The accumulation of erythrocytes quantified and visualized by Glycophorin C in carotid atherosclerotic plaque reflects intraplaque hemorrhage and pre-procedural neurological symptoms

Abstract

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. Hence, we theorized that the accumulation of erythrocytes quantified by glycophorin C could function as a marker for plaque vulnerability, possibly reflecting intraplaque hemorrhage (IPH), and offering predictive value for pre-procedural neurological symptoms. We employed the CellProfiler-integrated slideToolKit workflow to visualize and quantify glycophorin C, defined as the total plaque area that is positive for glycophorin C, in single slides of culprit lesions obtained from the Athero-Express Biobank of 1819 consecutive asymptomatic and symptomatic patients who underwent carotid endarterectomy. Our assessment included the evaluation of various parameters such as lipid core, calcifications, collagen content, SMC content, and macrophage burden. These parameters were evaluated using a semi-quantitative scoring method, and the resulting data was dichotomized as predefined criteria into categories of no/minor or moderate/heavy staining. In addition, the presence or absence of IPH was also scored. The prevalence of IPH and pre-procedural neurological symptoms were 62.4% and 87.1%, respectively. The amount of glycophorin staining was significantly higher in samples from men compared to samples of women (median 7.15 (IQR:3.37, 13.41) versus median 4.06 (IQR:1.98, 8.32), p < 0.001). Glycophorin C was associated with IPH adjusted for clinical confounders (OR 1.90; 95% CI 1.63, 2.21; p = < 0.001). Glycophorin C was significantly associated with ipsilateral pre-procedural neurological symptoms (OR:1.27, 95%CI:1.06-1.41, p = 0.005). Sex-stratified analysis, showed that this was also the case for men (OR 1.37; 95%CI 1.12, 1.69; p = 0.003), but not for women (OR 1.15; 95%CI 0.77, 1.73; p = 0.27). Glycophorin C was associated with classical features of a vulnerable plaque, such as a larger lipid core, a higher macrophage burden, less calcifications, a lower collagen and SMC content. There were marked sex differences, in men, glycophorin C was associated with calcifications and collagen while these associations were not found in women. To conclude, the accumulation of erythrocytes in atherosclerotic plaque quantified and visualized by glycophorin C was independently associated with the presence of IPH, preprocedural symptoms in men, and with a more vulnerable plaque composition in both men and women. These results strengthen the notion that the accumulation of erythrocytes quantified by glycophorin C can be used as a marker for plaque vulnerability.

Figure 1

Study design and flowchart of study participants. (A) Graphical illustration of the study design. Created with BioRender.com. (B) Flowchart of number of individuals included in the analyses of the current study. A total of 1068 patients were not included in the current study since they underwent iliofemoral endarterectomy. Additionally, 763 patients did not have available WSI’s with a glycophorin C staining.

Figure 2

Example of slides with glycophorin C (GLYCC) and H&E staining. Displayed are two examples samples: left image is representative of a ‘diffuse’ glycophorin C staining in women, the right image shows a more ‘localized’ ‘core’-staining with some staining outside the ‘core’ in men. The percentage of glycophorin C (inverse rank transformed) was respectively 32.0% and 26.1% of total plaque area in the above slides. Be aware of the slight differences in scale and form between HE- and glycophorin C-stained samples from the same patients, these are sequential cuts.

Figure 3

Univariable association between glycophorin C expressed as percentage glycophorin of total plaque surface with presence of intraplaque hemorrhage (IPH). P-values are derived from Kruskal Wallis test. Shown are the median values (central line), the upper and lower quartiles (box limits), and the 1.5 × interquartile range (whiskers). The percentage of glycophorin C (inverse rank transformed) was significantly higher in samples with intraplaque hemorrhage in comparison to samples without IPH (p < 0.001).

Figure 4

Example of slides with different levels of glycophorin C (GLYCC) staining compared to the presence of intraplaque hemorrhage (IPH). Displayed are example 6 individual patient samples for low, moderate and high glycophorin C (upper panel) staining in samples from patients in which IPH was scored as present or not present (displayed a HE stain, lower panel). The percentage of glycophorin C (inverse rank transformed) was respectively 0.80%, 28,74%, and 43.76% top row and 0.69%, 9.85% and 50.56% bottom row. Be aware of the slight differences between HE- and glycophorin C-stained samples from the same patients, these are sequential cuts.

Figure 5

Multivariable associations between plaque glycophorin C (1 SD increment) with IPH and pre-procedural neurological symptoms. Associations adjusted for plaque size (Model 1) and adjusted for plaque size and confounders (Model 2) are shown. Shown are odd ratios (OR) or hazard ratios (HR) and error bars corresponding to their 95% confidence intervals (CI). The confounders included in the Model 2 associations can be found in Supplemental Table S1. (A) Multivariable associations of glycophorin C with IPH, as derived from logistic regression analyses. (B) Multivariable associations of glycophorin C with pre-procedural symptoms (asymptomatic versus symptomatic), as derived from logistic regression analyses.

Figure 6

Univariable association between glycophorin C expressed as percentage glycophorin of total plaque surface with overall plaque phenotype. P-values are derived from pairwise comparisons using Wilcoxon rank sum test. Shown are the median values (central line), the upper and lower quartiles (box limits), and the 1.5 × interquartile range (whiskers). The percentage of glycophorin C (inverse rank transformed) was significantly higher in fibroatheromatous and atheromatous plaques in comparison to fibrous plaques. Additionally, the percentage of glycophorin C (inverse rank transformed) was significantly higher in Atheromatous plaques in comparison to fibroatheromatous plaques.

Figure 7

Association of plaque glycophorin C with (1 SD increment) with the individual (semi)-quantitative histopathological features (binary traits). Shown are odds ratios (OR) and error bars correspond to their 95% CI, as derived from logistic regression analyses. Blue error bars are the full cohort, and the green and red error bars are male and female subset, respectively.