Quantifying Arterial Plaque Composition

Carotid cross sections were stained for SR (Direct Red 80, Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany; 365548), ki67 (with the antibody M7248 from Dako, Agilent Technologies, Santa Clara, CA, USA; 1:10 diluted) and SMA (M0851; Dako; 1:100 diluted). These sections were scanned. Subsequent analysis was conducted with QuPath software (version 0.2.3, Queen’s University, Belfast, UK) [61 (link)]: once more, the areas of media and neointima were first designated manually before the subsequent parameters were automatically quantified for these two areas. The positive cell detection command in QuPath was used for cell detection, counting and classification. In the ki67-stained sections, the overall cell number, as well as the cell density per mm² and the number of ki67+ cells per cross-section were determined. The threshold for positive cell detection was set using the pixel intensity histogram in order to ensure a standardized classification while still taking staining variation between slides into account. The formula used was:
The relative proportion of SMA+ and SR+ areas was measured. For quantification of SMA-positive cells, it is common to analyze the stained areas [62 (link),63 (link)], as a reliable assignment to individual cells is not possible.