OR44 HLA expression following lymphocyte enrichment: Tissue source still matters for deceased donor crossmatching

Aim We previously reported differences in HLA expression on lymphocytes isolated from different tissues and the impact tissue source can have on reactivity. In the current study, we re-examined HLA expression and the impact of lymphocyte enrichment using multiple class I and II specific monoclonal antibodies and different fluorochromes. Methods Deceased donor lymphocytes were isolated from spleen, nodes, and peripheral blood. Following ficoll hypaque (FH) isolation, the EasySep™ Total Lymphocyte Enrichment Kit was used to increase purity of lymphocytes by negative selection. Cells were stained with four sets of monoclonal antibody-fluorochrome combinations. Data were acquired on either the BD FACSCanto™ II or the BD FACSAria™ using FACSDiva™ software. Results Lymphocytes isolated from deceased donors were assessed for variability of HLA expression among different tissues and mean values are presented in Table 1. Lymphocytes isolated from spleen had twofold greater class I expression as PBLs and twofold greater class II expression as nodes. Results using different combinations of monoclonal antibodies for class I and II show similar findings. HLA expression measurements were higher for lymphocytes isolated with bead-enrichment compared to FH isolation alone (not shown). Paired analysis using class I specific FITC labeled antibodies revealed lower than expected class I expression on B cells indicative of a possible autoquenching artifact due to similar excitation and emission spectra (not shown). Conclusions Similar to our previously published results, the spleen derived lymphocytes consistently yielded the highest HLA expression across T and B cells. We observed similar results across antibody clones but identified a possible FITC autoquenching artifact. Given increased transplant rates for highly sensitized candidates and the role of crossmatch tests to confirm compatibility, these HLA expression data should be corroborated in a larger dataset and used to guide deceased donor procurement protocols. Download high-res image (151KB) Download full-size image A.M. Jackson: 3. Speaker’s Bureau; Company/Organization; ThermoFisher.