Biology of Hirschsprung Disease: Pathomorphological, Histochemical, Immunohistochemical and Genetic (RET Gene) Study of the Enteric Nervous System
Creators: name: Yadav, Lokendra
name: KIni, Usha
name: Das, Kanishka
Publisher: name: figshare
Year: 2022
Subjects: subject: 110316 Pathology (excl. Oral Pathology)
subject: FOS: Clinical medicine

description: The present study titled " Biology of Hirschsprung Disease: Pathomorphological, Histochemical, Immunohistochemical and Genetic (RET Gene) Study of the Enteric Nervous System" is a prospective cross-sectional study on patients from the Indian subpopulation with Hirschsprung disease over three and a half years (Nov 2011 to June 2015) at the Department of Pathology, St. John’s Medical College, Bangalore - a national referral centre for diagnosis of Hirschsprung disease in India. The study with five chapters has explored pathobiology of Hirschsprung disease (HD) in the Indian context with emphasis on rapid reliable user friendly diagnostic modalities and also made an attempt at investigating the molecular basis of the HD to gain insight into its possible pathogenic mechanisms. Their summary is as follows: 1. The rapid modified agar-paraffin block technique designed in the first study titled "Improvised double-embedding technique of minute biopsies: A mega boon to histopathology laboratory" has revolutionized the processing of multiple minute mucosal and seromuscular biopsies which mandate proper orientation to visualize neuronal plexuses, especially when sampled from neonates. The simple reliable user friendly method has improved the quality of diagnostic information by optimal orientation, better quality of sections, faster turnaround time, cost-effectiveness by economizing on the number of paraffin blocks, manpower, chemical reagents and laboratory infrastructure. The modified tissue blocks are also best suited for enzyme and immunohistochemistry in addition to routine histochemistry. 2. The second study titled "Improvised rapid Acetylcholinesterase histochemistry versus calretinin immunohistochemistry in the evaluation of colorectal biopsies for 203 Hirschsprung disease" evaluated calretinin, a Vitamin D dependent calcium binding protein expressed in central and peripheral neural system, on formalin fixed biopsies and compared the results with the improvised modified rapid AChE histochemistry (designed in the Department) on their corresponding fresh rectal biopsies taken for the primary diagnosis of HD. Calretinin proved as a reliable immune marker in ruling out the diagnosis of HD on formalin fixed rectal mucosal biopsy by highlighting granular staining of intrinsic fibres in the mucosa and submucosa in suspect cases of HD. The study also proved that the accuracy of diagnosis in Hirschsprung disease could be improved by employing both AChE and Calretinin stains. 3. The detailed evaluation of Synaptophysin immunohistochemistry as a labelling immunohistochemical method in the third study titled "Role of Synaptophysin in the Intra-Operative Assessment of Quadrantic Innervation of the Proximal Doughnut in Hirschsprung Disease" assessed proximal doughnut for innervation abnormalities intraoperatively to find its suitability for anastomosis for pull through surgeries. The marker specific for the synaptic vesicles in the central and peripheral nervous system and the main constituent of AChE storage compartments, and an important neuromuscular junction marker, highlighted the morphology of ganglion cells, indirectly reflected their functional status by demonstrating synapses at the level of muscle fibers on frozen sections and mapped the ganglionic –aganglionic interface with the pattern and intensity of the SY-positive fibre distribution in the muscularis propria. 4. The forth study "The quest for a positive diagnostic marker for Hirschsprung Disease in formalin fixed rectal biopsies: A detailed seven marker IHC study" describes in detail the hunt for a positive diagnostic marker on formalin fixed rectal 204 biopsy in the diagnosis for HD from among the panel of seven neural markers namely Calretinin, GFAP, Synaptophysin, PGP 9.5, CD 56, NF and S100. None of these markers specifically stained and differentiated the hyperplastic-hypertrophic nerve bundles of Hirschsprung disease from the normal nerve bundles and extrinsic serosal nerves. Though CD 56 and S-100 failed to stain ganglion cells, they were not specific for hypertrophic nerve bundles and hence, these markers could not be considered as markers for HD. Thus, the quest of a novel marker for abnormal enteric nervous system continues with the proposal for the next panel of markers which may attempt to highlight pathology in perineurium. 5. The fifth study "Diversity of RET Proto-oncogene Mutation in an Indian sub population of Hirschsprung disease: A Pilot Study " highlights the association of RET gene in Hirschsprung disease. Sequencing of six exons namely, 10, 11, 13, 14, 15 and 16 of RET gene in 30 samples comprising of tests and control of Indian subpopulation in this pilot study reports the occurrence of a novel mutation, D624N in exon 10 of a single patient with LSHD. The variations seen in RSHD, and TCA were also seen in the control group. However, the reported mutations in other study population were not seen in the limited samples studied here and this could also because of limited number of exons scanned. Hence, this calls for complete RET gene sequencing of a large sample size to strengthen the data and to study its relevance.