P014 A simple and fast method for enrichment of lymphocyte subsets for complement dependent cytotoxicity assays

Abstract

Aim Serological crossmatch analysis (i.e. complement-dependent cytotoxicity (CDC)) is commonly done before solid organ transplantation to detect donor specific antibodies that may lead to graft rejection or dysfunction. Routine analysis is often performed on isolated donor lymphocyte subpopulations together with recipient serum. Enrichment of cells for CDC can be exceedingly time consuming or results in co-enrichment of non-target or dead cells. We aimed to develop a fast and convenient method for enrichment of donor lymphocyte subsets directly from whole blood. Methods Using MACSxpress technology, untouched human leukocyte subsets can be isolated within only 20 min from up to 30 ml of anticoagulated whole blood. For the enrichment of B, T or a mixture of BT cells whole blood or cell suspensions obtained from rinsed donor spleens were incubated with the respective bead cocktails for 5 min and the tube placed for 15 min in the magnetic field of a separator. With the tube inside the magnetic field, the supernatant, containing the enriched target cells, was collected. Magnetically labeled non-target cells as well as aggregated erythrocytes are retained in the tube. Purity and viability of the enriched cell populations were determined by flow cytometry and functionality was shown in CDC assays. Results MACSxpress-enriched lymphocyte subsets (B/T/BT cells) from whole blood had average purities of 87/96/98% with yields of 60/54/38% ( n = 27/29/23). Cell enrichment from spleen single cell suspensions resulted in purities of 97/96/98% and recoveries of 27/32/29% ( n = 5) (B/T/BT cells). CDC analysis of positive and negative control sera with enriched cells showed no influence of fluorescent readout by the enrichment process. Conclusion Using MACSprep HLA Isolation Kits, lymphocytes for crossmatch analysis can be enriched within 20 min from whole blood without the need of expensive lab equipment or preparation of PBMC by density gradient centrifugation. G. Winkels: Employee; Company/Organization; Miltenyi Biotec.