A technique for dual determination of cytotoxic and helper lymphocyte precursor frequency by a miniaturized dye release method

Helper (HTLPf) and cytotoxic (CTLPf) lymphocyte precursor frequency assays are increasingly used in bone marrow stem cell and organ transplant compati bility testing, Current techniques require large cell numbers and radioisot opes. To improve the technique, we developed a miniaturized fluorescent rea d-out combined HTLPf/CTLPf limiting dilution assay. The assay requires only 5 x 10(6) stimulators, 2 x 10(6) responders and 0.24 x 10(6) target cells in Terasaki plates (40 mu l/well). For the HTLPf, culture supernatants from each well were assayed for IL-2 production. The IL-2-dependent proliferati on of the mouse 9.12 cell line was detected by a semi-automated fluorescent dye technique. After addition of rhIL-2 (recombinant human IL-2) on days 3 and 7, CTLPs were detected on day 10 by measuring the lysis of dye-labeled targets, Results were comparable to standard radioisotope-based techniques . The assay had a coefficient of variation of approximately 30%, The assay detected helper CD4 cells, pure cytotoxic CD8, helper CD8 cells and helper/ cytotoxic CDS cells. Discrimination was demonstrated between HLA-matched re lated and non-related pairs. The ease of testing and small cell numbers req uired should facilitate further evaluation of HTLPf and CTLPf for compatibi lity testing in unrelated donor transplantation and monitoring immune respo nses following adoptive transfer of lymphocytes.