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HISTOPATHOLOGICAL STUDY
INTRODUCTION:
Children are vulnerable to a vast number of diseases including gastrointestinal disorders, which
may be associated with life threatening complications. There is limited information on the
epidemiology of pediatric gastrointestinal diseases in developing countries like India due to the
paucity of vital health statistics1,2.
The age and sex distribution of gastrointestinal diseases in children are influenced by many
factors including geographical location, types of lesion and etiological factors. There are a
number of gastrointestinal diseases, some of which include: Appendiceal lesions, Hirschsprung’s
disease, intussusception, enterocolitis, adenocarcinoma, jejunal atresia, and lymphoma.3,4,5
Hirschsprung’s disease (HD), a genetic disorder with a complex pattern of inheritance, is
the major cause of pediatric intestinal obstruction6. Failure of migration of ganglion cells from
neural crest leads to the development of aganglionic segments which varies from ultra‑short to
total intestinal involvement7,8. The incidence of HD is 1 in 5343 live births with male
preponderance9.
Acute appendicitis is one of the most common surgical causes of acute abdomen. The
lifetime risk for appendicitis is 7%; commonly occurring in adolescents and young adults 10.
Despite of advances in technology and imaging modalities, there is dilemma in the clinical
diagnosis of acute appendicitis. Histopathological examination still remains the gold standard
method for the confirmation of the appendicitis. Not only the pathologic diagnosis of acute
inflammation, at times unusual findings such as incidental tumours noted in the appendix
highlights the importance of the pathologic analyses of every single resected appendix.
Intestinal atresia or stenosis can occur anywhere along the gastrointestinal (GI) tract,
and the anatomic location of the obstruction determines the clinical presentation. Most new borns
with intestinal obstruction present with abdominal distention and bilious emesis in the first 2 days
of life, though the presentation can be delayed for weeks in infants with stenosis. Bilious
vomiting in the neonate should be considered secondary to a mechanical obstruction until proved
otherwise, and emergency surgical evaluation is warranted in every new born with this symptom.
Gastrointestinal (GI) polyps are defined as grossly visible protrusions from the mucosal
surface. Most polyps are asymptomatic and remain unrecognized, but in symptomatic cases, the
1
major clinical presentations are GI bleeding, abdominal pain, intestinal obstruction and rectal
prolapse11.
There are few studies documented in literature regarding the overview of lesions
involving the gastrointestinal tract in pediatric age group.
• To know the distribution of various gastrointestinal lesions in pediatric age group with
respect to age and sex.
• To know the role of histopathology, special stains and immunohistochemistry in
diagnosing Hirshsprung’s disease.
STUDY DESIGN:
• Exclusion criteria: Cases on therapy, recurrent lesions and above 18 years will be
excluded from study.
METHODOLOGY:
• The excised histopathology specimens were thoroughly grossed and data recorded.
• Tissues routinely processed as per paraffin embedded processing and stained with H&E.
In cases with clinical diagnosis of Hirshsprung’s disease: All cases were subjected to special
stains.
2
• 1% Toluidine blue stain to see mast cells in submucosa.
The mast cells in the submucosa was graded as per Hadeel A.Yasseen et al (Toulidine blue stain)
in both ganglionic and aganglionic segment.
• 2+ Easy to identify
Statistical analysis:
RESULTS
• Total number of cases analysed :443
• Neoplastic lesion: 10
3
Neoplastic lesion 10
4
Distribution of GIT lesions in relevance with Age and Sex distribution
Tuberculous ileitis 2 0 2 1 1
Lymphocytic enterocolitis 2 0 2 2 0
Gangrenous intestine 1 0 1 1 0
Hirschsprung disease 31 24 7 24 7
DEVELOPMENTAL ABNORMALITIES- 22
Intestinal atresia 13 13 0 8 5
Chylolymphatic cyst 2 2 0 1 1
Hamartoma 1 0 1 1 0
Meckel's diverticulum 3 2 1 2 1
Meconium pseudocyst 1 1 0 1 0
CARCINOID in appendix 1 0 1 1 0
Juvenile polyp 9 5 4 4 5
5
In GIT lesions
Number of cases-372
400
299
300
200
100 Number of cases
36 35 2
0
Acute Chronic Acute on Eosinophilic
suppurative appendicitis Chronic appendicitis
appendicitis appendicitis
6
Clinical features in GIT lesions
Disorders of 31 - 5 - 26
intestinal
motility
Developmental 22 - - - 22
anomalies
Neoplastic 10 1 - - 9
lesions
Typical clinical presentation in GIT lesions are pain abdomen in 376/443(84.87%) and intestinal
obstruction 59/443(13.31%).
7
Age and Sex distribution of Hirschsprung’s disease (HD) and Non-Hirschsprung’
disease (Non-HD) – 44 cases
Age in months
Mean age 31.33(2.5y) 20.15(1.6y) 0.964
SD 30.88 19.96 >0.05 (not
significant)
Minimum 1 2
Maximum 90(7.5y) 72(6y)
Sex
Male 23 6 .0498
Female 7 8 <0.05 (significant)
Samples subjected to special stain (Cresyl violet stain& Toulidine blue stain)
• 13 cases with clinical diagnosis of ileal atresia to rule out Hirschsprung’ disease.
• In 27 cases with clinical diagnosis of Hirschsprung’ disease: H&E and Cresyl violet
showed absence of ganglion cells.
8
• In 13 cases with clinical diagnosis of Ileal atresia to rule out Hirschsprung’ disease: H&E
and Cresyl violet stain showed presence of ganglion cells confirming the diagnosis of
Ileal atresia.
• Toulidine blue stain: In the present study there is increased number of mast cells in the
aganglionic segment when compared to ganglionic segment in Hirschsprung’ disease.
27 HD Absent Absent HD
4 HD Suspicious of Absent HD
ganglion cells
Toluidine blue stain: In the present study there is increased number of mast cells in the
aganglionic segment when compared to ganglionic segment in Hirschsprung’ disease.
• Due to cost effectiveness the following 17 samples were subjected to Calretenin IHC.
• 13 cases with clinical diagnosis of Ileal atresia to rule out Hirschsprung’ disease: H&E
and Cresyl violet stain showing presence of ganglion cells.
9
Results of CALRETININ Expression
• 4 cases with clinical diagnosis of Hirschsprung’ disease, H&E showing suspicious
ganglion cells and cresyl violet showing absence of ganglion cells: CALRETININ
NEGATIVE FOR GANGLION CELLS confirming the diagnosis of HD.
• 13 cases with clinical diagnosis of Ileal atresia to rule out Hirschsprung’ disease, H&E
and Cresyl violet stain showing presence of ganglion cells: CALRETININ POSITIVE
FOR GANGLION CELLS ruling out HD.
10
PHOTOGRAPHS
Ganglion cell
Ganglion cell
Ganglion cell
11
Ganglion cell
Fig 3:1% Cresyl violet stain Positive for Ganglion cells in muscularis propria (CV,400X)
MAST CELLS
Fig 4:1% Toulidine blue stain for mast cells in submucosa (Perivascular)(TB,400X)
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Fig 5: Calretinin, positive ganglion cells in submucosa (IHC,400X )
13
Fig 7 Calretinin, positive ganglion cells in Muscularis Propria (IHC,400X)
14
Fig 9 Calretinin, positive ganglion cells in Muscularis Propria (IHC,400X)
Fig 10: Hirschsprung disease: Hypertrophic nerve fibres with absence of ganglion
cells(H&E,400X)
15
Fig 11: Hirschsprung disease: Calretinin stain: Absence of ganglion cells in all layers of intestine
(IHC,100X)
16
17
DISCUSSION
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Abudu Emmanuel Kunle et al. analyzed seventy-four cases of gastrointestinal diseases in
children aged 14 years and below. The majority of cases were from the gastrointestinal tract.
Appendiceal lesions were (39.2%), Hirschsprung intussusceptions, enterocolitis and jejunal atresia
accounted for 29.7%, 10.8%, 6.8% and 4.1% of cases respectively. The common age group was
from 2 to 14 years, with a mean age of 8.6years. Male children were more frequently affected than
female children with the exception of appendiceal lesions, which occurred more in females (M: F
ratio= 1.6:1.0). Acute appendicitis was the commonest lesion of the appendix (72.4%) acute
suppurative appendicitis being more common.
13
Shrestha R et al. analyzed 930 specimens of the appendix; appendicitis accounted for 88.8%
with peak age incidence in the age group of 11 in both sexes. Histopathologic diagnoses included
acute appendicitis (45.6%), acute suppurative (20.8%), gangrenous (16.3%), perforated (1.7%),
resolving /recurrent/nonspecific chronic appendicitis (2.5%), acute eosinophilic appendicitis
(1.2%), peri appendicitis (0.2%), and carcinoid tumour (0.1%). Other important coexisting
pathologies were parasitic infestation (0.2%).
In English literature, Intestinal atresiais one of the most common causes of neonatal intestinal
obstruction, with an incidence of 1 in 5,000 newborns. Burjonrappa Set al14 analyzed 147 cases of
neonatal intestinal obstruction, out of which 39 cases were due to intestinal atresia, which is
observed in various other studies. Small intestinal atresia accounts for the majority of the cases,
while colonic atresia’s are quite rare.15
Hirschsprung disease is a genetic disorder of the enteric nervous system, often called congenital
aganglionic megacolon, and shows the absence of enteric neurons along a variable length of the
intestine causes chronic constipation. The first case of Hirschsprung disease was reported in the
year 1691, but the disease was named after Harald Hirschsprung, a Danish physician who first
described two infants who died of this disorder in 1888. The widely accepted theory of the cause
of Hirschsprung’s disease is a defect in the craniocaudal migration of neuroblasts originating from
the neural crest that occurs during the first 12 weeks of gestation. Defects in the differentiation of
neuroblasts into ganglion cells and augmented ganglion cell destruction within the intestine may
also contribute to the disorder. Hirschsprung’s Disease occurs in one in 5000 births and diagnosed
before the patient is ten years of age. The gold standard for definitive diagnosis of HD is a rectal
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biopsy with hypertrophied nerve trunks and a lack of ganglion cells. The biopsy is taken 2-3 cm
above the dentate line on the posterior wall of the rectum. Distal biopsies may result in a false-
positive diagnosis of HD because ganglion cells are normally absent in the anal canal.16
The ultimate diagnosis of Hirschsprung disease depends on histologic and histochemical staining
of sections. Short segment aganglionosis constitute 80% of the lesions, involving only the rectum
and the sigmoid colon, where as Long segment aganglionosis is seen in 20% of cases and involves
more proximal bowel. In rare cases the entire colon is affected (Total colonic aganglionosis)17
In 1884 Franz Nissl (1860-1919), a medical student in Munich, discovered that methylene blue, a
cationic (basic) dye stains granules in neuronal perikaryonof the brain giving a dark-colored hue.18
Haematoxylin and Eosin (H&E) staining is a commonly used stain for detecting ganglion cells in
muscular propria but has a lot of limitations as immature ganglion cells cannot be identified easily,
occur in clusters mimicking histiocytes, endothelial cells, fibroblasts, and needs expertise.
Acetylcholinesterase staining (AChE) is essential for confirming the presence of hypertrophic
nerve bundles and nerve trunks in the submucosa and lamina propria. The procedure is
cumbersome and needs a lot of time, hence not advisable in all the cases and not a specific marker
for ganglion cell19There are reports of false positive and false negative results using this
technique.20
The Cresyl Violet dye stains cell bodies of neurons and has abundant rough ER and ribosomes
(rRNA) or Nissl substance. Nissl substance is basophilic and will stain with basic aniline dyes like
Cresyl violet. DNA in the nucleus takes the same stain. In the neurons, cytoplasmic basophilia is
seen as a broad, dense band lying near the cell periphery. This peripheral basophilic ring is granular
and shows variations in density. Small rounded basophilic masses are seen against the nuclear
membrane forming a nuclear cap21
Toluidine blue stain (Tb): is a synthetic, acidophilic metachromatic dye that has an affinity for
nucleic acids and binds to nuclear material with a high DNA and RNA content, in chromatin or
Nissl substance and stains nucleus blue and cytoplasm light blue. Toluidine blue stains mast cells
with metachromatic violet. Metachromasia is due to stacking of dye cations at the sites of the high
density of anionic groups in the tissue which shortens the wavelength of maximum absorption so
19
that the maximum wavelength in the spectrum of the transmitted light makes the observed color
red instead of blue.22,
Cresyl violet is used in many studies as the main stains to highlight the ganglion cells in suspected
Hirschsprung’s disease by the principle of metachromasia.
Hadeel A. Yasseen23 stained all 50 cases of chronic constipation with a clinical diagnosis of
Hirschsprung disease with H&E, Toluidine blue, and Cresyl violet
for identification of ganglion cells spatially. His observations were Toluidine blue and
Cresyl violet stain is superior to H&E for the identification of ganglion cells in rectal specimens.
This is partially in agreement with HM. Canil et al. 24 who documented that Toluidine blue method
is a dependable method of demonstrating ganglion cells in frozen rectal biopsies. This method
provides quicker and easier identification of ganglion cells than with H&E staining and does not
need a counter stain.
Recent studies document an increase in the number of mast cells in Hirschsprung disease.
Kobayashi et al. 25 described an increased number of mast cells in the aganglionic segment of the
colon in patients with Hirsch sprung disease. Similar findings were documented by Demirbilek et
26
al. and Amit et al27.Themast cells are distributed around blood vessels, nerve fibers, and
28
transmurally.In the study done by Hermanowicz et al. , his observations were increased mast
cells in the mucosa and lamina propria of HD.
Immunohistochemistry (IHC) is the major ancillary method to detect ganglion cells and nerve
hypertrophy. Calretinin is a calcium-binding protein seen in intrinsic nerves of the muscular
mucosae and lamina propria and is used as a simple and reliable tool for the diagnosis of HD. Loss
of calretinin immunoreactive in the ganglion cells or nerve fibers correlates spatially with
aganglionosis. Calretinin IHC has numerous advantages such as it uses paraffin embedded
biopsies, is simple and has a high specificity and sensitivity and identify immature ganglion cells.
29
Nasser Rakhshaniet al. documented in their study that IHC for calretinin provided highly
compatible results with hematoxylin-eosin findings in the diagnosis of Hirschsprung disease with
higher specificity and accuracy values than H&E staining (P <0.0001). Similarly, Gonzalo et al.
112
found in their retrospective study that all patients without HD had a positive
20
immunohistochemical expression of nerve fibers in lamina propria or muscularis mucosae, and all
Hirschsprung patients showed negative calretinin expression concluding that
immunohistochemical testing of calretinin is quite supportive in triaging additional workup based
on clinical suspicion.
Neoplastic lesions were similar in both age and sex groups. Male: female ratio in non-neoplastic
lesions are 303:140(2.16:1).
Common clinical presentation in GIT lesions are pain abdomen in 376/443(84.87%) and intestinal
obstruction 59/443(13.31%).
In the present study, 31 cases with a clinical diagnosis of Hirschsprung’ disease and 13 cases with
the clinical diagnosis of ileal atresia to rule out Hirschsprung’ disease was subjected to special
stainCresyl violet stain for ganglion cells &Toluidine blue stain for grading mast cells in the
aganglionic segment. In 27 cases with a clinical diagnosis of Hirschsprung' disease: H&E and
Cresyl violet stain showed an absence of ganglion cells, in 4 cases with the clinical diagnosis of
Hirschsprung' disease: H&E showed suspicious ganglion cells and cresyl violet showed an absence
of ganglion cells.
In 13 cases with a clinical diagnosis of Ileal atresia to rule out Hirschsprung' disease: H&E and
Cresyl violet stain showed the presence of ganglion cells confirming the diagnosis of Ileal atresia.
The overall accuracy of identifying ganglion cells on H&E stain was 90.9% and 100% by Cresyl
violet stain.
21
In the present study there is an increased number of mast cells in the submucosa of the aganglionic
segment (2+ and 3+) when compared to the ganglionic segment in Hirschsprung' disease by
Toluidine blue stain
Due to cost-effectiveness the following 17 samples were subjected to Calretinin IHC:4 cases with
clinical diagnosis of Hirschsprung’ disease, H&E showing suspicious ganglion cells and cresyl
violet showing absence of ganglion cells and 13 cases with clinical diagnosis of Ileal atresia to rule
out Hirschsprung’ disease, H&E and Cresyl violet stain showing presence of ganglion cells.
CONCLUSION
• Cresyl violet stain should to be used as the routine stain to highlight the ganglion cells in
suspected Hirschsprung’s disease cases.
• In cases of Hirschsprung’s disease every case should be subjected to Cresyl violet stain
to confirm the presence of ganglion cells as it is cheap and cost effective and also helps in
differentiating ileal atresia from Hirschsprung’s disease.
• Calretinin IHC is a very reliable adjunctive test in identification of ganglion cells and
differentiating between pure Ileal atresia from Hirsch sprung disease, issue of accurate
report and reducing the requirement for repetitive biopsies.
22
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