Unstimulated platelets do not bind to intact endothelium. However, exposure to thrombin or other stimuli induces platelet activation and subsequent platelet adhesion to intact endothelium. Analysis of platelet-endothelial cell adhesion is possible by the LSC with the ability to detect individually bound platelets. LSC imaging of bound platelets to endothelial cells allows analysis of individual platelet populations and ability to re-scan areas of interest. Analysis at the single cell level provides assurance of even distribution of platelet binding.

Screen 1. Illustrates uniform distribution of adherent platelets on the endothelial cell monolayer.   Screen 2. Orange maximum pixel signal depicted in red dots, represents platelets fluorescing. Background luminescence from endothelial cells depicted in white dots can be gated out.   Screen 3. Clumping of platelets and artifacts can be gated out by excluding events larger than a single platelet. Total Count. Events are counted, and total number are displayed.

Scan Data Displays. Left. Laser scan light scatter images of endothelial cell monolayers with individual platelets marked by red and blue circles. Right. More platelets are adherent if previously incubated with a strong agonist (e.g., thrombin).

Laser scanned images utilizing forward scatter and fluorescence showing activated platelets adhering to endothelial cells. The laser-scanned images have been colorized with CompuColor™, a software feature that automatically applies multiple pseudo colors to data displays for enhanced visualization.   Contoured images of monolayers of endothelial cells with individual platelets marked by blue and green circles. The blue and green circles allow one to quantify both the numbers of platelets adhering as well as the level of fluorescence expressed per platelet.

Sample Preparation Endothelial cells are grown to confluence on glass cover slips. Platelets are washed and biotinylated. Manipulation of platelet activity and receptor occupancy can be implemented at this stage. Platelets are incubated with the endothelial cells and labeled with phycoerythrin prior to washing away unbound platelets. Glass cover slips are inverted and edges sealed. LSC Analysis Slides with inverted cover slips are scanned at 20X using 5 mW Laser power. Total area scanned is 16 square millimeters. Fluorescently labeled platelets are identified and counted. Individual display fields can be viewed during scanning for quality control purposes. Procedure and data from R. Brannon Claytor, MD and Alan Michelson, MD, University of Massachusetts Medical Center, Worcester, MA. Ref. XVIIth Congress International Society for Thrombosis & Haemostasis.

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