ABSTRACT
BACKGROUND: A diagnosis of Hirschsprung disease requires the demonstration of acetylcholinesterase fibres on frozen sections obtained from snap frozen biopsies of the rectum. This histochemical technique is generally not available in laboratories in developing countries. We improvised on the methodology of tissue preservation to make the staining technique more user-friendly, economical and reliable in demonstrating acetylcholinesterase activity in fresh rectal mucosal biopsies for the diagnosis of Hirschsprung disease. METHODS: Between June 1999 and May 2002 fresh rectal biopsies from 40 suspected cases of Hirschsprung disease were processed for routine frozen section (not snap frozen by liquid nitrogen) and stained by the Karnovsky and Roots method. These sections were assessed for the staining pattern of acetylcholinesterase fibres. The thickness of the nerve fibres and muscularis mucosa was assessed morphometrically. These were compared with biopsies obtained from 6 age-matched controls undergoing surgery for unrelated complaints. RESULTS: The sections stained for acetylcholinesterase by this improvised method of tissue fixation were good and crisp. A definite diagnosis of Hirschsprung disease was made in 25 cases and intestinal neuronal dysplasia in 1. The remaining 14 cases showed an equivocal staining pattern with no hypertrophic nerve bundles, thus excluding a diagnosis of Hirschsprung disease. The mean thickness of the submucosal nerve trunks measured in these enzyme-stained sections was found to be inversely proportional to the mean thickness of the muscularis mucosa. CONCLUSION: Our study on cryostat-cut sections suggests an inverse relationship between the thickness of the muscularis mucosa and the calibre of the nerve trunk--thinner the nerve trunk, thicker the muscularis mucosa and vice versa. Also, routine frozen sections, instead of snap frozen ones taken from a fresh rectal biopsy and stained by the Karnovsky and Roots method for acetylcholinesterase activity, are reliable for the diagnosis of Hirschsprung disease and are within the capability of a simple histopathology laboratory in a developing country.