Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • On the Market
  • Published:

Flow cytometry counting of CD34+ cells in whole blood

Nature Medicine volume 6, pages 833–836 (2000)Cite this article

Hematopoietic stem cells are able to self-renew and to differentiate into progenitors of all hematopoietic lineages1,2; these processes are regulated by many cytokines3. A subset of hematopoietic cells, presumably containing hematopoietic stem cells, expresses the cell-surface antigen CD34. CD34+-purified fractions are enriched in colony-forming units and long-term culture initiating cells, whereas CD34− fractions are depleted4. CD34+ cells are commonly used in hematopoietic stem cell transplantation. They can be mobilized from bone marrow into peripheral blood by means of chemotherapy and/or cytokine stimulatory treatments, and then collected for use in cancer therapy5, hematopoietic stem cell expansion studies and gene therapy6. The widening use of stem cell mobilization means that a method that accurately and rapidly quantifies CD34+ cells is very much needed. Although several methods for CD34+ quantification have been proposed, there is a lack of consensus on a method to count this rare population7.

Current flow cytometry methods for counting CD34 + cells

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: a, Kinetic determination of SYTO-13 uptake.
Figure 2: Analysis of CD34+ cells in unlysed whole blood, stained with PE-CD34 and PC5-CD45 monoclonal antibodies, and SYTO-13 dye at a final concentration of 50 nM.
Figure 3: Bland–Altman plots for method comparisons for 80 hematopoietic samples.

References

  1. Metcalf, D. in Haemopoietic Cells 550 (North Holland Publishing, Amsterdam, 1971).

    Google Scholar 

  2. Quesenberry, P. & Levitt, L. Hematopoietic stem cells. N. Engl. J. Med. 301, 755– 760 (1979).

    Article  CAS  Google Scholar 

  3. Spangrude, G.J. Biological and clinical aspects of hematopoietic stem cells. Ann. Rev. Med. 45, 93–104 ( 1994).

    Article  CAS  Google Scholar 

  4. Krause, D.S., Fackler, M.J., Civin, C.I. & May, W.S. CD34: Structure, biology, and clinical utility. Blood 87, 1–13 (1996).

    CAS  PubMed  Google Scholar 

  5. Siena, S. et al. Circulation of CD34+ hematopoietic stem cells in the peripheral blood of high-dose cyclophosphamide-treated patients: Enhancement by intravenous recombinant human granulocyte-macrophage colony-stimulating factor. Blood 74, 1905– 1914 (1989).

    CAS  PubMed  Google Scholar 

  6. Brenner, M.K. et al. Gene marking to determine whether autologous marrow infusion restores long-term haemopoiesis in cancer patients. Lancet 6, 1134–1137 (1993).

    Article  Google Scholar 

  7. Gratama, J.W. et al. Analysis of variation in results of flow cytometric lymphocyte immunophenotyping in a multicenter study. Cytometry 15, 166–177 (1997).

    Article  Google Scholar 

  8. Macey, M.G., McCarthy, D.A., van Agthoven, A. & Newland, A.C. How should CD34+ cells be analysed? A study of three classes of antibody and five leucocyte preparation procedures. J. Immunol. Meth. 26, 175–188 ( 1997).

    Article  Google Scholar 

  9. Cannavo, N., Bossy, D. & Hirn, J. in Leucocyte Typing VI. 978–982 (Garland Publishing, New York, 1998).

    Google Scholar 

  10. Steltzer, G.T., Shults, K.E. & Loken, M.R. CD45 gating for routine flow cytometric analysis of human bone marrow specimens. Ann. NY Acad. Sci. 677 , 265–280 (1993).

    Article  Google Scholar 

  11. Fritsch, G. et al. Quantification of CD34+ cells: Comparison of methods. Transfusion 37, 775– 784 (1997).

    Article  CAS  Google Scholar 

  12. Keeney, M. et al. Single platform flow cytometric absolute CD34+ cell counts based on the ISHAGE guidelines. International Society of Hematotherapy and Graft Engineering. Cytometry 15, 61– 70 (1998).

    Article  Google Scholar 

  13. Hubl, W. et al. Measurement of absolute concentration and viability of CD34+ cells in cord blood and cord blood products using fluorescent beads and cyanine nucleic acid dyes. Cytometry 15, 121–127 (1998).

    Article  Google Scholar 

  14. Barnett, D., Granger, V., Whitby, L., Storie, I. & Reilly, J.T. Absolute CD4+ T-lymphocyte and CD34+ stem cell counts by single-platform flow cytometry: The way forward . Br. J. Haematol. 106, 1059– 1062 (1999).

    Article  CAS  Google Scholar 

  15. Sutherland, D.R., Keating, A., Nayar, R., Anania, S. & Stewart, A.K. Sensitive detection and enumeration of CD34+ cells in peripheral and cord blood by flow cytometry. Exp. Hematol. 10, 1003–1010 (1994).

    Google Scholar 

  16. Yang, H., Acker, J., Chen, A. & McGann, L. In situ assessment of cell viability. Cell. Transplant. 7, 443–451 (1998).

    Article  CAS  Google Scholar 

  17. Chaudhary, P.M. & Roninson, I.B. Expression and activity of P-glycoprotein, a multidrug efflux pump, in human hematopoietic stem cells. Cell 12, 85– 94 (1991).

    Article  Google Scholar 

  18. Goodell, M.A. et al. Dye efflux studies suggest that hematopoietic stem cells expressing low or undetectable levels of CD34 antigen exist in multiple species. Nature Med. 3, 1337–1345 (1997).

    Article  CAS  Google Scholar 

  19. Broxterman, H.J. et al. Detection of P-glycoprotein function for haematological and solid tumour cells using a novel nucleic acid stain. Br. J. Cancer 76, 1029–1034 ( 1997).

    Article  CAS  Google Scholar 

  20. McCarthy, D.A. & Macey, M.G. A simple flow cytometric procedure for the determination of surface antigens on unfixed leucocytes in whole blood. J. Immunol. Meth. 163, 155–160 (1993).

    Article  CAS  Google Scholar 

  21. Bland, J.M. & Altman, D.G. Statistical methods for assessing agreement between two methods of clinical measurement. Lancet 1, 307–310 (1986).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The authors thank D.I.K. Martin for critical review of this report. We thank Ll. Jover for statistical analysis.

Author information

Author notes

  1. Jordi Petriz

    Present address: J.P. present address: Laboratory of Cryobiology, Hospital Clinic de Barcelona, Villarroel, 170, 08036 Barcelona, Spain,

Authors and Affiliations

  1. Department of Cryobiology and Cell Therapy, Cancer Research Institute, Gran Via km 2.7, Barcelona, 08907, Spain

    Oscar Fornas, Joan Garcia & Jordi Petriz

Authors
  1. Oscar Fornas
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Joan Garcia
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jordi Petriz
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jordi Petriz.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Fornas, O., Garcia, J. & Petriz, J. Flow cytometry counting of CD34+ cells in whole blood . Nat Med 6, 833–836 (2000). https://doi.org/10.1038/77571

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1038/77571

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing