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Original Articles: Gastroenterology

Laboratory Procedures Update on Hirschsprung Disease

Takawira, Catherine; D’Agostini, Stephanie; Shenouda, Suzan*; Persad, Rabin; Sergi, Consolato*

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Journal of Pediatric Gastroenterology and Nutrition: May 2015 - Volume 60 - Issue 5 - p 598–605
doi: 10.1097/MPG.0000000000000679
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Hirschsprung disease (HD), also known as congenital megacolon, is a life-threatening disease of infancy leading to bowel obstruction, perforation, and constipation (1). The first case of HD was reported in the literature in 1691, but Harald Hirschsprung's work is accredited as the first detailed description of this intriguing disease that took place approximately 200 years later (2). HD has a prevalence of 1 in 5000 live births, with a slight male-skewed boy-to-girl ratio of 3:1 to 4:1 (1,3,4). There are 2 types of HD. Type 1, also known as short-segment HD, is more common with 60% to 85% of patients with HD. In type 1, the lack of ganglion cells (aganglionosis) is restricted to the rectum and, in continuity, to a short portion of the colon situated distally to the splenic flexure (4). Type 2, termed long-segment HD, is rarer than type 1. Type 2 occurs in 15% to 25% of all of the patients with HD. In type 2, there is an extensive aganglionosis of the rectum and colon. In the gastrointestinal tract, aganglionosis is specifically defined by the absence of the ganglion cells in the submucosal and myenteric plexuses of the bowel wall (1). In most patients, HD has been found to be the result of the failure of neural crest cells, which give rise to the enteric nervous system of the gastrointestinal tract. There is a failure to fully migrate and colonize the gut during embryogenesis. It has been theorized that this is because of genetic susceptibility factors (5). In fact, the enteric nervous system is part of the peripheral nervous system that coordinates the complex behavior of the gut (6). This crucial defect in the craniocaudal migration of neural crest cells seems to occur during the first 12 weeks of gestation (4).

HD has been characterized as a sex-linked heterogeneous disorder that has a variable pattern of inheritance. The severity of the resulting phenotype seems in part because of the more or less incomplete migration of the neural crest cells (1). In addition, it has been indicated that HD is a multifactorial disease, and several genes have been implicated with possible variable reciprocal interaction (1). Studies with animal models and studies in humans have identified more than 10 different genes and 5 chromosomal loci in HD (1). During the past decade, multiple studies have been undertaken to understand the genetic basis of HD that may result in syndromic or nonsyndromic phenotypes. In fact, the possible occurrence of HD has also been considered in the setting of genetic syndromes, for example, trisomy 21 syndrome (Down syndrome). The most important genes that have been implicated and thoroughly investigated in HD include RET, SOX10, EDN3, GDNF, and EDNRB(6). These genes are believed to be involved in the pathogenesis of HD, but it is uncertain how much of each single gene is expressed. It is also unknown whether and how extensive an epigenetic regulation plays a role in the single patient. RET, a proto-oncogene that codes for the proteins that assist cells of the neural crest in their movement, is indeed the most well-studied gene in HD and is known to be affected in the majority of nonsyndromic patients with HD. Considering that HD is a multigenic disorder, alterations of other genes such as the endothelin receptor type B may play a role (5,7). The protein derived from EDNRB is G protein–coupled receptor, which activates a phosphatidylinositol–calcium second messenger system located on the surface of cells and functions as a signaling mechanism, transmitting information from extracellular to intracellular environment. The receptor interacts with endothelins to regulate several critical biological processes, including the stimulation of cell growth and division, the production of certain hormones, and, more important, the development and function of blood vessels. Endothelin 3, which derives from the EDN3 gene, is one of the proteins that interacts with EDNRB. During early embryonic development, EDN3 and EDNRB together play probably an important role in the differentiation of neural crest cells. EDN3 and EDNRB are indeed essential for the normal formation of enteric nerves and melanocytes, which contribute to skin, hair, and eye color. Diseases associated with EDNRB include HD type 2 and EDNRB-related HD.

A study by Carter et al looking at RET variants sequenced RET to identify coding and splice site variants in a population-based case group study. The authors tested for associations between HD and common variants in RET and other candidate genes. Through this study, they confirmed associations with common variants in HOXB5 and PHOX2B. HOXB5 gene is a member of the Antp homeobox family and encodes a nuclear protein with a homeobox DNA-binding domain. The encoded protein plays a functional role as a sequence-specific transcription factor that is involved in lung and gut development. Increased expression of HOXB5 is associated with acute myeloid leukemia, bronchopulmonary sequestration, and congenital pulmonary airway malformation, formerly known as congenital cystic adenomatoid malformation. Gastroschisis has also been associated with abnormalities of HOXB5. Paired-like homeobox 2b is encoded by the PHOX2B gene. PHOX2B is expressed exclusively in the nervous system, in most nerve cells that control the visceral organs (cardiovascular, digestive, and respiratory systems), and is essential for the phenotypic differentiation of nerve cells. In particular, PHOX2B functions as a transcription factor involved in the development of several noradrenergic neuron populations and the specific determination of neurotransmitter phenotype. Typically, PHOX2B-associated diseases include adrenal neuroblastoma.

It seems that common RET variants may not substantially contribute to HD in all of the ethnic groups. It seems increasingly evident that genes regulating the proliferation, migration, and differentiation of enteric neural crest cells could be considered the “true” risk factors for HD (8). At present, more discussion is actually focused on the lack of an adequate mutational analysis. This situation has obviously limited the practical value of HD susceptibility gene screening. In patients in whom family history or clinical findings strongly suggest syndromic HD, which is rare, gene mutation screening and genome editing tools may acquire some remarkable value (5). Whole genome expression studies that have been recently conducted to identify differences in gene expression between normal and abnormal segments of bowel in patients with HD have identified new interesting pathways that may also be relevant in the pathogenesis of HD. In the future, such studies will be crucial in identifying networks between already known genes and most recently discovered genes, including RET, GDNF, NRTN, PSPN, EDNRB, EDN3, ECE1, HOXB5, SOX10, PHOX2B, KIAA1279, and TCF4(1,9,10).

HD cannot be diagnosed in utero. It is commonly diagnosed shortly after birth or within the first year of life. Suction biopsy of the distal narrow rectal segment is the common practice when it comes to diagnose HD in neonates and infants (5). Histopathological analysis of these biopsies stained with hematoxylin and eosin (H&E) and/or acetylcholinesterase (AChE) shows submucosal ganglion cells being absent from the distal rectum of patients with HD and an increase in nerve fibers in the submucosa and an increase in nervous filaments in the lamina propria. This submucosal phenotype correlates with the absence of myenteric ganglion cells (5). A number of studies have shown that the distal 1 to 2 cm of the rectum is normally hypoganglionic. Thus, a biopsy taken at 2 to 3 cm proximally to the pectinate line (toward the oral direction) is required to avoid false-positive results of aganglionosis (5). In consideration of some technical aspects that have been recently discussed in gastroenterology and pathology platforms and of the terrific utility emphasized in some centers worldwide, a specific search was performed for calretinin immunostaining. Calretinin is a vitamin D–dependent calcium-binding protein with expression in nerve fibrils and ganglion cells in the submucosa and myenteric plexus throughout the unaffected colon and bowel. Calretinin has been described as an adjunctive or primary diagnostic test in HD (11).

Here, we highlight image-based clinical techniques and laboratory procedures indicating the specificity and sensitivity for each procedure, according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines.


Stollery Pediatric Gastroenterology investigative research group aims to perform systematic reviews on laboratory procedures according to the PRISMA guidelines (12) ( The process follows an a priori established protocol and is well standardized worldwide to uniform systematic reviews. Using the clinical queries tool, a search was performed using specific search queries and limitations. The key terms “Hirschsprung disease” and “diagnosis” were searched alongside the following limitations: “English language,” “human studies,” and “full text available.” The search was refined working on the category of “Diagnosis.” This search resulted in 21 studies. After reviewing the generated list, we removed studies that did not focus specifically on HD, or studies that did not focus on the involved diagnostic investigations. This excluded 5 studies that were irrelevant to the present research, and there were 16 remaining studies that were the object of the present systematic review. A search with the words “Calretinin” and “Hirschsprung” identified initially 19 articles up to April 2014 according to the same concordant PRISMA criteria used for the other diagnostic procedures. Only articles written in English were considered (N = 17). The exclusion of general reviews/not relevant articles left only 13 articles to be considered (Fig. 1).

Selection process for papers included in the calretinin table review.

Selection Criteria

We included randomized controlled trials or observational studies (cohort and case-control design) that met the following inclusion criteria: HD and diagnosis and/or laboratory procedures. Previous systematic reviews were also counted and helped highlighting diagnostic procedures. Language was restricted to English only. Inclusion was not otherwise restricted by study size or publication type. We excluded cross-sectional studies. When there were multiple publications from the same population, we included only the data arising from the most recent comprehensive report.

Data Sources and Search Strategy

First, we conducted a systematic literature search of PubMed, Embase, Web of Science, and Scopus, from inception to April 2014. Title and abstract of studies identified in the search were examined to exclude studies that did not answer the research question of interest, based on prespecified inclusion and exclusion criteria. The full text of the remaining articles was also reviewed to determine whether it contained relevant information. Next, we manually searched the bibliographies of the selected articles on the topic for additional material, in case some articles were missing. We did not review proceedings of gastroenterological associations.

Data Abstraction and Quality Assessment

After study identification, data on study and patient characteristics, exposure and outcome assessment, potential confounding variables, and estimates of association were abstracted into a standardized form. Conflicts in data abstraction were resolved by consensus, referring back to the original article. The methodological quality of each was, however, not assessed.

Outcomes Assessed

The primary analysis focused on assessing the specificity and sensitivity of each method proposed in the literature.


Compiled sensitivities and specificities for 3 major index tests from 2 studies (13,14) comparing contrast enema, anorectal manometry, and biopsy as diagnostic investigations for the diagnosis of HD are shown in Table 1. These studies looked in particular at rectal suction biopsies as the initial index test followed by either a full-thickness biopsy or a clinical follow-up as the reference standard. In the study published in 2005 (14), participants underwent all of the 3 index studies (contrast enema, anorectal manometry, and rectal suction biopsy). Those with 2 or more positive index tests or who continued to experience severe bowel problems underwent a full-thickness biopsy as the reference standard. In the study published in 2006 (13), participants underwent at least one of the index studies (contrast enema, anorectal manometry, and rectal suction biopsy), followed by a full-thickness biopsy in all of the participants as the reference standard. Neither of these studies included jumbo biopsies. The rates of sensitivity and specificity for anorectal manometry from 4 studies (15–18) targeting solely at this technique for the diagnostic investigation of HD are shown in Table 2. Table 3 illustrates the rates of sensitivity and specificity for AChE staining from 4 studies (19–22) targeting exclusively at this technique for the diagnostic investigation of HD.

Compiled sensitivities and specificities for 3 major index tests from 2 studies(13,14) comparing contrast enema, anorectal manometry, and biopsy as diagnostic investigations for the diagnosis of Hirschsprung disease
Sensitivities and specificities for anorectal manometry from 4 studies(15–18) looking solely at this technique for the diagnostic investigation of Hirschsprung disease
Sensitivities and specificities for acetylcholinesterase staining from 4 studies(19–22) looking solely at this technique for the diagnostic investigation of Hirschsprung disease

A 10-year (2004–2014) overview of calretinin investigations on HD was also performed. The details of data have been extrapolated from the original articles and are summarized in Table 4. To the best of our knowledge, this table is the most comprehensive representation of data updating meticulously calretinin investigations for the diagnostic process of HD. No absolute congruence of results from the studies has been found, probably because of the heterogeneity of the investigations. It may be suggested that a potential rate of 0.1% of false-positive results may occur, but no uniformity has been established with regard to guidelines worldwide and these data cannot be effectively supported.

10-y Calretinin Investigations on Hirschsprung Disease, chronologically presented (2004–2014)


The most commonly used diagnostic investigations in the diagnosis of HD are contrast enema, anorectal manometry, and biopsy with histology (23). The contrast enema is often the first tool used when physicians think about HD. A positive test result is based on the finding of a change in caliber of the colon. The distal, aganglionic tissue often appears normal or small in caliber size, whereas the more proximal, ganglionic tissue is large in caliber because of the distention of the colon. The more or less sharp demarcation of the 2 zones is referred to as the aganglionic–ganglionic transition zone. It is important to note that in approximately 10% of patients, the aganglionic tissue may be extensive in size (24). In fact, it has been suggested that to ensure the correct identification of the transition zone, multiple techniques should be used. According to our compiled data in the present 2014 PRISMA systematic review, contrast enema has the lowest sensitivity and specificity of all of the 3 index investigations (Table 1). To explain false-negative findings of contrast enema, the age of patients (in neonates, probably related to gestational age at birth) and subtotal colonic aganglionosis have been suggested. Digital rectal examinations and bowel washouts can also provide false-negative findings because they can decompress the bowel (13). Rosenfield et al (25) reported some explanation for false-positive results. These authors suggested the presence of a meconium plug near the splenic flexure that can actually mimic the transition zone seen in HD (25). O’Donovan et al also pointed to low-osmolality contrast agents that can be used to reveal the characteristic features of HD. In fact, water-soluble contrast enema may be used to reduce the rates of false-negative results compared with the barium enema (26). Anorectal manometry is the next index investigation we reviewed for the diagnosis of HD. This test places a balloon into the rectum, which is then inflated with small amounts of air. There is a manometer placed at the sphincter complex to identify the rectoanal inhibitory reflex (RAIR) that leads to the relaxation of the sphincter in response to rectal distention. RAIR is based on the function of the internal sphincter and occurs when the internal anal sphincter relaxes in response to rectal distension. It is present when there is a decrease of at least 5 mmHg in the internal sphincter pressure following the rectal balloon distension (27). In HD, because of the failure of migration of the neural crest cells to the more distal portions of the bowel, the lack of parasympathetic nerve innervation results in the loss of the RAIR. Thus, defecation is impaired. Our systematic review calculated the sensitivity and specificity to be 88% and 94%, respectively (Table 2). According to Wu et al, the reported rate of false-negative results for this investigation varies from 0% to 24% and the reported rate of false-positive results varies from 0% to 62% (28). We can detect false-positive results in children with megarectum, in whom we underestimate the degree to which the balloon must be inflated, or whether there is an air leak from the balloon. False-positive and -negative results can also be seen if the catheter is not properly positioned in the sphincter complex (29).

In terms of biopsies, there are different approaches that can be taken. Rectal suction biopsy, full-thickness biopsy, and, more recently, “jumbo” forceps biopsy are a few of the techniques that can be used. Although our literature search has not clearly revealed studies comparing these different techniques, some data suggest that full-thickness biopsy or jumbo forceps biopsy may be more adequate in older children, whereas rectal suction biopsy is most efficacious in neonates and younger infants (30,31). Because there was no apparent consensus for the original articles found through our systematic review, we decided to evaluate results considering the term biopsy “not otherwise specified.” One of the most common and routinely performed staining techniques used for the detection of ganglion cells is a classic H&E staining of the tissue biopsy. Some studies compare other staining techniques with the H&E such as the studies by Karim et al (3) and Fernandez et al (31a). In this study, the authors compared the use of H&E with RET oncoprotein staining for the diagnosis of HD. RET is a proto-oncogene expressed in the developing central and peripheral nervous systems. Unlike in the cancer susceptibility syndromes, mutations in HD are inactivating. Therefore, these gene mutations lead to misfolding of the protein or failure to transport it to the cell surface. This means that only approximately half of the functioning protein we need is being produced. This dose is not sufficient for the normal development of the bowel innervation and its good functionality (31). This loss of RET expression resulting in aganglionosis can be exploited to establish the diagnosis of HD (31). In the above-mentioned study, all of the slides demonstrating ganglion cells on H&E staining were also positive for RET staining. RET staining was able to pick up 3 histological slides containing ganglion cells, which were missed by regular H&E staining. Thus, according to this study, immunoreactivity for RET staining has comparable specificity but slightly higher sensitivity than for H&E staining (3). AChE staining was the most prominent type of staining studied in the papers identified in our systematic review. Looking at Table 3, we can detect that overall the specificity of this test is reported at 98% and the sensitivity at 81%. In HD, AChE staining is enhanced, because there is often hypertrophy of the parasympathetic fibers, which are stained by the AChE. More important, occasional false-negative results and, similarly, false-positive results have been reported in neonates and in patients with total colonic aganglionosis. In fact, it has been suggested that false-negative results commonly occur in neonates because AChE-positive fibers proliferate into the lamina propria within the first few weeks of life (32). The nature of the false-negative results seen in people with total colonic aganglionosis is unclear, but an explanation may be found on embryologic grounds (33,34). When compared with nerve growth factor receptor staining, the specificities and sensitivities were found to be similar to those of AChE (35). When looking at the actual results, however, the nerve growth factor receptor staining was less prominent in each slide viewed based on a 4-tiered scale of staining. Comparing AChE with H&E staining, AChE is found to have slightly higher rates of specificity and sensitivity. On the contrary, it must be emphasized that the majority of these studies point to technical difficulties and pathologist-related interpretation (interobserver variability and, theoretically, intraindividual variability), which seem to play a significant role (17). Indeed, detecting ganglion cells can prove a challenging task for the pediatric pathologist and years of diagnostic routine may be necessary to reach a good level of confidence. This task is even tougher considering that the evaluation of histological slides deals with samples that are obtained from preterm or term newborns instead of fully developed tissue samples from older infants. In many institutions, like ours, a consensus between 2 pediatric pathologists or gastrointestinal pathologists is always provided before a diagnosis is given to either a pediatric surgeon or a pediatric gastroenterologist. In case of discordance, the histological slides should be sent outside of the department for an extra-institutional opinion. Both the “primum non nocere” dictum and the “loss aversion heuristic” need to be satisfied harmoniously by preventing harm from unnecessary surgical interventions.

Calretinin is a vitamin D–dependent calcium-binding protein with expression in nerve fibrils and ganglion cells in the submucosa and myenteric plexus throughout the unaffected colon and bowel. It has been described as an adjunctive or primary diagnostic test in HD. Calretinin staining of formalin-fixed and paraffin-embedded tissue biopsy may be supportive for the diagnosis, although false-positivity may be of some concern, as identified recently (36–48). In fact, the number of potential false-positive results may limit the increasing advocacy for calretinin staining and some concern has been raised because of the potential overtreatment.

In our 10-year overview on calretinin and HD, no 100% congruent findings have been found. Our table is probably the most comprehensive representation of meticulously updating data composed of calretinin investigations for the diagnostic process of HD. It is, however, clear that most of the studies are heterogeneous. This relies mainly on different standards of practice for HD in several institutions worldwide. In the future decades, it would be interesting to evaluate the outcome of patients with HD who are identified with molecular biology tools, specifically when such tools will become available for practical use and evidence-based medicine (49).

In conclusion, although biopsy (H&E) is the criterion standard for diagnosis of HD, contrast enema and anorectal manometry are remarkable and useful tools that can be used to aid in the diagnosis. Intraindividual and interindividual variability may be the target of future studies, particularly considering immunohistochemical markers. Calretinin may be a useful diagnostic laboratory tool, but needs to be used cautiously. Although our search did not involve the use of genetic markers to aid in the diagnosis of HD, the present review is the first PRISMA-based systematic review on laboratory diagnostic procedures of HD and may be the basis to explore future areas of research on HD.


We acknowledge the contribution of pathologists (pediatric pathologists and gastrointestinal pathologists) of Department of Laboratory Medicine and Pathology at the University of Alberta, Edmonton, Canada, who offered second opinion and good comments and suggestions in the diagnostic procedures performed for Hirschsprung disease (


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anorectal manometry; biopsy; contrast enema; diagnosis; Hirschsprung disease

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