W188 - Improving Lymphocyte Purity for Flow Cytometric Crossmatch Assays: A Comparison of Cell Isolation Methods

Alexandra Manchón – Hospital Clínic; Nuria Exposito – Hospital Clínic; Jose Luis Caro – Hospital Clínic; Eduard Palou – Hospital Clínic; Manel Juan – Hospital Clínic; Juan Torres Canizales – Hospital Clínic

Abstract Text:

Introduction: The flow cytometric crossmatch (FCXM) assay is used to determine whether donor specific HLA antibodies (HLA-DSA) are present in recipient sera. Isolated cell purity is a widely recognized critical factor in the test outcome. Our goal was to compare the performance of two cell isolation methods in FCXM assays carried out with peripheral blood from living donors for kidney transplant.

Materials and methods: FCXM assays carried out after processing donor samples by erythrocyte aggregation and total lymphocyte enrichment through negative magnetic selection (EA&TLE) and gradient density centrifugation (Ficoll) were compared and correlated with HLA-DSA detection using the Luminex single-antigen bead assay (LSA).

Results: The mean lymphocyte percentage of samples processed using EA&TLE was 94% (95% confidence interval [95% CI] 89% to 97%), with 24 to 50% of the lymphocytes present before isolation retained. Analysis of 65 FCXM assays performed with EA&TLE and 66 with Ficoll demonstrated that EA&TLE results in higher sensibility (67% EA&TLE vs 43% Ficoll) and higher kappa correlation coefficient (0.61, 95% IC 0.318 to 0.90 EA&TLE vs 0.40, 95% IC 0.01 to 0.82 Ficoll) with HLA-DSA detection by LSA. Furthermore, FCXM assays performed with EA&TLE showed a lower detection threshold for class II HLA-DSA in comparison to FCXM assays performed with Ficoll (lowest mean fluorescence intensity of 5193 for EA&TLE vs 10289 for Ficoll).

Conclusions: Implementation of EA&TLE for cell isolation from whole blood increased lymphocyte purity in the samples, improved FCXM assay sensitivity and resulted in a greater correlation with HLA-DSA identification by LSA.