A flow cytometric crossmatch test for simultaneous detection of antibodies against donor lymphocytes and endothelial precursor cells

Complement-dependent cytotoxicity or flow cytometric lymphocyte crossmatch (LXM) tests may fail to detect clinically significant antibodies (Abs) against non-human leukocyte antigen (HLA). A flow cytometric endothelial precursor cell crossmatch (EPCXM) test (XM-ONE (R)) is available for detection of Abs against donor endothelial precursor cells (EPCs). We showed that lymphocytes co-purified with EPCs can be used in LXM tests allowing simultaneous detection of Abs reactive with donor EPCs and lymphocytes. The lymphocyte population co-purified with EPCs on anti-Tie-2 Ab-coupled magnetic beads contained CD 8+ and CD 4+ T-cells, B-cells, and natural killer (NK)- and natural killer T (NKT)-cells. HLA class I antigen expression was slightly higher on CD 3+ lymphocytes co-purified on Tie-2 Ab beads than on unseparated lymphocytes, whereas HLA class I and II antigen levels on CD 19+ lymphocytes were not significantly different. Sera from 10 patients with panel-reactive Abs were tested on cells from nine donors using flow cytometric LXM and EPCXM tests. There was a very good correlation (R2 = 0.94) between the channel shift values obtained on unseparated and Tie-2 Ab bead-isolated T-lymphocytes, whereas the correlation between the channel shift values obtained on the two B-lymphocyte populations was lower (R2 = 0.71). T- and B-lymphocytes co-purified with EPCs can be used in LXM tests enabling simultaneous detection of donor lymphocyte- and EPC-reactive Abs in a single-tube XM-ONE (R) assay.