Pronase treatment improves flow cytometry crossmatching results

Flow cytometry crossmatching (FC-XM) is the most sensitive cell-based method for detecting donor-specific antibodies in clinical organ transplantation. Unfortunately, background FC-XM reactivity is elevated in assays with B lymphocytes-partly because of nonspecific immunoglobulin binding by Fc receptors and B-cell surface immunoglobulins. To reduce the background reactivity in a B-cell FC-XM assay, we treated lymphocytes with pronase (1 mg/mL for 30 minutes). This treatment drastically reduced the presence of kappa light chains and Fc receptors (CD32b), while the concomitant decrease in CD19, CD20 and major histocompatibility complex (MHC) I and II expression on B-cells was acceptable. Higher pronase concentrations (>2 mg/mL) started to significantly affect CD19, CD20, MHC-I and -II expression on B-cells. In subsequent prospective experiments (on 42 donor cells tested with 102 sera), we found that pronase treatment was associated with a relative increase of the sensitivity and specificity in our B-cell FC-XM assay.