A new approach for rapid and reliable enumeration of circulating endothelial cells in patients

J Thromb Haemost. 2012 May;10(5):931-9. doi: 10.1111/j.1538-7836.2012.04681.x.


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.

Publication types

  • Comparative Study

MeSH terms

  • Antigens, CD34 / analysis
  • Biomarkers / analysis
  • CD146 Antigen / analysis
  • Case-Control Studies
  • Cell Count / methods*
  • Endothelial Cells / immunology
  • Endothelial Cells / pathology*
  • Flow Cytometry*
  • Gene Expression Regulation
  • Genotype
  • Humans
  • Immunohistochemistry
  • Immunophenotyping*
  • Leukocyte Common Antigens / analysis
  • Neoplasms / blood
  • Neoplasms / genetics
  • Neoplasms / immunology
  • Neoplasms / pathology*
  • Netherlands
  • Phenotype
  • Predictive Value of Tests
  • Prognosis
  • Real-Time Polymerase Chain Reaction
  • Reproducibility of Results
  • Reverse Transcriptase Polymerase Chain Reaction


  • Antigens, CD34
  • Biomarkers
  • CD146 Antigen
  • MCAM protein, human
  • Leukocyte Common Antigens
  • PTPRC protein, human