GASTROINTESTINAL PATHOLOGY

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CHAPTER 14 GASTROINTESTINAL PATHOLOGY

Hypertrophic pyloric stenosis

Gastric volvulus

Graft-versus-host disease

Spontaneous perforation of the GI tract

Colon

Chronic inflammatory bowel disease

Fibrosing colonopathy

Miscellaneous GI specimens

Anorectal anomalies

Currarino triad

Vermiform appendix

Acute appendicitis

Persistent hyperinsulinemic hypoglycemia of infancy

INTRODUCTION

• Specimens from the gastrointestinal tract comprise a significant proportion of the workload of many pediatric surgical pathology departments

number is increasing with advances in endoscopic techniques

• Complexity of GI disease is increasing since

newer forms of treatment becoming available which may alter classical histological appearances

– such as monoclonal anti-inflammatory antibodies

newer forms of treatment for non-GI disease may also cause changes in the GI tract

GI involvement in diseases previously not suspected to have GI pathology

– such as enteropathy in Shwachmann–Diamond syndrome

• Entities provided in this chapter are primarily those seen with reasonable frequency as surgical pathology specimens

most organized by main site of involvement

– some entities may affect multiple sites but are documented according to the usual site of disease

ESOPHAGUS

ESOPHAGEAL ATRESIA AND TRACHEO-ESOPHAGEAL FISTULA

• 1 : 3000–5000

• Associated maternal polyhydramnios (50%)

• Around half of cases preterm with associated abnormalities

cardiac defects

imperforate anus

duodenal atresia

Histopathological features (Dutta, Mathur, Bhatnagar 2000)

• Unusual to get surgical specimen

• When submitted tissue comprises elements of tracheal and esophageal wall in variable combinations according to case (Figs 14.2, 14.3)

Fig 14.2 One-day-old girl with esophageal atresia and tracheo-esophageal fistula. The specimen is the tip of the proximal esophageal pouch. Photomicrograph of the specimen demonstrating the pouch comprising the usual elements of esophageal wall. The mucosa shows some ciliated epithelium. Unusually, no striated muscle was present in the muscular coat.

Fig 14.3 One-year-old girl with previous EA repair. Stricture below anastomosis excised. Photomicrograph of the excised piece of esophagus with thickened and disorganized muscle coat and containing bronchial type glands extending deeply into the muscle coat.

DUPLICATION CYSTS

• Developmental defects

can affect any site in the GI tract from esophagus to rectum

• Variable size

• Tubal or spherical

• Mass effects or incidental

may be associated with vertebral defects

may communicate with spinal canal

– neuroenteric cyst

• Present with

respiratory distress

feeding difficulty

Histopathological features

• Two layered muscularis propria

• Variable gastrointestinal mucosal lining type (Figs 14.4, 14.5)

Fig 14.4 Nine-month-old boy. Antenatal diagnosis of thoracic cyst. Posterior mediastinal cyst (3.5 × 3 × 2.5 cm) attached to wall of esophagus beneath the right vagus nerve with a fibrous attachment to the vertebral body. It contained clear fluid. Photomicrograph of the cyst wall demonstrating double muscle layer lined by simple gastric cardiac type mucosa. Focally there was ulceration.

Fig 14.5 Three-year-old boy with an incidental finding of a para-esophageal cyst (1.7 × 1 × 1 cm) during fundoplication. Photomicrograph of the cyst wall showing a smooth muscle coat with a lining of ciliated columnar epithelium. There is no cartilage – an important point of distinction from a bronchogenic cyst.

ESOPHAGEAL DIVERTICULUM

• Congenital diverticula very rare

• Most are acquired

• Histologically consist of all layers of esophageal wall

ACHALASIA

• See below under motility disorders

GASTRO-ESOPHAGEAL REFLUX DISEASE

• Some degree of gastro-esophageal reflux (GER) nearly universal in children

• GER of sufficient severity to cause symptoms or disease is termed gastro-esophageal reflux disease (GERD)

• Common in infants and children

• Most common under 6 months of age

• Presents with

feeding difficulties

failure to thrive

respiratory symptoms

• Many mild cases treated without endoscopy

• Endoscopy and biopsy

• pH monitoring in difficult cases and differentiation from primary eosinophilic esophagitis

Histopathological features

• Changes can be patchy

most severe in distal esophagus

multiple biopsies required

• Basal cell hyperplasia (Fig 14.6)

• Intercellular edema

• Elongation of connective tissue papillae

• Congestion of capillaries

• Increased numbers of intraepithelial

mononuclear inflammatory cells (’squiggle’ cells)

neutrophil polymorphs (unusual)

– if numerous consider fungal disease also

• Ulceration uncommon

• Non-specific stromal inflammation

Fig 14.6 Reflux esophagitis. Eight-year-old boy with short gut, on parenteral nutrition. Biopsy close to gastro-esophageal junction. Photomicrograph of esophageal biopsy showing basal cell hyperplasia with elongation of the connective tissue papillae that demonstrate vascular engorgement and perivascular hemorrhage, intercellular edema and an infiltrate within the epithelium of lymphocytes and eosinophils. The infiltrate is confined largely to the epithelium around the connective tissue papillae.

Differential diagnosis

• Eosinophilic esophagitis

• Infectious esophagitis

• Drug and chemical injury

• Crohn’s disease

COLUMNAR EPITHELIAL LINED ESOPHAGUS (CELE)

• Columnar epithelium replaces normal squamous epithelial covering of lower esophagus

metaplastic process

• Irregular junction with squamous lined esophagus

• Requires certainty regarding exact site of biopsy

• Usually gastric cardiac type in children

• Barrett’s esophagus defined as CELE with goblet cells

• Barrett’s esophagus very uncommon in children

• More common in

cases with severe longstanding GERD

cases following TEF repair

cystic fibrosis

EOSINOPHILIC ESOPHAGITIS

• Relatively common in children (Furuta et al 2007)

• Frequently associated with allergic disease

• Normal pH studies

• Symptoms include

food intolerance

food ‘sticking’

• May have upper airway diseases such as

laryngeal edema

subglottic stenosis

• Diagnosis based on histological appearance in the correct clinical context

not on histology alone

• Corrugated mucosa on endoscopy

• Do not respond to anti-reflux treatment

• Complications

Differential diagnosis and diagnostic pitfalls

requires clinicopathological correlation

INFECTIVE ESOPHAGITIS

• Almost always in the setting of immunocompromise

• In severely immunocompromised infections with multiple organisms occur

CROHN’S DISEASE OF THE ESOPHAGUS

• Esophageal involvement relatively common

can occur in isolation

• Usually lymphocytic esophagitis with (Fig 14.11)

basal cell hyperplasia

intercellular edema

infiltration of epithelium by mononuclear cells

• May have well formed non-caseating epithelioid granulomas in lamina propria beneath epithelium (Fig 14.12)

• May be ulceration or stricture

Fig 14.11 Lymphocytic esophagitis in Crohn’s disease. Aphthous ulcers in duodenum and colon. Macroscopically normal ileum but granulomatous inflammation on biopsy. Photomicrograph of esophagus shows mild lymphocytic esophagitis with increased numbers of intraepithelial mononuclear cells associated with mild basal cell hyperplasia and intercellular edema. This appearance is very common in Crohn’s disease but is not specific.

Fig 14.12 Sixteen-year-old boy with Crohn’s disease treated with Infliximab. Stricture of esophagitis noted on endoscopy. Granulomas present in esophagus, stomach, ileum and colon. Photomicrograph of esophageal biopsy showing epithelial hyperplasia with a well formed epithelioid granuloma in the lamina propria immediately beneath the epithelium. A multinucleated giant cell is present. There is a scattering of lymphocytes in the surrounding connective tissue.

CHEMICAL AND DRUG-INDUCED ESOPHAGITIS

• Accidental swallowing of corrosive liquids in children

• Initial tissue necrosis edema and inflammation followed by repair with stricture formation (Figs 14.13, 14.14)

• Drug-induced inflammation tends to occur

in the middle one-third

at the site of strictures or other narrowings

• Not uncommon with high dose chemotherapy

• Superficial erosion

• Non-specific lymphocytic esophagitis

• May follow ingestion of iron containing medicines (Abraham 1999)

crystalline iron deposition in the tissue

• May also occur in stomach and duodenum (Figs 14.15, 14.16)

Fig 14.13 Caustic stricture of the esophagus from a 7-year-old boy. A tubular segment of esophagus cut longitudinally to demonstrate the severe fibrous thickening of the wall with occlusion of the lumen. The lumen is visible on the left hand side of the picture. The muscle coat visible as a slightly glistening darker layer on the left of the picture is replaced by white fibrous tissue on the right.

Fig 14.14 Caustic stricture of the esophagus. Photomicrograph showing intense hyperplasia of the epithelium. The underlying tissue consists of dense collagenous fibrous tissue with obliteration of muscle.

Fig 14.15 Photomicrograph of gastric mucosa showing crystalline iron deposition in gastric mucosa, evident as brown refractile material both on the surface and within the superficial lamina propria.

Fig 14.16 Same case as previous showing positive Perls’ reaction in the surface iron (to the right of the picture).

STOMACH

• Endoscopic biopsies for assessment of gastritis by far the most common gastric specimen for pediatric surgical pathologists

• Other entities such as Menetrier’s disease, dysplasia, GAVE and many other specific histological lesions seen in adults are exceptionally uncommon in children and are not described here

see standard adult texts

GASTRITIS

• An important cause of abdominal pain in children

• Several different types

• Non-specific gastric changes may also be seen in a range of pediatric enteropathies

see small intestine section for diagnostic features

• Any severely ill patient may also show non-specific acute gastritis

clinically appears to be less common than adults

Helicobacter pylori gastritis

• Common in children but unusual in tertiary level practice as most detected and treated locally

• May not be apparent endoscopically

• H. heilmanni in minority

organism is longer with tighter spiral (Qualia et al 2007)

Fig 14.17 Helicobacter gastritis. Giemsa stained preparation at high power showing the organisms in the surface mucus. The organisms can be seen on H&E but are much more evident in the Giemsa preparations. They are confined to the mucus of the surface and the pits.

Fig 14.18 Helicobacter gastritis. Photomicrograph of a high power view of the lamina propria. Although neutrophil polymorphs are present, they are not as prominent as in adult patients with Helicobacter gastritis.

Fig 14.19 Helicobacter gastritis. Low power photomicrograph of gastric antral mucosal biopsy showing a diffuse inflammatory cell infiltrate in the lamina propria with lymphoid follicle and germinal center. The presence of germinal centers should always prompt a careful search for Helicobacter. They were present in this case.

Lymphocytic gastritis

• Lymphocytic infiltrate in surface and crypt epithelium

>20 lymphocytes / 100 surface epithelial cells (Fig 14.21)

• Association with untreated celiac disease

More frequent in younger patients

Often associated with vomiting

Resolves with gluten-free diet

• Not all cases associated with gluten-enteropathy

minority of HLO-associated gastritis

minority of Crohn’s gastritis

other autoimmune disease

some apparently isolated

• Collagenous gastritis may be an associated entity

subsurface epithelial band of collagenous ‘fibrosis’

Fig 14.21 Celiac disease with lymphocytic gastritis. Photomicrograph of a gastric biopsy from a case of celiac disease showing increased numbers of intraepithelial lymphocytes in both surface and glandular epithelium, akin to the appearance in the duodenum. Such patients tend to have vomiting as a symptom of their disease.

Autoimmune gastritis

• Relatively uncommon in children

• Usually associated with autoimmune enteropathy and/or other autoimmune disease

• Histological features variable

non-specific inactive gastritis

active gastritis

relative neuroendocrine cell hyperplasia

HYPERTROPHIC PYLORIC STENOSIS

• See below under motility disorders

GASTRIC VOLVULUS

• Very rare in children

• Can be associated with apslenia

• Unusual to receive surgical specimens from such cases

non-specific secondary features

GRAFT-VERSUS-HOST DISEASE

• See specific section below

GASTRIC TUMORS

• Very rare in childhood

• Gastric carcinoma almost never occurs

• Stromal tumors may occur

see specific section below

• Polyps rare

see small intestine

SPONTANEOUS PERFORATION OF THE GI TRACT

• In this context means isolated perforation with no other detectable underlying disease process

does not include those with demonstrable acute ischemia, necrotizing enterocolitis (NEC), etc

• Reported more in low birth weight infants

even those without NEC

• Probably ischemic in nature

• Usually gastric

can affect colon, Meckel’s diverticulum

• May be associated with congenital focal defective musculature

• May rarely be associated with underlying genetic condition

Ehlers–Danlos syndrome

Histopathological features

• Non-specific features secondary to perforation (Fig 14.22)

• No detectable underlying cause such as acute ischemia

• Other causes such as vasculitis etc must always be excluded

Fig 14.22 Spontaneous perforation of the stomach. Photomicrograph from the edge of the perforation showing hemorrhagic necrosis of the tissues. This is a non-specific appearance of ischemic-type damage.

SMALL INTESTINE

INTESTINAL ATRESIA

• Intrauterine process resulting in incomplete bowel lumen

• Duodenal atresia

presumed developmental

mainly associated with trisomy 21

rarely submitted for pathological assessment

– usually treated with duodenoduodenostomy

• Intestinal atresia

presumed due to intrauterine insult

ischemic / hypotensive episode

jejunum and ileum affected

isolated or multiple

more commonly submitted

– treated with resection and anastomosis

Histopathological features

• Intestinal atresia

discontinuous lumen

atretic segment (Fig 14.23)

fibrosis (Fig 14.24)

– focal muscularis replacement (Fig 14.25)

dystrophic calcification

pigment-laden macrophages

Fig 14.23 Ileal atresia. Resected specimen showing a thread-like segment of stenotic bowel with a more proximal part with intense dilatation because of the obstruction.

Fig 14.24 Ileal atresia. Photomicrograph showing the junction of atretic and patent segment of intestine. The bowel ends blindly with fusion of all the layers into a fibrovascular band without recognizable bowel wall structures. Hemosiderin is frequently present at such sites indicating previous hemorrhage.

Fig 14.25 Stenosis of bowel following in-utero ischemia. Photomicrograph demonstrating the thickened submucosa with focal fresh hemorrhage. The muscularis mucosae is largely absent – a sign of previous ulceration. The muscular coat is also focally deficient.

MECKEL’S DIVERTICULUM

• 1–4% of population

• May be completely asymptomatic

Histopathological features

• Antimesenteric border of terminal ileum approx 30cm from ileo-cecal (IC) valve

• Up to about 8cm long (Fig 14.26)

• May be long and tubular or squat and almost cystic

• Tip may be bifid or nodular

• All components of normal bowel wall present

• Muscle coat may be very attenuated at the tip or distorted by heterotopic tissue

• Lined usually by

may have gastric body mucosa lining in minority (Fig 14.27)

– Helicobacter may colonize

rarely colonic tissue

• May see ulceration or bleeding point

• May have nodules of pancreatic tissue (including islets) in wall (Fig 14.27)

• Serosa may show fibrosis or inflammation

Fig 14.26 Meckel’s diverticulum. Photograph demonstrating the excised specimen of piece of ileum with the diverticulum arising from its antimesenteric border, being long and tubular with slightly bulbous end.

Fig 14.27 Meckel’s diverticulum. Photomicrograph showing gastric body mucosa lining the distal part of the diverticulum. In the submucosa there is a nodule of heterotopic pancreatic tissue.

INTUSSUSCEPTION

• Invagination of bowel into the bowel distal to it

• Mainly ileocecal

• Mainly (>90%) infants

when occurring in other ages or at other sites more likely to be secondary to another specific pathology

• Results in secondary ischemic damage to bowel

• Presents with

’red-currant jelly’ stool

palpable abdominal mass

• Many cases can be reduced medically

only those not responding come to surgery and resection

Histopathological features

• Resected mass comprises

outer layer of thinned distended bowel – the intussuscipiens

inner layer of everted invaginated bowel – the intussusceptum

• Mesentery of intussusceptum is stretched and congested

• Intussusceptum is frequently infarcted and hemorrhagic

• Histologically the intussusceptum shows

hemorrhagic infarction

viral features may be seen in hyperplastic bowel surface epithelium or lymphoid tissue

– especially adenovirus

other pathology associated with the cause of the mass

the cause may not be identified other than non-specific lymphoid hyperplasia

• Intussuscipiens shows

thinning of wall

Fig 14.30 Intussusception. Lymphoid hyperplasia secondary to infection with adenovirus. The wall of the intussusceptum is edematous and it is seen protruding from the intussucipiens on the right of the picture. There is enlargement of mesenteric lymph nodes.

Fig 14.31 Intussusception. Photomicrograph showing mucosa of non-necrotic part of terminal ileal mucosa. The epithelium is disorganized and there are the typical brick red nuclear inclusions of adenovirus with peripheral margination of chromatin.

Fig 14.32 Intussusception. Resected specimen showing intussusception due to everted Meckel’s diverticulum. The diverticulum is the pale mass in the center of the specimen.

MECONIUM ILEUS

• Neonatal bowel obstruction due to impacted meconium

usually terminal ileum

• May present as intrauterine obstruction / perforation

• Associated with cystic fibrosis (CF)

affects 15% of CF neonates

may be marker of more severe CF phenotype

• About 10–20% of apparent meconium ileus cases do not have CF

clinically may be mimicked by total colonic aganglionosis

• Treated medically or surgically

– gastrograffin enema

– enterotomy, irrigation and primary closure

Histopathological features

• Specimens received in surgically treated cases

may show secondary features of perforation (Fig 14.34)

• Intestinal lumen filled with inspissated meconium (Fig 14.35)

characteristically, mucus extends deep into crypts (plugging) (Fig 14.36)

Fig 14.34 Cystic fibrosis – meconium peritonitis. Photomicrograph of the peritoneal surface of a case of meconium peritonitis cased by rupture of the intestine. There is a foreign-body reaction with dystrophic calcification.

Fig 14.35 Meconium ileus in cystic fibrosis. A resected segment of bowel cut longitudinally and opened to display the thick green meconium, filling and obstructing the lumen. The upper part of the picture shows the meconium removed from the other half of the specimen. It forms a cast of the bowel lumen. It had the texture and consistency of soft rubber.

Fig 14.36 Photomicrograph of a case of cystic fibrosis with meconium ileus. The resected small bowel shows plugging of the lumen by thick eosinophilic mucus. The mucus extends into the crypts as far as their bases – a characteristic feature of meconium in cases of cystic fibrosis.

Other features of cystic fibrosis in the gut

• Plugging of crypts by mucus in otherwise normal intestine (Fig 14.37)

• Intestinal atresia

• Rectal prolapse

• Giant inflammatory polyposis in the presence of Crohn’s disease

Fig 14.37 A case of cystic fibrosis without meconium ileus. It still shows the characteristic mucous plugging of the crypt bases. While mucous plugging can be seen in bowel obstruction from many causes, the inspissated mucus rarely has the opaque ‘hard’ quality of that in cystic fibrosis. If this appearance is seen in a specimen, the clinician should be alerted to the possibility of cystic fibrosis.

CROHN’S DISEASE

• Inflammatory bowel disease is relatively common in children

• May affect any age

more common in older children

• About one-quarter of patients have extraintestinal manifestations

Histopathological features on biopsy of small intestine

• Duodenal involvement common (Fig 14.38)

• Ileal involvement very common

• Focal active inflammation (Fig 14.39)

• Villous blunting

• Aphthous ulceration

• Granulomas (Fig 14.40)

with or without giant cells

granulomas may be present in the lamina propria or in the lymphoid tissue of Peyer’s patches

Fig 14.38 Duodenal Crohn’s disease. Five-year-old girl with colonic Crohn’s disease. Granulomas are not present in the duodenum but there is focal active duodenitis, with villous broadening and a neutrophil and eosinophil infiltrate in lamina propria and surface epithelium.

Fig 14.39 Ileal Crohn’s disease. Photomicrograph showing active inflammation, with neutrophils and eosinophils in the lamina propria and in the epithelium.

Fig 14.40 Ileal Crohn’s disease. Same case as Fig 14.39 showing a small epithelioid granuloma in the lymphoid tissue, a common site for granulomas.

Differential diagnosis on biopsy of duodenum

• Non-specific duodenitis

• Celiac disease

• Ulcerative colitis associated inflammation

• Infective duodenitis

• Chronic granulomatous disease

Differential diagnosis on biopsy of terminal ileum

• Ulcerative colitis

• Tuberculosis

• Infective enteritis

• Chronic granulomatous disease

Resected specimen (Fig 14.41)

• Thickened small bowel wall

• Thickened mesentery

• Fat wrapping of intestine

• Enlarged mesenteric lymph nodes

• Serosal adhesions

• Mucosal changes

linear ulceration

– may show pale adherent exudate

reddening with polyps

may be intervening normal areas

– may show small aphthous ulcers

• May be stricture with fibrous thickening of the wall

Fig 14.41 Ileal Crohn’s disease. Photograph of an opened specimen of terminal ileum showing the mural thickening so characteristic of Crohn’s disease. There is extensive mucosal ulceration and also cobblestoning.

Complications

• Strictures with obstruction

INTESTINAL INFECTIONS

Cytomegalovirus

• Usually in the setting of immunosuppression

• May infect areas of ulceration from other causes

• Frequently co-exists with other pathogens

• Typical CMV inclusions seen in endothelial and other mesenchymal cells (Fig 14.46)

Fig 14.46 CMV small bowel. Photomicrograph showing typical CMV nuclear inclusions and basophilic small cytoplasmic inclusions. The infected cells are probably endothelial.

Astrovirus

• Astroviruses are single stranded RNA viruses

• Rare cause of diarrhea and vomiting in humans

especially infants and elderly

especially in immunodeficient or immunocompromised

• Virus infects mature enterocytes

• May rarely present as intussusception

• Morphological abnormalities relatively minor and non-specific

despite severe symptoms

mild small intestinal non-specific inflammation

partial villous atrophy may be present (Fig 14.47)

• Astrovirus identified using immunostaining and / or electron microscopy (Fig 14.48)

Fig 14.47 Astrovirus infection jejunum. Photomicrograph of small bowel showing hyperplasia and irregularity of the epithelium with a mild inflammatory cell infiltrate in the lamina propria.

Fig 14.48 Astrovirus infection jejunum. Photomicrograph showing immunostaining of individual enterocytes with antibody to astrovirus.

NEONATAL NECROTIZING ENTEROCOLITIS

• Common medical emergency in neonates (Lin et al 2006, Epelman et al 2007)

1–5% neonatal intensive care unit admissions

10–15% low birth weight infants

• Pathogenesis remains uncertain but associated with risk factors

prematurity and low birth weight

– >90% are preterm

enteral feeding

– especially milk formula vs breast milk

• Pathophysiology is complex and unresolved but involves

abnormal / immature GI motility and digestion

abnormal / immature GI circulatory regulation

abnormal intestinal bacterial colonization

abnormal / immature intestinal mucosal barrier function

abnormal / immature intestinal immunity

Histopathological features

• Can affect any site of GI tract

mainly distal small intestine and right colon

• Patchy areas of wall thinning and dilation (Fig 14.50)

• Partial thickness and full thickness ischemic type (coagulative) necrosis

may show pneumatosis intestinalis (Figs 14.51, 14.52)

– intramural gas

• Often associated with reparative type changes (Fig 14.53)

epithelial regeneration

granulation tissue

• Secondary perforation related changes

• May be complicated by / associated with, fungal infection

• Histological changes are essentially those seen with ischemia

probable final common pathway of microvasculature damage

• Late submucosal fibrosis and fibrous replacement of muscularis propria

strictures and dysmotility

• Histopathological examination mainly to confirm diagnosis and exclude other pathologies

• Comment should be made on margin viability

Fig 14.50 Necrotizing enterocolitis. Photograph of a specimen removed from a premature neonate demonstrating a yellow irregular area necrotic bowel wall. The necrosis does not involve the full circumference of the wall. Such lesions may be multiple and multiple areas of perforation are not uncommon.

Fig 14.51 Necrotizing enterocolitis. Photomicrograph demonstrating hemorrhagic necrosis of the bowel wall with the pathognomonic feature of gas bubbles in the wall – so-called pneumatosis.

Fig 14.52 Necrotizing enterocolitis. Photomicrograph at a higher power of the same lesion as in Fig 14.51 demonstrating the edge of one of the gas-filled areas. There is hemorrhage and a neutrophil polymorph infiltrate.

Fig 14.53 Necrotizing enterocolitis lesser degree of damage. Photomicrograph of a case showing an intact (largely) muscle coat but with marked submucosal edema and loss of much of the mucosa. The pathologic features in these cases do not differ significantly form those of other ischemic causes of bowel injury.

SPONTANEOUS PERFORATION OF THE SMALL INTESTINE

• See discussion under stomach (Figs 14.54, 14.55)

Fig 14.54 Spontaneous perforation of the ileum. Photomicrograph of a limited right hemicolectomy showing normal cecum and colon. The ileum shows disruption of its wall visible at the top of the photo. This case shows the edge of the perforated area. There is ischemic-type necrosis of the bowel wall with some fresh hemorrhage.

Fig 14.55 Spontaneous perforation of the ileum. Photomicrograph of the edge of the preparation seen in Fig 14.54 showing hemorrhagic ischemic necrosis of the bowel wall.

FOOD ALLERGY

• Usually cows milk allergy (CMA)

starts in infancy

– improves with age

• Other allergies develop later

• May be IgE or non IgE mediated processes

most believed non IgE mediated

– delayed onset from exposure

– mainly affects GIT

– no acute systemic life threatening effects

• May present as pain, loose stools, blood in stool or severe constipation

PRIMARY INTESTINAL FAILURE / NEONATAL-INFANTILE ENTEROPATHIES

• Group of conditions present with

intractable diarrhea

failure to thrive

– protein losing enteropathy

• May require parenteral nutritional support

associated long-term complications

• Classical causes are inherited intestinal epithelial defects (present early)

tufting enteropathy (intestinal epithelial dysplasia)

microvillous inclusion disease

• Many additional etiologies now recognized (present later; see below)

include mainly infantile autoimmune enteropathy

CELIAC DISEASE (GLUTEN SENSITIVE ENTEROPATHY)

• T-cell immunological response in small intestine (Green & Cellier 2007)

• Inflammatory reaction in proximal small intestine to gliadin

• More frequent than initially thought and increasing incidence

1 in 100 on screening

• Increased frequency in insulin-dependent diabetes mellitus (IDDM), Down and Turner syndrome

• May be precipitated by infection etc

• Breast feeding reduces risk

• Early introduction of gluten (<4 months) increases risk

• Patients have increased risk of other autoimmune disease

Genetics

• Necessary but insufficient for pathogenesis

• HLA DQ2 90–95%

• HLA DQ8 5–10%

• 50% susceptibility HLA related

Histopathological features

• Biopsies now with endoscopic forceps not suction capsules

orientation important

more fragmentation / crush artefact

• 4–6 biopsies required

• Features are characteristic but not specific

• Other causes of inflammatory changes with villous atrophy are possible

see other enteropathies

• Normal villous/crypt ratio >2.5/1

• Enteropathy associated T cell lymphoma may develop

almost never during childhood

infiltration by sheets of T cells

Fig 14.58 Celiac disease. Photomicrograph of a biopsy of duodenum showing effacement of the villi with hyperplasia of the crypts. There is an increase in plasma cells and eosinophils in the lamina propria and a marked increase in intraepithelial lymphocytes in the surface epithelium.

Fig 14.59 Celiac disease. Photomicrograph showing a section stained for CD8, demonstrating the intraepithelial lymphocytes are exclusively of the cytoxic / suppressor subtype.

TUFTING ENTEROPATHY

• Neonatal enteropathy due to abnormal enterocyte adhesion

• May be familial

epithelial cell adhesion molecule (EpCAM) mutations (Sivagnanam et al 2008)

• With or without other syndromic features

choanal, ophthalmic, hematological, syndactyly (Bird et al 2007)

– especially familial cases

• Initially thought incompatible with life

small bowel transplantation for severe cases

milder cases now also reported

• Probably represents a spectrum of diseases related to abnormal epithelial adhesion molecules

Histopathological features

• Variable villous atrophy

• Inflammation

variable temporally and spatially

• Epithelial ‘tufting’

focal clumps of surface hobnail enterocytes

nuclei away from basal area

surface epithelial irregularity

• Ultrastructurally abnormal desmosomes (Goulet et al 2007; Figs 14.60–14.65)

Fig 14.60 Tufting enteropathy inflammation. Photomicrograph demonstrating partial villous atrophy and an acute and chronic inflammatory cell infiltrate in the mucosa. No tufts are present in this field. The degree of inflammation present is very variable, but some degree is usually present.

Fig 14.61 Tufting enteropathy small bowel. Photomicrograph demonstrating the diagnostic tufts of epithelial cell on the villus surface.

Fig 14.62 Tufting enteropathy duodenum. Photomicrograph of small bowel from a known case of tufting. The abnormalities can be very subtle, especially in the small intestine. It would be very difficult to make a diagnosis on this field alone.

Fig 14.63 Tufting enteropathy colon. Photomicrograph showing large numbers of tufts on the surface. Diagnosis is relatively easy in such cases.

Fig 14.64 Tufting enteropathy colon. Photomicrograph of colonic mucosa showing very few tufts. There is inflammation in the underlying lamina propria.

Fig 14.65 Tufting enteropathy colon. Photomicrograph at high power showing the tufts to comprise rounded up enterocytes without stroma.

MICROVILLOUS INCLUSION DISEASE

• Neonatal enteropathy / intestinal failure due to defective formation of intestinal microvillous brush border

• Initially thought incompatible with life (microvillous atrophy)

less severe forms now described (microvillous dystrophy)

• Autosomal recessive

mainly MYO5B mutations (Erickson et al 2008)

Histopathological features

• Villous atrophy (Fig 14.67)

• Characteristic abnormality of apical brush border

loss of normal thin apical microvillus brush border pattern

abnormal apical cytoplasmic ‘smudging’ of microvilli

– best seen with PAS, alkaline phosphatase and CD10 stains (Figs 14.68–14.70)

• Electron microscopy characteristic

absence / reduction of normal apical microvilli (Figs 14.71–14.73)

abnormal cytoplasmic microvillous inclusions

– may be present in small bowel and colon

Fig 14.67 Microvillous inclusion disease. Photomicrograph of a small bowel specimen with partial villous atrophy.

Fig 14.68 Microvillous inclusion disease. Photomicrograph of a PAS-stained preparation. The normal staining of the microvillous border is abolished and there is excess PAS-positive material in the apices on the enterocytes giving a very irregular appearance to the specimen.

Fig 14.69 Normal small bowel. Photomicrograph showing alkaline phosphatase staining confined to the microvillous border of the enterocytes.

Fig 14.70 Microvillous inclusion disease. Photomicrograph (alkaline phosphatase) demonstrating the same pattern of irregularity as seen on the PAS preparation.

Fig 14.71 Normal enterocytes. Electronmicrograph showing normal microvilli and normal supranuclear cytoplasm in the enterocytes.

Fig 14.72 Microvillous inclusion disease. Electronmicrograph demonstrating patchy loss of microvilli from one cell surface and patchy loss from an adjacent cell. This cell shows a vacuole in the apical cytoplasm containing internalized microvilli.

Fig 14.73 Microvillous inclusion disease. Electronmicrograph demonstrating complete loss of microvilli from the cell surfaces and vacuoles in the apical cytoplasm showing internal microvilli.

INTRACTABLE ENTEROCOLITIS OF INFANCY

• Familial consanguinity (Thapar et al 2005)

• Onset of disease in the neonatal period

• Uncertain etiology

• Small bowel enteropathy

villous atrophy

• Transmural colitis with ulceration (Fig 14.76)

sharply demarcated

overhanging edges (Figs 14.77, 14.78)

• Oro-anal disease

• Poor response to immunosuppressive therapy

• Increased risk of lymphoid malignancies

appear mainly EBV-related

Fig 14.76 Intractable enterocolitis of infancy. Nine-month-old male infant. Photomicrograph of resected colon showing the extensive mucosal ulceration and edematous surviving mucosa.

Fig 14.77 Intractable enterocolitis of infancy. Photomicrograph of the same specimen. Note the transmural nature of the infiltrate and the deep ulcer with overhanging mucosal margins.

Fig 14.78 Intractable enterocolitis of infancy. Photomicrograph showing the inflammatory cell infiltrate in the mucosa adjacent to one of the ulcers.

ABETALIPOPROTEINEMIA

• Autosomal recessive

• Defect in transport of triglyceride from enterocyte to lymphatics with fat malabsorption

• Prolonged malabsorption of fat soluble vitamins leads in later life to

abnormal erythrocytes (acanthocytes)

CNS disease resembling Friedreich’s ataxia

• Very low serum cholesterol and complete absence of low density lipoproteins

• Histological features

normal villous morphology on low power

lipid accumulates in enterocytes

– vacuolated cytoplasm (Fig 14.79)

– demonstrated on Oil Red-O staining (Fig 14.80)

Fig 14.79 Abetalipoproteinemia. Photomicrograph of a duodenal biopsy specimen showing the marked vacuolation of the enterocyte cytoplasm. The vacuoles are predominantly supranuclear but there are some subnuclear vacuoles.

Fig 14.80 Abetalipoproteinemia. Photomicrograph stained for fat showing the marked accumulation of lipid in the enterocytes, corresponding to the vacuoles seen on the H&E stained preparation. (Sudan)

PATHOLOGY OF INTESTINAL TRANSPLANTATION

• Used for patients with short bowel or intestinal failure

• May be patchy

multiple biopsies required

• Subclinical rejection identified on biopsy associated with worse graft survival (Takahashi et al 2007)

• Chronic rejection

obliterative arteriopathy

mucosal fibrosis (Tryphonopoulos et al 2006)

sclerosing peritonitis

• Main differential diagnoses are infectious

EBV-related LPD

• Immuno / ISH required

INTESTINAL GRAFT-VERSUS-HOST DISEASE

• Usually post bone marrow transplantation

• May affect upper or lower GI tract

• Thrombotic microangiopathy now recognized (Nishida et al 2004)

• CMV/adenovirus infection must be excluded in all cases

Fig 14.81 Intestinal graft-versus-host disease. Photomicrograph of large bowel mucosa showing apoptotic cells in the epithelium of the crypt bases and a scattering of small lymphocytes in the lamina propria.

GASTROINTESTINAL NEUROMUSCULAR / MOTILITY DISEASES

Introduction

• Encompass a range of conditions

can occur anywhere throughout the luminal gastrointestinal tract

• Requires multidisciplinary approach

integration of molecular and cell biology, physiology, and clinical observations

• Smooth muscle cells, enteric neurons, interstitial cells of Cajal, hormones, gut–brain pathways, and the central nervous system continuously interact with each other

may all contribute to disturbance of gut motility

• International Working Group on Gastrointestinal Neuromuscular Diseases recommendations for investigation of these disorders (Knowles et al 2009)

advises on handling of tissue samples

suggests investigations to be carried out

– in local centers

– in centers with a special interest and expertise

• In general, gastrointestinal motility disorders are due to pathology affecting

enteric nervous system (ENS)

enteric smooth muscle

congenital absence or malformation of enteric ganglion cells or smooth muscle

inflammatory processes which may be autoimmune

degenerative processes resulting in fibrosis

• In approximately one-third of cases no morphological abnormality is detected

Investigation of intestinal motility disorders

• Apart from Hirschsprung’s disease (where the diagnosis is made on rectal suction biopsy) full thickness intestinal biopsy is recommended

specimen should be received fresh in the laboratory

– full thickness sample should be frozen

– sample taken for electron microscopy

– sample for tissue bank if possible

ACHALASIA OF ESOPHAGUS

• Poorly relaxing lower esophageal sphincter

functional obstruction

• Most cases idiopathic

• Chagas’ disease most frequent etiology in Brazil

after acute infestation with Trypanosoma cruzi, some develop chronic megacolon and mega-esophagus

HYPERTROPHIC PYLORIC STENOSIS

• Affects infants

• Pyloric portion of the stomach becomes abnormally thickened

• Manifests as gastric outlet obstruction

Genetics

• Paradigm for the sex-modified model of multifactorial inheritance

males four times more frequent than females

uncommon in black infants

Clinical features

• Progressive projectile non-bilious vomiting in first few months of life

• Treated with pyloromyotomy

Histopathological features

• Few pathological descriptions (usually treated with pyloromyotomy only)

• Morphologically unremarkable smooth muscle

• Possible absence of sarcoglycans within hypertrophied pyloric muscle (Romeo et al 2007)

HIRSCHSPRUNG’S DISEASE (HSCR)

• Developmental disorder with absence of ganglion cells in the distal rectum and variable length of contiguous bowel

• Due to failure of neural crest cells to migrate, proliferate and/or differentiate during development of the enteric nervous system (ENS)

Genetics

• Genetically heterogeneous

• 1 in 5000 births

• Multifactorial, non-Mendelian pattern of inheritance

several genes playing role in pathogenesis

at least 9 known susceptibility genes

• Affected families carry 200 times higher risk

• Genetic counseling via pedigree analysis difficult

• Significance of genetic variations unclear

• To date, 10 susceptibility genes and five loci

main susceptibility gene is RET gene on chromosome 10

– loss-of-function mutations, haploinsufficiency or polymorphisms and haplotypes of the RET promoter region

– 50% of familial cases of HSCR

– 70% of total colonic aganglionosis

– 15–20% of sporadic cases

• About 30% of cases of HSCR are syndromic

about 1% of patients with Down syndrome have HSCR

• Frequent low penetrant predisposing allele of RET gene is risk factor for HSCR phenotype in

• Shah–Waardenburg syndrome – Type 4 Waardenburg syndrome (WS4)

autosomal dominant

Sox10 gene mutation

pigmentary anomalies

mental retardation

peripheral neuropathy

Hirschsprung’s disease

• Cartilage hair hypoplasia syndrome

mutations in ribonuclease mitochondrial RNA processing (RMRP) gene

chondrodysplasia with growth failure

impaired cellular immunity

predisposition to malignancy

high incidence of Hirschsprung’s disease

• Rare association of HSCR with disorders due to mutations of L1CAM genes – acrocallosal syndrome

Histopathological features

• Histopathological evaluation of biopsies for HSCR is one of the most fraught in pediatric pathology practice

gives rise to most litigation

meticulous examination and documentation is essential

• In the majority of cases the diagnosis is straightforward

in about 20% the diagnosis can be difficult

– these cases have many pitfalls

• Three stages in the diagnosis and treatment of HSCR in which there is histopathological involvement

primary diagnosis

intraoperative guidance of resection of the aganglionic segment

evaluation of the resected bowel

• Histopathologist may also be involved in further evaluation if the patient continues to have symptoms after the definitive surgical pull-through

Primary diagnosis on rectal suction biopsy

• HSCR diagnosed or excluded by rectal suction biopsy

• 2– 4mm portion of rectal mucosa and submucosa obtained by suction technique using specifically designed forceps

• Quality of sample depends on skill and experience of operator

• Diagnosis of HSCR depends on

absence of submucosal ganglion cells in an adequate sample

± hypertrophic submucosal nerves (diameter ≥ 40 µm)

abnormal acetylcholinesterase (AChE) staining (Figs 14.84, 14.85)

– increased numbers of coarse fibers in and across muscularis mucosae ± lamina propria

– transversely running nerve fibers in lamina propria

• Different approaches to these biopsies used in different laboratories

AChE not used in all laboratories

• The following describes the protocol in our institution (the Great Ormond Street Protocol)

in use for >20 years

highly reliable

• Relies on combination of

clinical features

H&E stained serial sections (frozen sections if sick neonate needing urgent surgery) (Figs 14.86, 14.87)

AChE staining pattern

• At least two rectal suction biopsy specimens received on saline-moistened filter paper

different levels above the dentate line (e.g. 2 cm, 3 cm, 5 cm)

if 3 specimens received, biopsy from middle level fixed in formalin, processed in paraffin and 60–150 serial sections cut

Fig 14.84 Photomicrograph of frozen section of normal rectal suction biopsy stained to display acetylcholinesterase activity demonstrating occasional fine nerves in the muscularis mucosae and lamina propria.

Fig 14.85 Photomicrograph of frozen section of rectal suction biopsy stained to display acetylcholinesterase activity demonstrating thick ‘knotty’ fibers in the muscularis mucosae and coarse fibers in the lamina propria. Note transversely running fibers in the lamina propria. The appearances are of Hirschsprung’s disease.

Fig 14.86 Photograph demonstrating 60 serial sections of paraffin-embedded rectal suction biopsy. Note orientation to display maximum submucosa (pale-staining central area of section).

Fig 14.87 Photomicrograph of adequate rectal suction biopsy demonstrating large submucosal surface area.

Fig 14.88 Photomicrograph of frozen section of rectal suction biopsy from a normal child, stained for acetylcholinesterase demonstrating fine nerve fibers around blood vessels. The presence of these fibers serves as an internal control to assess whether an acetyl cholinesterase preparation is adequate.

Urgent

• If urgent diagnosis required, specimens processed immediately

• Specimen from lowest level (distal) always cut first

• Orientated with submucosa side up, frozen rapidly on CARDICE, and serial sections cut without trimming

• Rapid H&E performed using Harris’s hematoxylin

• If submucosa not apparent on first set of frozen sections

further serial sections cut and stained with H&E

• When many sections with submucosa present

slides passed to pathologist

• Two frozen sections then cut at 12 µm and mounted on coverslips

• If Hirschsprung’s disease suspected

rapid acetylcholinesterase (AChE) stain performed (45 minutes)

• If specimen ganglionic

standard AChE stain performed later

• Report issued <1 hour from receipt of specimen

• After AChE reported

remainder of specimen fixed in 10% buffered formalin for paraffin processing

ribbon of at least 60 serial sections cut with specimen orientated in usual way and stained with H&E

Differential diagnosis and diagnostic pitfalls

Identification of ganglion cells

• With practice and experience it is possible to identify ganglion cells on the AChE preparation and to distinguish them from nerves

• However, because of the potential for error, a rule for reporting is that ganglion cells must be identified on H&E stained sections (Fig 14.89)

• Report as ‘ganglionic at the level of this biopsy’

especially if only one specimen received

allows for the possibility of short segment disease

– may become apparent if symptoms continue and further biopsies are taken at lower levels

• In the presence of large hypertrophic nerves associated with ganglion cells

possibility of intestinal ganglioneuromatosis should be suggested (see below)

CMV infected cells in the submucosa can be mistaken for ganglion cells (Dimmick, Bove 1984)

– they show surrounding inflammatory cells (Figs 14.90, 14.91)

Fig 14.89 Photomicrograph of frozen section of rectal suction biopsy demonstrating normal submucosal ganglion cells. Note cluster of six cells with basophilic cytoplasm and prominent eccentric nucleus. Most do not show a nucleolus, a feature which is typical of neonatal specimens. Note also fine fibrillary neuropil and satellite cell nuclei.

Fig 14.90 Photomicrograph of H&E stained section of rectal suction biopsy in a patient with Hirschsprung’s disease who also had cytomegalovirus infection, to demonstrate submucosal CMV-infected cells that could easily be mistaken for ganglion cells. Note surrounding lymphocytes which give a clue to the infection.

Fig 14.91 Photomicrograph of same case as shown in Fig 14.90 showing positive immunostaining for CMV.

Variability of AChE staining patterns

• Neonatal HSCR frequently does not demonstrate the classical marked increase in fibers in the lamina propria

only abnormal in the muscularis mucosae

• Rarely with HSCR (particularly in total colonic aganglionosis) there are no submucosal hypertrophic nerves and the AChE staining pattern may appear almost normal (Fig 14.95)

no ganglion cells in at least 60 serial sections of adequate biopsies

serial sections right through the block

– ‘No ganglion cells identified in x serial sections through the block of an adequate specimen. Normal AChE pattern. Repeat biopsy advised’

possibility of long segment HSCR or total colonic aganglionosis should be discussed with the surgeon

if a repeat biopsy shows the same appearance the biopsy is reported as ‘suggestive of long segment Hirschsprung’s disease or total colonic aganglionosis’

• In some patients with HSCR there is staining pattern in muscularis mucosae of a few coarse nerve fibers present only on the deep aspect of the muscularis mucosae (Fig 14.96)

reported as Hirschsprung’s disease

• Approximately 20% of patients with HSCR have at least 1mm of normal muscularis mucosae (Pacheco et al 2008)

• 10% of normal biopsies have at least one submucosal nerve >35 µm in diameter

• Intraspecimen variability in AChE patterns can usually be resolved by findings elsewhere in an adequate specimen, minimizing the need for repeat procedures

• Inflammation can also cause a mild increase in thin vertical fibers in the lamina propria

pattern is different from the coarse transversely running fibers seen in lamina propria in post-neonatal HSCR

Fig 14.95 Photomicrograph of frozen section of rectal suction biopsy from patient with long segment Hirschsprung’s disease stained for acetylcholinesterase demonstrating a mild increase in abnormal fibers on the inferior aspect of the muscularis mucosa and no increase in fibers in the lamina propria. This pattern is suggestive of long segment disease.

Fig 14.96 Photomicrograph of frozen section of rectal suction biopsy from patient with Hirschsprung’s disease stained for acetylcholinesterase demonstrating an increase in abnormal fibers on the inferior aspect of the muscularis mucosa and no increase in fibers in the lamina propria. This pattern is a variant seen in Hirschsprung’s disease.

Short segment HSCR

• Definitions vary: together with our surgeons we define short segment HSCR as HSCR in which the length of aganglionic bowel is less than 2 cm

• Diagnosis made when only the most distal rectal suction biopsy or part of the biopsy shows the appearances of HSCR

more proximal biopsies show normal ganglionic bowel with normal AChE staining

– may respond to anorectal myectomy

Ultra-short segment HSCR (see also below: achalasia of internal anal sphincter)

• Definitions vary: together with our surgeons we define this as a clinical syndrome with functional abnormality of internal anal sphincter (internal sphincter achalasia)

diagnosed on anorectal manometry

clinical features of HSCR

no detectable morphological abnormality on rectal suction biopsy

treated by dilatation of the internal anal sphincter ± internal sphincter myectomy

Hypoganglionosis

• Hypoganglionosis cannot be diagnosed on a rectal suction biopsy

• Requires ganglion cell counts along a length of bowel

Intestinal neuronal dysplasia (INDB)

• Very controversial over the past 30 years (Fig 14.97)

• Now recognized as a morphological description of a normal variant of the submucosal plexus (Bruder, Meier-Ruge et al 2007b, Meier-Ruge et al 2006)

• Finding of ‘giant ganglia’ containing more than seven nerve cells is a normal feature of biopsies in infants under the age of one year

• The only situation in which the changes of IND may be of clinical significance is proximal to an aganglionic segment in a patient with HSCR (see below)

Fig 14.97 Photomicrograph of rectal suction biopsy stained for acetylcholinesterase demonstrating an intramucosal ganglion cell. This finding is of no pathological significance. Such cells are found occasionally.

Stains used in evaluation of rectal suction biopsies in other institutions

• Many laboratories do not use AChE staining

rely on H&E staining of serial paraffin sections (Gomez et al 2009)

• In Switzerland, enzyme histochemical staining for lactic and succinic dehydrogenase and nitroxide synthase are also used (Bruder, Knecht et al 2007)

• Some groups use enzyme histochemical staining for nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase (Tomita et al 2004)

• Most may facilitate recognition of ganglion cells, but in practice add little to the H&E-based interpretation of an experienced pathologist

• Recent interest in Calretinin immunohistochemistry (Kapur et al 2009)

• Loss of calretinin immunoreactive nerves correlates spatially with aganglionosis

Reporting on frozen sections of intra-operative biopsies

• Treatment of HSCR is by surgical removal of the aganglionic segment and endorectal pull-through of ganglionic bowel

• Usually primary repair

in some cases, staged repair with formation of stoma

– closed later

• May be performed laparoscopically

• Transanal pull-through consists of rectal mucosectomy, resection of the aganglionic bowel and a colo-anal anastomosis

• Procedure is guided by intraoperative frozen section diagnosis

to determine the level at which bowel is ganglionic

• Seromuscular biopsies consisting of two layers of muscularis propria and intervening plexus submitted for urgent frozen section

level from which the biopsy was taken should be indicated

submission of appendix for identification of ganglion cells not recommended

– appendix does not always have well defined myenteric plexus

– ganglion cells can be difficult to locate in frozen section

orientation such that the two muscle layers can be easily distinguished

ribbon of serial sections is cut and stained with H&E

• Fully ganglionic specimen should contain clusters of readily identifiable ganglion cells with associated neuropil and no nerves report as (Fig 14.98)

‘Intraoperative seromuscular biopsy from (cite level): fully ganglionic’

• If ganglion cells are present but are accompanied by hypertrophic nerves, report as:

‘Intraoperative seromuscular biopsy from (cite level): transitional zone. Biopsy from more proximal level advised’ (Fig 14.99)

• If ganglion cells appear sparse (ie clusters are small or more widely spaced than usual), report as:

‘Intraoperative seromuscular biopsy from (cite level): ganglion cells present but appear reduced in number. Possible transitional zone. Biopsy from more proximal level advised’

• If ganglion cells are absent, and there are hypertrophic nerves, report as:

‘Intraoperative seromuscular biopsy from (cite level): no ganglion cells present. Hypertrophic nerves. Biopsy from more proximal level advised’ (Fig 14.100)

• If there are no ganglion cells present, but no hypertrophic nerves and the muscle layers are closely apposed, report as:

‘Intraoperative seromuscular biopsy from (cite level): no ganglion cells present. No hypertrophic nerves. Closely apposed muscle layers, suggestive of long segment disease or total colonic aganglionosis. Biopsy from more proximal level advised’ (Fig 14.101)

• Note that the aganglionic segment in total colonic aganglionosis may extend into the small intestine, sometimes for a considerable length

Fig 14.98 Photomicrograph of frozen section of intraoperative seromuscular biopsy demonstrating a normal myenteric plexus, reported as ‘Biopsy from (cite level): fully ganglionic.’

Fig 14.99 Photomicrograph of frozen section of intraoperative seromuscular biopsy demonstrating a hypertrophic nerve together with the presence of ganglionic cells. Report as ‘Biopsy from (cite level): transitional zone biopsy. Further biopsy recommended.’

Fig 14.100 Photomicrograph of frozen section of intraoperative seromuscular biopsy demonstrating a large hypertrophic nerve and no ganglion cells. Report as ‘Biopsy from (cite level): no ganglion cells present. Large hypertrophic nerve. Further biopsy recommended.’

Fig 14.101 Photomicrograph of frozen section of intraoperative seromuscular biopsy demonstrating closely apposed muscle layers and no ganglion cells. This appearance is typical of long segment or total colonic aganglionosis. Report as ‘ Biopsy from (cite level): no ganglion cells present. Closely apposed muscle layers. Further biopsy recommended.’

Differential diagnosis and diagnostic pitfalls

• Must be absolutely confident that two muscle layers can be distinguished

serosa can be mistaken for intermyenteric layer

• As a rule of thumb, ganglion cells should be of quality that could be photographed convincingly

if pathologist ‘not sure’ if something is a ganglion cell assume it is not, cut deeper levels and/or ask for another biopsy

• Beware mistaking florid neuropil for a hypertrophic nerve

nerve has wavy fibers

neuropil is fluffy and wispy

Evaluation of resected bowel

• 80% of HSCR patients have rectosigmoid aganglionosis

• 10% have long segment disease

• 5% short segment disease

• 5% total colonic aganglionosis

may involve small intestine

• Transitional zone

shows hypoganglionosis and occasional hypertrophic nerves

may be highly variable length

• Depending on the clinical circumstances, resected bowel may be received all in one piece or in multiple parts

sutures should be applied by the surgeon to indicate orientation

• Ideally the specimen should be received fresh, opened on arrival and circumferential samples of proximal margin taken for frozen sections and AChE staining

remainder pinned out and fixed in formalin

• Sampling of the proximal margin is most important

we perform circumferential sampling of the proximal end

since neuronal population may have spiral architecture, despite intraoperative sections showing ganglionic bowel at one point, may occasionally reveal transitional zone

– up to one-third with transitional zone pull through will require revisional surgery for intractable symptoms (Farrugia et al 2003)

acetylcholinesterase activity in the proximal resection margins useful indicator (Huang et al 2001)

• Further sampling of complete longitudinal full thickness strip of entire resected bowel

document lengths of

– aganglionic segment

– transitional zone

· hypoganglionosis and occasional hypertrophic nerves

– fully ganglionic bowel

• Skip segments may be present

segment of ganglionic bowel in an otherwise aganglionic segment

• Note appearance of apparent intestinal neuronal dysplasia in segment proximal to aganglionic segment

large clusters of submucosal ganglion cells

ectopic ganglion cells in the lamina propria

Procedures in other institutions

• Not all laboratories carry out circumferential sampling of proximal resection margin

• Some laboratories take representative samples along resected bowel rather than sampling entire length (Gomez et al 2009)

Assessment of biopsies from patients with failed pull-through procedure for Hirschsprung’s disease

• One of the reasons for a failed pull -through procedure for Hirschsprung’s disease is retained aganglionic segment

• Biopsies may be taken to assess whether or not the aganglionic segment has been fully excised

pathologist must be familiar with the surgical pull-through procedure which has been performed

exact site of biopsy is important

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