Low Expression Loci and the Use of Pronase in Flow Cytometry Crossmatch

Pronase treatment of lymphocytes has been used to improve the specificity and sensitivity of flow cytometric crossmatch, especially B-cell flow cytometric crossmatch, due to the presence of Fc receptors on the cell surface. Some limitations have been reported in the literature: false negatives due to the reduction of major histocompatibility complex expression and false-positive T cells in HIV+ patients due to exposure to cryptic epitopes. This study aimed to evaluate the effect of pronase in our assays, using untreated and treated cells with 2.35 U/mL of pronase to improve flow cytometric crossmatch specificity and sensitivity. The study was carried out with donor-specific IgG antibodies (DSAs) to low expression loci (HLA-C, -DQ, or -DP) because, in our laboratory practice, patients with virtual crossmatch (LABScreen single antigen assays) to DSA against antigen HLA-A, B, and DR are excluded from cellular crossmatch. Our results showed that, for T-cell flow cytometry crossmatch (FCXM), a cutoff value of 1171 median fluorescence intensity (MFI), an area under the curve (AUC) of 0.926 (P < .0001), and 0.834 (P < .0001), a sensitivity of 100% and 85.7%, and a specificity of 77.5% and 74.4%, without and with pronase treatment, respectively. For B-cell FCXM without pronase treatment, the best cutoff was 2766 MFI, an AUC of 0.731 (P < .0001), a sensitivity of 69.6%, and a specificity of 66.7%, whereas for B cells treated with pronase, the cutoff value was 4496 MFI, an AUC of 0.852 (P < .0001), a sensitivity of 86.4%, and a specificity of 77.8%. Our analysis of 128 FCXM showed a better performance using the untreated lymphocytes for FCXM with the prerequisite of a higher cutoff value (≈5000 MFI) to reach a better sensitivity and specificity due to the loss of HLA expression.