title: Imaging flow cytometry for morphologic and phenotypic characterization of rare circulating endothelial cells.
abstract: Endothelial cells in the peripheral circulation are rare events that require technically rigorous approaches for detection by flow cytometry. Visualization of these cells has been even more demanding, as this has historically required extensive enrichment and processing prior to attempting imaging. As a result, few, if any, examples exist on images of peripheral blood circulating endothelial cells (CECs) that include verification of the cell lineage both phenotypically and genomically. In this study, we have devised a method whereby CECs can be directly visualized after lysis of red blood cells and staining, without pre-enrichment or additional processing. Peripheral blood is stained with CD45, CD146, CD3, Hoechst, and DAPI to permit identification of CD146 positive, nonleukocyte, nucleated, and live cells that fit the description of CECs. These cells are imaged using the Amnis ImageStream(X), an imaging flow cytometer. Genomic verification of the endothelial nature of these cells is accomplished by using an aliquot of the same stained samples for sorting CECs using similar gating strategies. This proof of principle of direct imaging of CECs by imaging flow cytometry will permit studies to be conducted heretofore not possible, as the ImageStream(X) has the capability of detecting additional fluorochromes other than those used to identify the CECs. Such potential investigations include antigen colocalization or capping, autophagy and apoptosis, morphologic changes in response to therapy, and others. Thus, this method will enable a broad range of novel studies to be conducted using CECs as surrogates of the endothelium.
authors.affiliation: Flow Cytometry Core Facility, National Heart Lung and Blood Institute, NIH, Bethesda, Maryland.
authors.forename: Leigh
authors.initials: L
authors.lastname: Samsel
authors.forename: Pradeep K
authors.initials: PK
authors.lastname: Dagur
authors.forename: Nalini
authors.initials: N
authors.lastname: Raghavachari
authors.forename: Catherine
authors.initials: C
authors.lastname: Seamon
authors.forename: Gregory J
authors.initials: GJ
authors.lastname: Kato
authors.forename: J Philip
authors.initials: JP
authors.lastname: McCoy
mesh_terms: D051929:CD146 Antigen / Q000097:blood; D002452:Cell Count; D019070:Cell Lineage; D003952:Diagnostic Imaging; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D004912:Erythrocytes / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans
chemical_list: D051929:CD146 Antigen; C497902:MCAM protein, human
keywords: circulating endothelial cells; imaging cytometry; rare event cytometry
references.citation: Cytometry B Clin Cytom. 2005 Mar;64(1):1-8
references.pmid: 15668988
references.citation: Cytometry A. 2007 Feb;71(2):105-13
references.pmid: 17226859
references.citation: J Am Coll Cardiol. 2012 Jul 24;60(4):324-31
references.pmid: 22813611
references.citation: Thromb Haemost. 2006 Jul;96(1):45-52
references.pmid: 16807650
references.citation: Cancer Res. 2006 Mar 15;66(6):2918-22
references.pmid: 16540638
references.citation: Am J Physiol Cell Physiol. 2012 Jan 15;302(2):C383-91
references.pmid: 22031599
references.citation: Clin Lab Med. 2007 Sep;27(3):653-70, viii
references.pmid: 17658411
references.citation: Nat Commun. 2012 Feb 14;3:671
references.pmid: 22334074
references.citation: Methods. 2001 Dec;25(4):402-8
references.pmid: 11846609
references.citation: J Autoimmun. 2011 Dec;37(4):319-27
references.pmid: 21959269
references.citation: Clin Cancer Res. 2009 Jan 1;15(1):267-73
references.pmid: 19118054
references.citation: Thromb Haemost. 2003 Oct;90(4):698-703
references.pmid: 14515191
references.citation: Cytometry B Clin Cytom. 2011 Sep;80(5):335-8
references.pmid: 21567939
references.citation: Cancer. 2010 Apr 15;116(8):1926-37
references.pmid: 20166207
references.citation: Thromb Haemost. 2005 Feb;93(2):228-35
references.pmid: 15711737