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Biology of Hirschsprung Disease: Pathomorphological, Histochemical, Immunohistochemical and Genetic (RET Gene) Study of the Enteric Nervous System

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Title Biology of Hirschsprung Disease: Pathomorphological, Histochemical, Immunohistochemical and Genetic (RET Gene) Study of the Enteric Nervous System
 
Identifier https://doi.org/10.7910/DVN/T9DUZ5
 
Creator Yadav, Lokendra
 
Publisher Harvard Dataverse
 
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
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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
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.
 
Subject Medicine, Health and Life Sciences
Hirschsprung Disease, Pathomorphology, Histochemistry, Immunohistochemistry, Genetic (RET Gene), Enteric Nervous System
 
Contributor Yadav, Lokendra