Effects of Various Concentrations of Pronase on Flow Cytometric Crossmatching Patients Treated With Rituximab and Donor HLA-Specific Antibodies.

Background: Pronase pretreatment can reduce rituximab (RTX) interference by degrading CD20 in B-cell flow cytometry crossmatch (FCXM) testing. However, it may also reduce the assay sensitivity by degrading HLA molecules. We investigated the effects of various pronase concentrations on RTX interference and the analytical sensitivity of B-cell FCXM testing.

Methods: Using 59 patient serum samples and 38 donor lymphocyte samples, we designed 97 recipient-donor pairs and divided them into three groups according to RTX use and the presence of weak-to-moderate donor HLA-specific antibody (DSA) reactions: RTX+/DSA-, RTX+/DSA+, and RTX-/DSA+. FCXM was performed after pretreating lymphocytes with six different pronase concentrations (0, 0.5, 1, 2, 3, and 4 mg/mL).

Results: With B-FCXM testing, false-positive results due to RTX in the RTX+/DSA- group markedly decreased with increasing pronase concentrations. The median channel shift values in the RTX+/DSA+ and RTX-/DSA+ groups did not significantly decrease when the pronase concentration was increased from 1 mg/mL to 2 or 3 mg/mL. All eight RTX+/DSA+ cases that were positive at 1 mg/mL pronase but negative at 2 or 3 mg/mL had mean fluorescence intensity (MFI) DSA values of less than 3,000 except for DQ5 (MFI: 5,226). With T-cell FCXM, false-positive results were observed in 2.9% of 315 FCXM tests with pronase pretreatment.

Conclusions: Higher concentrations (2 or 3 mg/mL) of pronase effectively eliminated RTX interference but still carried a risk for false negativity for weak DSA reactions in B-cell FCXM. Higher pronase concentrations can be used as an auxiliary method to detect moderate-to-strong DSA reactions in RTX-treated patients.