Cytotoxic flow cytometric crossmatch in renal transplantation: A single assay to simultaneously detect antibody binding and cytotoxicity

Objectives: The complement-dependent lymphocytotoxicity crossmatch (CDC-XM) detects cytotoxic parameters of preformed antibodies. The flow cytometric crossmatch (FCXM) is used to detect the binding of recipient antibodies to donor cells. Because these two assays provide different information, both methods are often performed to assess the compatibility of donor-recipient pairs. The aim of this study was to develop a single assay that can simultaneously detect antibody binding and cytotoxicity. Methods: A procedure called cytotoxic flow cytometric crossmatch (cFCXM) that determines cell death and antibody binding simultaneously was developed. The assay was validated in parallel with extended incubation CDC-XM. Receiver operating characteristic analysis was used to determine the cut-off level. Furthermore, pretransplantation sera from seven recipients with pretransplantation donor-specific antibodies (DSA) and negative CDC-XM were retrospectively tested for cFCXM (4 without antibody-mediated rejection (AMR) and three with AMR). Results: The optimal method for the simultaneous detection of antibody binding and cytotoxicity in a single assay has been determined. Four of four patients (100%) with pretransplantation DSA and without AMR had negative cFCXM in both parameters. Of three patients with pretransplantation DSA who developed AMR, two patients (66.7%) had positive B-cell cFCXM in both parameters, and 1 patient (33.3%) had positive T-cell cFCXM in a binding parameter only. The first patient had anti-DR9, DR53, DQ9, the second patient had anti-A11, DR12 and the last one had an anti-B46 in their pretransplantation sera. These 3 cases experienced biopsy-proven AMR after living-donor kidney transplantation. Conclusion: The newly developed assay, cFCXM, can simultaneously determine cytoxicity and antibody binding using a single platform. Furthermore, this assay can detect clinically significant HLA alloantibodies undetectable by conventional crossmatches. The cFCXM could serve as a new tool for the detection of a recipient's alloantibodies.