Imaging flow cytometry for morphologic and phenotypic characterization of rare circulating endothelial cells

Leigh Samsel, Pradeep K. Dagur, Nalini Raghavachari, Catherine Seamon, Gregory J. Kato, J. Philip McCoy

Research output: Contribution to journalArticle

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 ImageStreamX, 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 ImageStreamX 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. Published 2013 Wiley-Periodicals, Inc.†

Original languageEnglish (US)
Pages (from-to)379-389
Number of pages11
JournalCytometry Part B - Clinical Cytometry
Volume84
Issue number6
DOIs
StatePublished - Nov 2013
Externally publishedYes

Fingerprint

Flow Cytometry
Endothelial Cells
Autophagy
Cell Lineage
Fluorescent Dyes
Endothelium
Erythrocytes
Apoptosis
Staining and Labeling
Antigens

Keywords

  • circulating endothelial cells
  • imaging cytometry
  • rare event cytometry

ASJC Scopus subject areas

  • Cell Biology
  • Histology
  • Pathology and Forensic Medicine

Cite this

Imaging flow cytometry for morphologic and phenotypic characterization of rare circulating endothelial cells. / Samsel, Leigh; Dagur, Pradeep K.; Raghavachari, Nalini; Seamon, Catherine; Kato, Gregory J.; McCoy, J. Philip.

In: Cytometry Part B - Clinical Cytometry, Vol. 84, No. 6, 11.2013, p. 379-389.

Research output: Contribution to journalArticle

Samsel, Leigh ; Dagur, Pradeep K. ; Raghavachari, Nalini ; Seamon, Catherine ; Kato, Gregory J. ; McCoy, J. Philip. / Imaging flow cytometry for morphologic and phenotypic characterization of rare circulating endothelial cells. In: Cytometry Part B - Clinical Cytometry. 2013 ; Vol. 84, No. 6. pp. 379-389.
@article{cdc26882d830477f85028a0e2227525a,
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 ImageStreamX, 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 ImageStreamX 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. Published 2013 Wiley-Periodicals, Inc.†",
keywords = "circulating endothelial cells, imaging cytometry, rare event cytometry",
author = "Leigh Samsel and Dagur, {Pradeep K.} and Nalini Raghavachari and Catherine Seamon and Kato, {Gregory J.} and McCoy, {J. Philip}",
year = "2013",
month = "11",
doi = "10.1002/cyto.b.21088",
language = "English (US)",
volume = "84",
pages = "379--389",
journal = "Cytometry Part B - Clinical Cytometry",
issn = "1552-4949",
publisher = "Wiley-Liss Inc.",
number = "6",

}

TY - JOUR

T1 - Imaging flow cytometry for morphologic and phenotypic characterization of rare circulating endothelial cells

AU - Samsel, Leigh

AU - Dagur, Pradeep K.

AU - Raghavachari, Nalini

AU - Seamon, Catherine

AU - Kato, Gregory J.

AU - McCoy, J. Philip

PY - 2013/11

Y1 - 2013/11

N2 - 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 ImageStreamX, 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 ImageStreamX 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. Published 2013 Wiley-Periodicals, Inc.†

AB - 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 ImageStreamX, 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 ImageStreamX 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. Published 2013 Wiley-Periodicals, Inc.†

KW - circulating endothelial cells

KW - imaging cytometry

KW - rare event cytometry

UR - http://www.scopus.com/inward/record.url?scp=84887498634&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84887498634&partnerID=8YFLogxK

U2 - 10.1002/cyto.b.21088

DO - 10.1002/cyto.b.21088

M3 - Article

VL - 84

SP - 379

EP - 389

JO - Cytometry Part B - Clinical Cytometry

JF - Cytometry Part B - Clinical Cytometry

SN - 1552-4949

IS - 6

ER -