1016-LBP: RosetteSep HLA – An exceptional method for isolation of lymphocytes from peripheral blood

Abstract

Aim The isolation of a sufficient quantity of lymphocytes from deceased donor peripheral blood can be problematic. As an OPO HLA lab that serves six different transplant centers, we routinely perform both complement dependent cytotoxicity (CDC) and flow cytometric crossmatching (FCXM) for 15 potential renal recipients plus additional non-renal recipients. Historically, we have used T/B Lympho-Kwik to isolate lymphocytes for FCXM, but this reagent is no longer available. As a replacement method, we adopted a protocol using CD14 and CD15 magnetic beads to negatively select non-lymphocyte cells. However, the lymphocyte yield and purity was frequently insufficient, so we sought to validate a method using RosetteSep HLA Total Lymphocyte Enrichment Cocktail. Methods RosetteSep contains bispecific antibodies which crosslink unwanted cells to RBCs, forming rosettes, which are removed by centrifugation. Buffy coats were pulled from ACD blood tubes and lymphocytes were isolated by centrifugation over IsoPrep and negative selection with CD14/CD15 Dynabeads or RosetteSep. The isolated lymphocytes were counted on a hemacytometer and either used directly for FCXM or further separated by positive selection of B cells using Class II Dynabeads for CDC testing. Results The average cell yield for the RosetteSep isolations was nearly double that of the CD14/CD15 isolations, lymphocyte purity was >95%, and there was an increase in the ratio of B cells to T cells. The viability and CDC reaction of the B cells was comparable between the two methods. The results of the FCXM were also comparable between the two methods, both in terms of T and B cell MFI with negative serum (NS) and T and B cell MCS with patient sera. The increased background on B cells with NS historically seen when using Lympho–Kwik was not observed with RosetteSep. Conclusions Our method using RosetteSep is cost efficient, fast, easy to perform, and significantly increases the yield of lymphocytes compared to CD14/CD15 magnetic bead negative selection.