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Indian Journal of Clinical Biochemistry

, Volume 19, Issue 2, pp 95–99 | Cite as

Enumeration of lymphocyte subsets using flow cytometry: Effect of storage before and after staining in a developing country setting

  • Sanju Jalla
  • Sunil Sazawal
  • Salkat Deb
  • robert E. Black
  • Satya Narayan Das
  • Archana Sarkar
  • Maharaj K. Bhan
Article

Abstract

Lymphocyte subset estimations by flow cytometry in population-based studies require transportation of samples from the field site to the laboratory. As samples arrive late in the day they have to wait overnight before being processed. The effect of two possible approaches, sample storage for 24 h before staining and immediate staining with analysis after 24 h and 48 h were evaluated. Two sets of experiments were performed with EDTA (ethylenediamine tetra-acetate) anticoagulated peripheral blood. In the first experiment, after collection, each sample was divided into two portions. One portion was stained at the time of blood collection and the other 24 h later after keeping it at room temperature (38–45°C). In the second experiment, blood samples were stained within 1–2 h. Each sample was analyzed immediately upon completion of staining process and subsequently after 24 h and 48 h of storage at 4°C. Results suggest that blood collected in EDTA can be processed using whole blood lysis method, after storage at room temperature (38–45°C) for 24 h with some but not significant alteration in T-cell subsets. Storage at 4°C after staining for 24 h results in a lesser and insignificant loss of cells or alteration of T-cell subsets and may be the method of choice.

Key Words

Lymphocyte storage flow cytometry leukocyte phenotypes 

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References

  1. 1.
    Ekong, T., Hill, A.M., Gomples, M., Brown, A. and Pinching, A.J. (1992) The effect of the temperature and duration of sample storage on the measurement of lymphocyte subpopulations from HIV-I-Positive and control subjects. J. Immunol. Methods 151: 217–225.PubMedCrossRefGoogle Scholar
  2. 2.
    Thornthwaite, J.T., Rosenthal, P.K., Vasquez, D.A. and Seckinger, D. (1984) The effects of anticoagulant and temperature on the measurement of helper and supresser cells. Diagn. Immunol. 2: 167–174.PubMedGoogle Scholar
  3. 3.
    Reinherz, E.L., Kung, P.Z., Goldstein, G. and Schlossman, S.F. (1979) A monoclonal antibody with selective reactivity with functionally mature human thymocytes and all peripheral human T cells. J. Immunol. 123: 1312–1317.PubMedGoogle Scholar
  4. 4.
    Thomas, Y., Sosman, J., Irigoyen, O., Friedman, S.M., Kung, P.C., Goldstein, G. and Chess, L. (1980) Expression of cell surface markers after human B lymphocyte activation. Immunol. 125: 2402–2408.Google Scholar
  5. 5.
    Stashenko P., Nadler, L.M., Hardy, R. and Schlossman, S.F. (1981) Expression of cell surface markers after human B lymphocyte activation. Proc. Natl. Acd. Sci. 78: 3848–3852.CrossRefGoogle Scholar
  6. 6.
    Liu, C., Muirhead, K.A., George, S.P. and Landay, A.L. (1989) Flow cytometric monitoring of HIV-infected patients: Simultaneous enumeration of five lymphocyte subsets. Am. J. Clin. Pathol. 92: 721–728.PubMedGoogle Scholar
  7. 7.
    Prince, H. and Jensen, E. (1991) Three colour cytofluorometric analysis of Cd8 cell substes in HIV-1 infection. J. Acquir. Immuno. Defic. Synd. 4: 1227–1232.Google Scholar
  8. 8.
    Ekong, T., Hill, A.M., Clark, C., Alun, D. and Pinching, A.J.P. (1993) Technical influences on immunophenotyping by flow cytometry the effect of time and temperature of storage on the viability of lymphocyte subsets. J. Immunol. Methods. 164: 263–273.PubMedCrossRefGoogle Scholar
  9. 9.
    Shield, C.F., Martlett, P. and Smith, A. (1983) Stability of human lymphocyte differentiation antigens when stored at room temperature. J. Immunol. Methods. 62: 347.PubMedCrossRefGoogle Scholar
  10. 10.
    Bongers, V. and Bertrams, J. (1984) The influence of common variables on T cell subset analysis by monoclonal antibodies. J. Immunol. Methods. 67 (2): 243–253.PubMedCrossRefGoogle Scholar
  11. 11.
    Parker, J.W., Adelsberg, B., Azen, S.P., Boone, D., Fletcher, M.A., Gjerset, G.F.,et al. (1990) Leucocyte immunophenotyping by flowcytometry in a multisite study: standardisation, quality control and normal values in the transfusion safety study. Clin. Immunal. Immunopathol. 55: 187–220.CrossRefGoogle Scholar
  12. 12.
    Nicholson, J.K.A., Jones, B.M. and Cross, D. (1984) Comparison of T and B cell analysis on fresh and aged blood. J. Immunol. Methods. 73: 29–40.PubMedCrossRefGoogle Scholar
  13. 13.
    Hensleigh, P.A., Waters, V.B. and Herzenberg, L.A. (1993) Human T lymphocyte differentiation antigens. Effects of blood sample storage of Leu antibody binding. Cytometry. 3: 453–455.CrossRefGoogle Scholar
  14. 14.
    Miller, C.H. and Levy, N.B. (1989) Effects of storage conditions on lymphocyte phenotypes from healthy and diseased persons. J. Clin. Lab. Anal. 3: 296–300.PubMedCrossRefGoogle Scholar
  15. 15.
    Nicholson, J.K.A. and Green, T.A. (1993) Selection of anticoagulants for lymphocyte immunophenotyping. J. Immunol. Methods. 165: 31–35.PubMedCrossRefGoogle Scholar
  16. 16.
    Paxton, H. and Bendele, T. (1993) Effect of Time, Temperature and Anticoagulant on Flow Cytometry and Hematological values. Annals of New York Academy of Sciences 677: 440–443.CrossRefGoogle Scholar
  17. 17.
    Lloyd, J.B., Gill, H.S. and Husband, A.J. (1995) The effect of storage on immunophenotyping of sheep peripheral blood lymphocytes by flow cytometry. Vet. Immunol. Immunopathol. 47: 135–142.PubMedCrossRefGoogle Scholar
  18. 18.
    Ruiz, A.R., Fernandez, I.P., Perea Baena, J.M., de La Torre, F.J. and Ramirez, G.R. (2000) Influence of sample storage time and temperature on lymphocyte subset count using a FACS count system. Haematologica. 85: 550–551.PubMedGoogle Scholar
  19. 19.
    Jalla, S., Sazawal, S., Deb, S., Black, R.E. and Bhan, M.K. (2002) Modifications in flow cytometric estimation of T cells subsets and B cells in peripheral blood to reduce the cost of investigation. Indian Journal of Clinical Biochemistry. 17 (1): 69–74.CrossRefGoogle Scholar

Copyright information

© Association of Clinical Biochemists of India 2004

Authors and Affiliations

  • Sanju Jalla
    • 1
  • Sunil Sazawal
    • 3
  • Salkat Deb
    • 3
  • robert E. Black
    • 2
  • Satya Narayan Das
    • 4
  • Archana Sarkar
    • 3
  • Maharaj K. Bhan
    • 5
  1. 1.Center for Cancer ResearchJohn Hopkins University School of MedicineBaltimoreUSA
  2. 2.Blumenberg School of Public HealthJohns Hopkins UniversityBaltimoreUSA
  3. 3.Center for Micronutrient ResearchAnnamalai UniversityChidambaram
  4. 4.Department of BiotechnologyAll India Institute of Medical SciencesNew Delhi
  5. 5.Department of PediatricsAll India Institute of Medical SciencesNew Delhi

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