Quantification of circulating endothelial progenitor cells: A methodological comparison of six flow cytometric approaches

Craenenbroeck, Emeline M. F. Van; Conraads, Viviane M.; Bockstaele, Dirk R. Van; Haine, Steven; Vermeulen, Katrien; Tendeloo, Viggo Van; Vrints, Christiaan J.; Hoymans, Vicky Y.

doi:10.1016/j.jim.2007.12.006

Cited by 75 publications

(57 citation statements)

References 43 publications

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“…We and others have described that CEC and CEP kinetics correlate with survival in cancer patients treated with antiangiogenic therapies and might help to stratify patients who are likely to receive clinical benefit from antiangiogenic or antivascular treatments (2 -11). In these clinical studies, CECs have been measured in most cases by multiparametric flow cytometry, an approach that requires accurate sequential gating and is prone to operator-induced variability (12). Moreover, antigenic overlap between CECs and platelets has generated the concern that some of the cells counted as CECs by flow cytometry might not be endothelial cells (13,14).…”

mentioning

confidence: 99%

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

Mancuso

Antoniotti

Quarna

et al. 2008

Clinical Cancer Research

150

140

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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.+ 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 F 171/mL in healthy subjects (n = 37) and 951 F1,876/mL in cancer patients (n = 78; P < 0.0001). The fraction of apoptotic/necrotic CECs was 77 F 14% in healthy subjects and 43 F 23% in cancer patients (P < 0.0001). CEPs were 181 F 167/mL in healthy donors and 429 F 507/mL in patients (P = 0.00019). Coefficients of variation were 4 F 4% (intrareader), 17 F 4% (interreader), and 17 F 7% (variability over 0-72 h), respectively. Parallel samples were frozen by a standardized protocol. After thawing, coefficients of variation were 12 F 8% (intrareader), 16 F 10% (interreader), and 26 F 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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mentioning

confidence: 99%

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

Mancuso

Antoniotti

Quarna

et al. 2008

Clinical Cancer Research

150

140

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“…A general agreement for measurement of circulating adult EPCs is still absent. To date, direct ex vivo flow cytometry from peripheral blood (with or without pre-enrichment of mononuclear cells) and in vitro enumeration of CFUs or spindle-shaped cells are the most commonly used methods for evaluation of EPCs (3,11,(17)(18)(19). However, such a divergent approach makes comparison of study outcomes very difficult and is the cause for some inconsistence and controversy.…”

Section: Discussionmentioning

confidence: 99%

An optimized flow cytometry protocol for analysis of angiogenic monocytes and endothelial progenitor cells in peripheral blood

et al. 2009

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Circulating adult CD341 VEGFR2 1 endothelial progenitor cells (EPCs) have been shown to differentiate into endothelial cells, thus contributing to vascular homeostasis. Furthermore, a subset of circulating CD141 monocytes coexpresses CD16 together with the angiopoietin receptor Tie2 and has been functionally implicated in tumor angiogenesis. However, clinically applicable protocols for flow cytometric quantification of EPCs and Tie2 1 monocytes in peripheral blood and a consensus on reference values remain elusive. The number of Tie2 1 CD14 1 CD16 mid angiogenic monocytes and CD34 1 VEGFR2 1 CD45 low/2 EPCs was assessed in the peripheral venous blood of patients with stable coronary artery disease by three-color flow cytometry using specific monoclonal antibodies conjugated to PerCP, PE, PE-Cy7, APC, and APC-Cy7. Scatter multigating with exclusion of dead cells was performed to dissect complex mononuclear cell populations. This analysis was further refined by matching bright fluorochromes (PE-Cy7, PE, APC) with dimly expressed markers (CD34, VEGFR2, Tie2), by automatic compensation for minimizing fluorescence spillover and by using fluorescence-minus-one (FMO) controls to determine positive/negative boundaries. Presuming a Gaussian distribution, we obtained average values (mean AE SD) of 1.45 AE 1.29% for Tie2 1 CD14 1 CD16 mid monocytes (n ¼ 11, range: 0.12-3.64%) and 0.019 AE 0.013% for CD34low/2 EPCs (n ¼ 17, range: 0.003-0.042%). The intra-and inter-assay variability was 1.6% and 4.5%, respectively. We have optimized a fast and sensitive assay for the flow cytometric quantification of circulating angiogenic monocytes and EPCs in cardiovascular medicine. This protocol may represent a basis for standardized analysis and monitoring of these cell subsets to define their normal range and prognostic/diagnostic value in clinical use. ' 2009 International Society for Advancement of Cytometry

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“…Specifically, two rare and important CD133ϩ cell populations in whole blood (found at Ͻ1% of the total cell population), namely endothelial progenitor cells (EPCs) and hematopoietic stem cells (HSCs) (37) were isolated, followed by deep proteomic analysis. EPCs possess the ability to differentiate into endothelial cells that make up the lining of blood vessels, and their concentration in blood is known to correlate with cardiovascular risk and clinical outcome (38,39). EPCs also play a role in tumor growth, metastasis and angiogenesis, and therefore, these poorly understood cells are of interest as targets for development of novel therapeutics in cancer research (40,41).…”

Section: Proteomic Profiling Of Hematopoietic Stem Cells (Hscs) and Ementioning

confidence: 99%

An Integrated Platform for Isolation, Processing, and Mass Spectrometry-based Proteomic Profiling of Rare Cells in Whole Blood*

Plouffe

Belov

et al. 2015

Molecular & Cellular Proteomics

148

173

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Quantification of circulating endothelial progenitor cells: A methodological comparison of six flow cytometric approaches

Cited by 75 publications

References 43 publications

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

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

An optimized flow cytometry protocol for analysis of angiogenic monocytes and endothelial progenitor cells in peripheral blood

An Integrated Platform for Isolation, Processing, and Mass Spectrometry-based Proteomic Profiling of Rare Cells in Whole Blood*

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