P137 Go pro(nase)! the importance of donor lymphocyte treatment with pronase in flow cytometric crossmatch testing

Abstract

Aim Flow cytometric crossmatch (FCXM) is performed to confirm donor/recipient histocompatibility. Previous multicenter study (Liwski et al. ASHI 2012) suggested that protocol differences contribute to FCXM result variability and that pronase treatment improves B cell FCXM. In this study we assess the impact of pronase on FCXM results. Methods Donor lymphocytes, isolated from spleens/lymph nodes, were treated with pronase (4.7 U/ml) or saline. FCXM were performed at Santa Casa Lab in Porto Alegre using the Halifax protocol, acquired on BD Canto II and analyzed using a median channel fluorescence (MCF; 1024 channel) scale. 3SD cutoffs were determined by testing neg ctrl (NC) sera against 63 donor cells. Pos ctrl (PC) sera were used in each FCXM. A total of 240 FCXM (122 predicted neg; 118 predicted pos) were performed in parallel using pronase treated vs untreated cells. Pos/Neg FCXMs were predicted based on LABScreen single antigen bead testing with a 1,000 MFI cutoff. Results NC serum testing shows that pronase significantly reduced B cell background reactivity (pronase MCF = 195 +/- 32 vs untreated MCF = 363 +/- 89, p Conclusions B cell FCXM sensitivity is unacceptably low (26.3%) when untreated cell are used. Pronase treatment greatly improves B cell FCXM sensitivity (74.6%) and decreases the rate of false negative B cell reactions by reducing the background and improving signal to noise ratio.