Quantitation of T lymphocytes in human bone marrow by a limiting dilution assay Academic Article uri icon

Overview

MeSH Major

  • Bone Marrow Cells
  • T-Lymphocytes

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 microliter. 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 consistent 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 X 10(5) T cells in a total of 10(9) 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 X 10(9) and in T-cell-depleted samples 4.3 X 10(5). 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 X 10(6) mononuclear cells for complete analysis and, therefore, compares favorably with previously published methods.

publication date

  • January 1985

Research

keywords

  • Academic Article

Identity

Language

  • eng

PubMed ID

  • 3875918

Additional Document Info

start page

  • 317

end page

  • 22

volume

  • 40

number

  • 3