Image cytometry as an alternative to flow cytometry for the transplant histocompatibility crossmatch assay

The lymphocyte crossmatch is currently the only cell-based compatibility assay performed by histocompatibility laboratories for transplant purposes. While in many transplant programs the complement-dependent cytotoxicity crossmatch (CDCXM) remains in use, when available, the flow cytometry crossmatch (FCXM) is the method of choice because of its superior sensitivity and specificity. Unfortunately, the maintenance and cost of a flow cytometer is a considerable limitation for small histocompatibility laboratories. Therefore, in this study, we evaluated the use of the Cellometer Vision CBA image cytometer (Nexcelom Bioscience LLC, Lawrence, Massachusetts) as an alternative instrument to perform the crossmatch assay. The 3-color FCXM protocol was modified into two separate 2-color panel image cytometry crossmatches (IXMs), one for T cells and one for B cells. After initial serum and cell incubation, a cocktail consisting of PE/Cy5-conjugated anti-human CD3 or CD19 and PE-conjugated anti-human IgG F(ab')₂ was added to the T cell and B cell panels, respectively. The final cell preparation was added to a separate counting chamber. Images were captured using the Cellometer Vision CBA, an image cytometer designed for cell counting, size analysis and fluorescence intensity measurement. Thirty-nine IXMs were performed and compared with the FCXM. We obtained a concordance sensitivity of 94.1% and 100% and specificity of 100% and 88.9% for T cells and B cells, respectively. The linearity of the system was verified using dilutions of a sample containing known donor-specific anti-HLA antibodies (DSA) against the target cells. This feasibility study demonstrates that the FCXM test could be easily adapted to the Cellometer Vision CBA image cytometer without compromising specificity and sensitivity. The low instrumentation cost, minimal maintenance, and simple operation allow for efficient implementation or transition from the FCXM to the IXM method.