LAUSR

Aim Flow cytometry crossmatch (FCXM) is an important tool in pre-transplant assessment of immunologic risk. Variation in HLA antigen density (AgD) may affect FCXM interpretation. The objectives of this study are to measure HLA AgD on lymphocytes and compare the differences in AgD between cell source, donor type, and pronase treatment. Methods AgD was measured with monoclonal antibodies (clones: W6/32, WR18 (pan-Class II), and HLA-DQ1 (binds DQB1 except DQ2)). Lymphocytes were isolated by immunomagnetic depletion from living donors (LD-PBL, n  = 39), deceased donors (DD-PBL, n  = 7), and spleen (SPL, n  = 20). Cells were pronase treated. AgD was assessed with and without pronase ( n  = 3). HLA-DQ AgD analyses were limited to DQ2-negative cells. Results There were no significant differences across the three cell sources between class I AgD on either T- or B-cells (Fig. 1A and 1B). In contrast, class II AgD was lower on SPL vs LD-PBL and DD-PBL. DQ AgD was lower on both DD-PBL and SPL (Fig. 1C and 1D). Pronase treatment enabled increased detection of class I AgD on T-cells but not B-cells with only slight increase in class II AgD (Table 1). Conclusions Although T or B cell class I AgD did not differ significantly between cell sources, B cell class II was substantially decreased, particularly in DD spleen while HLA-DQ expression was lower on all deceased donor cells. Our results suggest that FCXM results may be affected when assessing deceased donor FCXM in the context of class II DSA. Pronase treatment primarily affected the detection of class I on T cells with minimal impact on B cell AgD, but this was observed in a limited number of samples. We are continuing to collect data and will assess the impact of class/loci specific HLA antibody and the relationship to AgD. Download high-res image (167KB) Download full-size image Download high-res image (81KB) Download full-size image