A single [3H]thymidine-based limiting dilution analysis to determine HTLp and CTLp frequencies for bone marrow donor...

Histocompatibility between recipient and donor is a critical factor in allogeneic BMT which, to a large extent, determines the incidence of GVHD after BMT. Functional histocompatibility assays, such as the helper T lymphocyte precursor frequency assay (HTLp) and the cytotoxic T lymphocyte precursor frequency assay (CTLp), have proved to be helpful tools in facilitating donor selection procedures. However, a major drawback of these assays is that they are laborious and require large numbers of cells. We therefore adapted a [3H]thymidine-based assay, the 'JAM' test, as a read-out for CTLp frequencies, to replace the more cumbersome 51Cr-release assay. Furthermore, we applied an experimental setup that enables the assessment of HTLp and CTLp frequencies from a single limiting dilution assay to reduce the number of cells needed. The newly developed assay is relatively easy to perform and has the advantage that different subsets of T cells can be quantified in a single ongoing alloreaction. When the combined assay was applied in unrelated donor selection it proved to be a sensitive method that enables differentiation in suitability of distinct donors for a single patient. Therefore, the combined HTLp/CTLp assay appears to be a practical and sensitive method for identifying functional histocompatibility in related and unrelated donor/recipient combinations.