Effectiveness of calretinin immunohistochemistry with digital morphometry in mapping of different segments of Hirschsprung disease

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Definitive diagnosis of Hirschsprung disease (HD) is exclusively based on histological examination of rectal biopsies. Among the immunohistochemical markers, calretinin appears to be an encouraging marker for HD diagnosis. Calretinin expression needs to be more elaborated in the transition zone.


This work evaluates the usefulness of calretinin immunostaining with morphometric assessment of the nerve caliber in the diagnostic workup of HD, essentially the transitional zone.

Materials and methods

A retrospective study including 25 HD patients and 10 control full-thickness colonic biopsies was conducted. Different sections representing each segment were evaluated by calretinin immunostaining and digital morphometry.


A highly significant difference was demonstrated between aganglionic, transitional, and ganglionic areas as regards submucosal and myenteric nerve fiber caliber (P=0.001). There was overlap of nerve caliber in the transitional zone and the ganglionic segment. The cutoff value for submucosal and myenteric nerve caliber to delineate the transitional zone from the ganglionic area was ≥34.5 and ≥28.4 μm, respectively, and that from the aganglionic area was ≤62.25 and ≤80 μm, respectively. Calretinin immunostaining of ganglion cells and nerve fibers disclosed highly significant differences between aganglionic and ganglionic segments and between aganglionic and transitional segments (P=0.001), whereas there was no significant difference between transitional and ganglionic segments in submucosal and myenteric innervations by calretinin staining (P>0.05).


Morphometric analysis with calretinin immunohistochemical study is a better method for precisely characterizing the transitional zone and easily delimiting the proximal margin of the transitional zone, which is considered a critical issue in surgery.

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