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

Area of Science:

• Transplant immunology
• Histocompatibility testing
• Immunogenetics

Background:

• Histocompatibility laboratories utilize complement-dependent cytotoxicity (CDC) and antibody binding assays (e.g., flow cytometry crossmatch, FCXM) to detect HLA antibodies.
• Conventional CDC assays are insensitive and subjective, while FCXM is sensitive and objective.
• Discrepancies between CDC and FCXM results create controversy in transplant decisions.

Purpose of the Study:

• To develop a single assay, the cytotoxic flow crossmatch (cFCXM), for simultaneous detection of HLA antibody binding and cell death.
• To validate the cFCXM assay against conventional anti-human globulin (AHG) and FCXM methods.

Main Methods:

• Developed a comprehensive procedure for simultaneous cell death and antibody binding detection using the cFCXM assay.
• Validated the cFCXM assay in parallel with AHG and FCXM methodologies using a panel of eight cells and 15 sera.

Main Results:

• Optimized a single-platform method for simultaneous detection of HLA antibody binding and cytotoxicity.
• Identified three distinct HLA antibody reactivity patterns: AHG(pos)/cFCXM(pos)/FCXM(pos), AHG(neg)/cFCXM(neg)/FCXM(pos), and a novel AHG(neg)/cFCXM(pos)/FCXM(pos) category.

Conclusions:

• Cytotoxicity and antibody binding can be assessed simultaneously, demonstrating complement-dependent HLA antibodies undetectable by conventional cytotoxicity assays.
• Routine use of cFCXM may offer new insights for transplanting patients with AHG(neg)/FCXM(pos) results by classifying them based on cFCXM status.