- Rectal suction biopsy is used to diagnose Hirschsprung's disease by demonstrating the absence of ganglion cells in the submucosa and submucosal plexus.
- The biopsy specimen is handled carefully to avoid artifacts and is evaluated with hematoxylin and eosin staining as well as acetylcholinesterase staining to identify abnormal nerve fibers.
- Calretinin immunohistochemistry can also be used as it stains nerve fibers in normal tissue but not in biopsies from patients with Hirschsprung's disease, providing supportive evidence for the diagnosis.
- Rectal suction biopsy is used to diagnose Hirschsprung's disease by demonstrating the absence of ganglion cells in the submucosa and submucosal plexus.
- The biopsy specimen is handled carefully to avoid artifacts and is evaluated with hematoxylin and eosin staining as well as acetylcholinesterase staining to identify abnormal nerve fibers.
- Calretinin immunohistochemistry can also be used as it stains nerve fibers in normal tissue but not in biopsies from patients with Hirschsprung's disease, providing supportive evidence for the diagnosis.
- Rectal suction biopsy is used to diagnose Hirschsprung's disease by demonstrating the absence of ganglion cells in the submucosa and submucosal plexus.
- The biopsy specimen is handled carefully to avoid artifacts and is evaluated with hematoxylin and eosin staining as well as acetylcholinesterase staining to identify abnormal nerve fibers.
- Calretinin immunohistochemistry can also be used as it stains nerve fibers in normal tissue but not in biopsies from patients with Hirschsprung's disease, providing supportive evidence for the diagnosis.
- Rectal suction biopsy is used to diagnose Hirschsprung's disease by demonstrating the absence of ganglion cells in the submucosa and submucosal plexus.
- The biopsy specimen is handled carefully to avoid artifacts and is evaluated with hematoxylin and eosin staining as well as acetylcholinesterase staining to identify abnormal nerve fibers.
- Calretinin immunohistochemistry can also be used as it stains nerve fibers in normal tissue but not in biopsies from patients with Hirschsprung's disease, providing supportive evidence for the diagnosis.
submucosa plexus Hirschsprung’s disease occur in new born babies
M &moganglion enlarged colon
• congenital aganglionic megacolon V V
• absence of ganglia, aganglionosis
• between the circular and longitudinal smooth muscle junction (myenteric plexus) and in the submucosa • Due to failure of migration of primitive neuroblasts into the developing gut • marked increase in no. and thickening of non- argyrophilic nerve fibres in the submucosa and the lamina propria Hirschsprung’s disease • Diagnosis made on bx by demonstrating a lack of ganglia and the associated, unchecked proliferation of parasympathetic acetylcholinesterase-producing t nerves in the mucosa enzyme
• Acetyl cholinesterase method is used to
demonstrate the abnormal nerve fibres in the lamina propria • Identification of Hirschsprung’s is required to confirm initial diagnosis and before corrective surgery - Rectal suction biopsy Rectal biopsy handling • rectal suction biopsies has replaced full thickness biopsies • fresh tissues are wrapped by gauze moistened with normal saline and send to the lab • specimens are in a disc-like structure having a gross appearance of two concentric layers:
-> ➢a circle of mucosal tissue ➢a circle of muscularis mucosae with or without - submucosa Rectal biopsy handling :too small, immediate frozen section
• Transfer the tissue onto a dental wax plate with a
pair of fine forceps • Identify the presence of mucosa and submucosa with a dissecting microscope X contaminate w/fixative
• avoid inducing traumatic & drying artefacts
• specimen should be sufficient for both FS (enzyme histochem) & FFPE (HE, IHC) studies Rectal biopsy handling • Frozen sections: ➢to document absence of ganglion cells ➢acetylcholinesterase staining 31EE.) ganglion III (surgery cut part remore
& w/ganglion) ➢determine level of bowel transaction at surgery ↓ cutseveral from anus) pieces ( 10 am away 20130 am away
• Block freezing - "double embedding" technique
[OIIs embed ODEJS.embed &
5 paraffinsection microtome
• Cut one section (4um) for rapid HE to identify the
presence of mucosa and submucosa layers • Cut 10 µm cryostat sections for ACHE & demonstration enzyme histochem Rectal biopsy handling after than frozen block 5 • After frozen sectioning, the tissue is fixed in 10% NBF and processed • Routine HE sections (levels) are examined for the presence / absence of ganglion cells • IHC may also be used in detecting aganglionosis “Double embedding" technique • A small sheet of aluminium foil is placed onto the freezing stage of the cryostat with a blob of OCT added • When the OCT begin to solidify, the disc of rectal tissue is placed on top of the OCT • The tissue will easily fall flat and become frozen • Excess OCT is trimmed away at either ends of the frozen block using a razor blade • Turn the frozen block 90O by means of a pair of pre-cooled forceps and embed it into a new blob of OCT on a tissue chuck • The tissue is then in a plane perpendicular to the chuck & well-orientated transverse sections are easily obtained Acetylcholine Esterase (AChE) substrate of Achenzyme ↓
mix in correct order
1 X optional (inhibit pseudoAchE seducebg
brown precipitate Xferrocyanide sulphate Acetylcholine Esterase (AChE) • acetylcholine esterase hydrolyzes the substrate acetyl thiocholine to give thiocholine • thiocholine reduces the ferricyanide to ferrocyanide • ferrocyanide combines with the copper ions to form the insoluble brown granular copper ferrocyanide precipitate (Hatchett’s brown) at the site of enzyme activity Acetylcholine Esterase (AChE) • AChE stains the parasympathetic nerve fibers and trunks of fibers that increase dramatically in the lamina propria mucosa and sub mucous layer, but is not a specific marker for ganglion cell Intramuscular nerve plexus in HE staining, arrows indicate numerous ganglion cells
Normal rectal suction biopsy specimen including mucosa, muscularis mucosae and submucosa. Ganglion cells are visible with different stainings (arrowhead). a Hematoxylin and eosin staining (H&E). b Acetylcholinesterase staining (AChE). There is a minimal AChE activity in the lamina propria.
Pediatr Surg Int (2008) 24:785–792
↑ AchEactivity
Tonerrefiber thickness fibers) Chypertropic here
Hirschsprung's disease specimen including mucosa, muscularis mucosae and
submucosa. a Hematoxylin and eosin staining (H&E). Ganglion cells are absent in the submucosa. b Acetylcholinesterase staining (AChE). AChE activity is markedly increased in the muscularis mucosae and lamina propria of the mucosa. Thick nerve fibers are strongly stained in the submucosa. Pediatr Surg Int (2008) 24:785–792 Supportive evidence for the diagnosis of Hirschsprung disease submucosal are formally pale stained strongly stained
• absence of ganglion cells in both submucosal and
myenteric plexuses • presence of hypertrophic submucosal nerve fibers • presence of acetylcholinesterase positive cholinergic nerve fibers within the lamina propria (a feature not present in normal individuals) Calretinin [IHC AChEClessspecific • It has been suggested that absence of calretinin staining to be superior and more reliable marker than increased AChE staining for the diagnosis of Hirschsprung disease Calretinin • Vitamin D-dependent calcium-binding protein involved in calcium signaling • Important role in the organization and functioning of the central nervous system • Absence of calretinin expression in nerve fibers was correlated to aganglionosis Calretinin • Antibody to the calcium-binding protein calretinin stains delicate nerve fibers in the lamina propria of ganglionated rectal biopsies but not biopsies from HD • Advantages over AChE reaction – easier reproducibility and interpretation – use in paraffin-embedded tissues - autostainer can be used (more standardizedfrozen section is added
• Calretinin immunohistochemistry is an adjunct to
and not a substitute for traditional methods of examining RSB A, Calretinin immunohistochemistry highlights mucosal nerves (arrows) and a subset of superficial submucosal ganglion cells (g) in normal rectal tissue. B, Immunoreactive mucosal nerves and ganglion cells are completely CAL+ve absent in a biopsy from aganglionic rectum, although expression is present in enlarged submucosal nerves (asterisk) and mast cells (arrowhead). Acetylcholinesterase histochemistry (C and D) demonstrate sparse or ⑤hypertropic nerve absent cholinergic mucosal nerves in coarse nerve fibers biopsies from normal ganglionic rectum (C), as opposed to numerous coarse mucosal nerves (arrows in D) in aganglionic rectum
SAL-Ve Pediatric and Developmental Pathology 2020, Vol. 23(1) 8–22 if ganglion cell -> report if ganglion cell requestdeepcut
