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

Area of Science:

• Cardiovascular Biology
• Cellular Biology
• Hematology

Background:

• Peripheral blood circulating endothelial cells (CECs) are rare and difficult to detect and visualize.
• Existing methods require extensive sample enrichment and processing, limiting detailed analysis.
• Few studies have provided both phenotypic and genomic verification of peripheral CECs.

Purpose of the Study:

• To develop a direct visualization method for peripheral CECs using imaging flow cytometry.
• To enable phenotypic and genomic verification of CECs without pre-enrichment.
• To establish CECs as viable surrogates for studying the endothelium in circulation.

Main Methods:

• Developed a protocol for direct imaging of CECs after red blood cell lysis and staining.
• Utilized specific markers (CD45, CD146, CD3, Hoechst, DAPI) for CEC identification.
• Employed Amnis ImageStream(X) imaging flow cytometry for cell visualization.
• Performed genomic verification via cell sorting using similar gating strategies.

Main Results:

• Successfully visualized CECs directly from peripheral blood without pre-enrichment.
• Confirmed CEC identification through combined phenotypic and genomic analysis.
• Demonstrated the capability of imaging flow cytometry for detailed CEC analysis.

Conclusions:

• The developed method allows direct, detailed visualization and verification of peripheral CECs.
• This technique overcomes previous limitations in CEC detection and imaging.
• Enables novel investigations into endothelial biology and disease using CECs as surrogates.