A new approach for rapid and reliable enumeration of circulating endothelial cells in patients
J Kraan1, M H Strijbos, A M Sieuwerts
1Department of Medical Oncology, Erasmus Medical Center, Daniel den Hoed Cancer Center, Rotterdam, Netherlands. j.kraan@erasmusmc.nl
Insights
A new, affordable flow cytometric assay accurately enumerates circulating endothelial cells (CECs), which are key markers of endothelial dysfunction. This method offers a reliable way to quantify CECs in healthy individuals and cancer patients.
Area of Science:
- Biomedical Engineering
- Clinical Diagnostics
- Flow Cytometry
Background:
- Mature circulating endothelial cells (CECs) serve as biomarkers for endothelial damage and dysfunction.
- Current methods for CEC enumeration lack standardization, leading to significant variability in reported cell counts.
Purpose of the Study:
- To develop a rapid, reliable, robust, and affordable assay for enumerating CECs.
- To establish a multi-parameter flow cytometric (FCM) method for CEC quantification without requiring immunological pre-enrichment.
Main Methods:
- CECs were identified as CD34+, CD45neg, CD146+, and DNA+ events, based on endothelial cell immunophenotype.
- The assay utilized absolute CD34 counts from a 4 mL blood volume for precise CEC enumeration, even at low frequencies (< 1 cell/µL).
Main Results:
- The endothelial origin of identified CECs was confirmed through morphology, immunohistochemistry, and gene expression analysis.
- The novel FCM assay demonstrated comparable results to the validated CellSearch assay.
- CEC levels were significantly elevated in patients with advanced solid and hematological malignancies compared to healthy individuals.
Conclusions:
- The developed flow cytometric method provides a fast and economical approach for enumerating and characterizing CECs.
- This assay has the potential to be a valuable tool in clinical diagnostics and research for assessing endothelial health.
Background:
Mature circulating endothelial cells (CECs) are surrogate markers of endothelial damage/dysfunction. A lack of standardized assays and consensus on CEC phenotype has resulted in a wide variation of reported CEC numbers (4-1300 per mL).
Objectives:
Given the need for a quick, reliable, robust and validated CEC assay at an affordable price, we present a novel approach to enumerate CECs using a multi-parameter flow cytometric (FCM) method without immunological pre-enrichment.
Methods:
CECs were defined as CD34+, CD45neg, CD146+ and DNA+ events based on the immunophenotype of endothelial cells from vein-wall dissections. As CECs express high levels of CD34, we based our assay on absolute CD34 counts after analyzing all CD34 positive events in a total blood volume of 4 mL needed for a precise enumeration of CECs at a frequency of < 1 cell μL(-1).
Results:
The endothelial origin of CECs was confirmed by morphology, immunohistochemistry and gene expression. The new FCM assay was tested in parallel with a validated assay (i.e. CellSearch). CEC levels ranged from 4 to 79 CEC mL(-1) in healthy individuals and were significantly higher in patients with advanced solid malignancies (P = 0.0008) and in patients with hematological malignancies (P < 0.0001).
Conclusions:
This flow cytometric method should be useful as a fast and economical assay to enumerate and characterize CECs.
