6-P

Abstract

Flow cytometric crossmatches (FCXM) are used to detect low-level donor-specific antibody and determine transplantation eligibility. We measured HLA class I and II surface expression using different lymphocyte sources (peripheral blood (PBL), spleen, or nodes) to investigate the impact of HLA expression on detecting low-level donor specific antibody. HLA expression was measured on a BD FACSCanto II using fluorescently conjugated antibodies specific for monomorphic determinants on HLA class I and II; measurements were converted to Molecules of Soluble Fluorescence (MESF) units. FCXM reactivity was determined using the ratio of test serum to negative control serum and MESF units. Compared to Live Donor (LD) PBLs (n=249), mean HLA class I expression on lymphocytes isolated from Deceased Donor (DD) (n=25) spleen and lymph nodes was lower (5.8% and 10.4%, respectively). The mean HLA class II expression was significantly lower on DD splenic (90.4%, p < 0.05) and lymph node (125.6%, p < 0.05) lymphocytes. We then compared lymphocytes isolated from the same DD (n=4) to better assess the variability of HLA expression on cells derived from different tissues. Compared to DD PBL, HLA class I expression was higher on splenic (54%) and lymph node lymphocytes (34%). HLA class II expression on DD PBLs was lower than that from spleen (63%), but higher than the lymph node-derived lymphocytes (99%). Variation in HLA class II expression on cells from different tissues also impacted crossmatch reactivity. HLA class II donor specific sera, expected to yield a positive FCXM, consistently showed the highest reactivity with splenic lymphocytes; intermediate reactivity with PBL; and the lowest reactivity with lymph node cells. HLA expression may not be routinely analyzed; however, it can substantially impact crossmatch reactivity. Laboratories using a single positive threshold for all FCXMs should consider that splenic lymphocytes may yield reactivity most comparable to that of PBLs from LDs.