QUANTITATION OF T LYMPHOCYTES IN HUMAN BONE MARROW BY A LIMITING DILUTION ASSAY

Abstract

A limiting-dilution microculture assay (LDMA) for quantitation of T lymphocytes in human bone marrow is described. Phytohemagglutinin (PHA)-responsive T cells are maintained in interleukin 2 (IL-2)-containing medium with feeder cells in a total volume of 20 .mu.l. After 16 days of culture, each well is scored by microscopic examination as positive or negative based on the presence or absence of cell growth. A limiting dilution analysis of the relationship between the number of cells seeded per well and the fraction of wells without growth demonstrate that the data are consistant with single-hit kinetics. Minimum chi square statistics were used to establish the line of best fit to calculate the T lymphocyte frequency in a sample. This method for enumeration of T cells was applied to untreated samples of bone marrow, soybean-agglutinin-negative (SBA-) marrow, and soybean-agglutinin-negative marrow cells subjected to a single sheep red blood cell (SRBC) rosette (SBA-E-) or double SRBC rosette (SBA-E-E-) depletion. It was demonstrated that the LDMA can detect as few as 4.3 .times. 105 T cells in a total of 109 bone marrow mononuclear cells. The assay system also allows for a comparison of T lymphocytes in the untreated marrow with the T-cell-depleted marrow samples. The mean number of T cells in untreated marrow was 1 .times. 109 and in T-cell-depleted samples 4.3 .times. 105. This corresponds to a 3.5 log or 99.96% reduction in total T cell number by the SBA-E- rosette technique. The phenotypic analysis of single positive wells as well as pooled cells from all positive wells indicate that at least 95% of the wells scored microscopically as positive for T cell growth did in fact contain T cells. The assay requires only 1 .times. 106 mononuclear cells for complete analysis and, therefore, compares favorably with previously published methods.