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Rbgp-An-International-Study.Pdf IMMUNOHEMATOLOGY Recombinant blood group proteins facilitate the detection of alloantibodies to high-prevalence antigens and reveal underlying antibodies: results of an international study Axel Seltsam,1 Franz Wagner,1 Mark Lambert,2 Tom Bullock,3 Nicole Thornton,3 Erwin A. Scharberg,4 Daniela Grueger,5 Clemens Schneeweiss,5 and Rainer Blasczyk6 n current blood transfusion practice, red blood cell BACKGROUND: Alloantibodies to high-prevalence red (RBC) antibodies are identified using panels of blood cell (RBC) antigens are not easily identified by human RBCs pretyped for the most common blood routine serologic techniques. This multicenter study was group antigens. The specificity of a given antibody is conducted to test the effectiveness of recombinant Iidentified based on the pattern of reactivity observed blood group proteins (rBGPs) at regional and interna- when serum is tested with the cell panel. Since many anti- tional RBC reference laboratories. gens are expressed on RBCs, antibody identification in STUDY DESIGN AND METHODS: Single or mixed RBC-based assays relies on nonreactivity of an antibody soluble rBGPs (Lu, Yt, Kn, JMH, Sc, Rg, Ch, Do, and with panel cells lacking the corresponding antigen. This Cr) were assessed for their ability to inhibit the reactiv- method of antibody identification is challenged if autoan- ity of antibodies to specific antigens. Initially, the effect tibodies, multiple antibodies, or antibodies to high- of rBGPs was validated by testing panels of well- prevalence antigens are present. In such cases, rare characterized patient serum samples containing antisera and cells as well as specially trained personnel antibodies to high-prevalence antigens in the hemagglu- not available to routine laboratories are required for tination inhibition assay. Subsequently, the rBGPs were prospectively used for routine antibody identification and the results were compared to those obtained with ABBREVIATIONS: CR1 = Complement Receptor 1; RBC-based diagnostics. HIA = hemagglutination inhibition assay; IBGRL = International RESULTS: Panels of predefined antibodies to high- Blood Group Reference Laboratory; LHR = long homologous prevalence antigens were completely and specifically repeat; MAEIA = monoclonal antibody–specific immobilization neutralized by the corresponding rBGP specificities. For of erythrocyte antigens; rBGP(s) = recombinant blood group prospective identification, antibodies to high-prevalence protein(s). antigens (n = 62) were specifically inhibited by the cor- 1 responding rBGP specificities except for some Comple- From the German Red Cross Blood Service NSTOB, Springe, 2 ment Receptor 1–related antibodies, which may be Germany; the National Blood Centre, Irish Blood Transfusion 3 directed to epitopes not expressed on the truncated Service, Dublin, Ireland; the International Blood Group recombinant Kn. In 14 cases, additional clinically rel- Reference Laboratory, NHSBT Filton Blood Centre, Bristol, 4 evant alloantibodies were identified. In cross-matching, UK; the Institute for Transfusion Medicine and the rBGPs were successfully used to inhibit the reactiv- Immunohematology, German Red Cross Blood Service 5 ity of clinically irrelevant antibodies to high-prevalence Baden-Württemberg–Hessen, Baden-Baden, Germany; Imusyn, 6 antigens to determine compatibility between donor and Hannover, Germany; and the Institute for Transfusion recipient. Medicine, Hannover Medical School, Hannover, Germany. CONCLUSION: rBGPs enable the identification of anti- Address reprint requests to: Axel Seltsam, MD, MHBA, bodies to high-prevalence antigens without the need for German Red Cross Blood Service NSTOB, Institute Springe, rare RBC reagents, which are often unavailable. Under- Eldagsener Strasse 38, 31832 Springe, Germany; e-mail: lying antibodies can be reliably detected and cross- [email protected]. matching results validated, resulting in a more efficient Received for publication September 30, 2013; revision blood supply for immunized patients. received November 18, 2013, and accepted November 25, 2013. doi: 10.1111/trf.12553 © 2014 AABB TRANSFUSION 2014;54:1823-1830. Volume 54, July 2014 TRANSFUSION 1823 SELTSAM ET AL. antibody identification. This often results in the referral of Initially, the inhibitory effect of rBGPs was validated samples to specialized reference laboratories, ultimately by testing panels of well-characterized patient serum leading to a delay in antibody identification and a possible samples containing antibodies to high-prevalence anti- delay in patient care. gens in the hemagglutination inhibition assay (HIA). A The availability of recombinant blood group proteins total of 40 antiserum samples with predefined antibodies (rBGPs) enables the development of new antibody detec- to high-prevalence antigens obtained either from immu- tion assays based on the use of defined antigens for each nized in-house patients or from Serum, Cells and Rare reaction.1 Unlike current procedures, this novel approach Fluid (SCARF) exchange program members were used to directly indicates antibody specificities when an antibody test the specificity and activity of the single rBGP specifici- reacts with its respective recombinant antigen. Moreover, ties and the rBGP cocktail. Patient serum samples con- it allows detection and identification of blood group anti- taining RBC alloantibodies of other common or rare bodies in a single step, without the need for time- specificities (e.g., anti-K, anti-D, anti-c, anti-Jka, anti-Fya, consuming additional tests. In particular, rBGPs may have anti-Fyb, anti-S, anti-Vel, and anti-Lan) were used as ref- wide applications in blood group serology as they can be erence samples. used with different technical platforms, such as enzyme- Subsequently, the rBGPs were prospectively used by linked immunosorbent assay, color-coded microspheres, all participating laboratories for routine antibody identi- and protein microarrays.2-5 Previous studies have shown fication, and the results were compared to those obtained that rBGPs may be of great value for identification of with RBC-based diagnostics. At the two laboratories in alloantibodies to high-prevalence blood group antigens, Germany, where pretransfusion serologic cross-matching which is still a major challenge for immunohematology is mandatory, the rBGPs were used to inhibit the reactivity laboratories.2-12 If rBGPs are used as soluble protein of clinically irrelevant high-prevalence antibodies to reagents in the hemagglutinin inhibition assay, their determine compatibility between donor and recipient. implementation in blood group serology is straightfor- ward, even in routine laboratories. Such reagents would enable serologists to identify antibodies directed against a Recombinant RBC proteins host of distinct high-prevalence blood group antigens. Ten soluble rBGPs derived from eukaryotic expression In this study, a panel of rBGPs was assessed by systems (Imusyn GmbH, Hannover, Germany) were used national and international blood group serology reference in the study (Table 1). In earlier published and unpub- laboratories for their ability and usefulness to detect and lished studies, they were designed and validated to identify antibodies to high-prevalence blood group anti- express the most relevant polymorphic and high- gens. First, the inhibitory effect of rBGPs was validated prevalence antigens of the respective blood group using predefined antisera. Subsequently, the recombinant systems.3-5,8,10 A cocktail of five different rBGP specificities proteins were prospectively used for routine antibody (rPromix) containing Sc, Rg, Ch, Kn, and JMH was pre- identification, and the results were compared to those pared by mixing equal volumes of the respective single- obtained with RBC-based diagnostics. rBGP solutions. The single-rBGP and rPromix products were supplied in storage buffer (150 mmol/L NaCl, MATERIALS AND METHODS 7 mmol/L sodium phosphate, pH 7.8, 0.1% ProClin 300, Sigma-Aldrich, St Louis, MO) at a concentration of 0.5 mg/ Study design mL, with a purity of more than 85% and a shelf life of 6 Over a 6-month period, single soluble rBGPs (Lu, Yt, Sc, months. All protein solutions were stored at 2 to 8°C Doa,Dob, Rg, Ch, Cr, Kn, and JMH) or cocktails thereof throughout the study. The single and mixed rBGPs were were assessed for their ability to inhibit the reactivity of stored from 0 to 7 months before the start of validation antibodies to the corresponding antigens. Two national testing. reference laboratories in Germany (the German Red Cross Blood Service Lower Saxony, Saxony-Anhalt, Thuringia, Oldenburg, and Bremen (NSTOB)/Institute Springe in Antibody detection procedures Springe and the German Red Cross Blood Service Baden- Single and mixed rBGPs were used in the HIA for antibody Württemberg–Hessen/Institute for Transfusion Medicine detection, which is based on the principle that reactions and Immunohematology in Baden-Baden), a national ref- between RBC antibodies and test cells carrying the corre- erence laboratory in Ireland (National Blood Centre, Irish sponding antigens can be neutralized by incubating test Blood Transfusion Service in Dublin), and an international samples with reagents containing soluble antigenic pro- reference laboratory in the United Kingdom (Interna- teins. The ability of rBGPs to inhibit patient serum reac- tional Blood Group Reference Laboratory [IBGRL], tivity was assessed in an HIA using RBCs that tested National Health Service Blood and Transplant Filton positive for the respective high-prevalence
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