Flow Cytometry–Based Cytotoxicity and Antibody Binding Assay

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. Curr. Protoc. Cytom. 66:6.34.1-6.34.11. © 2013 by John Wiley & Sons, Inc.