Intraplaque haemorrhage quantification and molecular characterisation using attention based multiple instance learning

Intraplaque haemorrhage (IPH) represents a critical feature of plaque vulnerability as it is robustly associated with adverse cardiovascular events, including stroke and myocardial infarction. How IPH drives plaque instability is unknown. However, its identification and quantification in atherosclerotic plaques is currently performed manually, with high interobserver variability, limiting its accurate assessment in large cohorts. Leveraging the Athero-Express biobank, an ongoing study comprising a comprehensive dataset of histological, transcriptional, and clinical information from 2,595 carotid endarterectomy patients, we developed an attention-based additive multiple instance learning (MIL) framework to automate the detection and quantification of IPH across whole-slide images of nine distinct histological stains. We demonstrate that routinely available Haematoxylin and Eosin (H&E) staining outperformed all other plaque relevant Immunohistochemistry (IHC) stains tested (AUROC = 0.86), underscoring its utility in quantifying IPH. When combining stains through ensemble models, we see that H&E + CD68 (a macrophage marker) as well as H&E + Verhoeff-Van Gieson elastic fibers staining (EVG) leads to a substantial improvement (AUROC = 0.92). Using our model, we could derive IPH area from the MIL-derived patch-level attention scores, enabling not only classification but precise localisation and quantification of IPH area in each plaque, facilitating downstream analyses of its association and cellular composition with clinical outcomes. By doing so, we demonstrate that IPH presence and area are the most significant predictors of both preoperative symptom presentation and major adverse cardiovascular events (MACE), outperforming manual scoring methods. Automating IPH detection also allowed us to characterise IPH on a molecular level at scale. Pairing IPH measurements with single-cell transcriptomic analyses revealed key molecular pathways involved in IPH, including TNF-α signalling, extracellular matrix remodelling and the presence of foam cells. This study represents the largest effort in the cardiovascular field to integrate digital pathology, machine learning, and molecular data to predict and characterize IPH which leads to better understanding how it drives symptoms and MACE. Our model provides a scalable, interpretable, and reproducible method for plaque phenotyping, enabling the derivation of plaque phenotypes for predictive modelling of MACE outcomes.