Pronase treatment enhances the sensitivity of B cell flow cytometry crossmatch: A single-center data analysis

The Flow Cytometry Crossmatch (FCXM) is a vital tool in HLA laboratories for assessing transplantation immunological risk. This study evaluates FCXM results using data from 2018 to 2023, with the aim to assess the efficacy of pronase treatment in FCXM assay by comparing control serum samples in the FCXM assays performed during this time period. Tests followed our standard protocol using the BD FACSLyric™ Flow Cytometry System and BD FACSCalibur™ Flow Cytometry System, employing normal human serum (NHS) as negative control and pooled patient serum samples with strong anti-HLA antibodies as positive control. Pronase and non-pronase tests were performed and compared to rule out any false T cell positive crossmatches that could be attributable to pronase treatment. Results showed no significant difference in delta values for T cell crossmatches between pronase-treated (MCS 256 ± 112) and non-treated (MCS 247 ± 116) groups (p = 0.253). However, B cell crossmatches had significantly higher delta values with pronase treatment (MCS 353 ± 129) versus non-treatment (MCS 300 ± 117), indicating enhanced sensitivity with pronase (p < 0.000001). Pronase-treated B cell negative controls had lower signal (MCS 245 ± 61) compared to non-treated (MCS 300 ± 73), with positive control values consistent across both groups. Anti-CD23 analysis demonstrated a reduction in all the tests performed (MCS 372 ± 64 vs 217 ± 50), confirming efficacy of pronase treatment. These findings highlight the importance of pronase in improving FCXM sensitivity for B cells, enhancing the accuracy and reliability of FCXM protocols for better transplantation outcomes.