Tissue eosinophil count on whole slide image
As an alternative method for histopathological assessment of samples, glass slides were prepared for whole slide image (WSI) acquisition using previously described methods.19 The slides were digitalised (Ventana DP 200, Roche, Basel, Switzerland). Component TIF image tiles from each slide were then imported into the open source digital pathology software QuPath version 0.2.3.20 In order to more precisely quantify tissue eosinophils, two 0.1mm2 fields in sub-epithelial layers in each H&E-stained tissue section with high eosinophil density were selected on QuPath. Areas of crush were not selected. Crush areas were defined as abnormally elongated dark thinned nuclei in stroma.21Eosinophils were independently counted on H&E-stained tissue sections by the first author (SW) and a senior anatomical pathology registrar (SL) in a blinded manner. Eosinophils in the epithelium and intravascular space were excluded.
The eosinophil cell count for each of the two reviewers was calculated as the average number of eosinophil cells across the two 0.1mm2 areas and was expressed as eosinophils per 0.1mm2. The final eosinophil cell count was calculated as the mean of the two reviewers’ scores.