Calretinin versus Hematoxylin and Eosin Stain for Diagnosis of Hirschsprung’s Disease; Comparison in Ganglionic, Transitional, and Aganglionic Zones

Abstract

Objective: To compare the results of calretinin stain with hematoxylin and eosin (H&E) stain for diagnosis of Hirschsprung’s disease. Materials and Methods: A prospective double-blind diagnostic study was done. Hirschsprung’s patients who underwent transanal endorectal pull through (TERPT) surgery between February 2015 and February 2017 were included. The pulled-through specimens were sampled at the ganglionic, transitional, and aganglionic zones. Each specimen was stained with calretinin and H&E. Agreement and kappa analysis were done. Results: Fifty-one Hirschsprung’s patients (153 specimens) were included for analysis. There were 37 males (72.5%) and 14 females (27.5%) with a median age at surgery of four months. Thirty-one specimens showed a negative stain for calretinin (aganglionic bowel) and 33 specimens showed no ganglion cells in the H&E stain. One hundred twenty-two specimens in calretinin stain were consistently positive with ganglionic bowel and 120 specimens in the H&E stain showed ganglion cells. Agreement and Cohen’s kappa coefficient were 97.4% and 0.921 (95% confidence interval 0.845 to 0.997), respectively. Disconcordance was found in four specimens. Three out of four were in the transitional zone. One was in the aganglionic zone, which has no muscular layer attached. (Thirty-six specimens had no muscular layer.) Conclusion: Calretinin stain was found to be comparable with the H&E stain and could be used for diagnosis of Hirschsprung’s disease. In rectal suction biopsy specimen in which the muscular layer was not included, both calretinin and H&E can be used. Keywords: Megacolon, Hematoxylin and eosin (H&E), Calretinin, Rectal suction biopsy (RSB), Full thickness biopsy