Cytotoxicity and antibody binding by flow cytometry: A single assay to simultaneously assess two parameters

Abstract

Cell death, as determined by complement-dependent cytotoxicity or antibody binding, as assessed by flow cytometry, are the two main methods histocompatibility laboratories use to identify HLA antibodies. Conventional cytotoxicity assays, even anti-human globulin (AHG) enhanced, are relatively insensitive, subjective and require complement fixation. In contrast, antibody binding assays (e.g.: flow cytometry crossmatch, FCXM) are sensitive, objective, and complement independent. When sera from potential transplant recipients test positive by both methods, most centers would not proceed to transplant unless patients undergo desensitization. However, when the CDC crossmatch is negative but the FCXM positive, how or whether to proceed is controversial. Frequently, both assays are performed. In this study, we developed a comprehensive procedure that determines cell death, and antibody binding in a single assay: the cytotoxic flow crossmatch (cFCXM). The assay was validated in parallel studies with two other conventional methodologies, namely AHG and FCXM. Using a combination of eight cells and 15 sera, we have determined an optimal method for the simultaneous detection of HLA antibody binding and cytotoxicity using a single platform. Additionally, we have identified three distinct patterns of HLA antibody reactivity: (1) AHG(pos)/cFCXM(pos)/FCXM(pos), (2) AHG(neg)/cFCXM(neg)/FCXM(pos), and (3) a previously unrecognized category, namely, AHG(neg)/cFCXM(pos)/FCXM(pos). These studies document that cytotoxicity and antibody binding can be simultaneously assessed and marks the first demonstration of complement-dependent HLA antibodies undetectable by conventional cytotoxicity. Routine application of this assay may provide new insights regarding transplantation of patients presenting as AHG(neg)/FCXM(pos) by classifying them according to their cFCXM status.