Calretinin, β‐Tubulin Immunohistochemistry, and Submucosal Nerve Trunks Morphology in Hirschsprung Disease

ABSTRACT Objectives: The aim of this study was to investigate calretinin and β‐tubulin immunohistochemical expression together with submucosal nerve trunks morphology in differently innervated segments of Hirschsprung disease (HD) and total colonic aganglionosis (TCA). Methods: A total of 25 cases (22 HD, 3 TCA) and 18 controls were processed for calretinin and β‐tubulin immunohistochemistry. Sections representative of distal aganglionic, transition, and proximal ganglionic segments were evaluated by a visual grading score; β‐tubulin was evaluated also by image analysis. Submucosal nerve trunks hypertrophy and hyperplasia were measured by citomorphology. The length of proximal segment was correlated to postoperative bowel function. Results: Controls showed intense calretinin and β‐tubulin staining. In HD and TCA, calretinin staining was related to the presence of ganglion cells: negative in distal, faint in transition, intense in proximal segment. β‐Tubulin staining was weak in all of the segments of HD and negative in TCA. Hypertrophic and hyperplastic nerve trunks characterized aganglionic segment, and progressively decreasing nerve size was observed in transition and ganglionic segments. Transient postoperative constipation, soiling, or enterocolitis was present in 59% of patients with HD without clear relation to proximal segment length or presence of hypertrophic nerve trunks. Conclusions: Calretinin is a reliable marker of the presence of ganglion cells, and, together with nerve hypertrophy, it helps to identify the transition zone. Length and nerve size of proximal segment in resected specimen did not affect the postsurgical intestinal function. Reduced β‐tubulin expression along the entire colonic tract, included proximal ganglionic segments, may represent a potential impairing factor for the enteric neural transmission.