IMMUNOHEMATOLOGY Implication of transfected cell lines for the detection of alloantibodies against human neutrophil antigen-3 Behnaz Bayat,* Yudy Tjahjono,* Silke Werth, Heike Berghöfer, Angelika Reil, Hartmut Kroll, Ulrich J. Sachs, and Sentot Santoso BACKGROUND: Alloantibodies against human neutro- phil antigen-3 (HNA-3) are responsible for the fatalities reported in transfusion-related acute lung injury. Conse- quently, reliable detection of these alloantibodies is mandatory to improve blood transfusion safety. In this study, we developed stable cell lines for the detection of HNA-3 antibodies. STUDY DESIGN AND METHODS: HEK293T were transfected with HNA-3a or HNA-3b constructs and sorted by flow cytometry according to high surface expression. Transfected cells were tested with sera containing HNA-3 antibodies in flow cytometry and anti- body capture assay (ACA). The results were compared with granulocyte agglutination test and granulocyte immunofluorescence test. RESULTS: In flow cytometry, 12 of 14 HNA-3a sera reacted specifically with HNA-3aa cells. One serum sample showed positive reaction with HNA-3bb cells. All HNA-3b sera recognized HNA-3bb cells. No reaction was observed with broad reactive antibodies against HLA Class I. In ACA, all HNA-3a sera (12/12) showed positive reactivity with HNA-3aa cells with no cross- reactivity with HNA-3bb cells. Again, all HNA-3b sera reacted with HNA-3bb cells only. Furthermore, genotyp- ing of 249 individuals detected a new HNA-3 allele caused by a nucleotide substitution C>T at Position 457 leading to L153F mutation in choline transporter-like protein-2. This mutation impairs polymerase chain reaction with sequence-specific primers based HNA-3a typing. However, analysis with cells expressing F153 isoform showed that this mutation did not alter the binding of HNA-3 antibodies. CONCLUSIONS: This study demonstrated that HEK293T cells expressing stable recombinant HNA-3 are suitable for the detection of HNA-3 alloantibodies allowing reliable screening of blood products. H uman neutrophil antigens (HNAs) are expressed on the neutrophil surface and play thereby an important role in the pathophysi- ologic mechanism of alloimmune-mediated neutropenia and transfusion-related acute lung injury (TRALI). TRALI is defined as an acute lung injury that is related to a blood transfusion. It is typically associated with fresh-frozen plasma, although TRALI cases caused by blood cells (platelets, red blood cells) have been reported. 1 Besides alloantibodies against HNAs, antibodies toward HLA Class I and II antigens are implicated in TRALI. 2,3 Currently, five HNA systems including HNA-1, -2, -3, -4, and -5 have been described. 4 Among HNAs, antibodies against HNA-3a cause more severe and fatal cases of TRALI. 5,6 Thus, reliable identification of HNA-3a and HNA-3b alloantibodies in plasma of blood donors is indispensible to improve blood transfusion safety. 7 HNA-3a is a high-frequency antigen in Caucasian and Asian populations with phenotype frequencies of 0.792 to 0.738. HNA-3b has a frequency of 0.207 to 0.262 in ABBREVIATIONS: ACA = antibody capture assay; CTL-2 = choline transporter-like protein-2; GAT = granulocyte agglutina- tion test; GIFT = granulocyte immunofluorescence test; MAIGA = monoclonal antibody immobilization of granulocyte antigens; PCR-SSP = polymerase chain reaction with sequence- specific primers; PVDF = polyvinyl difluoride. From the Institute for Clinical Immunology and Transfusion Medicine, Justus Liebig University Giessen, Giessen; the German Red Cross Blood Service West, Hagen; and German Red Cross Blood Transfusion Service NSTOB, Dessau, Germany. Address reprint requests to: Sentot Santoso, PhD, Institute for Clinical Immunology and Transfusion Medicine, Justus Liebig University Giessen, Langhansstrasse 7, 35385 Giessen, Germany; e-mail: sentot.santoso@immunologie.med.uni- giessen.de. *Both authors contributed equally in this study. Received for publication April 19, 2011; revision received July 27, 2011, and accepted July 29, 2011. doi: 10.1111/j.1537-2995.2011.03303.x TRANSFUSION 2012;52:613-621. Volume 52, March 2012 TRANSFUSION 613