Abstracts Background and aims The accumulation of erythrocyte membranes within an atherosclerotic plaque may contribute to the deposition of free cholesterol and thereby the enlargement of the necrotic core. Erythrocyte membranes can be visualized and quantified in the plaque by immunostaining for the erythrocyte marker glycophorin C. We therefore hypothesized, that glycophorin C is a marker for plaque vulnerability and may therefore reflect intraplaque hemorrhage (IPH), vulnerability of plaques and predict pre-procedural neurological symptoms.[

Methods We quantified glycophorin C in human atherosclerotic plaque samples from 1,819 consecutive asymptomatic and symptomatic patients undergoing carotid endarterectomy from the Athero-Express Biobank with the slideToolKit method.

Results The prevalence of IPH was 62.4%, and the prevalence of pre-procedural neurological symptoms was 87.1%. The percentage glycophorin staining was significantly higher in male patients compared to female patients (median 7.15 (IQR:3.37, 13.41) versus median 4.06 (IQR:1.98, 8.32), p<0.001). Quantified glycophorin C was associated with IPH (OR 1.90; 95% CI 1.63, 2.21; p=<0.001) when corrected for clinical confounders. Glycophorin C was significantly associated with ipsilateral pre-procedural neurological symptoms (OR:1.27, 95%CI:1.06-1.41, p=0.005), and upon sex-stratified analyses, specifically in male patients (OR 1.37; 95%CI 1.12, 1.69; p=0.003), but not in female patients (OR 1.15; 95%CI 0.77, 1.73; p=0.27). Glycophorin was associated with classical features of a vulnerable plaque, such as a larger lipid core (OR 1.85; 95%CI 1.60, 2.15; p<0.001), a higher macrophage burden (OR 1.87; 95%CI 1.63, 2.14; p<0.001), less calcifications (OR 0.81; 95%CI 0.71, 0.91; p<0.001), a lower collagen and SMC content (OR 0.70; 95%CI 0.60, 0.82; p<0.001 and OR 0.60; 95%CI 0.52, 0.68; p<0.001, respectively). There were marked sex differences, in male patients glycophorin was associated with calcifications and collagen (OR 0.75; 95%CI 0.64, 0.87; p<0.001 and OR 0.65; 95%CI 0.53, 0.79; p<0.001, respectively) while these associations were not found in female patients.

Conclusions Quantified glycophorin C was independently associated with the presence of IPH, symptomatic preprocedural symptoms in male, and with a more vulnerable plaque composition in both women and men. Which strengthens the hypothesize that plaque glycophorin C is a marker of plaque vulnerability.

Project specific data For this project we share the unedited raw results that came directly from ExpressHist and CellProfiler through the slideToolKit pipeline. These data are stored on UMC Utrecht server for reference and sharing with others (see below Important notice on availability of data).

_MasksTileCrossedImages: Folder containing information on the crossed-tiles and whether they were kept/used for analysis. Each images was manually checked on whether or not the ExpressHist algorithm had properly assigned a cross to the whole whole-slide image. This was done by the authors (Van der Laan and Mekke).

_ndpi: Results per individual sample, including all the used tiles, analysis-reports, and output.

_ndpi_with_20x_error: Results per individual sample, including all the used tiles, analysis-reports, and output of .ndpi-images which were first exported to 20x resolution .tiff-images.

_tif: Results per individual sample, including all the used tiles, analysis-reports, and output of the .TIF image-analyses.

slideQuantify: Analysis script for the .TIF-images.

slideQuantifyNDPI: Analysis script for the .ndpi-images.

AEDB_March_2022.sav: Athero-Express Biobank Study dataset used for all the analyses in the paper.

About Athero-Express Biobank The AE started in 2002 and now includes over 3,500 patients who underwent surgery to remove atherosclerotic plaques (endarterectomy) from one (or more) of their major arteries (majority carotids and femorals). The AAA started in 2003 and now includes over 1,000 patients who underwent open surgery on arterial aneurysms, the majority on aortic aneurysms. The staining protocols are described by Verhoeven et al. (AE) and Hurks et al. (AAA).

About ExpressScan The ExpressScan is an ongoing, unfunded project to scan pathological slides of atherosclerotic plaques and aneurysm tissues at high-resolution using pathology scanners into whole-slide images (WSI). Here we describe these histological WSI data available for the Athero-Express (AE) and Aneurysm-Express (AAA) Biobank Studies.

Whole-slide images are available for the several commonly used stains, but note that these are not available in all samples in both studies. A table showing the approximate numbers of available WSI is given here.

Associated ExpressScan projects - Glycophorin C: Mekke JM et al. and the associated GitHub repository. - slideEMask: entropy based tissue masker - slideNormalize: normalize WSIhttps://github.com/swvanderlaan/slideNormalize - ExpressScan-Unlock: local, on-campus webportal to inspect WSI (private, ongoing and unpublished) - ExpressScan_QC: quality control procedures to process results from slideToolKit (private, ongoing and unpublished) - EntropyMasker: improved method to automatically mask WSI using entropy (private, ongoing and unpublished); preprint available here. - CONVOCALS: a deep-learning classification project on plaques (private, ongoing and unpublished) - DEEP-ENIGMA: a deep-learning image-segmentation project on plaques (private, ongoing and unpublished)

About slideToolKit These data are also used for slideToolKit and other ExpressScan projects (see below). Depending on the content of the project, a list of slides used is available to enable reproducible science. A link to the public GitHub repository for slideToolKit can be found here: https://github.com/swvanderlaan/slideToolKit.

Important notice on availability of data The amount of data is huge: over 25,000 WSI on average 1Gb size per WSI. There are also restrictions on use by commercial parties, and on sharing openly based on (inter)national laws and regulations and the written informed consent. Therefore these data (and additional clinical data) are only available upon discussion and signing a Data Sharing Agreement (see Terms of Access) and within a specially designed UMC Utrecht provided environment.