Flow cytometric technology is used to perform pre-transplant crossmatching with donor lymphocytes and recipient sera. The traditional B lymphocyte (B-cell) and T lymphocyte (T-cell) flow cytometric crossmatch (FCXM) detects anti-Human Leukocyte Antigen (HLA) class I and class II antibodies bound to lymphocytes (Bray, Terasaki). Traditionally the C4d fragment generated during the antibody-mediated classical complement activation pathway is diagnostic of humoral renal allograft rejection (Moll). Complement activating anti-HLA class I antibodies are defined as a high risk factor for acute renal transplant rejection (Constance). Anti-HLA class II antibodies are of interest because they also contribute to allograft loss (Campos).

Current FCXM techniques do not discriminate between complement and non-complement activating anti-HLA antibodies. The goal of this research was to develop a four color FCXM which displays antibody binding of B-cells and T-cells indicating class I and class II response, which also displays whether or not the bound antibody is complement activating or cytotoxic. This cytotoxic flow cytometric crossmatch (CFCXM) procedure was designed to mimic the traditional B-cell and T-cell FCXM, but with the addition of complement and the viability dye 7-Amino-Actinomycin D (7-AAD) to detect cytotoxicity. The proposed procedure can separate HLA class I from class II antibodies bound to B-cells and T-cells, while simultaneously determining their cytotoxic potential.