Redefining histological cell counts using a standardized method: The Leuven Intestinal Counting Protocol

Abstract

Objectives:The diagnosis of eosinophilic gastrointestinal diseases is largely based on mucosal eosinophil counts, but thresholds and normal ranges beyond the esophagus are debated, calling for much-needed methodological standardization. We aimed to develop a standardized workflow for duodenal cell quantification and estimate duodenal eosinophil and mast cell numbers in healthy controls.Methods:Software-based histological cell quantification using free-sized or fixed-sized regions was developed and applied to digitized hematoxylin and eosin (H&E) stained slides from 58 individuals (healthy controls (HC) and functional dyspepsia (FD) patients). Intraclass correlation coefficients (ICC) compared interrater reliability between software-based and microscopic quantification. Reproducibility of the software-based method was validated in an independent cohort of 37 control and FD subjects. Eosinophil identification on H&E staining was compared to immunohistochemistry (IHC). Normal eosinophil (H&E) and mast cell (cKit) ranges were determined in 70 adult HC.Results:Eosinophil quantification on digitized slides demonstrated excellent (ICC: 0.909) and significantly improved reproducibility over microscopic evaluation (ICC: 0.796, P =.0014), validated in an independent cohort (ICC: 0.910). Duodenal eosinophils were more abundant around crypts than in villi (P