EVALUATION OF A NOVEL MONONUCLEAR CELL ISOLATION PROCEDURE FOR SEROLOGICAL HLA TYPING

Despite recent advances in DNA-based genotyping, the microcytotoxicity test is still broadly used for the determination of human leukocyte c lass I antigens in patients as well as organ donors and also for the d etection of HLA antibodies, Excellent purity and viability of peripher al blood mononuclear cells (PBMC) are essential fur reliable HLA typin g results. Background staining and cell loss can contribute to impaire d typing results or even cause misinterpretations. A novel isolation p rocedure using cell preparation tubes (CPT) with prefilled Ficoll was compared with the standard Ficoll gradient. We determined the recovery , purity, and viability of the PBMC after several periods of storage. Finally, the isolated cells were used for HLA class I typing, and back ground reactivities were scored. By using the CPT method, the recovery of PBMC was significantly higher than recovery with the standard tech nique (P less than or equal to 0.001). Contamination by granulocytes i ncreased considerably during the storage time for the standard protoco l, whereas purity remained stable when CPT were used (P less than or e qual to 0.001), With both methods, lymphocyte viability declined marke dly over time. We found significantly more dead cells by using the CPT methods. Due to high background scores, HLA typing was impossible aft er 48 h, The isolation of PBMC by the CPT method resulted in a higher yield and improved purity compared to those obtained with the standard gradient technique. The decreasing viability after 48 h limits the us e of both methods for HLA typing and HLA antibody screening.