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Flow cytometric identification and functional characterization of immature and mature circulating endothelial cells.

ABSTRACT: We sought to identify and characterize 2 distinct populations of bona fide circulating endothelial cells, including the endothelial colony-forming cell (ECFC), by polychromatic flow cytometry (PFC), colony assays, immunomagnetic selection, and electron microscopy.Mononuclear cells from human umbilical cord blood and peripheral blood were analyzed using our recently published PFC protocol. A population of cells containing both ECFCs and mature circulating endothelial cells was determined by varying expressions of CD34, CD31, and CD146 but not AC133 and CD45. After immunomagnetic separation, these cells failed to form hematopoietic colonies, yet clonogenic endothelial colonies with proliferative potential were obtained, thus verifying their identity as ECFCs. The frequency of ECFCs were increased in cord blood and were extremely rare in the peripheral blood of healthy adults. We also detected another mature endothelial cell population in the circulation that was apoptotic. Finally, when comparing this new protocol with a prior method, we determined that the present protocol identifies circulating endothelial cells, whereas the earlier protocol identified extracellular vesicles.Two populations of circulating endothelial cells, including the functionally characterized ECFC, are now identifiable in human cord blood and peripheral blood by PFC.

SUBMITTER: Mund JA

PROVIDER: S-EPMC3306529 | biostudies-literature | 2012 Apr

REPOSITORIES: biostudies-literature

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Flow cytometric identification and functional characterization of immature and mature circulating endothelial cells.

Mund Julie A JA Estes Myka L ML Yoder Mervin C MC Ingram David A DA Case Jamie J

Arteriosclerosis, thrombosis, and vascular biology 20120126 4

<h4>Objective</h4>We sought to identify and characterize 2 distinct populations of bona fide circulating endothelial cells, including the endothelial colony-forming cell (ECFC), by polychromatic flow cytometry (PFC), colony assays, immunomagnetic selection, and electron microscopy.<h4>Methods and results</h4>Mononuclear cells from human umbilical cord blood and peripheral blood were analyzed using our recently published PFC protocol. A population of cells containing both ECFCs and mature circula ...[more]

PMID: 22282356

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Project description:Background:Circulating endothelial cells (CECs) and their subpopulations could be potential novel biomarkers for various malignancies. However, reliable enumerable methods are warranted to further improve their clinical utility. This study aimed to optimize a flow cytometric method (FCM) assay for CECs and subpopulations in peripheral blood for patients with solid cancers. Patients and methods:An FCM assay was used to detect and identify CECs. A panel of 60 blood samples, including 44 metastatic cancer patients and 16 healthy controls, were used in this study. Some key issues of CEC enumeration, including sample material and anticoagulant selection, optimal titration of antibodies, lysis/wash procedures of blood sample preparation, conditions of sample storage, sufficient cell events to enhance the signal, fluorescence-minus-one controls instead of isotype controls to reduce background noise, optimal selection of cell surface markers, and evaluating the reproducibility of our method, were integrated and investigated. Wilcoxon and Mann-Whitney U tests were used to determine statistically significant differences. Results:In this validation study, we refined a five-color FCM method to detect CECs and their subpopulations in peripheral blood of patients with solid tumors. Several key technical issues regarding preanalytical elements, FCM data acquisition, and analysis were addressed. Furthermore, we clinically validated the utility of our method. The baseline levels of mature CECs, endothelial progenitor cells, and activated CECs were higher in cancer patients than healthy subjects (P<0.01). However, there was no significant difference in resting CEC levels between healthy subjects and cancer patients (P=0.193). Conclusion:We integrated and comprehensively addressed significant technical issues found in previously published assays and validated the reproducibility and sensitivity of our proposed method. Future work is required to explore the potential of our optimized method in clinical oncologic applications.

Dataset Information

Flow cytometric identification and functional characterization of immature and mature circulating endothelial cells.

Publications

Flow cytometric identification and functional characterization of immature and mature circulating endothelial cells.

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