PRONASE IMPROVES DETECTION

PRONASE IMPROVES DETECTION OF HLA ANTIBODIES IN FLOW CROSSMATCHES.
S Vaidya, T Cooper, D Stewart and RA Bray. University of Texas Medical Branch, Galveston, Texas, Oschner Clinic, New Orleans, Louisiana, Emory University, Atlanta, Georgia.

The flow cytomeric crossmatch (FCXM) has grown in popularity and, for many programs, has become the "standard of practice." However B-cell FCXM has been problematic due to Fc-receptors present on B-cells. In an attempt to address these issues, we utilized the proteolytic enzyme, pronase to remove Fc-receptors from the surface of lymphocytes prior to their use in FCXM. Five to ten million lymphocytes were incubated with pronase (conc 1 mg/ml, sp.act.4. 3 x 10^-3units/ml) in a 37°C in waterbath for 30 minutes. Following a single wash, these pronase-digested lymphocytes were used in a 3-color FCXM assay. Our results demonstrate statistically significant improvement in sensitivity and specificity in both T and B cell FCXM. A total of 167 T and B cell FCXMs were performed using serial dilutions of HLA allosera (22 Class I and 6 Class II sera). The HLA reactivity of each serum was determined by testing against a panel of T cells (N=200) and B cells (N=150) for cytotoxicity and confirmed by testing against the HLA-specific microparticles, FlowPRA^TM. The titer of each antibody used in FCXM was one dilution below the titer at which CDC-AHG crossmatch became negative. Following pronase digestion, the channel values of negative control in T and B FCXM declined from 78 to 57 (paired t test p<0.05) and 107 to 49 (paired t test p<0.00001)(256 channel scale) respectively. Following pronase treatment, the sensitivity of T cell and B cell FCXM improved from 80% to 100%, respectively, for the 22 Class I antisera (p<0.0001). The increase in sensitivity was observed as an increase in the end-point titer by at least 2 serial dilutions. In no instance, a false positive reaction was noted. Pronase digestion improved specificity of B FCXM in detection HLA Class II allosera. On the basis of these observations, T and B FCXMs of 3 primary transplant patients who lost their allografts due to accelerated acute rejection were re-evaluated. These 3 patients were transplanted across positive T FCXM but negative B FCXM and CDC crossmatches, based upon assumption that if the antibody binds with only T but not B cells, it is unlikely to be HLA antibody. Following pronase digestion, T and B crossmatches of each patient became strongly positive and donor-specific anti Class I HLA antibody was identified in each case. In summary pronase digestion is a simple and short assay that improves both sensitivity and specificity of T and B cell flow crossmatches.