title: Preservation and enumeration of endothelial progenitor and endothelial cells from peripheral blood for clinical trials.
abstract: OBJECTIVE
  Endothelial progenitor cells (EPCs) are markers of vascular repair. Increased numbers of circulating endothelial cells (ECs) are associated with endothelial damage.
  
  
  METHODS
  We enumerated EPC-EC by using Enrichment kit with addition of anti-human CD146-PE/Cy7 from peripheral blood mononuclear cell (PBMC) isolated either by red blood cell (RBC) lysing solution or by Ficoll centrifugation, and from fresh and preserved samples. PBMCs were quantified by flow cytometry.
  
  
  RESULTS
  RBC lysis yielded higher percentage of PBMC (p = 0.0242) and higher numbers of PBMC/ml (p = 0.0039) than Ficoll. Absolute numbers of CD34(+)CD133(+)VEGFR2(+) and CD146(+)CD34(+)VEGFR2(+) were higher (p = 0.0117 for both), when isolated by RBC lysis than by Ficoll, when no difference in other subsets was found. Cryopreservation at -160°C and -80°C and short-term preservation at room temperature decreased EPC-EC.
  
  
  CONCLUSIONS
  Our data support use of fresh samples and isolation of PBMC from human blood by RBC lysis for enumeration of EPC and EC.
authors.affiliation: Center for Neuroscience & Regenerative Medicine, Uniformed Services University of Health Sciences, 12725 Twinbrook Pkwy, Rockville, MD 20852, USA.|National Institute of Neurological Disorders and Stroke, Stroke Branch, 10 Center Drive, Bethesda, MD 20814, USA.
authors.forename: Tanya
authors.initials: T
authors.lastname: Bogoslovsky
authors.affiliation: National Institute of Neurological Disorders and Stroke, Flow Cytometry Core Facility, 49 Convent Drive, Bethesda, MD 20814, USA.
authors.forename: Dragan
authors.initials: D
authors.lastname: Maric
authors.affiliation: Center for Neuroscience & Regenerative Medicine, Uniformed Services University of Health Sciences, 12725 Twinbrook Pkwy, Rockville, MD 20852, USA.
authors.forename: Yunhua
authors.initials: Y
authors.lastname: Gong
authors.affiliation: Center for Neuroscience & Regenerative Medicine, Uniformed Services University of Health Sciences, 12725 Twinbrook Pkwy, Rockville, MD 20852, USA.
authors.forename: Baoxi
authors.initials: B
authors.lastname: Qu
authors.affiliation: National Institute of Neurological Disorders and Stroke, 10 Center Drive, Bethesda, MD 20814, USA.
authors.forename: Kelly
authors.initials: K
authors.lastname: Yang
authors.affiliation: National Institute of Neurological Disorders and Stroke, Stroke Branch, 10 Center Drive, Bethesda, MD 20814, USA.
authors.forename: Maria
authors.initials: M
authors.lastname: Spatz
authors.affiliation: National Institute of Neurological Disorders and Stroke, Stroke Branch, 10 Center Drive, Bethesda, MD 20814, USA.
authors.forename: John
authors.initials: J
authors.lastname: Hallenbeck
authors.affiliation: Center for Neuroscience & Regenerative Medicine, Uniformed Services University of Health Sciences, 12725 Twinbrook Pkwy, Rockville, MD 20852, USA.
authors.forename: Ramon
authors.initials: R
authors.lastname: Diaz-Arrastia
mesh_terms: D000071916:AC133 Antigen; D000328:Adult; D000368:Aged; D015703:Antigens, CD / Q000378:metabolism; D018952:Antigens, CD34 / Q000378:metabolism; D051929:CD146 Antigen / Q000378:metabolism; D015925:Cryopreservation; D042783:Endothelial Cells / Q000166:cytology*; D004912:Erythrocytes / Q000166:cytology; D005260:Female; D005362:Ficoll / Q000737:chemistry; D005434:Flow Cytometry; D006023:Glycoproteins / Q000378:metabolism; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000166:cytology* / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D010455:Peptides / Q000378:metabolism; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism
chemical_list: D000071916:AC133 Antigen; D015703:Antigens, CD; D018952:Antigens, CD34; D051929:CD146 Antigen; D006023:Glycoproteins; C000606023:PROM1 protein, human; D010455:Peptides; D005362:Ficoll; D040301:Vascular Endothelial Growth Factor Receptor-2
keywords: angiogenesis; cryopreservation; endothelial cells; endothelial progenitor cells; flow cytometry