Whereas a contrast enema may show findings suggestive of HSCR, a normal contrast enema is not sufficient to formally exclude the diagnosis of HSCR. In patients with a normal contrast enema but strong clinical suspicion of HSCR, the diagnosis will be confirmed or excluded using a rectal biopsy.

Although HSCR is a congenital condition, it can be suspected in patients beyond infancy, and even in adults who have severe chronic constipation, although such late diagnosis remains rare114. A careful history and physical examination should look for delayed meconium passage, failure to thrive, abdominal distension and dependence on enemas. A plain abdominal radiography would show extensive faecal impaction and a contrast enema may reveal massive colonic distension and a transition zone.

Anorectal manometry can be used to assess the reflex relaxation of the internal anal sphincter in response to rectal distension, typically observed in the normal population115. The presence of rectoanal inhibitory reflex (RAIR) essentially rules out HSCR116. However, the absence of RAIR is not enough to diagnose HSCR as it has a positive predictive value of ~74%117. In settings with limited resources, diagnosis of HSCR is primarily based on history, and physical and radiological findings, as expertise in histopathology may not be available118.

Rectal biopsy.The definitive diagnosis of HSCRis typicallyestablished via suction or incisional rectal biopsy6,119,120. Suction biopsy involves using a specialized instrument to capture a small piece of mucosa attached to submucosa, measuring 3 mm2. In young infants, this procedure can be performed without anaesthesia121,122. The instrument’s barrel is inserted through the anus to a measured distance of at least 2.5 cm from the anal verge (where non-keratinized squamous mucosa in the anal canal meets the skin). In routine practice, suction biopsies are obtained for evaluation at multiple levels, such as 2 cm, 3 cm and 4 cm above the anal verge, which provides spatial data enabling the diagnosis of a very short segment of aganglionosis120,123,124.

Incisional biopsy requires general anaesthesia and is performed usually 1 cm above the dentate line, with direct visualization of the anorectal canal and the rectum. Incisional biopsy provides a larger tissue sample, ideally including the myenteric plexus, and is the preferred option in patients older than 2 or 3 years in some medical centres125,126.

The main objective of a rectal biopsy is to obtain a sufficient sample of submucosa, possibly including the myenteric plexus, for histological evaluation and identification of aganglionosis features. Consistent practices for the pathological study of rectal biopsies in HSCR is essential but practices can vary substantially across centres. Effective coordination between the surgeon, pathologist and the laboratory is crucial to ensure proper handling of specimens127.

Histopathological evaluation.If ganglion cells are observed in a distal rectal biopsy, a diagnosis of HSCR can be almost completely ruled out. On the other hand, if ganglion cells are not identified, the adequacy of a rectal biopsy for diagnosing HSCR is determined by both appropriate mucosa and sufficient submucosa. The surface of the biopsy specimen should be covered by colonic mucosa rather than entirely or partially by anal canal mucosa (squamous or transitional). The biopsy is deemed inadequate if it exhibits anal canal mucosa as physiological hypoganglionosis is observed in the most distal rectum and anal canal128–130. A suction biopsy sample should ideally measure 2–3 mm in its largest dimension and contain at least 50% of the submucosal layer for diagnosing HSCR. Extensive sampling is necessary and tissue sectioning is performed routinely to exclude the presence of ganglion cells in the

submucosa131,132. Depending on the initial findings, additional sections may be necessary to reach a conclusive diagnosis.

The hallmark histological features of HSCR are the absence of ganglion cells in the submucosa and the presence of evident submucosal nerve hypertrophy (Fig. 4). In neonates, the diagnostic criterion fornervehypertrophyisthepresenceofsubmucosalnerveswithadiameter of >40 µm. Similar to peripheral non-enteric nerves, these hypertrophied nerve fibres express GLUT1 in the perineurium133. However, encountering nerves with a diameter of >40 µm, including some with GLUT1-positive perineurium is considered normal in children older than 1 year. In patients with total colonic aganglionosis and very young patients (<3 months), nerve hypertrophy may be absent103.

Histological identification of a ganglion cell with surrounding neuropil and glia within the plexus is generally straightforward for an experienced pathologist (Fig. 4). However, it can be challenging for less experienced pathologists, particularly when assessing very young or premature infants in whom the classic cytological features of ganglion cells (such as abundant cytoplasm, eccentric round nucleus, conspicuous nucleolus) may be absent. When histological findings are ambiguous or if the biopsy sample is inadequate, immunohistochemical confirmation with markers such as PHOX2B, which specifically labels immature and mature ganglion cells, can be beneficial and facilitate an accurate diagnosis of HSCR134.

One of the historically widely used ancillary stains in HSCR diagnosticworkflowisacetylcholinesteraseenzyme(AChE)histochemistry. In the aganglionic rectum, the typically sparse AChE-positive mucosal nervesarereplacedbyanextensivenetworkofabnormalAChE-positive cholinergic fibres in the muscularis mucosae and lamina propria (Fig. 4). Although AChE histochemistry is known to be sensitive and specific, equivocal or false-negative staining may occur, particularly in very young patients or in those with total colonic aganglionosis.

Currently, calretinin immunohistochemistry has replaced AChE histochemistry as the most commonly used ancillary diagnostic technique in HSCR. Calretinin is a calcium-binding protein that is expressed by a specific subset of enteric neurons. In normal bowel, calretinin-immunoreactive fibres are consistently present in the muscularis mucosae and the lamina propria, extending from the colonic mucosa up to the transition at the anal canal135. Hence, a complete absence of calretinin-positive mucosal nerves indicates aganglionic rectal tissue, although immunoreactive fibres may still be present in the submucosa in patients with very short-segment HSCR136 (Fig. 4).

A combined approach involving histopathology and immunohistochemistry is typically sufficient to diagnose or exclude HSCR in the majority of patients. However, in situations in which the biopsies are inadequateorhistologicalandimmunohistochemicalfindingsareinconclusive, additional biopsies may be necessary to further examine the tissue and perform additional tests to reach a conclusive diagnosis.

Differential diagnosis

Othercausesofneonataldistalintestinalobstructionincludelowerilealor colonicatresia,meconiumileusresultingfromcysticfibrosis,meconium plugsecondarytomaternalgestationaldiabetes,ENSanomalies,suchas chronicintestinalpseudo-obstructionandfunctionalintestinalobstruc- tionorparalyticileusresultingfromsepsis,congenitalhypothyroidism, electrolytic imbalance or maternal intoxication110 (Table 3).

Management

The primary goals of treatment are to alleviate symptoms (such as, constipation, abdominal distension and failure to thrive), correct the

d

e

f

g

Fig. 4 | Anorectal anatomy, rectal biopsy and histopathological diagnosis of Hirschsprung disease. a, The distal rectum is lined by colonic mucosa (blue), which transitions to squamous mucosa in the anal canal at the dentate line. Ganglion cells (blue dots) are located in the rectal submucosa and myenteric plexus but become sparser within 1–2 cm above the dentate line. b, A full-thickness rectal biopsy captures the entire submucosa and myenteric

plexus and provides ample opportunity to identify ganglion cells in both areas. c, A suction biopsy retrieves only mucosa and submucosa. Blue dots in the submucosa and myenteric plexus indicate ganglion cells. d, An adequate biopsy is 2–3 mm in size and contains 50% or more submucosa (magnification ×5). e, In a normal rectal biopsy, submucosal ganglia are present with unequivocal ganglion cells (magnification ×40). In an aganglionic rectum, unequivocal hypertrophy of

submucosal nerves with perineuria (black arrows) is present (magnification ×30). f, Calretinin immunohistochemistry demonstrates positive nerve fibres within the lamina propria and muscularis mucosae (red arrows) and some ganglion cell bodiesinthesubmucosa(blackarrows)inanormalrectalbiopsy(magnification×15, ×35 for insert), whereas it shows complete absence of immunoreactive

nerves in an aganglionic rectum (magnification ×20). g, In a normal rectum, acetylcholinesterase (AChE) enzyme histochemistry shows only thick and scattered dark brown positive fibres in the muscularis mucosae and none in the lamina propria. In an aganglionic rectum, AChE enzyme histochemistry demonstrates numerous coarse cholinergic nerves in the muscularis mucosae and lamina propria (black arrows) (magnification ×15, ×40 for insert).

Part d, haematoxylin & eosin staining.

underlying bowel obstruction, and improve the overall quality of life of affected individuals. The specific treatment plan for HSCR varies depending on the individual’s age, the extent of the affected bowel and other factors. Close collaboration between the patients, families and the health-care team, including paediatric surgeons, paediatric gastroenterologists and nurses, is crucial to develop and implement a personalized treatment and management plan that best addresses the patient’s needs. Treatment for HSCR can vary globally owing to

several factors, including the availability of health-care resources, medical expertise, cultural differences and health-care system variations. Following surgery, management typically involves nutritional support when needed to ensure adequate growth and development, regular follow-up to monitor bowel function and overall health, education and support to patients and their families to help them manage the condition and make informed decisions, and psychological and emotional support to ensure long-term quality of life.