Table 1. Description of the image analysis process.

			Analysis
Element	Staining (IHC, HC)	Digitisation	Material	Software	Algorithm (segmentation)
Blood vessels	Anti CD31 (Dako, clone JC70A, 1/50)	-Scanner: Aperio ScanScope XT (Aperio, Vista, CA) − × 40 magnification -TIF format	-Individual images -TIFF format -HSV colour model	AngioPatha
Ret Fs	Gomori		-Individual images -JPEG format, quality 80 -RGB colour model	Image-Pro Plus (Media Cybernetics, Inc., Silver Spring, MD, USA)	Customised macro: black and dark brown Ret Fs -Colour adjustments: None -Segmentation: R 0–181, G 0–178, B 0–172 -Restriction of the shape and size of the objects detected (roundness >3, area >7.5 μm2, length >8 μm, width >1.5 μm) to avoid unspecific signal-form cell nuclei
Col I Fs	Masson's trichrome				Customised macro: specifically detect thick collagen type I bundles and distinguish them from other thinner blue-stained collagen -Colour adjustments: colour hues intensified (brightness: 68, contrast: 81, gamma: 1) -Segmentation: R 0–17, G 0–90, B 255–255 -Restriction of the shape and size of the objects detected (roundness >2, area >7.5 μm2, length >8 μm, width >10 μm).
Elastic fibres	Orcein		No image analysis was performed: a subset of 69 samples was only subjectively assesed by a pathologist
GAGs	Alcian blue pH 2.5 Stain Kit (Artisan, Dako)		-Whole-digitised slide (identification of cylinders corresponding to the different samples) -TIFF format -RGB colour model	Aperio ImageScope	Positive pixel count: -Colour adjustments: None -Segmentation: RGB >221 -Remaining parameters set by default

Abbreviations: Col I Fs=collagen type I fibres; GAGs=glycosaminoglycans; HC=histochemistry ; HSV=hue, saturation and value colour model; IHC=immunohistochemistry; JPEG=joint photographic expert group format; Ret Fs=reticulin fibres; RGB=red, green and blue colour model; TIFF=tagged image file format.

^aAngiopath: self-designed tool (Fernandez-Carrobles et al, 2013) with minor modifications. In all cases, large trabeculae were excluded.