Quantitation of T lymphocytes in human bone marrow by a limiting dilution assay Journal Article

  


Authors: Kernan, N. A.; Flomenberg, N.; Collins, N. H.; O'Reilly, R. J.; Dupont, B.
Article Title: 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 μl. 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 4.3×105 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 1×109 and in T-cell-depleted samples 4.3×105. 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 1×106 mononuclear cells for complete analysis and, therefore, compares favorably with previously published methods. © 1985 by The Williams and Wilkins Co.
Keywords: human cell; t lymphocyte; t-lymphocytes; bone marrow cells; interleukin 2; bone marrow; in vitro study; graft versus host reaction; hematopoietic stem cells; rosette formation; leukocyte count; monocytes; bone marrow transplantation; erythrocytes; phytohemagglutinin; humans; human; priority journal; blood and hemopoietic system
Journal Title: Transplantation
Volume: 40
Issue: 3
ISSN: 0041-1337
Publisher: Lippincott Williams & Wilkins  
Date Published: 1985-09-01
Start Page: 317
End Page: 322
Language: English
DOI: 10.1097/00007890-198509000-00019
PROVIDER: scopus
PUBMED: 3875918
DOI/URL:

https://www.scopus.com/inward/record.uri?eid=2-s2.0-0021816219&doi=10.1097%2f00007890-198509000-00019&partnerID=40&md5=1fe4835b7d34ce68743e8dc4e8a4e28a
Notes: Article -- Export Date: 26 October 2021 -- Source: Scopus
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MSK Authors
  1. Nancy Kernan
    512 Kernan
  2. Richard O'Reilly
    747 O'Reilly
  3. Bo Dupont
    264 Dupont
  4. Nancy Collins
    90 Collins
  5. Neal Flomenberg
    56 Flomenberg
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