curvers, PhD (corresponding author), Resident in Clinical As has previously been suggested, blood group Chemistry, Volkher Scharnhorst, PhD, Resident in Clinical genotyping can be helpful in acute situations, when serologic Chemistry, Clinical Laboratory Department of the Catharina Hospital, Michelangelolaan 2, 5623 EJ Eindhoven, The Netherlands; (hemagglutination with antisera) blood group typing is Masja de Haas, MD, PhD, Head of the Diagnostic and Research not possible or yields inconclusive results (i.e., mixed-field Laboratory of Immunohematology, Sanquin Blood Supply 6,8 agglutination, discrepant results). The success of genotyping Foundation, Amsterdam, The Netherlands; Loes Warnier-Wandel, in cases of emergency depends on the speed of processing and BSc, Head of the Immunohematology Department of the Blood Bank the turnaround time. Improvements in these will eventually Southwest Region, Sanquin Blood Supply Foundation, Dordrecht, lead to fewer unmatched transfusions. The blood transfusion The Netherlands; and Daan van de Kerkhof, PhD, Resident in Clinical Chemistry, Clinical Laboratory Department of the Catharina laboratory should, however, always be aware of the limitations Hospital, Michelangelolaan 2, 5623 EJ Eindhoven, The Netherlands. of genotyping (vs. serologic determination of blood groups). In a trauma center, genotyping a patient’s sample to determine ABO and D might be a suitable alternative to transfusing group O, D– RBCs, thus reducing the unnecessary use of such blood products.

IMMUNOHEMATOLOGY, Volume 28, Number 3, 2012 87 Warm autoadsorption with enzyme-treated red blood cells

F. Tsimba-Chitsva, S. Bishop, and K. Kezeor

Patients demonstrating warm autoantibody specificity present serologic challenges for laboratory staff performing antibody Reagents/Supplies identification in the blood bank. Autoantibody can be removed from plasma or serum by adsorption onto autologous red blood Enzyme Treatment cells (RBCs) provided the patient has not been transfused in the Method Reagents Supplies previous 3 months. The adsorption process can be enhanced by enzyme pretreatment of autologous RBCs. Immunohematology Papain 1% • Isotonic • 1 mL graduated pipettes cysteine- saline • 37°C water bath 2012;28:88–90. activated • Patient papain • Large bore test tubes autologous Ficin 1% ficin red blood • Calibrated centrifuge Key Words: warm autoadsorption, ficin, papain, autologous cells

Principle Procedural Steps

Performing pretransfusion testing in the presence of Treatment • Mix enzyme with packed red cells of Cells • Incubate mixture warm autoantibodies optimally reactive at 37°C presents • Wash mixture to remove enzyme serologic challenges when attempting to determine the • Centrifuge to pack the red cells • Remove supernatant presence of underlying clinically significant alloantibodies.1 Adsorption • Mix enzyme-treated cells with patient plasma/serum Warm-reactive autoantibodies may mask the presence of other • Incubate the mixture antibodies because of their agglutination with all cells tested, • Centrifuge the mixture • Remove plasma/serum including autologous red blood cells (RBCs). To determine the presence of clinically significant antibodies, the removal of autoantibody may be achieved by adsorption of patient plasma or serum with autologous RBCs.2–4 Allogeneic RBCs may be these methods and reagents, see the applicable procedural

SEROLOGIC METHOD REVIEW used for adsorption if the patient has been recently transfused reference documents or manufacturer’s package insert.) After or when the quantity of autologous RBCs is insufficient for dissociation of the autoantibody from the autologous RBCs, the autoadsorption.2 antigen sites should be available for binding the autoantibody from the plasma or serum during the adsorption process. Indications To further enhance the adsorption process, autologous RBCs can be treated with proteolytic enzymes such as ficin or As circulating autologous RBCs are coated with papain. This additional treatment enhances antibody uptake autoantibody, antigen sites on the autologous cells may onto the autologous cells by cleaving some of the glycoprotein become blocked with immunoglobulin. To efficiently achieve chains extending from the cell membrane, leading to improved autologous adsorption of warm-reactive autoantibodies, accessibility of the antigens not removed by the dissociation autoantibody must be dissociated from the RBC before method.4 the adsorption process.2 Autoantibody dissociation can be accomplished by several methods, including partial heat elution Procedure at 45°C, gentle heat elution at 56°C, a combination of 0.2 M dithiothreitol (DTT) and papain or ficin treatment, acid-EDTA For papain or ficin (1%) treatment of RBCs, one volume treatment, ZZAP (a mixture of cysteine-activated proteolytic of 1% cysteine-activated papain or 1% ficin is added to two papain and DTT) treatment, and chloroquine diphosphate volumes of packed RBCs. For example, if 2 mL of packed (CDP) treatment.5 (For specific procedures concerning RBCs is treated, 1 mL of ficin or papain is added. The mixture

88 IMMUNOHEMATOLOGY, Volume 28, Number 3, 2012 Warm autoadsorptions with enzyme-treated RBCs

is incubated at 37°C for a predetermined time to allow Limitations for the most effective enzyme treatment. If commercially prepared enzyme is used, the manufacturer’s insert is used A recently transfused patient (within the last 3 months) to determine the incubation time. If enzyme is prepared in- is not a candidate for autologous adsorption. Circulating house, qualification studies are performed to determine the transfused RBCs may adsorb clinically significant plasma or appropriate treatment time.6 serum alloantibodies.2 It has been demonstrated that very small The enzyme-treated RBCs are washed with copious amounts of antigen-positive RBC are capable of eliminating amounts of isotonic saline a minimum of three times, or until alloantibody reactivity from the plasma or serum.2,10 the supernatant saline is clear, to ensure complete removal of Large volumes of autologous RBCs from the patient are enzyme. The last wash is centrifuged a minimum of 5 minutes needed to perform autoadsorption. In the case of a low patient at 900 to 1000× g to completely pack the RBCs.7 This step hematocrit, it may be difficult to obtain a sufficient volume is critical to achieve proper packing of RBCs and removal of of autologous cells for testing without exacerbating clinical maximum amounts of saline. To avoid plasma or serum dilution symptoms of anemia. during adsorption, as much saline as possible is carefully Cell treatment may be unsuccessful in stripping removed after the final wash. This can be accomplished with autoantibody from the autologous, coated RBCs. As a result, suction using a small-bore pipette or filter paper.4 adsorption may be less efficient in removing autoantibody. REVIEW METHOD SEROLOGIC To perform adsorption, one volume of enzyme-treated Enzyme treatment of RBCs will destroy antigen sites on RBCs is mixed with an equal volume of the patient’s plasma or the RBCs. Enzymes remove sialic acid from the RBCs and will serum. To enhance antibody uptake, the proportion of RBCs to destroy some antigens (-M, -N, -S, -s, -Fya, -Fyb, -Ena, -Ge, plasma or serum can be increased. Adsorption is more effective -JMH, -Ch/Rg, -Inb). Autoantibodies specific for enzyme- if the area of contact between the RBCs and plasma or serum sensitive antigens will not be removed by this method.4 is large; use of a large-bore test tube (16 × 100 mm) increases Potential dilution of adsorbed plasma or serum must be the surface area and allows for optimum plasma or serum and considered a consequence of preparation of enzyme-treated RBC contact. The mixture is incubated at 37°C for 10 to 60 cells. If the saline is not properly removed after the final wash, minutes.4 The mixture is then centrifuged and the plasma or the residual saline will dilute the plasma or serum when serum is carefully removed. If the harvested adsorbed plasma adsorptions are performed. The more adsorptions performed, or serum will be used for additional adsorptions, disturbing the greater the dilution factor. As a result, diluted antibody the packed RBCs to achieve maximum plasma removal is may become undetectable in the adsorbed plasma or serum. acceptable. If, however, the harvested adsorbed plasma or Enzyme treatment of autologous RBCs can cause serum will be used for antibody detection, the packed RBCs hemolysis. If the RBCs are hemolyzed by enzyme treatment, should not be disturbed in an attempt to recover additional there may be an insufficient quantity of RBCs remaining to plasma. perform adsorptions. The number of adsorptions required to exhaust autoantibody from the plasma or serum is proportionally Acknowledgment related to the strength of its reactivity in plasma or serum; however, in some instances, additional adsorptions may be The authors thank Kerry Burright-Hittner, MT(ASCP) required to remove the antibody.7–9 If the original plasma or SBB, Director IRL, American Red Cross, Midwest Region, for serum reactivity is 1+ with the indirect antiglobulin test (IAT), providing technical assistance and expertise, as well as for her the first aliquot of adsorbed plasma or serum should be tested critical reading and editing of the manuscript. against group O reagent RBCs or direct antiglobulin test (DAT) negative autologous RBCs, if available. If reactivity persists, References the adsorption procedure should be repeated, as needed, using . 1 Leger RM, Garratty G. Evaluation of methods for detecting a fresh volume of enzyme-treated RBCs. Once reactivity is alloantibodies underlying warm autoantibodies. Transfusion abolished, the adsorbed plasma or serum is tested against the 1999; 39:11–16. appropriate group O reagent panel RBCs to exclude underlying . 2 Leger, RM. Positive DAT and immune-mediated hemolysis. In: Roback JD (ed). Technical manual. 17th ed. Bethesda, MD: alloantibodies. American Association of Blood Banks, 2011:505–6.

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. 3 Morel PA, Bergren MO, Frank BA. A simple method for the . 8 Garratty G. What do you do when all units are incompatible? detection of alloantibody in the presence of autoantibody 43. Instituto Mexicano del Seguro Social, 2005:S107–11. [abstract]. Transfusion 1978;18:388. . 9 Method 3-12: Adsorption procedure. In: Roback JD (ed). . 4 Judd J, Johnson ST, Storry JR. Judd’s methods in Technical manual. 17th ed. Bethesda, MD: American immunohematology. 3rd ed. Bethesda, MD: American Association of Blood Banks, 2011:914. Association of Blood Banks, 2008:45, 457–9. 10. Garratty G, Telen MJ, Petz LD. Red cell antigens as functional . 5 Beattie K, Crawford M, Martin, J, et al. Immunohematology molecules and obstacles to transfusion. Hematology AmSoc methods. Rockville, MD: American Red Cross, 1993:44-1, 48- Hematol Educ Program 2002:445–50. 1, 51-1. . 6 Method 3-5-1: Preparing ficin enzyme stock, 1% W/V; Method Farai Tsimba-Chitsva, MLS(ASCP)SBBCM, Reference Technologist, 3-5-2: Preparing papain enzyme stock, 1% W/V; Method Susanne Bishop, MLS(ASCP)SBBCM, Reference Technologist, 3-5-3: Standardizing enzyme procedures. In: Roback JD (ed). Technical manual. 17th ed. Bethesda, MD: American and Kelly Kezeor, MT(ASCP), (corresponding author) Reference Association of Blood Banks, 2011:902–3. Technologist, Midwest Region American Red Cross Reference . 7 Method 4-8: Adsorbing warm-reactive autoantibodies using Laboratory. American Red Cross, 3838 Dewey Avenue, Omaha, NE autologous red cells. In: Roback JD (ed). Technical manual. 68105. 17th ed. Bethesda, MD: American Association of Blood Banks, 2011:924–5. SEROLOGIC METHOD REVIEW

For information concerning the National Reference Laboratory for Blood Group Serology, including the American Rare Donor Program, contact Sandra Nance, by phone at (215) 451-4362, by fax at (215) 451-2538, or by e-mail at [email protected]

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90 IMMUNOHEMATOLOGY, Volume 28, Number 3, 2012 R e p o r t Implications of the Kidd blood group system in renal transplantation

A. Rourk and J.E. Squires

The association of the Kidd blood group system with hemolytic patient was discharged from the hospital but, as a result of transfusion reactions and hemolytic disease of the newborn progressive renal disease, required peritoneal dialysis 48 is well known. The Kidd antigens, which are localized to the months later. He did well on dialysis for 4 months but then HUT/UT-B urea transport protein, are found on red blood cells and the endothelial cells of the blood vessels of the medulla of underwent an uncomplicated cadaveric renal transplant the kidney. Recently it has been suggested that these antigens procedure requiring no transfusions. For approximately might play a role as minor histocompatibility antigens in renal 10 years, the patient did well and was maintained on a transplantation. In the current case, the appearance of an anti-Jkb 10 years after renal transplantation associated with early renal routine immunosuppressive regimen including tacrolimus, allograft rejection further supports the potential importance of mycophenolate, and prednisone. Then, during a routine follow- these antigens in renal transplantation and allograft rejection. up visit, he was found to have a slightly elevated creatinine (1.7 Immunohematology 2012;28:91–4. mg/dL compared with his usual baseline of 1.2 mg/dL). On further questioning, he admitted to oliguria and to “missing Key Words: Kidd blood group, renal transplantation, several doses” of his immunosuppressive medications. In allograft rejection preparation for a renal biopsy, a blood type and antibody screen was performed. His blood type was confirmed as O+, The Kidd blood group antigens Jka and Jkb are expressed but for the first time his antibody screen was strongly positive at position 280 of the membrane glycoprotein HUT11/UT-B. (3+); anti-Jkb was identified. Kidney biopsy was consistent This protein is thought to function as a urea transporter that with acute allograft rejection, Banff classification type IA, protects the red blood cell (RBC) from osmotic gradients, plasma cell rich.6 The patient’s immunosuppressive regimen particularly as it passes through the kidney.1 The UT-B protein was reestablished while he was in the hospital, and now, 7 has also been localized to endothelial cells of the vasa recta of months later, his creatinine has returned to his usual baseline the medulla of the kidney where it facilitates the excretion of and his antibody screen is again negative. urea and concentration of the urine.2 More recently, the UT-B protein has been identified in a variety of other tissues such as Materials and Methods testis3 and colon.4 Antibodies to Jka and Jkb have been associated with both Current blood type and antibody identification were hemolytic transfusion reactions (HTR) and hemolytic disease performed by solid-phase red blood cell adherence assay using of the fetus and newborn (HDFN). More recently, they have the Galileo immunohematology analyzer (Immucor Gamma, also been associated with several cases of acute or delayed renal Norcross, GA). allograft rejection.5 We report a case in which the appearance of anti-Jkb is associated with early renal allograft rejection Results more than 10 years after the initial renal transplant surgery. During the course of the patient’s treatment for HUS at the Case Report age of 14 months, he received 5 group O+ RBC transfusions and one group O+ platelet transfusion. His antibody screen at At 14 months of age, an African American boy was treated this time remained negative during the 8 days during which for pneumonia caused by Streptococcus pneumoniae and he was transfused. No further blood type or antibody screen associated hemolytic uremic syndrome (HUS). At that time, was performed until the time of his renal transplant procedure the patient received five group O+ RBC transfusions and one 48 months later; his antibody screen at that time continued group O+ platelet transfusion; his antibody screen remained to be negative and he received no transfusions during or after negative throughout the 8 days of his hospitalization. The this surgical procedure. The patient was routinely evaluated

IMMUNOHEMATOLOGY, Volume 28, Number 3, 2012 91 A. Rourk and J.E. Squires

Table 1. Summary of patient information

Date Purpose Antibody screen Antibody identification Transfusion Creatinine Nov. 1996 Treatment; HUS Negative 5 RBC, 1 platelet Not available Dec. 2000 Clinic visit; follow-up Negative None Not available April 2001 Hospitalization; pre-renal transplant Negative None Not available Aug. 10, 2010 Clinic visit; follow-up Not Done None 1.7 mg/dL Aug. 13, 2010 Hospitalization; treatment of acute rejection Positive Anti-Jkb None 1.5 mg/dL Aug. 15, 2010 Hospital discharge Not Done None 1.6 mg/dL March 2011 Clinic visit; follow-up Negative None 1.3 mg/dL HUS = hemolytic uremic syndrome; RBC = red blood cell. during the subsequent 10 years and apparently was doing well referred to as UT-B, is expressed on both the RBC membrane until, at the time of a routine follow-up visit, he was found to and the endothelial cells of the vasa recta of the medulla of the have an elevated creatinine level. When the patient admitted kidney.7 to oliguria and failing to adhere to his immunosuppressive We report a case of renal allograft rejection occurring 10 regimen, a renal biopsy was scheduled. In preparation for this years after transplantation. Transplant rejection in this case procedure, an antibody screen was performed and was found was heralded by a slight rise in serum creatinine and was to be strongly positive (3+). The antibody was identified as probably related to the patient’s recent poor compliance with anti-Jkb. Table 1 summarizes these results. The renal biopsy his immunosuppressive medications. Unexpectedly, anti-Jkb (Fig. 1) was considered diagnostic of acute allograft rejection was identified when a routine blood type and antibody screen (Banff type IA, plasma cell rich). was performed in preparation for the renal biopsy. Because the patient had only been transfused with RBCs approximately 10 Discussion years before the appearance of anti-Jkb and had had a negative antibody screen during that period, it seems safe to assume Antibodies to the Kidd blood group antigens, Jka and Jkb, that the transplanted kidney stimulated the development of this are well known to cause HTR and HDFN. However, recently antibody. It is also important to note that the antibody appeared a small number of reports have also suggested the importance at a time when the patient admitted to poor compliance with of these antibodies in renal transplantation. The importance his immunosuppressive medications. It is possible that anti- of these antibodies in renal transplantation presumably arises Jkb first appeared in this patient at the time of his transfusions from the fact that the protein carrying the Kidd antigens, at the age of 14 months and the reappearance of the antibody 10 years later is simply an anamnestic response to the exposure to the Jkb antigen presumably present on the transplanted kidney. Alternatively, it seems possible that the Kidd antigen on the transplanted kidney stimulated the de novo development of the antibody once the patient’s immunosuppressive regimen was interrupted. This observation is supported by several recent case reports. In one of these, Hamilton et al.8 report the case of a 19-year-old woman who underwent a cadaveric kidney transplant procedure owing to a diagnosis of focal segmental glomerulosclerosis. The patient was noncompliant with her immunosuppressive medications, and approximately 24 months after her transplant she exhibited renal failure that ultimately necessitated dialysis and transplant nephrectomy. During her hospitalization she was found to have a positive Fig. 1 Photomicrograph of kidney biopsy specimen demonstrates antibody screen, and anti-Jkb was identified. Because this a prominent inflammatory infiltrate with prominent plasma cells patient had no history of blood transfusion or pregnancy, the consistent with Banff type 1 acute rejection, plasma cell rich. authors hypothesized that the antigen on the transplanted

92 IMMUNOHEMATOLOGY, Volume 28, Number 3, 2012 Kidd blood group in renal transplants

kidney served as the immunizing antigen causing the kidney transplants. With respect to the Kidd antigens, development of the anti-Jkb. In this case, a diagnosis of plasma those transplants in which the donor and recipient were cell-rich acute cellular rejection (PCAR) was made by renal mismatched for Kidd antigens showed a greater degree of biopsy. It has been suggested that in PCAR, which is a rather interstitial inflammation than those cases in which the donor uncommon inflammatory manifestation of allograft rejection, and recipient were matched, thus suggesting the potential patient-derived plasma cells infiltrate the renal allograft and importance of these antigens in clinical transplantation. are responsible for the production of antibody in situ.9 Hamilton Clearly, then, each of these case reports supports the et al.8 make the suggestion that the anti-Jkb was synthesized suggestion that Kidd antigens do, in fact, act as minor by the plasma cells identified in the kidney. Hamilton et al.10 histocompatibility antigens that are uncommon but potentially described another 19-year-old woman who exhibited anti- important causes of renal allograft rejection. Functionally, the Jka 7 years after renal transplantation. Again, the patient explanation for this lies in the observation that the protein was described as noncompliant with her immunosuppressive carrying the Kidd antigens derives from a single gene and is regimen. In this case, however, at the time of original renal found on both the RBC and the endothelial cells of the vasa transplantation the patient received two units of RBCs that recta of the medulla of the kidney. The question that remains were known to be Jk(a+). in the current case is whether the Kidd antigens on the In each of these three cases a cadaveric renal transplant transplanted kidney served as the primary stimulus for the was carried out on a patient with a negative antibody screen and development of the Jkb antibody (as suggested by the report 2 to 10 years later, in a setting of noncompliance with a routine of Hamilton et al.8) or whether it served as the stimulus for immunosuppressive regimen, an antibody to the Kidd blood an anamnestic antibody response of an anti-Jkb that originally group system (anti-Jka or anti-Jkb) appeared in conjunction arose because of the RBC transfusions 10 years earlier. In either with acute kidney transplant rejection. A comparison of these event the important effect of the Kidd antigens on the kidney cases is found in Table 2. is apparent as the transplanted kidney must have either served

Table 2. Comparison of case reports of renal allograft rejection occurring after transplantation

Case Patient Transfusion Kidd antibody Diagnosis: kidney rejection Contributing factors to rejection Current Male 5 RBC; 1 platelet Jkb Acute rejection Noncompliance with immunosuppressive medications Hamilton, 2006 Female None Jkb Acute rejection Noncompliance with immunosuppressive medications Hamilton, 2008 Female 2 RBC Jka Acute rejection Noncompliance with immunosuppressive medications Holt, 2004 Female Yes (total number not Jka Acute rejection RBC transfusion reaction caused by anti-Jka reported)

The critical role that antibodies of the Kidd system as an immunogen in stimulating an anamnestic development may play in renal allograft rejection is further suggested by of anti-Jka or been the primary immunizing agent. Holt et al.11 In this case a 29-year-old patient experienced a hyperacute renal allograft rejection presumably caused by a Acknowledgment complement-fixing anti-Jka. In this case it was suspected that the patient had developed anti-Jka as a result of a series of RBC The authors would like to acknowledge the technical staff transfusions that she had received approximately 18 years in Transfusion Medicine at the Medical University of South earlier. After her renal transplant procedure, she received Carolina. three units of RBCs. Within 2 hours of this transfusion, the patient exhibited a fever and urine output slowed. Eventually References the direct antiglobulin test was found to be positive, and an . 1 Westhoff CM, Reid ME. Review: the Kell, Duffy, and Kidd a eluate demonstrated anti-Jk . The authors suggested that the blood group systems. Immunohematology 2004;20:37–49. transfusions shortly after her renal transplant stimulated an . 2 Daniels G. Structure and function of red cell surface antigens. anamnestic anti-Jka, which was then responsible for both the ISBT Science Series 2006;1:3–8. transfusion reaction and ultimately failure of her graft. . 3 Fenton RA, Cooper GJ, Morris ID, Smith CP. Coordinated expression of UT-A and UT-B urea transporters in rat testis. 5 Lerut et al. compared the matching versus mismatching Am J Physiol Cell Physiol 2002;282:C1492–501. status for Kidd and Duffy polymorphisms in 370 consecutive

IMMUNOHEMATOLOGY, Volume 28, Number 3, 2012 93 A. Rourk and J.E. Squires

. 4 Inoue H, Jackson SD, Vikulina T, Klein JD, Tomita K, Bagnasco . 9 Charney DA, Nadasdy T, Lo AW-H, Racusen LC. Plasma SM. Identification and characterization of a Kidd antigen/UT-B cell-rich acute renal allograft rejection. Transplantation urea transporter expressed in human colon. Am J Physiol Cell 1999;68:791–7. Physiol 2004;287:C30–5. 10. Hamilton MS, Singh, V, Warady BA. Additional case of acute . 5 Lerut E, Van Damme B, Noizat-Pirenne F, et al. Duffy and cellular kidney rejection associated with the presence of Kidd blood group antigens: minor histocompatibility antigens antibodies to the red blood cell Kidd antigen. Pediatr Transplant involved in renal allograft rejection? Transfusion 2007;47: 2008;12:918–9. 28–40. 11. Holt S, Donaldson H, Hazlehurst, et al. Acute transplant . 6 Bogman MJJT, Dooper IMM. Banff classification for the rejection induced by blood transfusion reaction to the Kidd histological diagnosis of renal graft rejection: what are the blood group system. Nephrol Dial Transplant 2004;19: advantages? Nephrol Dial Transplant 1995;10:1291–3. 2403–6. . 7 Timmer RT, Klein JD, Bagnasco SM, et al. Localization of the urea transporter UT-B protein in human and rat erythrocytes Angela Rourk, MT (ASCP), Medical Technologist, and Jerry E. and tissues. Am J Physiol Cell Physiol 2001;281:C1318–25. Squires, MD (corresponding author), Medical Director, Transfusion . 8 Hamilton MS, Singh V, Warady BA. Plasma cell-rich acute Service, Department of Pathology and Laboratory Medicine, cellular rejection of a transplanted kidney associated with antibody to the red cell Kidd antigen. Pediatr Transplant Medical University of South Carolina, 171 Ashley Avenue, MSC 908, 20 06;10:974 –7. Charleston, SC 29425-9080.

94 IMMUNOHEMATOLOGY, Volume 28, Number 3, 2012 EDTA glycine acid treatment of red blood cells

J. Kosanke

IgG dissociation is necessary when a sample is direct antiglobulin test (DAT) positive and antigen testing using blood grouping Reagents/Supplies serum reactive by the antiglobulin test is performed. Exposure of IgG-coated red blood cells (RBCs) to a low pH of 3.0 with EDTA Reagents Supplies glycine acid successfully dissociates the IgG, rendering the RBCs Saline Centrifuge DAT negative 82 to 85 percent of the time. The procedure takes Reagents for EDTA-GA solution Test tubes one minute or less and leaves RBC antigens intact and able to be TRIS buffer Pipettes typed except for those antigens in the Kell blood group system and for the high-prevalence antigen Era. Immunohematology Reagents for DAT Agglutination viewer 2012;28:95–6. GA = glycine acid; DAT = direct antiglobulin test. SEROLOGIC METHOD REVIEW METHOD SEROLOGIC Key Words: EGA, EDTA glycine acid, IgG dissociation, Procedural Steps direct antiglobulin test, DAT Mix EDTA-GA with packed red blood cells Add buffering solution Principle Process Wash cells The chemical bonds in antibody-antigen reactions are Perform DAT on treated cells weak noncovalent bonds. The reactions between an antibody GA = glycine acid; DAT = direct antiglobulin test. and antigen are often described as a lock and key fit and are held together by van der Waals, electrostatic, and hydrophobic forces. These forces are easily dissociated by extremes in pH. Indications EDTA glycine acid (GA) treatment uses an acidic pH of 1.5 to dissociate IgG from red blood cells (RBCs). EDTA-GA is used to dissociate IgG from a patient’s The effectiveness of EDTA-GA was first described in autologous RBCs to prepare them for antigen testing with an abstract by Louie, Jiang, and Zaroulis in 1986.1 Fifty blood grouping reagents. An autologous RBC sample would be samples were treated using an EDTA-GA method after in defined minimally as a sample collected more than 3 months vitro sensitization with alloantibodies. After one treatment, 48 from the last transfusion. If a patient has been transfused samples had a negative direct antiglobulin test (DAT) and the within this 3-month period, a cell separation to recover remaining two had a negative DAT after a second treatment. autologous cells should be performed before the EDTA-GA When 30 in vivo strongly sensitized samples were treated with treatment of the RBCs. EDTA-GA and compared with treatment with chloroquine RBCs sensitized with IgG invalidate test results with diphosphate (CDP), the results indicated the EDTA-GA blood grouping reagents that require an indirect antiglobulin method was more effective than CDP. All 30 samples had test (IAT). Some monoclonal blood grouping reagents that negative DATs after EDTA-GA treatment, and 26 had negative do not require an IAT still may be considered invalid unless DATs with CDP treatment. EDTA-GA has the added benefit one of the antigens of the tested allelic pair is nonreactive. of only requiring a 1-minute incubation instead of a minimum IgG dissociation by EDTA-GA treatment leaves cells intact to of 30 minutes with CDP. Additional testing of 100 consecutive perform antigen typing. If the autologous cells have a negative samples had IgG dissociated from the patient’s RBCs 85 DAT after treatment, a valid antigen typing can be obtained percent of the time. Another abstract, in 1989, reported the for common antigens except for K. EDTA-GA is known to findings with 45 patient samples with DAT reactivity ranging denature all antigens in the Kell blood group system and the from 1+ to 4+ with a success rate of 82 percent dissociation of high-prevalence antigen Era.3 IgG.2

IMMUNOHEMATOLOGY, Volume 28, Number 3, 2012 95 J. Kosanke

Materials Limitations

Because an acidic pH in normal saline would cause EDTA-GA is effective at dissociating IgG, but it does not hemolysis of RBCs, it is necessary to prepare the low-pH remove complement components from RBCs. Anti-IgG rather reagent in an EDTA glycine solution to maintain osmolarity of than polyspecific anti-human globulin reagent should be used the RBCs (Table 1). A high-pH buffer is needed to bring the pH when testing EDTA-GA–treated cells. to 7.0 to 7.5 after the RBCs are exposed to EDTA-GA. Normal If antigen typing the patient’s EDTA-GA–treated RBCs saline is used to wash the cells before and after treatment. and the effect of acid is unknown, RBCs known to be antigen- The reagents for preparing EDTA-GA–treated cells may positive should be EDTA-GA treated and tested in parallel be prepared from stock chemicals or purchased commercially with the patient’s cells to determine the effect of the acid. as a kit with three individual reagent vials. Two of the reagents EDTA-GA is not successful 100 percent of the time. If are used to prepare the EDTA-GA solution for addition to the treatment of RBCs from a patient who has not been transfused cells, and the third reagent is used to neutralize the pH before in the previous 3 months is unsuccessful, recovering the washing the treated cells. patient’s reticulocytes and treating them with EDTA-GA may yield successful results.4 Procedure Some patients’ RBCs may turn brown when incubated a full minute with the EDTA-GA solution. Preparing a new A volume of packed RBCs is exposed to EDTA-GA for 1 to suspension and incubating for a shorter amount of time may 2 minutes at room temperature (Table 2). prevent browning and still be effective at dissociating IgG. • Although washing the cells to prepare a cell suspension may have no impact on the effectiveness of the EDTA- Quality Control GA, standard laboratory practices often start with RBCs washed at least one time in normal saline, if indeed not An inert control such as 6 percent albumin should be three times. tested in parallel with blood grouping reagents to detect weak • The ratio of RBCs, EDTA, and GA reported by Louie, reactivity that may occur with EDTA-GA–treated DAT- Jiang, and Zaroulis is 1:1:4. Although the incubation negative cells. If the 6 percent albumin control is positive, time was up to 2 minutes, an incubation of 1 minute or antigen typing results are invalid. less is an effective time for the low pH to dissociate IgG Whenever implementing a new procedure, verification from the cells. studies should be performed as required by 42 CFR 493.1253. • As a reagent, EDTA-GA typically is used to disrupt

SEROLOGIC METHOD REVIEW the binding interaction of antibody and antigen and References also in immunoaffinity purification of monoclonal and . 1 Louie JE, Jiang AF, Zaroulis CG. Preparation of intact polyclonal antibodies. antibody-free red blood cells in autoimmune hemolytic anemia (abstract). Transfusion 1986;26(Suppl):S20. A few drops of buffering solution are added. . 2 Kosanke J, McDowell MA, Stocker I, Thompson D, Wissel M. Treatment of DAT positive red cells with EDTA-glycine acid for • Although EDTA-GA can disrupt the bonds without antigen typing (abstract). Transfusion 1989;29(Suppl):S208. denaturing either the antibody or the antigen, prolonged . 3 Liew YW, Uchikawa M. Loss of Era antigen in very low pH exposure to an acidic environment may damage some buffers (letter). Transfusion 1987;27:442–3. proteins; an adjustment to neutral pH should be . 4 Gammon RR, Delk A, Clarke A, et al. Use of EGA treated neocytes allowed phenotyping of DAT positive samples performed quickly. from patients not recently transfused (abstract). Transfusion 2006;46(Suppl):SP294. The cells are washed three to four times, and a DAT is performed on the treated cells to determine effectiveness of Joanne Kosanke, MT(ASCP)SBB, Director, Immunohematology the treatment. Reference Laboratory, American Red Cross-Central Ohio Blood Services Region, 995 East Broad Street, Columbus, OH 43205.

96 IMMUNOHEMATOLOGY, Volume 28, Number 3, 2012 R e p o r t Molecular background of RH in Bastiaan, the RH:–31,–34 index case, and two novel RHD alleles

M.E. Reid, C.H. Hipsky, R.W. Velliquette, C. Lomas-Francis, K. Larimore, and C. Olsen

Anti-hrB (-RH31) and anti-HrB (-RH34) were found nearly 40 Smart2 stated the two antibodies were not separable, it is now B years ago in the serum of a South African woman. The anti-hr was generally accepted that they are indeed discrete specificities. revealed after adsorption with DcE/DcE red blood cells (RBCs). Allen and Rosenfield3 applied the term anti-RH34 to describe Numerous anti-hrB, in the absence of anti-HrB, have since been B identified. We obtained a sample of blood from this index case the total immune response of Bastiaan (i.e., anti-Hr and anti- (Bastiaan) and report the molecular basis of her D+C–E–c+e+/–, hrB [anti-RH31]) but anti-RH34 has been generally accepted S B B V–VS+Hr+hr +hr –Hr – phenotype as well as results of testing to refer specifically to anti-HrB. Bastiaan’s RBCs are e+ hrB– her RBCs using currently available reagents. We tested a cohort of B B B African Americans to estimate the frequency of the RHCE*ce 48C, Hr –; however, e+ hr – Hr + RBCs exist, and people with 733G, 1006T allele, and in addition found two novel RHD alleles. this phenotype can make anti-hrB.4,5 Additionally, although Hemagglutination tests and DNA analyses were performed by these antibodies often exist concomitantly, they can also exist standard methods. Analyses revealed homozygosity for RHCE*ce separately in unabsorbed plasma from different patients.4–6 48C, 733G, 1006T in Bastiaan. RBCs from Bastiaan were strongly 7 agglutinated by three commercial anti-e reagents. Testing RBCs Before reporting Bastiaan, Shapiro reported a similar from people homozygous for RHCE*ce 48C, 733G, 1006T showed case, in which RBCs from a Bantu woman named Shabalala that anti-e MS16, MS17, and MS63 were weakly reactive or (after whom the antigen was named) had an Hr– hrS– (RH: nonreactive, MS21 was strongly reactive, and HIRO38, HIRO41, –18,–19) phenotype. Her plasma contained anti-Hr with an and HIRO43 were nonreactive. Results show that Bastiaan S has RHD*DIIIa150C and RHD*DIIIa-CE(4-7)-D. Tests on 605 underlying anti-hr that remained after absorption with R2R2 samples from random African Americans revealed a frequency RBCs.7 Issitt recorded that “when e+hrS– and e+hrB– RBCs of 0.036 for RHCE*ce 48C, 733G, 1006T and revealed two novel first became available for use in serological tests, some anti- alleles: RHD*186T and RHD*DIIIa 150C. The Bastiaan phenotype S B is encoded by RHD*DIIIa150C-RHCE*ce 48C, 733G, 1006T and e-like specificities were called anti-hr or anti-hr because of RHD*DIIIa-CE(4-7)-D-RHCE*ce 48C, 733G, 1006T; thus, this their failure to react with hrS– and hrB– RBCs, even when genotype is the gold standard for the hrB–HrB– phenotype. The tested with only one example of the antibody. Those sera S B r′ complex encodes VS, which explains why most hr – RBCs are were then used to classify, not necessarily accurately, other VS+. Immunohematology 2012;28:97–103. RBCs with e variants. Thus, the ‘new’ examples of anti-hrS In 1972, Shapiro and colleagues reported the first example and anti-hrB were not the same specificity as the originals. As of anti-hrB and anti-HrB in the plasma of a Cape Coloured South these inaccurately classified antibodies and RBC phenotypes African, Ann Bastiaan (the original publication used the full were used to ‘resolve’ further cases, the situation grew to name of the patient after whom the antigens [hrB, HrB] were where now it is difficult, serologically, to accurately define e named; in this report we will use Bastiaan).1 Her second child variants. While RBC samples and sera in the collections of had hemolytic disease of the fetus and newborn (HDFN). Her immunohematologists are labeled in terms of hrS and hrB, D+ red blood cells (RBCs) were not agglutinated by anti-RhD, they would be more accurately described as being RBCs or the anti-D made by people with the DIIIa phenotype, which sera from people with e variants. Terms anti-hrS-like and anti- showed that she had the partial DIIIa phenotype. However, hrB-like should be abandoned.”4 Issitt concluded that “at the because her husband and child were both D–, it was thought serological level the situation is remarkably complex and is that anti-D was not the cause of the HDFN. unlikely soon (or easily) to be resolved. . . . It is clear that some The antibody in Bastiaan’s plasma reacted with all RBCs of the apparent heterogeneity of anti-hrS and anti-hrB relates on antibody identification panels, but reacted more weakly directly to the labels that have been applied to various similar 4 with e– (R2R2) RBCs than with e+ RBCs. Absorption of her but not identical specificity antibodies.” Added to this situation B plasma with R2R2 RBCs removed the anti-Hr and left the and complexity is the fact that the e antigen can have altered anti-hrB, which can resemble anti-Ce. Although Moores and expression not only in inherited variants (alloanti-e-like)

IMMUNOHEMATOLOGY, Volume 28, Number 3, 2012 97 M.E. Reid et al.

but also on RBCs from patients with warm autoantibodies. basis of the serologically defined index cases is valuable. Furthermore, e+ people with alloanti-e are frequently D+ with Unfortunately, despite considerable effort, we have been unable alloanti-D.8,9 to obtain a blood (or DNA) sample from Shabalala, the index As of August 2011, there were 291 RHD and 135 RHCE Hr– hrS– case. However, we did succeed in obtaining a sample alleles listed in two databases.10,11 The numerous alleles occur from Bastiaan, the index hrB– HrB– case. We have established because of the high degree of homology between RHD and that her DIIIa, C+E–c+e+/–, V– VS+, hrS+, hrB–, HrB– RHCE, their close proximity at chromosome 1p36.11, and phenotype is owing to the homozygous presence of RHCE*ce their opposite orientation.12 The variant alleles result from 48C, 733G, 1006T (ceS) and heterozygosity for RHD*DIIIa single and multiple inserts of RHD into RHCE or vice versa, 150C/DIIIa-CE(4-7)-D. We performed hemagglutination novel nucleotide changes, deletion of nucleotide(s), or insertion tests on RBCs from Bastiaan and others with the same RHCE of nucleotide(s). genotype, using currently available anti-e reagents. We also With knowledge of the DNA sequence of alleles encoding tested samples from a cohort of people of African descent to e variants, we now have tools to more accurately predict an estimate the frequency of the RHCE allele and the RHD alleles RBC phenotype and therefore determine the specificity of an associated with them, and these analyses revealed two novel antibody. The e/E polymorphism is fundamentally defined RHD alleles: RHD*D186T (accession number JN635688) and by a single amino acid change (Ala226Pro) on the RhCE RHD*DIIIa 150C (accession number JN635689). No ISBT protein13; however, amino acid changes at other positions on allele name was obtained because these novel RHD alleles do the Rhce protein affect expression of e. Noizat-Pirenne et al.6 not, apparently, exhibit a phenotype. reported the molecular bases of several e variants, including two cases of anti-RH34 in patients with the r′S phenotype. Materials and Methods Pham et al.14 showed that in addition to the existence of the r′S haplotype with exons 1 to 3 of the RHD hybrid allele derived Whole blood samples were freshly collected as part of from RHD*DIIIa (r′S type 1),15 the haplotype also exists with antibody identification or recovered from storage in liquid exon 1 to 3 being derived from RHD (r′S type 2). These workers nitrogen. (The names of donors are codes and not related performed DNA and hemagglutination testing to show that to the donor’s actual name; they are used for the benefit of RBCs from people with either of these r′S phenotypes lacked immunohematologists who have blood samples from these hrB and HrB. Among the samples tested, they found four that donors in their collections). Hemagglutination tests were contained anti-hrB plus anti-HrB, two with anti-hrB, and two performed by standard methods using reagents from our with anti-HrB.5 libraries of reagents and from Bio-Rad (Dreieich, Germany), The hrB– phenotype is often, but not invariably, Immucor (Norcross, GA), and Ortho Clinical Diagnostics VS+, which led to the speculation that hrB and VS may (Raritan, NJ) according to the manufacturer’s instructions. be antithetical.16–18 Four RHCE alleles with 48G>C and 733C>G changes (ISBT provisional name RHCE*01.20.02 Genomic DNA and RNA Extraction, Amplification, [RHCE*ceVS.02], RHCE*01.20.03 [RHCE*ceVS.03], RHCE* Cloning, Sequencing, and Restriction Fragment 01.20.04 [RHCE*ceVS.04], and RHCE*01.20.08 [RHCE* Length Polymorphism ceVS.08]) encode the VS+ and hrB– phenotype, three alleles Genomic DNA was extracted by standard methods from (ISBT name RHCE*01.20.01 [RHCE*ceVS.01], RHCE* peripheral white blood cells with a commercial kit (QIAamp; 01.20.06 [RHCE*ceCF], and RHCE*01.20.07 [RHCE*ceJAL]) Qiagen Inc., Valencia, CA). DNA was subjected to polymerase encode altered VS and altered hrB, and one allele (ISBT name chain reaction (PCR) amplification of specific exons ofRHD RHCE*01.20.05 [RHCE*ceVS.05]) encodes VS, but the hrB or RHCE with HotStarTaq DNA Master Mix (Qiagen, Inc.) status is unknown (www.isbt-web.org).19,20 The RHCE*ceMO according to the manufacturer’s instructions, using primers (ISBT name RHCE*01.07.01) encodes a partial e antigen and and conditions as described previously.20 the VS– hrS– hrB– phenotype.18 Thus, it is now known that hrB RNA was isolated from reticulocytes in peripheral blood and VS are not, in the classical sense, antithetical. samples by standard methods (TriZol, and PureLink RNA The understanding of the underlying molecular bases of Mini kit; Invitrogen, Carlsbad, CA). Reverse transcription numerous e variants is beginning to provide a tool, based on (RT) was carried out with gene-specific RHD and RHCE DNA analyses, to more precisely match blood to a patient. To aid primers and Superscript III according to the manufacturer’s the antibody identification process, determining the molecular instructions (Superscript III first-strand synthesis SuperMix;

98 IMMUNOHEMATOLOGY, Volume 28, Number 3, 2012 Molecular background of RHCE in Bastiaan

Invitrogen). PCR amplification ofRHD and RHCE cDNA was false-negative result in people of African origin because of the carried out with a kit (HotStarTaq DNA Master Mix, Qiagen, altered nucleotide sequence of their hybrid Rhesus boxes.23–25 Inc.) according to the manufacturer’s instructions, with primers to amplify exons 1–4 and exons 5–10 in RHD and DNA Array Testing RHCE, as described previously.21 DNA arrays containing multiple probes either for RHD PCR and RT-PCR products were checked for purity on or for RHCE were tested and analyzed, and interpretations agarose gels, cleaned (ExoSAP-IT Affymetrix, Santa Clara, were determined (BioArray Solutions, Warren, NJ) using the CA) according to the manufacturer’s instructions, and directly manufacturer’s BeadChip technology. sequenced through contract service provided by Genewiz Inc. (South Plainfield, NJ). Cloning and sequencing of clones of Results RHD-specific cDNA or genomic DNA were also carried out by Genewiz Inc. Hemagglutination DNA sequence results were aligned to reference alleles RBCs from Bastiaan were strongly agglutinated (4+) NM_020485 for RHCE and NM_016124 for RHD, and by three commercial anti-e reagents (Bio-Rad, Seraclone; predicted protein sequence comparisons were performed Immucor, Gamma-clone; and Ortho BioClone, MTS (Sequencher v4.9, GeneCodes, Ann Arbor, MI). monoclonal gel card), each of which contains a blend of MscI digestion of RHD exon 2 products from Bastiaan and three monoclonal anti-e clones: namely MS16, MS21, and a donor, Alson, was conducted at 37°C for 2 hours and run MS63. Testing RBCs from Bastiaan and four other probands on a 2 percent agarose gel with a 100-bp DNA ladder. RHD homozygous for RHCE*ce 48C, 733G, 1006T with single clone zygosity analyses were performed to detect the presence or anti-e showed that MS16 and MS63 were weakly reactive or absence of the hybrid Rhesus box in Bastiaan and Alson as nonreactive and that MS21 was strongly reactive (Table 1). described previously.22 No hybrid box was detected in either Additional tests with other monoclonal anti-e supernatant sample, indicating that both samples carry two copies of fluids showed weak reactivity or nonreactivity with MS17 and RHD. However, it should be noted that this assay can give a nonreactivity with HIRO38, HIRO41, and HIRO43. Anti-hrB

Table 1. Testing RBC samples from people with RHCE*ce 48C, 733G, 1006T. All five test samples were D+E–c+DAK+

RHD* RHCE* cDNA gDNA by LDT MS MS MS MS HIRO HIRO HIRO Euro Name C e hrS hrB V VS V/VS† by LDT and BAS LDT gDNA by BAS 16 17 21 63 38 41 43 Bio Bastiaan + + + 0 0 NT NT No cDNA DIIIa, ceS 48C, (C)ceS, 0 0 4+ 0 0 0 0 0 available DIIIa‡ ceS 48C (C)ceS MA212-08, NYBC +W + +W 0 0 + NT DIIIa, DIIIa, ceS 48C, (C)ceS, 1+S 0 3+S 1+ 0 0 0 0 donor DIIIa-CE DIIIa ceS 48C (C)ceS (4-7)-D MG89-01, SCD + + NT 0 NT + NT DIIIa, DIIIa, ceS 48C, (C)ceS, weak 0 3+W 2+W 0 0 0 NT patient with anti-e-like DIIIa-CE DIIIa ceS 48C (C)ceS (4-7)-D MA141-09, NYBC 0 + 0§ 0 NT NT + DIIIa, DIIIa, ceS 48C, (C)ceS, 2+W 1+ 3+W 1+ 0 0 0 0 donor DIIIa DIIIa ceS 48C (C)ceS MA156-08, SCD 0 + +W 0 0 + + DIIIa, DIIIa, ceS 48C, (C)ceS, 1+ 0 4+ 1+ 0 0 0 NT patient with anti-D, DIIIa DIIIa ceS 48C (C)ceS -C, -E, -hrB rʹʹr 0 + + + 0 0 0 1+ 2+W 2+ 4+ 1+ 1+ 1+ NT

R1R2 + + + + 0 0 0 2+ 4+ 4+ 3+ 3+ 4+ 4+ 3+

R2R2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

R1Rz + + + + 0 0 0 ± 4+ 4+ 3+ 4+ 3+ 4+ 3+

W R1R1 + + + + 0 0 0 NT NT NT NT NT NT NT 3+ BAS = BioArray Solutions; LDT = laboratory developed test; NT = not tested; NYBC = New York Blood Center; SCD = sickle cell disease. †Red blood cells tested with plasma containing both anti-V and anti-VS. ‡Based on a large family pedigree, Bastiaan’s RHD genotype has to be DIIIa/DIIIa-CE(4-7)-D. §Negative reaction when the control reacted 1+W.

IMMUNOHEMATOLOGY, Volume 28, Number 3, 2012 99 M.E. Reid et al.

(EuroBio, France) was also nonreactive. The epitope detected Table 3. Red blood cell typings on Bastiaan family from Shapiro et al.1 with phenotype interpretation from Shapiro (1972) and from by MS21 is the only one that is unaffected by the nucleotide the authors of this manuscript (2011) S changes encoding the r′ phenotype. Phenotype interpretation RBCs from Bastiaan typed D+ DAK+, as would be Person RBC typing* 1972 2011 26 expected with the presence of the DIIIa phenotype. ndH S S Ann D+, C+, E–, c+, e+, G+, f+, V–, Rh1 DIIIa-ce /rʹ Bastiaan hrS+, Rh28+, RhD– Molecular Analyses of RHCE Father D–, C–, E–, c+, e+, G–, f+, V+, rhVH ce/ceV+ hrS+, Rh28+, RhD– Analyses of RHCE gDNA from the Bastiaan sample, by Child D–, C+, E–, c+, e+, G–, f+, V–, rhʹnH rʹS/ce both PCR amplification and sequencing, and by the RHCE DNA hrS+, Rh28+, RhD– array, showed homozygosity for RHCE*ce 48C, 733G, 1006T *Antigen names translated from the Wiener terminology used by Shapiro et al. (ce S, ISBT allele name RHCE*ceVS.03 or RHCE*01.20.03; Table 2). This allele encodes a V–VS+hrB–HrB– phenotype, and our findings are consistent with the RBC typings on Bastiaan, DIIIa phenotype) and C+hrB– (Table 3), one can predict that her second child, and his father, as reported by Shapiro et al. her RHD alleles are RHD*DIIIa 150C and RHD*DIIIa-CE(4- (Table 3).1 7)-D, and her RH haplotypes are RHD*DIIIa 150C/RHCE*ce In analyzing the sequences of RHD and RHCE alleles in 48C, 733G, 1006T (which encodes a D+ [DIIIa] C–V–VS+hrB– 605 samples from random African Americans (donors and HrB– phenotype) and RHD*DIIIa-CE(4-7)-D/RHCE*ce 48C, patients with sickle cell disease), we found 43 samples and 733G, 1006T (previously known as r′S; now as r′S type 1, 44 alleles (one sample had a homozygous change) exhibiting which encodes a D–C+V–VS+hrB– HrB– phenotype).27 RHCE*ce 48C, 733G, 1006T, which corresponds to an allele As several attempts to extract cDNA from the Bastiaan frequency of 0.036. Of these, 31 were assumed to be in cis to sample failed, we were limited to analyzing gDNA to determine RHD*DIIIa-CE(4-7)-D, 12 in cis to RHD*DIIIa, and one in cis the alleles present in this sample. As stated, gDNA revealed an to RHD. The latter haplotype is rare, if not novel. unexpected RHD*DIIIa/RHD*DIIIa result and no detection of a hybrid allele. However, we have previously noticed the inability to Molecular Analyses of RHD detect an RHD*DIIIa-CE(4-7)-D allele in trans to an RHD*DIIIa Sequence analyses of RHD gDNA from the Bastiaan allele using genomic DNA. In contrast, analyses of cDNA clearly sample showed RHD*DIIIa (ISBT allele name RHD*03.01) show the hybrid allele. In earlier investigations, three samples and RHD*DIIIa 150C, a novel allele reported here (Table 2). that gave a cDNA sequencing result of RHD*DIIIa/RHD*DIIIa- Testing gDNA on the RHD DNA array showed RHD*DIIIa/ CE(4-7)-D were followed up with gDNA analyses, which gave a RHD*DIIIa, which is consistent given that the array does not sequencing result of RHD*DIIIa/RHD*DIIIa. The RHD DNA probe for the nucleotide at position 150. Our DNA result is BeadChip (which uses gDNA) gave the same RHD*DIIIa/ inconsistent with the RBC typings on Bastiaan, her second RHD*DIIIa result. This discrepancy between gDNA and cDNA child, and his father that were reported by Shapiro et al. (Table has also been demonstrated by others.28 3).1 As the father was D–C–, the child must have inherited the After an upgrade by BioArray Solutions of their BeadChip D–C+ phenotype from his mother. As Bastiaan typed D+ but interpretative software, RHD*DIIIa and hybrid samples RhD– (i.e., nonreactive with an anti-D from a person with the were more accurately detected on the RHD BeadChip and the interpretation was in concordance with cDNA results: Table 2. RHD and RHCE alleles in Bastiaan and the other three the DIIIa-CE(4-7)-D hybrid15 was detected when paired with samples with novel RHD alleles normal D (n = 15), DIIIa-CE(4-7)-D (n = 10), DAU-0 (n = Donor code name RHD* RHCE* 5), DAU-3 (n = 2), DOL (n = 1), and pseudo D (n = 1), but Bastiaan DIIIa 150C, DIIIa-CE(4-7)-D† ceS, ceS still not when paired with DIIIa (n = 3). Three samples that Alson DIIIa 150C, homozygous or hemizygous‡ ceS, ceS were typed by cDNA as RHD*DIIIa-CE(4-7)-D/RHD*DIIIa Dennis D 186T, weak D type 4.2.2 ceAR, ceSM were still called RHD*DIIIa/RHD*DIIIa by RHD BeadChip Sybil D 186T, weak D type 4.2.2 ceAR, ceSM using the new software and by sequencing of gDNA (Table 4). †Although DNA analyses predicted RHD*DIIIa/RHD*DIIIa 150C, the Thus, without access to cDNA for Bastiaan, it is not possible allele given here is an interpretation based on both DNA analyses and to verify our interpretation that, based on both gDNA and hemagglutination results on the proband and her family. hemagglutination results, Bastiaan is actually RHD*DIIIa- ‡It was not possible to determine whether two copies of this allele were present or there was one copy in trans to a deleted RHD allele. CE(4-7)-D/RHD*DIIIa 150C.

100 IMMUNOHEMATOLOGY, Volume 28, Number 3, 2012 Molecular background of RHCE in Bastiaan

Table 4. RHD*DIIIa and RHD*DIIIa-CE-D hybrid samples tested (Dennis and Sybil) had the 186G>T change. Both samples also by both cDNA sequencing and by gDNA analysis. carried a Weak D Type 4.2.2 allele (602C>G, 667T>G, 744C>T, No. of samples tested RHD* alleles by cDNA and gDNA Concordance 957G>A, 1025T>C). All changes were discovered by the DIIIa/D 13 13 concordant sequencing of RHD-specific cDNA. To determine which allele DIIIa/DIIIa 12 12 concordant carried the 186T change, the RHD exons 1–4 cDNA product DIIIa/other D allele 4 4 concordant from Dennis was cloned. Clones with 186G (in exon 2) had DIIIa-CE(4-7)-D/D 15 15 concordant 602C>G (in exon 4), whereas clones with 186G>T had 602C, DIIIa-CE(4-7)-D/DIIIa-CE(4-7)-D 10 10 concordant confirming that the 186G>T was carried byRHD and not by DIIIa-CE(4-7)-D/other D allele 8 8 concordant RHD*Weak D Type 4.2.2. DIIIa-CE(4-7)-D/DIIIa 3 3 not concordant Samples with these novel RHD alleles were further investigated to determine their corresponding RHCE alleles. Interestingly, both samples with RHD*DIIIa 150C had homozygous RHCE*ceS (48G>C; 733C>G; 1006G>T), and Discovery of Novel RHD Alleles both samples with RHD*D 186T had RHCE*ceSM (48G>C, We observed that RHD*DIIIa from Bastiaan had a novel 712A>G, 818C>T) and RHCE*ceAR (48G>C; 712A>G; nucleotide change: RHD*DIIIa 150C. This RHD 150T>C is a 733C>G; 787A>G; 800T>A; 916A>G; Table 1). silent nucleotide change from RHCE (Val[GTT]50Val[GTC]). Of the 605 samples from random African Americans (blood Discussion donors or patients with sickle cell disease screened for another study), none had an RHD*150T>C change even though 38 Testing the sample from Bastiaan, the original proband carried an RHD*DIIIa (186G>T, 410C>T, 455A>C, 602C>G, with anti-hrB and anti-HrB, has shown that her V–VS+hrB– 667T>G, 819G>A) and 29 carried an RHD*DIIIa-CE-D hybrid HrB– phenotype is encoded by RHCE*ce 48C, 733G, 1006T allele (186G>T, 410C>T, 455A>C, RHCE exons 4–7). Of the regardless of whether it is in trans to RHD*DIIIa or to 118 samples from patients or donors of African ancestry who RHD*DIIIa-CE(4-7)-D. As the effect of the Rh complex were selected because they were known to have diverse Rh constituents in the RBC membrane on the expression of phenotypes (based on typing discrepancies, the presence of antigens is not known, it should rather be stated that Bastiaan’s alloantibodies in antigen-positive people, or the presence of genotype (RHD*DIIIa-RHCE*ceS and RHD*DIIIa-CE(4-7)-D- a low-prevalence antigen marker), 25 carried an RHD*DIIIa RHCE*ceS) is the gold standard for the hrB–HrB– phenotype. of which one (Alson) had an RHD*150T>C change, and 19 The RHCE*ce 48G>C, 733C>G, 1006G>T allele is frequently carried an RHD*DIIIa-CE(4-7)-D. To confirm the presence of in cis to three differentRHD alleles: RHD*DIIIa-CE(4-7)-D, the 150T>C change found in sequencing genomic DNA from RHD*D-CE(4-7)-D, and RHD*DIIIa (previously DIII type Bastiaan and RHD-specific cDNA from Alson, the genomic 5),5,27 which explains why many patients with this haplotype RHD exon 2 was amplified (as described previously20) from are stimulated to produce alloanti-D as well as alloanti-e, or both samples and cloned. Further verification was obtained by alloanti-hrB. Such cases can be complex to resolve. digesting the genomic RHD exon 2 products with MscI, which As the antibodies in Bastiaan’s plasma caused HDFN in her loses a cut site in the presence of 150C. Bastiaan showed a second child, Shapiro et al.1 tested and reported phenotyping heterozygous banding pattern for 150T/C, and Alson showed results not only on Bastiaan’s husband and affected child a homozygous (or hemizygous) banding pattern for 150C/C but also on 98 other family members in three generations. (Table 2). The pedigree allowed the Rh phenotype of Bastiaan to be In analyzing samples for the presence of RHD*DIIIa determined. Although the phenotype denotations were in (186G>T, 410C>T, 455A>C, 602C>G, 667 T>G, 819G>A), the Wiener terminology system, which is no longer familiar, we discovered a new RHD allele that contained only one the RBC typings given by Shapiro et al.1 were sufficient nucleotide change (186G>T; Leu62Phe). Of the 605 samples to allow us to interpret the Rh phenotypes using current from the random African American donors and patients with terminology (Table 2). That the father was D–C– made the sickle cell disease, 470 carried at least one wild-type D allele, child’s phenotype particularly informative. The child’s D–C+ of which none had the lone 186G>T change. However, of the phenotype most likely was a consequence of the r′S haplotype 118 selected patient or donor samples with known diverse (D–C+V–VS+) inherited from Bastiaan. Clearly, Bastiaan has Rh phenotypes, 60 had a wild-type D allele, and of these, two the DIIIa 150C-ceS/RHD*DIIIa-CE(4-7)-D haplotype.

IMMUNOHEMATOLOGY, Volume 28, Number 3, 2012 101 M.E. Reid et al.

We report here the first examples of alleles that had either a . 8 Reid ME, Lomas-Francis C. The blood group antigen factsbook. single 186G>T change (Leu62Phe) in RHD or a single 150T>C 2nd ed. San Diego, CA: Academic Press, 2004. change in RHD*DIIIa. RHD alleles known to harbor a 186G>T . 9 Flegel WA. Molecular genetics and clinical applications for RH. Transfus Apher Sci 2011;44:81-91. S change (silent) include ce -type 1, DIIIa (DIII type 5), DIII type 10. Patnaik SK, Helmberg W, Blumenfeld OO. BGMUT: NCBI 4, and DIVa (DIVa.2). RHD alleles known to harbor a 150T>C dbRBC database of allelic variations of genes encoding antigens silent nucleotide change include RHD*DIIIb, RHD*DIII type 7, of blood group systems. Nucleic Acids Res 2012;40:D1023-9. RHD*DKK, and other hybrid alleles with exon 2 from RHCE. 11. Flegel WA. The Rhesus site: explore the intricacies of the most complex blood group gene locus. Ulm, Germany: Institut für In conclusion, the gold standard RHCE allele that is Klinische Transfusionsmedizin und Immungenetik Ulm 2007. associated with the hrB– HrB– phenotype is r′S; the genotype http://www.uni-ulm.de/%7Ewflegel/RH/. is RHD*DIIIa-RHCE*ceS/RHD*DIIIa-CE(4-7)-D-RHCE*ceS. 12. Wagner FF, Frohmajer A, Flegel WA. RHD positive haplotypes B B W in D negative Europeans. BMC Genet 2001;2:10-24. Phenotypes that lack hr and Hr are Rhnull, D– –, Dc–, DC –, S S S 13. Simsek S, de Jong CA, Cuijpers HT, et al. Sequence analysis r′ type 1, r′ type 2, and DIIIa-ce . of cDNA derived from reticulocyte mRNAs coding for Rh polypeptides and demonstration of of E/e and C/c Acknowledgments polymorphisms. Vox Sang 1994;67:203-9. 14. Pham BN, Peyrard T, Juszczak G, et al. Heterogeneous molecular background of the weak C, VS+, hr B–, Hr B– We thank Sheryl L. Bruns, from Hoxworth Blood Center, phenotype in black persons. Transfusion 2009;49:495-504. University of Cincinnati, for technical assistance and Bob 15. Faas BHW, Beckers EAM, Wildoer P, et al. Molecular Ratner for help in the preparation of this manuscript. This background of VS and weak C expression in blacks. Transfusion 1997;37:38-44. study was supported in part by grant NIH HL091030 (CHH, 16. Reid ME, Beal C, Mallory D, Combs MR, Issitt PD. The RH MER). The authors certify that they have no affiliation with antigens hrB (RH31) and VS (RH20) may be antithetical. or financial involvement in any organization or entity with (Abstract) Transfusion 1995;35:51S. a direct financial interest in the subject matter or materials 17. Reid ME, Storry JR, Issitt PD, et al. Rh haplotypes that make e B discussed in this manuscript. but not hr usually make VS. Vox Sang 1997;72:41-4. 18. Beal CL, Oliver CK, Mallory DM, Issitt PD. The r’ gene is overrepresented in hrB-negative individuals. References Immunohematology 1996;11:74-7. 19. Westhoff CM, Vege S, Hue-Roye K, Nickle P, Lomas-Francis . 1 Shapiro M, Le Roux M, Brink S. Serology and genetics of a new C, Reid M. The RHCE*ceMO allele is linked to RHD*DAU0 blood factor: hrB. Haematologia 1972;6:121-8. and encodes an hrS– and hrB– red cell phenotype (abstract). . 2 Moores P, Smart E. Serology and genetics of the red blood cell Transfusion 2007;47(Suppl):155A-6A. factor Rh34. Vox Sang 1991;61:122-9. 20. Hipsky CH, Hue-Roye K, Lomas-Francis C, Huang C-H, Reid . 3 Rosenfield RE, Allen FH, Jr., Swisher SN, Kochwa S. A ME. Molecular basis of the rare gene complex, DIV(C)–, which review of Rh serology and presentation of a new terminology. encodes four low-prevalence antigens in the Rh blood group Transfusion 1962;2: 287-312. system. Vox Sang 2012;102:167-70. . 4 Issitt PD. An invited review: the Rh antigen e, its variants, 21. Hipsky CH, Lomas-Francis C, Fuchisawa A, et al. RHCE*ceCF and some closely related serological observations. encodes partial c and partial e but not CELO an antigen Immunohematology 1991;7:29-36. antithetical to Crawford. Transfusion 2011;51:25-31. B b . 5 Pham B-N, Peyrard T, Tourret S, et al. Anti-Hr and anti-hr 22. Perco P, Shao CP, Mayr WR, Panzer S, Legler TJ. Testing revisited. Transfusion 2009;49:2400-5. for the D zygosity with three different methods revealed . 6 Noizat-Pirenne F, Lee K, Le Pennec P-Y, et al. Rare RHCE altered Rhesus boxes and a new weak D type. Transfusion phenotypes in black individuals of Afro-Caribbean origin: 2003;43:335-9. identification and transfusion safety. Blood 2002;100:4223-31. 23. Denomme G, Fernandes BJ. The assignment of RHD . 7 Shapiro M. Serology and genetics of a new blood factor hrS. J gene zygosity by densitometric quantification (Abstract). Forensic Med 1960;7:96-105. Transfusion 1999;39(Suppl 1):51s.

102 IMMUNOHEMATOLOGY, Volume 28, Number 3, 2012 Molecular background of RHCE in Bastiaan

24. Wagner FF, Moulds JM, Tounkara A, Kouriba B, Flegel WA. 28. Vege S, Westhoff CM. Identification of altered RHD and RHCE RHD allele distribution in Africans of Mali. BMC Genet alleles: A comparison of manual and automated molecular 2003;4:14-20. methods. In: Moulds JM, Ness PM, Sloan SR, eds. BeadChip 25. Matheson KA, Denomme GA. Novel 3’Rhesus box sequences molecular immunohematology. New York, NY: Springer confound RHD zygosity assignment. Transfusion 2002;42:645- Science+Business Media, 2010:121-31. 50. 26. Reid ME, Storry JR, Sausais L, et al. DAK, a new low- Marion E. Reid, PhD, DSc (Hon.), (corresponding author), and incidence antigen in the Rh blood group system. Transfusion Christine Halter Hipsky MS, Laboratory of Immunochemistry, 2003;43:1394-7. and Randall W. Velliquette, MS, and Christine Lomas-Francis, BS, 27. Westhoff CM, Vege S, Halter-Hipsky C, et al. DIIIa and DIII FIBMS, Laboratory of Immunohematology and Genomics, New York Type 5 are encoded by the same allele and are associated with Blood Center, 310 East 67th Street, New York, NY 10065; Kathleen altered RHCE*ce alleles: clinical implications. Transfusion 2010;50:1303-11. Larimore, MT(ASCP)SBB, Hoxworth Blood Center, Cincinnati, OH; and Coral Olsen, BS, Specialized Laboratory Services, South African National Blood Services, Durban, South Africa.

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Letter from the Editor

Serologic Method Reviews—A New Feature

The editors of Immunohematology are excited to announce compile them and publish them as a collection. We anticipate a new review series, Serologic Method Reviews. Authors that the individual method reviews will be a resource for have been recruited from the immunohematology reference incumbent and new laboratory staff and that a copy of the laboratory community to draft reviews of specific serologic collected methods will be a must-have for transfusion medicine methods. (See table for a sampling of methods.) Each review will facilities, libraries, and personal bookshelves. address aspects of the method such as its principle, indications, specific reagents and supplies, high-level procedural steps in a Sandra Nance table format, detailed procedural information, limitations, and Editor-in-Chief references. We will be publishing these reviews individually Immunohematology in successive issues of Immunohematology, and we expect to

Acid elution Acidification of serum Adsorption of cold agglutinins with rabbit RBCs (REST) Allogeneic red cell adsorption for removal of warm autoantibody Antibody titration using master dilution CDP dissociation of IgG from RBCs Determination of antigen type of RBCs with a positive DAT EDTA glycine acid treatment of RBCs Freeze-thaw (Lui) elution Freezing and recovering rare RBCs using glycerol Identification of high titer, low avidity-like antibodies Polyethylene glycol antiglobulin test (PEG-AGT) Prewarm saline antiglobulin test Recovery of autologous neocytes by microhematocrit centrifugation Recovery of autologous sickle RBCs by hypotonic wash Thermal amplitude tests (quick screens) Use of enzymes—ficin, papain, trypsin Warm autoadsorption with enzyme-treated RBCs ZZAP treatment of RBCs

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Specialist in Blood Bank (SBB) Program The Department of Transfusion Medicine, National Institutes of Health, is accepting applications for its 1-year Specialist in Blood Bank Technology Program. Students are federal employees who work 32 hours/week. This program introduces students to all areas of transfusion medicine including reference serology, cell processing, HLA, and infectious disease testing. Students also design and conduct a research project. NIH is an Equal Opportunity Organization. Application deadline is December 31, 2012, for the July 2013 class. See www.cc.nih.gov/dtm > education for brochure and application. For further information contact Karen M. Byrne at (301) 451-8645 or [email protected]

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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 2013 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.

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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 28, Number 3, 2012 111 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 ×7ʺ 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]

112 IMMUNOHEMATOLOGY, Volume 28, Number 3, 2012

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