Utility of Image Processing and Analysis (IPA) for Diagnosis of Very Short Segment Hirschsprung Disease (vsHD)

Abstract Introduction The diagnosis of vsHD, HD with distal rectal aganglionic segment of ≤2 cm, is extremely difficult as the aganglionic zone (AZ) and physiologic hypoganglionic zone may overlap. Also, proximal AZ may show calretinin-positive mucosal nerve fibers originating from ganglion cells (GCs) in the distal transition zone (TZ), resulting in false negativity. The proximal 2 cm of AZ and distal TZ in pull-through specimens may represent a surrogate model to study vsHD. Methods Ten HD pull-through specimens were retrieved, and sections containing proximal AZ and distal TZ were subject to calretinin immunohistochemistry. Immunostained slides were scanned and consecutive images were captured (×100 in JPEG) to include up to 2.0 cm of proximal AZ and up to 1.2 cm of distal TZ. 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. A cutoff of PC 0.06% in the AZ (false negative), extending up to 12.1 (range, 6.9 to 12.1) mm distal to the distal-most GC. Seven cases (70%) showed PC