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Validation of a Standardized Method for Enumerating Circulating Endothelial Cells and Progenitors: Flow Cytometry and Molecular and Ultrastructural Analyses

Authors' Affiliations: ¹ Division of Hematology-Oncology, Department of Medicine, European Institute of Oncology; ² FIRC Institute of Molecular Oncology Foundation; ³ University of Milan, Milan, Italy; ⁴ The University of Texas M. D. Anderson Cancer Center, Houston, Texas; and ⁵ Pfizer Global Research and Development, La Jolla, California

Requests for reprints: Francesco Bertolini, Division of Hematology-Oncology, Department of Medicine, European Institute of Oncology, via Ripamonti 435, 20141 Milan, Italy. Phone: 39-2-57489535; Fax: 39-2-57489537; E-mail: francesco.bertolini{at}ieo.it.

Purpose: Antigenic overlap among circulating endothelial cells (CEC) and progenitors (CEP), platelets, and other blood cells led to the need to develop a reliable standardized method for CEC and CEP quantification. These cells are emerging as promising preclinical/clinical tools to define optimal biological doses of antiangiogenic therapies and to help stratify patients in clinical trials.

Experimental Design: We report the experimental validation of a novel flow cytometry method that precisely dissects CEC/CEP from platelets and other cell populations and provides information about CEC/CEP viability.

Results: Sorted DNA/Syto16⁺CD45^–CD31⁺CD146⁺ CECs, investigated by electron microscopy, were found to be bona fide endothelial cells by the presence of Weibel-Palade bodies. More than 75% of the circulating mRNAs of the endothelial-specific gene, VE-cadherin, found in the blood were present in the sorted population. CECs were 140 ± 171/mL in healthy subjects (n = 37) and 951 ± 1,876/mL in cancer patients (n = 78; P < 0.0001). The fraction of apoptotic/necrotic CECs was 77 ± 14% in healthy subjects and 43 ± 23% in cancer patients (P < 0.0001). CEPs were 181 ± 167/mL in healthy donors and 429 ± 507/mL in patients (P = 0.00019). Coefficients of variation were 4 ± 4% (intrareader), 17 ± 4% (interreader), and 17 ± 7% (variability over 0-72 h), respectively. Parallel samples were frozen by a standardized protocol. After thawing, coefficients of variation were 12 ± 8% (intrareader), 16 ± 10% (interreader), and 26 ± 16% (variability over 0-14 days of frozen storage), respectively.

Conclusions: This procedure enumerates a truly endothelial cell population with limited intrareader and interreader variability. It appears possible to freeze samples for large-scale CEC enumeration during clinical trials. This approach could be enlarged to investigate other angiogenic cell populations as well.

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				M. H. Strijbos, J. Kraan, C. H.J. Lamers, S. Sleijfer, and J. W. Gratama Quantification of Circulating Endothelial Cells by Flow Cytometry Clin. Cancer Res., May 15, 2009; 15(10): 3640 - 3640. [Full Text] [PDF]