THE USE OF PRONASE-DIGESTED HUMAN-LEUKOCYTES TO IMPROVE SPECIFICITY OF THE FLOW CYTOMETRIC CROSS-MATCH

Two-color fluorescence cytometry (FCXM) has recently been introduced t o improve the detection of anti-HLA antibodies that react to donor cel ls, especially in recipients receiving kidney allografts. Although thi s assay system is highly sensitive, it lacks specificity. Between 70 % and 90 % of potential kidney recipients with a positive FCXM would ha ve been denied transplant if such an assay had been used alone to dete ct antidonor antibodies. Lack of specificity is principally due to nor mal or irrelevant IgG in aggregates or immune complexes binding to Fc gamma R receptors on lymphocytes including B cells and a significant s ubset of T cells. To circumvent this problem, we digested Fc gamma R r eceptors on lymphocytes with pronase. We present data demonstrating th at pronase digestion of lymphocytes does not alter HLA antigenicity. I n addition, pronased lymphocytes allow one to use either single- or tw o-color FCXM. With single-color FCXM, one can quantitate antibody reac tivity to lymphocytes via a cursor (on the fluorescence histogram) tha t separates lymphocytes that do not bind to antibodies. We present dat a demonstrating that this modification renders FCXM highly sensitive a nd specific. In addition, one can discriminate between IgG and IgM ant ibodies that react to lymphocytes.