Skip to main content
NCBI home page
Clinical and Experimental Immunology logoLink to Clinical and Experimental Immunology
. 1974 Sep;18(1):155–167.

A rapid micro-method for the phytohaemagglutinin-induced human lymphocyte transformation test

W J Penhale, Anne Farmer, A C Maccuish, W J Irvine
PMCID: PMC1537979  PMID: 4468190

Abstract

A quantitative in vitro method for phytohaemagglutinin (PHA) induced lymphocyte transformation is described. This method has the following advantages.

Cultures require as few as 105 lymphocytes and are performed in microtrays, over a short incubation period (48 hr).

A short pulse time of 4 hr with [3H]thymidine is utilized. This enables high levels of [3H]thymidine to be maintained throughout the labelling period, ensures maximum incorporation of thymidine into cellular DNA, and diminishes cellular damage by internal irradiation.

The extraction process has been simplified by using a communal washing procedure after drying the cultures on glass-fibre filter discs. The procedure is both quicker and more reproducible (coefficient of variation 6%) than extraction and drying on filtration manifolds (coefficient of variation 23%).

The effect of adjusting variables such as the number of cells, incubation time, concentration of [3H]thymidine, concentration of PHA, specific activity of [3H]-thymidine and duration of [3H]thymidine pulse has been studied in order to approach optimal labelling conditions for the assay system.

The reproducibility of the method has been investigated by repeated testing of normal individuals on a day-to-day basis and over extended periods. The mean coefficient of variation for samples repeated daily and over longer time intervals (weeks or months between samples) was 15%.

Full text

PDF
155

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. DREW R. M., PAINTER R. B. Further studies on the clonal growth of HeLa S3 cells treated with tritiated thymidine. Radiat Res. 1962 Mar;16:303–311. [PubMed] [Google Scholar]
  2. Fitzgerald M. G. The establishment of a normal human population dose-response curve for lymphocytes cultured with PHA (phytohaemagglutinin). Clin Exp Immunol. 1971 Mar;8(3):421–425. [PMC free article] [PubMed] [Google Scholar]
  3. Gotoff S. P. Lymphocytes in congenital immunological deficiency diseases. Clin Exp Immunol. 1968 Oct;3(8):843–856. [PMC free article] [PubMed] [Google Scholar]
  4. Han T., Takita H. Immunologic impairment in bronchogenic carcinoma: a study of lymphocyte response to phytohemagglutinin. Cancer. 1972 Sep;30(3):616–620. doi: 10.1002/1097-0142(197209)30:3<616::aid-cncr2820300304>3.0.co;2-q. [DOI] [PubMed] [Google Scholar]
  5. Hosking C. S., Fitzgerald M. G., Simons M. J. Quantified deficiency of lymphocyte response to phytohaemagglutinin in immune deficiency diseases. Clin Exp Immunol. 1971 Oct;9(4):467–476. [PMC free article] [PubMed] [Google Scholar]
  6. Oppenheim J. J., Blaese R. M., Waldmann T. A. Defective lymphocyte transformation and delayed hypersensitivity in Wiskott-Aldrich syndrome. J Immunol. 1970 Apr;104(4):835–844. [PubMed] [Google Scholar]
  7. Paty D. W., Bone G. Response to P.H.A. in cancer patients. Lancet. 1973 Mar 24;1(7804):668–669. doi: 10.1016/s0140-6736(73)92235-6. [DOI] [PubMed] [Google Scholar]
  8. Perper R. J., Zee T. W., Mickelson M. M. Purification of lymphocytes and platelets by gradient centrifugation. J Lab Clin Med. 1968 Nov;72(5):842–848. [PubMed] [Google Scholar]
  9. Phillips B., Roitt I. M. Evidence for transformation of human B lymphocytes by PHA. Nat New Biol. 1973 Feb 21;241(112):254–256. doi: 10.1038/newbio241254a0. [DOI] [PubMed] [Google Scholar]
  10. Sample W. F., Chretien P. B. Thymidine kinetics in human lymphocyte transformation: determination of optimal labelling conditions. Clin Exp Immunol. 1971 Sep;9(3):419–427. [PMC free article] [PubMed] [Google Scholar]
  11. Schellekens P. T., Eijsvoogel V. P. Lymphocyte transformation in vitro. I. Tissue culture conditions and quantitative measurements. Clin Exp Immunol. 1968 Jul;3(6):571–584. [PMC free article] [PubMed] [Google Scholar]
  12. Trubowitz S., Masek B., Del Rosario A. Lymphocyte response to phytohemagglutinin in Hodgkin's disease, lymphatic leukemia and lymphosarcoma. Cancer. 1966 Dec;19(12):2019–2023. doi: 10.1002/1097-0142(196612)19:12<2019::aid-cncr2820191228>3.0.co;2-h. [DOI] [PubMed] [Google Scholar]
  13. Yamamura M. Standardization of lymphocyte transformation to phytohaemagglutinin. Clin Exp Immunol. 1973 Jul;14(3):457–467. [PMC free article] [PubMed] [Google Scholar]

Articles from Clinical and Experimental Immunology are provided here courtesy of British Society for Immunology

RESOURCES