title: Improved flow cytometry based cytotoxicity and binding assay for clinical antibody HLA crossmatching.
abstract: The presence of preformed donor-specific HLA antibodies leads to early antibody mediated kidney allograft rejection. Therefore, detection and avoidance of donor reactive HLA antibodies prior to transplantation is of outmost importance in order to minimize the risk of rejection. Detection of pre-formed HLA antibodies is currently performed using complement-dependent cytotoxicity (CDC) assay alone or together with a flow cytometry based crossmatch (FCXM). This study was initiated to further evaluate our recently developed flow cytometry based procedure for determination of both cytotoxicity of and IgG binding to donor-derived lymphocytes by HLA antibodies. Highly enriched immuno-magnetic bead purified T and B lymphocytes were used as target cells for patient sera using 96-well plates. Importantly, the assay shows high sensitivity and specificity as determined by HLA typed donor cells and serum with defined HLA antibody IgG and C1q. Based on this and additional data generated in this paper, such as evaluation of appropriate serum and complements incubation times and assay reproducibility and stability, will enable us to more rapidly implement this assay in our clinical laboratory routines. In addition, we demonstrate that FCtox crossmatching of deceased donor cells has superior specificity compared to conventional CDC assay especially regarding high frequencies of false-positive reactions.
authors.affiliation: Department of Laboratory Medicine, Division of Clinical Immunology and Transfusion Medicine, Karolinska Institutet and Karolinska University Hospital, Stockholm, Sweden. Electronic address: mats.alheim@karolinska.se.
authors.forename: Mats
authors.initials: M
authors.lastname: Alheim
authors.affiliation: Department of Laboratory Medicine, Division of Clinical Immunology and Transfusion Medicine, Karolinska Institutet and Karolinska University Hospital, Stockholm, Sweden.
authors.forename: Prashanta Kumer
authors.initials: PK
authors.lastname: Paul
authors.affiliation: Department of Laboratory Medicine, Division of Clinical Immunology and Transfusion Medicine, Karolinska Institutet and Karolinska University Hospital, Stockholm, Sweden.
authors.forename: Dan-Mikael
authors.initials: DM
authors.lastname: Hauzenberger
authors.affiliation: Department of Laboratory Medicine, Division of Clinical Immunology and Transfusion Medicine, Karolinska Institutet and Karolinska University Hospital, Stockholm, Sweden; Department of Biosciences and Nutrition, Karolinska Institutet, NOVUM, SE-141 57 Stockholm, Sweden.
authors.forename: Ann-Charlotte
authors.initials: AC
authors.lastname: Wikström
mesh_terms: D000906:Antibodies / Q000097:blood / Q000276:immunology*; D003165:Complement System Proteins / Q000276:immunology; D003601:Cytotoxicity Tests, Immunologic / Q000379:methods; D005434:Flow Cytometry / Q000379:methods; D006680:HLA Antigens / Q000276:immunology*; D006650:Histocompatibility Testing / Q000379:methods; D006801:Humans; D018189:Immunomagnetic Separation / Q000379:methods; D007963:Leukocytes, Mononuclear / Q000276:immunology; D008214:Lymphocytes / Q000276:immunology / Q000378:metabolism; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D000906:Antibodies; D006680:HLA Antigens; D003165:Complement System Proteins
keywords: C1q assay; Complement-dependent lymphocytotoxicity; Flow cytometry crossmatch; HLA antibodies; Transplantation