DIGITAL MORPHOMETRIC MEASUREMENT OF QUANTITATIVE MORPHOLOGICAL CHANGES WITH IMMUNOHISTOCHEMISTRY IN HIRSCHSPRUNG DISEASE

Objective: Hirschsprung disease is an important childhood disease with an incidence of one per 5000 births. Morphological diagnosis is the gold standard and plays a crucial role on the prognosis of surgical treatment. The purpose of this study is to quantitate the morphological changes using digital morphometry and improve the histological diagnostic impact for this disease. Material and Method: This retrospective study enrolled seven cases of resection material due to the diagnosis of Hirschsprung disease. The mean maximum diameter and number of nerve plexuses in the submucosal area and muscularis propria, the number of eosinophil leukocytes and the presence of vascular changes in the transition zone were evaluated morphometrically using immunohistochemistry. Results: PGP 9.5 immunostaining showed that the mean ganglion cell number per submucosal plexus ranged between 1.1 and 12.9. The mean ganglion cell diameter in the submucosal and myenteric plexus in ganglionic is greater than the mean ganglion cell diameter in the hypoganglionic plexus (p=0.002, p=0.002). The number of ganglion is also higher in the ganglionic zone than in the hypoganglionic zone (p=0.003, p=0.002). H&E stained sections revealed that the mean submucosal plexus diameter in the ganglionic, transition and aganglionic zones was 32.8μ, 58.9μ and 84.0μ, respectively(p<0.001); whereas for the myenterik plexus 42.2μ,77.4μ and 96.1μ respectively(p<0.001). Eosinophilic infiltrate in the myenterik plexus of the transition zone was found in six cases (85.7%). Cajal cell density evaluated with c-kit immunohistochemistry in the muscularis propria was not significantly different in any of three zones. Calretinin immunohistochemical staining showed that cholinergic nerve fibrils both in the lamina propria and submucosa of the aganglionic zone were significantly absent (p<0.001). Conclusion: Morphometric quantification and calretinin immunohistochemistry is a useful method in the diagnosis of Hirschsprung disease. Especially evaluation of the submucosal ganglion cell number and plexus diameter appears to be valuable