P099 The impact of lymphocyte purity on flow cytometry crossmatch (FCXM) assay. It’s not purely theoretical

Abstract

Aim Lymphocyte flow cytometric crossmatch (FCXM) is used in pre-transplant risk assessment to detect donor specific antibodies (DSA). Many labs use density medium centrifugation to isolate donor cells for FCXM, which yields variable lymphocyte purity (15–90%) depending on the original concentration of lymphocytes and other leukocytes in donor blood, as well as sample quality and age. However, it is unclear whether variation in lymphocyte purity can effect FCXM assay. The goal of this study was to assess the impact of donor lymphocyte purity on FCXM results. Methods Enriched lymphocytes (Ly; >97%), neutrophils (Nu; >98%) and monocytes (Mo; >92%) were isolated from 5 volunteer donors using subset specific EasySep Direct kits (STEMCELL). Whole leukocyte (WL) cell preps were obtained by mixing enriched Ly, Nu and Mo in equal proportion (approx. 33% each) to mimic poor quality cell preps. Ly and WL cell preps were treated with pronase (2.35 kunitz units/ml) or control (PBS) and used as targets in FCXM against negative control (NC) and several dilutions of positive control (PC) sera. Median channel fluorescence (MCF) shifts from NC obtained with Ly vs WL target cell preps were compared. Results T and B cell FCXM NC reactivity was similar with Ly and WL cells. Pronase treatment was equally effective at reducing B cell background in Ly and WL cell preps (191 vs 198 MCF shift). Interestingly, both T and B cells FCXM performed with WL targets showed significantly reduced PC reactivity compared to Ly cells (Fig. 1). This was true at all PC concentrations (1/50–1/400) and was seen with both pronase treated and untreated cells. Average MCF decrease in pronase treated Ly vs WL preps was 49 MCF for T and 68 MCF for B cell FCXM, and in untreated preps it was 41 MCF for T and 61 MCF for B cell FCXM. Conclusion Lymphocyte purity has significant impact on FCXM results. Weak DSA could be missed if low purity lymphocyte preps are used in FCXM assay. Use of highly enriched lymphocytes will improve detection of DSA and reduce variability of FCXM results. Download : Download high-res image (220KB) Download : Download full-size image