title: A new approach for rapid and reliable enumeration of circulating endothelial cells in patients.
abstract: 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).
  
  
  OBJECTIVE
  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.
authors.affiliation: Department of Medical Oncology, Erasmus Medical Center, Daniel den Hoed Cancer Center, Rotterdam, Netherlands. j.kraan@erasmusmc.nl
authors.forename: J
authors.initials: J
authors.lastname: Kraan
authors.forename: M H
authors.initials: MH
authors.lastname: Strijbos
authors.forename: A M
authors.initials: AM
authors.lastname: Sieuwerts
authors.forename: J A
authors.initials: JA
authors.lastname: Foekens
authors.forename: M A
authors.initials: MA
authors.lastname: den Bakker
authors.forename: C
authors.initials: C
authors.lastname: Verhoef
authors.forename: S
authors.initials: S
authors.lastname: Sleijfer
authors.forename: J W
authors.initials: JW
authors.lastname: Gratama
mesh_terms: D018952:Antigens, CD34 / Q000032:analysis; D015415:Biomarkers / Q000032:analysis; D051929:CD146 Antigen / Q000032:analysis; D016022:Case-Control Studies; D002452:Cell Count / Q000379:methods*; D042783:Endothelial Cells / Q000276:immunology / Q000473:pathology*; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D005838:Genotype; D006801:Humans; D007150:Immunohistochemistry; D016130:Immunophenotyping; D017493:Leukocyte Common Antigens / Q000032:analysis; D009369:Neoplasms / Q000097:blood / Q000235:genetics / Q000276:immunology / Q000473:pathology*; D009426:Netherlands; D010641:Phenotype; D011237:Predictive Value of Tests; D011379:Prognosis; D060888:Real-Time Polymerase Chain Reaction; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D018952:Antigens, CD34; D015415:Biomarkers; D051929:CD146 Antigen; C497902:MCAM protein, human; D017493:Leukocyte Common Antigens; C516769:PTPRC protein, human