title: Flow cytometry-based cytotoxicity and antibody binding assay.
abstract: Human leukocyte antigen (HLA) antibodies with the ability to activate complement are associated with an increased risk of early antibody-mediated graft rejection in kidney transplantation (KTx). Detection of these potentially harmful complement-fixing HLA antibodies is commonly performed via the complement-dependent cytotoxicity (CDC) assay according to protocols that were developed as early as 40 years ago. The read-out for this assay is based on manual scoring by visual inspection of cells under a fluorescence microscope. CDC is often used in combination with the flow cytometry-based lymphocyte crossmatch assay (FCXM), which, with high sensitivity, detects HLA antibody binding. Here we describe a new approach wherein both cytotoxicity and antibody binding can be simultaneously assessed with flow cytometry. Two strategies are described, using either magnetic bead-enriched T and B lymphocytes or bulk peripheral blood mononuclear cells (PBMC) as donor target cells.
authors: Department of Laboratory Medicine, Division of Clinical Immunology and Transfusion Medicine, Karolinska University Hospital, Stockholm, Sweden.
authors: Mats
authors: M
authors: Alheim
mesh_terms: D000906:Antibodies / Q000378:metabolism*; D001402:B-Lymphocytes / Q000276:immunology; D001788:Blood Grouping and Crossmatching; D003601:Cytotoxicity Tests, Immunologic / Q000379:methods*; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D008863:Microspheres; D013601:T-Lymphocytes / Q000276:immunology; D014019:Tissue Donors
chemical_list: D000906:Antibodies
keywords: HLA antibodies; complement dependent cytotoxicity; crossmatching; flow cytometry; kidney transplantation