Diagnostic Utility of Calretinin-Positive Mucosal Nerve Fiber Quantification in Hirschsprung Disease (HD): an Image Processing and Analysis (IPA) Study

Transition (TZ) and normal zone (GZ) in HD pull-through specimens show variable density of calretinin-positive mucosal nerve fibers. This variability may also be present in GZ in non-HD. Despite the variability, IPA may aid in defining a cutoff that separates the aganglionic segment (AZ) in HD from the GZ in non-HD. Rectal biopsies from HD and non-HD and HD pull-through specimens were retrieved (2010–2017), and calretinin immunohistochemistry was performed. The immunostained slides were scanned and multiple images were captured (×100, JPEG format). Pixel count (PC), defined as the percentage of calretinin-stained pixels in the mucosa calculated by IPA as previously described, was measured for each image. Pearson’s correlation coefficient was calculated between the PC and the location of the biopsies. In the non-HD group, 62 biopsies taken at 0 to 4 cm (median 2 cm) from the dentate line were collected from 28 patients (age 4 days to 273 months, median 37 months), and 243 images (2–10 per biopsy, mean 3.5 images/case) were captured. In the HD group, 46 biopsies/segments were collected from 13 patients (age 2 days to 88 months, median 0.5 months) and 110 images (2–13 images/case, mean 2.4 images/case) were captured. Average PC was 0.482% in non-HD and 0.0153% in HD group, respectively (P < .0001). The average coefficient of variation in the non-HD group was 0.45. No correlation was found between the PC and location of the biopsies. All (100%) non-HD PCs were >0.06%, and 45 of 46 (98%) HD PCs were <0.06%. Although PC varies along the distance in non-HD patients, PC in non-HD is almost always higher than AZ in HD. Thus, defining a cutoff by IPA would aid in HD diagnosis. Defining the TZ in HD remains a challenge given the variation of the PC even in non-HD biopsies. Further study is warranted.