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

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 P 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.