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A protocol for phenotypic detection and enumeration of circulating endothelial cells and circulating progenitor cells in human blood

Nature Protocols volume 2, pages 805–810 (2007)Cite this article

Abstract

Blood circulating endothelial cells (CECs) and circulating hematopoietic progenitor cells (CPCs) represent two cell populations that are thought to play important roles in tissue vascularization. CECs and CPCs are currently studied as surrogate markers in patients for more than a dozen pathologies, including heart disease and cancer. However, data interpretation has often been difficult because of multiple definitions, methods and protocols used to evaluate and count these cells by different laboratories. Here, we propose a cytometry protocol for phenotypic identification and enumeration of CECs and CPCs in human blood using four surface markers: CD31, CD34, CD133 and CD45. This method allows further phenotypic analyses to explore the biology of these cells. In addition, it offers a platform for longitudinal studies of these cells in patients with different pathologies. The protocol is relatively simple, inexpensive and can be adapted for multiple flow cytometer types or software. The procedure should take 2–2.5 h, and is expected to detect 0.1–6.0% viable CECs and 0.01–0.20% CPCs within blood mononuclear cell population.

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Figure 1: Multicolor flow cytometric analyses of human mononuclear cells separated from whole blood samples.
Figure 2: Multicolor flow cytometric analyses of human mononuclear cells in whole blood samples.

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Acknowledgements

We thank Drs. C. Willett, T. Batchelor, G. Sorensen and M. Ancukiewicz for collaborations on correlative clinical studies, and S. Roberge and G. Gorospe for technical assistance. This work was supported by US National Cancer Institute grants P01-CA80124 and R01-CA115767 to R.K.J. and an American Association for Cancer Research Award to D.G.D.

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Author notes

  1. Dan G Duda and Kenneth S Cohen: These authors contributed equally to establishing this protocol.

Authors and Affiliations

  1. Department of Radiation Oncology, Steele Laboratory for Tumor Biology, Massachusetts General Hospital, Boston, 02114, Massachusetts, USA

    Dan G Duda & Rakesh K Jain

  2. Center for Regenerative Medicine, Massachusetts General Hospital and Harvard Stem Cell Institute, Boston, 02114, Massachusetts, USA

    Kenneth S Cohen & David T Scadden

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  1. Dan G Duda
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  2. Kenneth S Cohen
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  4. Rakesh K Jain
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Correspondence to Rakesh K Jain.

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The authors declare no competing financial interests.

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Duda, D., Cohen, K., Scadden, D. et al. A protocol for phenotypic detection and enumeration of circulating endothelial cells and circulating progenitor cells in human blood. Nat Protoc 2, 805–810 (2007). https://doi.org/10.1038/nprot.2007.111

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