Validation of a standardized method for enumerating circulating endothelial cells and progenitors: flow cytometry and molecular and ultrastructural analyses.
OBJECTIVE
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.


METHODS
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.
Mancuso
Patrizia
P
Division of Hematology-Oncology, Department of Medicine, European Institute of Oncology, Milan, Italy.
Antoniotti
Pierluigi
P
Quarna
Jessica
J
Calleri
Angelica
A
Rabascio
Cristina
C
Tacchetti
Carlo
C
Braidotti
Paola
P
Wu
Hua-Kang
HK
Zurita
Amado J
AJ
Saronni
Luca
L
Cheng
John B
JB
Shalinsky
David R
DR
Heymach
John V
JV
Bertolini
Francesco
F
D002452:Cell Count / Q000379:methods; D002470:Cell Survival; D042783:Endothelial Cells / Q000737:chemistry; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D008854:Microscopy, Electron; D009369:Neoplasms / Q000097:blood*; D015203:Reproducibility of Results; D013234:Stem Cells; D020302:Weibel-Palade Bodies