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 \ub1 171/mL in healthy subjects (n = 37) and 951 \ub1 1,876/mL in cancer patients (n - 78; P \uab 0.0001). The fraction of apoptotic/necrotic CECs was 77 \ub1 14% in healthy subjects and 43 \ub1 23% in cancer patients (P \uab 0.0001). CEPs were 181 \ub1 167/mL in healthy donors and 429 \ub1 507/mL in patients (P = 0.00019). Coefficients of variation were 4 \ub1 4% (intrareader), 17 \ub1 4% (interreader), and 17 \ub1 7% (variability over 0-72 h), respectively. Parallel samples were frozen by a standardized protocol. After thawing, coefficients of variation were 12 \ub1 8% (intrareader), 16 \ub1 10% (interreader), and 26 \ub1 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