245 BABU et al. : A MODIFIED TECHNIQUE FOR THE DIAGNOSIS OF HIRSCHSPRUNG DISEASE Original Articles © The National Medical Journal of India 2003 A modified technique for the diagnosis of Hirschsprung disease from rectal biopsies M. K. BABU, USHA KINI, KANISHKA DAS, ANAND ALLADI, ASHLEY J. D’CRUZ ABSTRACT Background.A diagnosis of Hirschsprung disease requires thedemonstrationofacetylcholinesterasefibresonfrozensec- tions obtained from snap frozen biopsies of the rectum. This histochemicaltechniqueisgenerallynotavailableinlaboratories indevelopingcountries.Weimprovisedonthemethodologyof tissue preservation to make the staining technique more user- friendly, economical and reliable in demonstrating acetyl- cholinesterase activity in fresh rectal mucosal biopsies for the diagnosisofHirschsprungdisease. Methods. Between June 1999 and May 2002 fresh rectal biopsiesfrom40suspectedcasesofHirschsprungdiseasewere processed for routine frozen section (not snap frozen by liquid nitrogen) and stained by the Karnovsky and Roots method. Thesesectionswereassessedforthestainingpatternofacetyl- cholinesterase fibres. The thickness of the nerve fibres and muscularismucosawasassessedmorphometrically.Thesewere comparedwithbiopsiesobtainedfrom6age-matchedcontrols undergoingsurgeryforunrelatedcomplaints. Results.Thesectionsstainedforacetylcholinesterasebythis improvised method of tissue fixation were good and crisp. A definitediagnosisofHirschsprungdiseasewasmadein25cases and intestinal neuronal dysplasia in 1. The remaining 14 cases showedanequivocalstainingpatternwithnohypertrophicnerve bundles,thusexcludingadiagnosisofHirschsprungdisease.The meanthicknessofthesubmucosalnervetrunksmeasuredinthese enzyme-stainedsectionswasfoundtobeinverselyproportional tothemeanthicknessofthemuscularismucosa. Conclusion .Ourstudyoncryostat-cutsectionssuggestsan inverse relationship between the thickness of the muscularis mucosa and the calibre of the nerve trunk—thinner the nerve trunk,thickerthemuscularismucosaand viceversa. Also , routine frozensections,insteadofsnapfrozenonestakenfromafresh rectalbiopsyandstainedbytheKarnovskyandRootsmethodfor acetylcholinesterase activity, are reliable for the diagnosis of Hirschsprung disease and are within the capability of a simple histopathologylaboratoryinadevelopingcountry. Natl Med J India 2003;16:245–8 INTRODUCTION The diagnosis of Hirschsprung disease (HD) in children is a complex and much debated subject. Histochemistry of fresh snap frozen tissue reveals a spectrum of pathology in HD. 1 We describe an improvisation in the method of conventional tissue preservation and fixation for acetylcholinesterase (AChE) staining of rectal mucosal biopsies in the diagnosis of HD and its variants. With this simplified technique aided by morphometry, a definite diagnosis of HD was possible. We believe this improvisation can be carried out in the simple laboratory set-up of a developing country. METHODS Between June 1999 and May 2002 rectal biopsies from 40 patients with suspected HD were studied. These children had symptoms of neonatal large bowel obstruction with/without delayed passage of meconium or chronic constipation in infancy/childhood and were subjected to rectal biopsy as part of the investigations. One to three rectal biopsy fragments were taken 2–3 cm above the dentate line so that a small amount of submucosa was included in each case. Biopsies were excluded from the study if they were taken from too low an area showing the presence of squamous epithelium/skeletal muscle, or those in which the submucosal tissue was scant or absent, and those in which the submucosal area was dominated by a lymphoid follicle. The test biopsies were collected on a 0.9% saline-soaked gauze piece and directly fresh frozen at –20 °C in a Leitz cryostat instead of the classical procedure of snap freezing by liquid nitrogen and/ or isopentane. Twenty serial 10 m thick cryostat sections were cut from the directly frozen tissue sample and concurrently stained with haematoxylin and eosin (H&E) for routine morphology, toluidine blue (Tb) for highlighting the ganglion cells by the principle of metachromasia and for acetylcholinesterase (AChE enzyme by the Karnovsky and Roots method 1 as detailed by Meier- Ruge et al. 2 ). The classical processing for AChE staining was thus improvised in this study. The sections stained with H&E were checked for adequacy of the tissue sample by assessing the amount of submucosal tissue. Those stained for AChE were analysed for the following: 1. Presence of hypertrophic nerve bundles in the submucosa; 2. Presence of submucosal ganglion cells, if any; 3. Hypertrophic AChE-positive fibres traversing the muscularis mucosa into the lamina propria; 4. AChE staining patterns in the mucosa (described below); and 5. Morphometric analysis of the thickness of the muscularis mucosa and AChE fibres in the submucosa with the Leitz oculometer attachment of the light microscope. St John’s Medical College and Hospital, Bangalore 560034, Karnataka, India M. K. BABU, USHA KINI Department of Pathology KANISHKA DAS, ANAND ALLADI, ASHLEY J. D’CRUZ Department of Paediatric Surgery Correspondence to USHA KINI; zipcb028@bgl.vsnl.net.in THE NATIONAL MEDICAL JOURNAL OF INDIA VOL. 16, NO. 5, 2003