title: CD146-based immunomagnetic enrichment followed by multiparameter flow cytometry: a new approach to counting circulating endothelial cells.
abstract: BACKGROUND
  Circulating endothelial cells (CECs) have emerged as non-invasive biomarkers of vascular dysfunction. The most widely used method for their detection is CD146-based immunomagnetic separation (IMS). Although this approach has provided consensus values in both normal and pathologic situations, it remains tedious and requires a trained operator.
  
  
  OBJECTIVE
  Our objective was to evaluate a new hybrid assay for CEC measurement using a combination of pre-enrichment of CD146+ circulating cells and multiparametric flow cytometry measurement (FCM).
  
  
  METHODS
  CECs were determined in peripheral blood from 20 healthy volunteers, 12 patients undergoing coronary angioplasty, and 30 renal transplant recipients, and blood spiked with cultured endothelial cells. CD146+ cells were isolated using CD146-coated magnetic nanoparticles and labeled using CD45-fluorescein isothiocyanate and CD146-PE or isotype control antibody and propidium iodide before FCM. The same samples were also processed using CD146-based immunomagnetic separation as the reference method.
  
  
  RESULTS
  The hybrid assay detected CECs, identified as CD45(dim)/CD146(bright)/propidium iodide(+), with high size-related scatter characteristics, and clearly discriminated these from CD45(bright)/CD146(dim) activated T lymphocytes. The method demonstrated both high recovery efficiency and good reproducibility. Both IMS and the hybrid assay similarly identified increased CEC levels in patients undergoing coronary angioplasty and renal transplantation, when compared to healthy controls. In patients, CEC values from these two methods were of the same order of magnitude and highly correlated. Bland-Altman analysis revealed poor statistical agreement between methods, flerrofluid-FCM providing higher values than IMS.
  
  
  CONCLUSIONS
  This new hybrid FCM assay constitutes an accurate alternative to visual counting of CECs following CD146-based IMS.
authors.affiliation: UMR-S 608 INSERM-Université de la Méditerranée Laboratoire d'Hématologie et d'Immunologie, UFR de Pharmacie, Université de la Méditerranée, Marseille, France.
authors.forename: A
authors.initials: A
authors.lastname: Widemann
authors.forename: F
authors.initials: F
authors.lastname: Sabatier
authors.forename: L
authors.initials: L
authors.lastname: Arnaud
authors.forename: L
authors.initials: L
authors.lastname: Bonello
authors.forename: G
authors.initials: G
authors.lastname: Al-Massarani
authors.forename: F
authors.initials: F
authors.lastname: Paganelli
authors.forename: P
authors.initials: P
authors.lastname: Poncelet
authors.forename: F
authors.initials: F
authors.lastname: Dignat-George
mesh_terms: D001775:Blood Circulation; D051929:CD146 Antigen; D002452:Cell Count; D042783:Endothelial Cells / Q000166:cytology*; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D018189:Immunomagnetic Separation
chemical_list: D051929:CD146 Antigen