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Immunohematology JOURNAL of BLOOD GROUP SEROLOGY and EDUCATION Immunohematology JOURNAL OF BLOOD GROUP SEROLOGY AND EDUCATION V OLUME 22, NUMBER 4, 2006 Immunohematology JOURNALOFBLOODGROUPSEROLOGYANDEDUCATION VOLUME 22, NUMBER 4, 2006 CONTENTS 161 Efficacy of murine monoclonal antibodies in RBC phenotyping of DAT-positive samples E. LEE, K. HART, G. BURGESS, G.R. HALVERSON,AND M.E. REID 166 Case report: moderate hemolytic disease of the newborn due to anti-G A.R. HUBER, G.T.LEONARD, R.W.DRIGGERS, S.B. LEARN,AND C.W.GILSTAD 171 Review: molecular basis of MNS blood group variants P.PALACAJORNSUK 183 Chimerism and mosaicism are important causes of ABO phenotyping discrepancies D. CHO, J.S. LEE, M.H.YAZER, J.W.SONG, M.G. SHIN, J.H. SHIN, S.P.SUH, M.J. JEON, J.Y.KIM, J.T.PARK,AND D.W.RYANG 188 Update on HDFN: new information on long-standing controversies A.F.EDER 196 In search of the Holy Grail: comparison of antibody screening methods T.S.CASINA 203 COMMUNICATIONS Letter to the editors Anti-Cra: pregnancy and transfusion support N.WIN AND M. NEEDS 205 Letter to the editors A reminder that ZZAP reagent removes complement in addition to IgG from coated RBCs R.M. LEGER AND G. GARRATTY 207 Letters from the editors Thank you to contributors to the 2006 issues Changes in the journal 209 211 212 ANNOUNCEMENTSCLASSIFIEDADADVERTISEMENTS 215 INSTRUCTIONSFORAUTHORS 216 INDEX — VOLUME 22, NOS. 1, 2, 3, 4, 2006 EDITORS-IN-CHIEF MANAGING EDITOR Sandra Nance, MS, MT(ASCP)SBB Cynthia Flickinger, MT(ASCP)SBB Philadelphia, Pennsylvania Philadelphia, Pennsylvania Connie M.Westhoff, MT(ASCP)SBB, PhD Philadelphia, Pennsylvania TECHNICAL EDITORS SENIOR MEDICAL EDITOR Christine Lomas-Francis, MSc Geralyn M. Meny, MD New York City, New York Philadelphia, Pennsylvania Dawn M. Rumsey,ART(CSMLT) Glen Allen, Virginia ASSOCIATE MEDICAL EDITORS David Moolton, MD Ralph R.Vassallo, MD Philadelphia, Pennsylvania Philadelphia, Pennsylvania EDITORIAL BOARD Patricia Arndt, MT(ASCP)SBB W.John Judd, FIBMS, MIBiol Mark Popovsky, MD Pomona, California Ann Arbor, Michigan Braintree, Massachusetts James P.AuBuchon, MD Christine Lomas-Francis, MSc Marion E. Reid, PhD, FIBMS Lebanon, New Hampshire New York City, New York New York City, New York Geoffrey Daniels, PhD Gary Moroff, PhD Susan Rolih, MS, MT(ASCP)SBB Bristol, United Kingdom Rockville, Maryland Cincinnati, Ohio Richard Davey, MD Ruth Mougey, MT(ASCP)SBB S. Gerald Sandler, MD Washington, District of Columbia Carrollton, Kentucky Washington, District of Columbia Sandra Ellisor, MS, MT(ASCP)SBB John J. Moulds, MT(ASCP)SBB David F.Stroncek, MD Anaheim, California Shreveport, Louisiana Bethesda, Maryland George Garratty, PhD, FRCPath Marilyn K. Moulds, MT(ASCP)SBB Marilyn J.Telen, MD Pomona, California Houston, Texas Durham, North Carolina Brenda J. Grossman, MD Paul M. Ness, MD St. Louis, Missouri Baltimore, Maryland EDITORIAL ASSISTANT PRODUCTION ASSISTANT Judith Abrams Marge Manigly COPY EDITOR ELECTRONIC PUBLISHER Lucy Oppenheim Paul Duquette Immunohematology is published quarterly (March, June, September, and December) by the American Red Cross, National Headquarters,Washington, DC 20006. Immunohematology is indexed and included in Index Medicus and MEDLINE on the MEDLARS system. The contents are also cited in the EBASE/Excerpta Medica and Elsevier BIOBASE/Current Awareness in Biological Sciences (CABS) databases. The subscription price is $30.00 (U.S.) and $35.00 (foreign) per year. Subscriptions, Change of Address, and Extra Copies: Immunohematology, P.O. Box 40325 Philadelphia, PA 19106 Or call (215) 451-4902 Web site: www.redcross.org/pubs/immuno Copyright 2006 by The American National Red Cross ISSN 0894-203X Efficacy of murine monoclonal antibodies in RBC phenotyping of DAT-positive samples E. LEE, K. HART, G. BURGESS, G.R. HALVERSON,AND M.E. REID Determining the phenotype of patient RBCs that are positive by the use chloroquine diphosphate (CDP), a combination DAT may prove problematic. Antigen typing of RBCs coated with enzyme/reducing agent (ZZAP,National Blood Service IgG requires direct agglutinating reagents or chemical treatment (such as chloroquine diphosphate [CDP] or citric acid) to remove Reagents, Birmingham, UK) which is composed of DTT sufficient IgG to permit testing with IAT-reactive reagents. The and papain, or EDTA/citric acid (Elu-Kit II, Gamma citric acid elution method is commonly used in the United States; Biologicals, Inc., Houston, TX). Several other methods however, antigens in the Kell system are altered to the extent that they may appear to be absent by this method. There are a limited are available to remove the antibody for serologic number of direct agglutinating monoclonal antibodies available. testing; however the RBCs are not suitable for testing Murine monoclonal antibodies provide an additional tool for typing after these treatments.1–5 RBCs with a positive DAT. Five murine monoclonal IgG antibodies (anti-K: MIMA-22, MIMA-23; anti-Kpa: MIMA-21, MIMA-27; anti-Fya: CDP and enzyme/reducing agent (ZZAP, National MIMA-19) were used in this study. Donor RBCs with known Blood Service Reagents) treatments can cause damage phenotypes were sensitized in vitro with alloanti-D, alloanti-c, and to the RBCs, resulting in the loss of some RBC antigens alloanti-K and with 20 autoantibodies (autoanti-D [n=3], autoanti-e 6–8 [n=5], autoanti-Ce/e [n=5], autoanti-e+D+E [n=1], autoanti-I [n=1], and possible invalid typing results. Additionally,CDP and nonspecific [n=5]) to simulate a positive in vivo DAT. The may not totally remove the coating autoantibody from sensitized RBCs were treated with CDP to remove IgG. To the RBCs and it does not remove complement determine the efficacy of the murine monoclonal antibodies when component 3 (C3).1 The proteolytic enzyme,papain,in testing DAT-positive samples, both sensitized and CDP-treated RBCs were tested with these monoclonal antibodies by the IAT using the enzyme/reducing agent product denatures some anti-mouse IgG. No discrepancies were noted with the MNS and Duffy antigens. The reducing agent, DTT, unsensitized, sensitized, or CDP-treated RBCs. An exception was denatures the antigens in Kell and Yt systems among noted with a potent autoanti-I, where direct agglutination of the sensitized RBCs was obtained.This study demonstrates the value of others. RBCs treated with the reagent combining both using murine monoclonal antibodies to determine the phenotype these chemicals (ZZAP, National Blood Service of RBCs with a positive DAT caused by autoantibodies (e.g., in Reagents), therefore, have limited applications for use autoimmune hemolytic anemia) and supports previous studies 9 showing that RBCs sensitized in vivo can be typed without in phenotyping studies. The citric acid elution chemical manipulation. Immunohematology 2006;22:161–165. method is commonly used in the United States, a major drawback being that antigens of the Kell system are Keys Words: blood groups, murine monoclonal significantly weakened by this method. The antibodies, anti-mouse IgG, autoantibodies, DAT, development of murine monoclonal antibodies has chloroquine diphosphate provided an additional tool to allow DAT-positive RBCs to be antigen typed.10,11 Determining an accurate RBC phenotype for patients whose RBCs are positive by the DAT can be problematic because these RBCs are already coated in Materials and Methods vivo with immunoglobulin, complement, or both; all Selected patient samples containing alloantibodies tests performed will be positive by IAT with antihuman and autoantibodies were obtained from the samples IgG reagents. There are very few IgM directly routinely referred to the Red Cell Immunohaematology agglutinating reagents available for the clinically Laboratory, National Blood Service, Colindale Centre, significant antibodies (i.e., anti-K, -Jka, -Jkb, -S, and -Fya). UK. Samples from three antenatal women with high One option is to chemically treat the DAT-positive RBCs titers of anti-D, -c, and –K, respectively, were selected to remove IgG autoantibodies. These treatments may for testing. A total of 20 autoantibodies from patients IMMUNOHEMATOLOGY, VOLUME 22, NUMBER 4, 2006 161 E. LEE ET AL. with autoimmune hemolytic anemia (AIHA) were also Bellshill, Scotland) at a 2% suspension. Both treated selected:anti-D (n=3),anti-e (n=5),anti-Ce/e (n=5),anti- and untreated RBCs were tested with murine e+D+E (n=1), anti-I (n=1), and nonspecific (n=5). monoclonal antibodies by IAT (40 µL:40 µL) and (See Table 1 as an example of phenotyping results incubated at 20°C for 30 minutes using a sheep-derived using murine monoclonal antibodies with IgG reagent directed at mouse IgG (sheep-anti-mouse unsensitized, sensitized, and CDP-treated RBCs for IgG,The Binding Site, Birmingham, UK, code PC271.X). sample 2). All samples were collected in EDTA and The sheep-derived reagent was diluted at a ratio of 1 in were tested within 5 days of collection. The murine 50 in a bovine serum albumin solution (1% BSA, monoclonal antibodies used included MIMA-19: Serologicals, Kankakee, IL) in PBS. (anti-Fya), MIMA-21: (anti-Kpa), MIMA-22: (anti-K), MIMA-23: (anti-K), and MIMA-27: a Table 1. An example of phenotype results of murine monoclonal antibodies with (anti-Kp ). Ten different commercial human unsensitized, sensitized, and CDP-treated RBCs (Sample 2)* monoclonal antibodies were also tested; anti-C, -c, -E, and -e (MS-24, MS-33, MS-258/ 906, MS-16/MS-21/MS-63 respectively, Biotest AG, Dreiech, Germany); anti-Cw, -Jka, b -Jk , and -S (MS-110, MS-15, MS-8, MS-94 MIMA-21 MIMA-27 Sample 2 Donor’s Treated or (5T72) Bio-Rad MIMA-19 a a respectively, Serologicals Ltd., Livingston, Alloanti-K Phenoytpe Untreated a a Scotland), anti-K (MS-56, Lorne Laboratories, RBCs Rh K Fya Kpa Anti-Fy Anti-Kp Anti-K MIMA-22 Anti-K MIMA-23 Anti-K (MS56) Lorne Anti-Fy Reading, UK), and anti-Fya (5T72, Bio-Rad, Anti-Kp DAT 0 Donor 1 R1R1 ++0Unsensitized 44404NT 0 Marines La Coquette, France).
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