Vessel Spatial Analysis (VeSpA): a tool for whole slide image segmentation, morphometry, and QuPath extension.

Quantifying vascular architecture in histological whole slide images is needed to study tissue organisation, tumour microenvironment biology, and diseaseassociated vascular remodelling. However, vessel analysis in routine immunohistochemistry remains challenging. Available workflows are often manual, require programming expertise, or lack direct integration with digital pathology platforms. We developed VeSpA (Vessel Spatial Analysis), an open-source pipeline and QuPath extension for automated vessel segmentation and morphometric quantification in CD31-stained whole slide images. VeSpA combines configurable signal extraction, using CMYK Yellow channel extraction by default and optional DAB stain deconvolution for H-DAB images, with automatic or percentile-based thresholding, morphological refinement, contour filtering, and lumen filling to generate vessel masks from standard DAB-stained sections. The QuPath extension includes a graphical interface for selecting annotations, TMA cores, or whole images, configuring segmentation parameters, running the Python backend, and importing vessel objects directly into the QuPath hierarchy. For each detected vessel, VeSpA extracts area, major axis length, minor axis length, eccentricity, centroid, and orientation, while also appending summary measurements to parent annotations and TMA cores. Validation against independent pathologist annotations showed that VeSpA achieved segmentation performance close to inter-rater agreement and outperformed yellow channel prompt-based SAM and zero-shot YOLOv8-seg on overlap-based metrics in the tested dataset. VeSpA integrates vessel segmentation, morphometric feature extraction, and QuPath-based visualisation into a single reproducible workflow for vascular quantification in computational pathology and spatial analysis of histological tissue architecture.