Indian Journal of Pathology and Microbiology
Home About us Instructions Submission Subscribe Advertise Contact e-Alerts Ahead Of Print Login 
Users Online: 530
Print this page  Email this page Bookmark this page Small font sizeDefault font sizeIncrease font size

  Table of Contents    
Year : 2014  |  Volume : 57  |  Issue : 3  |  Page : 369-375
Calretinin immunohistochemistry versus improvised rapid Acetylcholinesterase histochemistry in the evaluation of colorectal biopsies for Hirschsprung disease

1 Department of Pathology, St. John's Medical College and Hospital, St. John's National Academy of Health Sciences, Bengaluru, Karnataka, India
2 Department of Paediatric Surgery, St. John's Medical College and Hospital, St. John's National Academy of Health Sciences, Bengaluru, Karnataka, India

Click here for correspondence address and email

Date of Web Publication14-Aug-2014


Background: Acetylcholinesterase (AChE) histochemistry on rectal mucosal biopsies accurately diagnoses Hirschsprung disease (HD), but is not widely employed as it requires special tissue handling and pathologist expertise. Calretinin immunohistochemistry (IHC) has been reported to be comparable to AChE staining with the loss of expression correlating with aganglionosis. Aim: The aim was to evaluate calretinin IHC as a primary diagnostic tool in comparison to the improvised rapid AChE technique in the diagnosis of HD. Materials and Methods: A total of 74 rectal biopsies (18 fresh frozen - 18 cases, 56 formalin fixed - 33 cases) from 51 cases of suspect HD were evaluated with hematoxylin and eosin/AChE/Calretinin. Ten biopsies each from ganglionated and aganglionated segments served as positive and negative controls. Ileal (3), appendiceal (3) and ring bowel (2) biopsies were also included. Two pathologists blinded to the clinical details evaluated the histomorphology with AChE and calretinin. Observations were statistically analyzed and Cohen's k coefficient employed to assess agreement between two pathologists and calretinin and the AChE. Results: The study confirmed HD in 26 and non-HD in 25 cases. There were 7 neonates, 5 low level biopsies and 14 "inadequate" biopsies. The results of calretinin were comparable with AChE with a statistically significant measure of agreement of k = 0.973 between the two. One false-positive case of HD was noted with calretinin. The advantages and disadvantages of calretinin versus AChE are discussed. Conclusion: Calretinin is a reliable single immune marker for ruling out HD by its specific positive mucosal staining of formalin fixed rectal biopsy. The improvised AChE staining remains indispensable to confirm HD on fresh biopsies and thus, along with calretinin IHC maximizes the diagnostic accuracy of HD in difficult cases.

Keywords: Acetylcholinesterase staining, calretinin, frozen sections, Hirschsprung disease, mucosal biopsies

How to cite this article:
Yadav L, Kini U, Das K, Mohanty S, Puttegowda D. Calretinin immunohistochemistry versus improvised rapid Acetylcholinesterase histochemistry in the evaluation of colorectal biopsies for Hirschsprung disease. Indian J Pathol Microbiol 2014;57:369-75

How to cite this URL:
Yadav L, Kini U, Das K, Mohanty S, Puttegowda D. Calretinin immunohistochemistry versus improvised rapid Acetylcholinesterase histochemistry in the evaluation of colorectal biopsies for Hirschsprung disease. Indian J Pathol Microbiol [serial online] 2014 [cited 2021 Nov 27];57:369-75. Available from: https://www.ijpmonline.org/text.asp?2014/57/3/369/138717

   Introduction Top

Hirschsprung disease (HD), a congenital malformation of the enteric nervous system (ENS) characterized by aganglionosis commonly affects the rectosigmoid region and is a leading cause of neonatal large gut obstruction and childhood constipation. HD affects 1/4500-5500 live births in the West and Japan [1],[2] and 1/3000-3500 in Asiatics. [3] Though no published epidemiological data is available from India, a referral center for HD like ours that has screened over 3000 colorectal biopsies for suspect HD over 15 years currently diagnoses about 60 new cases annually.

Despite differences in the opinion across the Atlantic, acetylcholinesterase (AChE) histochemistry is considered to be a reliable method for the intra-operative frozen section diagnosis of HD and the subsequent leveling of aganglionosis. However, the laboratory facility and technical expertise for this exist in few centers across our country. We have trained pediatric surgeons and their pathologists from over 30 state and central tertiary health care centers across India over the last 4 years in annual hands-on workshops; yet only 3/30 institutions have implemented AChE histochemistry routinely for managing their patients. The optimal evaluation of multiple biopsies for diagnosis and leveling can interrupt the workflow of a surgical pathology laboratory, even with the rapid technique. These logistic problems in establishing AChE histochemistry has simultaneously fuelled our interest in immunohistochemistry (IHC) with a battery of markers such as S-100, neuron specific enolase (NSE), C-Kit, PGP 9.5, and synaptophysin. [4] However, none has proved to be a reliable single marker in clinching the diagnosis of HD on formalin fixed rectal mucosal biopsy.

Calretinin, a vitamin D dependent calcium binding protein expressed in central and peripheral nervous system, has been studied in the ENS too. Absence of calretinin expression in HD was first reported on resected specimens in 2004 [5] and subsequently on rectal mucosal biopsies. [6],[7],[8],[9],[10] In this study, we have compared calretinin IHC as a primary diagnostic tool in the diagnosis of HD with our standardized improvised rapid method of AChE histochemistry [11],[12],[13] for the 1 st time in India.

   Materials and Methods Top


This cross-sectional comparative study was conducted at the Department of Pathology of a tertiary teaching medical college hospital which serves as a referral center for the diagnosis of HD over a 3-month period.

The following were included:

  1. All rectal biopsies that were performed to diagnose HD or exclude a clinical diagnosis of HD. They were either fresh/fixed in formalin, adequate (mucosubmucosal)/inadequate (mucosa with scanty submucosa/totally replaced by lymphoid follicles/low level with anorectal junctional mucosa) and submitted for histological (hematoxylin and eosin [H and E]/histochemical (AChE) evaluation.
  2. Colorectal resection specimens obtained at definitive pull through surgery in previously confirmed cases of HD.
  3. Colonic, ileal and appendicular biopsies from suspect cases of long segment colonic Hirschsprung disease (LScHD) and total colonic aganglionosis (TCA).

Ethical clearance for this study was obtained from the Institutional Ethical Review Board.


Frozen sections were cut from fresh rectal biopsies at 10 μ thickness using Leica CM1510 cryostat at different levels. Two sections were stained with rapid H and E and two with AChE by employing our standardized modified rapid method with a staining time of 40 min. [13] A frozen section positive for HD was run as a positive control. The frozen tissue remains were preserved at –25°C for further use if necessary. Where no formalin fixed tissue was available, the frozen tissue remains were embedded in agar medium, fixed in formalin and processed for routine histopathology. In suspect LScHD/TCA, ileal biopsies and the appendix were similarly processed for AChE and thereafter fixed in formalin.

All the formalin fixed, paraffin embedded tissues were cut at 4 μ and stained with H and E. One section of full-thickness biopsies and 12 sections (two slides) of mucosal biopsies were taken. One section per slide was stained for calretinin IHC using Novolink Max Polymer Detection System and ready to use antibody (prediluted) from Dako (Clone-DAK Calret 1) with heat antigen retrieval in ethylene diamine tetra acetic acid(EDTA) buffer at pH 9.0. A full-thickness section of colon with ganglion cells was stained as a positive control while the mast cells in the mucosa were considered as an inbuilt positive control. A negative control involved the omission of the primary antibody. No AChE was attempted on formalin fixed tissue as it is not suitable for AChE enzyme studies. One section each from the ganglionic and aganglionic segment of the resected bowel at pull through was studied with H and E and calretinin staining. The sections thus stained (H and E, AChE and calretinin) were studied independently by two reporting pathologists who were blinded to the clinical details. A diagnosis of HD or non-Hirschsprung disease (NHD) was made after a consensus between the histopathologists and correlation with patient clinical details. The following diagnostic criteria were employed.

Diagnostic criteria for NHD in a rectal mucosal biopsy:

  • At least one ganglion cell is identified in one or more tissue sections and/or
  • The AChE stains an occasional nerve twig in the submucosa and highlights a ganglion cell on frozen section of fresh biopsy. However, no stainable AChE fibers are identifiable (negative staining) in the muscularis mucosa (MM) and lamina propria (LP) and/or
  • The calretinin immunostain shows distinct linear granular black fibers in the MM and LP, extending upwards to a variable distance in between the crypts (positive staining) and stains ganglion cells (both nuclear and cytoplasmic) in the submucosa if included in the biopsy ( formalin fixed) [Figure 1]a.
Figure 1: (a) Positive calretinin immunostain in a case of non-Hirschsprung disease showing ganglion cells (both nuclear and cytoplasmic staining) in the submucosa and positive fibers in the muscularis mucosa and lamina propria (×400), (b) positive Acetylcholinesterase (AChE) patt ern A in rectal mucosa of a case of
Hirschsprung disease. Note the tree like branching patt ern of AChE positive nerve fibers in the submucosa and in between the crypts in the lamina propria (×200)

Click here to view

Diagnostic criteria for HD in a rectal mucosal biopsy:

  • Absent ganglion cells, presence of hypertrophic nerve bundles in the submucosa and
  • Increased AChE activity with positive staining of hypertrophic nerve fibers as dark green- black staining in specific patterns (pattern A - nerve fibers in the submucosa extending through the MM into the LP akin to an arborizing tree trunk, pattern B - nerve fibers extending only up to the base of the crypts, equivocal pattern-hypertrophic nerve bundles in the submucosa alone with no specific pattern in the LP) [Figure 1]b on frozen section of fresh biopsy and /or
  • With calretinin IHC, the biopsy ( formalin fixed) demonstrates neither ganglion cells nor any stainable fibers in the MM and LP (negative staining).

The collated data were analyzed using SPSS version 16 (SPSS Inc, Chicago, IL) and P < 0.05 was considered as significant. Mean and standard deviation were obtained for continuous variables while Cohen's κ coefficient was used to assess agreement between calretinin and the AChE as read by two pathologists.

   Results Top

The test group comprised of 74 paraffin blocks from 51 suspect HD cases studied in addition to three ileal biopsies, three appendices and two circumferential full-thickness ring bowel biopsies (doughnuts) over 3 months. These included 18 frozen remains fixed in formalin from 18 fresh rectal mucosal biopsies after being processed for AChE (18 cases) and 56 biopsies in formalin (33 cases). Seven biopsies were from neonates (2 fresh, 5 in formalin). The study confirmed HD in 26 cases (22 rectosigmoid HD, 1 LScHD and 3 TCA) and NHD in 25.

The control group comprised of full-thickness formalin fixed rectal biopsies from ganglionic and aganglionic segments of 10 pull through segments which formed the negative and positive control group, respectively. The demographic data of the two groups are compared in [Table 1].
Table 1: Demographic data of 51 cases in the study

Click here to view

Control group

The H and E sections showed ganglion cell clusters placed at regular intervals in the submucosal and myenteric plexus and no hypertrophic nerve bundles [Figure 2]a. The calretinin stain showed distinct intensely granular positive fibers in the MM and mucosa between the crypts, reaching up to the surface lining at foci. Calretinin stained both the nucleus and cytoplasm of ganglion cell clusters along with the extrinsic nerve fibers in the serosa. The positively stained mast cells were seen scattered in all layers of the bowel, but predominantly in the LP.
Figure 2: Non-Hirschsprung disease: Normally innervated rectal mucosa (a) showing no increase in Acetylcholinesterase activity (b), and calretinin staining positive fibers in the mucosa and muscularis mucosa (c) (×200)

Click here to view

Test group

The findings in the 74 paraffin blocks from rectal biopsies (18 fresh, 56 formalin fixed) from 51 cases and their eight appendicular/ileal/doughnut biopsies received are discussed separately as follows.

The fresh and formalin fixed rectal biopsies [Figure 2]a-c and [Figure 3]a-c are described separately below.
Figure 3: Hirschsprung disease: Rectal mucosa (a) showing hypertrophic nerve bundle in the submucosa, increase in Acetylcholinesterase activity (b) of pattern A and negative staining with calretinin (c) note the negative staining of hypertrophic nerve fiber (arrow) with calretinin

Click here to view

Fresh rectal biopsies

Eighteen fresh rectal biopsies (3 full-thicknesses, 15 mucosal) from 18 cases (2 neonates) were studied.

H and E stain

Of 18 fresh rectal biopsies (3 full-thickness, 15 mucosal), 7 were conclusively diagnosed (3/3 full-thickness biopsies - HD [Figure 3]a, 4/15 mucosal biopsies - NHD). No diagnoses was possible in 11/15 mucosal biopsies (7-inadequate, 4-low level mucosal biopsies) [Table 2].
Table 2: Analysis of fresh rectal biopsies from 18 cases of suspect HD

Click here to view

AChE stain

AChE confirmed HD in 6/18 cases - 3 full-thickness, 3/11 mucosal where diagnosis was not possible with H and E (inadequate-2/7, low level-1/4) [Table 2] and [Table 3]. Of the six, two were neonates with an equivocal pattern and pattern A [Figure 3]b in one each. NHD with an equivocal pattern [Figure 2]b was proved in 11/18 cases; of these 3/11 were 'adequate' mucosal on H and E and 8/11 where no opinion was possible on H and E (5/7-inadequate biopsy, 3/4-low level biopsies). An 8 year old with onset of constipation since infancy was diagnosed as refractory constipation as the mucosal biopsy showed ganglion cells as well as the increased AChE activity in the mucosa with pattern B.
Table 3: Fresh biopsies: Comparison of sensitivity, specificity, predictive values (positive and negative) and accuracy of calretinin versus AChE in readings by the two pathologists

Click here to view

Calretinin stain

The calretinin staining showed positive staining [Figure 2]c in all the 12/18 biopsies diagnosed as NHD by AChE including the three low level biopsies and one of refractory constipation. Six biopsies with no stainable fibers [Figure 3]c (reported by the first pathologist) correlated with increased AChE of HD. Thus, the concordance of the calretinin readings with AChE was excellent with the first pathologist (sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and accuracy being 100%). Though the results obtained from the second pathologist were largely agreeing with the first, there was one false-positive diagnosis of HD where the minute, thin fibrillar fibers with calretinin were missed because of a lymphoid follicle obscuring the fibers. The sensitivity, specificity, PPV, NPV and accuracy are tabulated in [Table 3]. However, the hypertrophic nerve bundles seen in biopsies with HD were not stained with calretinin.

Formalin fixed biopsies

Fifty-six rectal biopsies (43 full-thicknesses, 13 mucosal) from 33cases (5 neonates) were studied and the results shown in [Table 4]. At times, there was more than one biopsy per case for evaluation with an average of 1.5 biopsies/case. Twenty cases were diagnosed as HD and 13 as NHD. One was a low level biopsy.
Table 4: Analysis of formalin fixed rectal biopsies from 33 cases of suspected HD

Click here to view

The full-thickness biopsies and mucosal biopsies are described separately below.

Full-thickness biopsies

Hematoxylin and eosin and calretinin stain: A definite diagnosis of HD was made on 26 biopsies (12 cases) and of NHD on 17 biopsies (8 cases, including 5 neonates). There was 100% concordance between the two pathologists in every instance.

Mucosal biopsies

H and E stain

On the basis of findings on the H and E, the 13 biopsies (13 cases) were classified as follows-NHD (1); suspect HD with hypertrophic nerves (4) or where diagnosis was not possible (8-7 inadequate and 1 low level biopsy).


All 13 were classified conclusively as HD (8) or NHD (5). The HD included 4 suspected on H and E and 4/7 labeled inadequate on H and E; these showed no expression. The 5 NHD cases which showed expression for calretinin included the remaining 3/7 inadequate on H and E and the single low level biopsy.

Biopsies from Ileum (3), appendix and doughnut (3):

Calretinin expression was noted in all three ileal biopsies and 1/3 appendices from suspect cases of LScHD indicating normal ganglion cell distribution. In the second appendix, ganglion cells were highlighted but no calretinin positive fibers were evident in the area of necrosis. The third appendix showed neither fibers nor ganglion cells and the AChE stain showed no ganglion cells, hence diagnostic of TCA. Interestingly, an earlier rectal biopsy of this case had showed increased AChE activity and no calretinin positive fibers, hence diagnosed as aganglionosis.

Two doughnuts from the left transverse colon assessed for circumferential innervation prior to a definitive pull through showed no increase in AChE activity, but few distinct calretinin stained fibers indicating the irregular leading ganglionated edge.

The above histologic and histochemistry findings of 74 rectal biopsies were statistically analyzed [Table 5] and [Table 6] and found to be significant with a measure of agreement of Kappa between the two pathologists being 0.973 and between calretinin and AChE with P < 0.001.
Table 5: Comparison of pathologists' readings on 74 rectal samples based on calretinin staining

Click here to view
Table 6: Cross tabulation of findings obtained by the two pathologists on 74 rectal biopsies suspected of HD

Click here to view

   Discussion Top

A conclusive histopathologic diagnosis of HD on rectal biopsy is a primary requisite before planning further patient management. Conventionally, in staged procedures for HD, mucosal biopsies are preferred over full-thickness ones as they result in minimal posterior rectal fibrosis and the subsequent pull-through is technically easier. [14] However, unlike full-thickness biopsies where plexuses are available for a detailed study, reporting on the minute mucosal biopsies is challenging as ganglion cells/submucosal neural plexus may not get sampled. Hence, H and E stained mucosal biopsies must be supplemented with auxiliary stains such as AChE which demonstrate typical histochemical patterns to clinch the diagnosis of HD on the frozen biopsies. Despite minor disagreements among experts, AChE staining is considered to be a reliable diagnostic modality for HD with 100% specificity and 85% sensitivity. [15] Over the last two decades, investigators have described and compared the role of various immunohistochemical markers (e.g., PGP 9.5, S-100, NSE, GLUT-1, glial fibrillary acidic protein, Synaptophysin, MAP-5, CD 56 and Bcl 2 ) to AChE stain, yet none have shown any significant advantages over the latter. [6],[16]

When a ganglion cell is not identified in a H and E stained rectal mucosal biopsy because of scanty submucosa, a pathologist may commit a diagnosis of HD even when hypertrophic nerve fibers are not evident. Such an error is avoided when a concurrent AChE staining is available on the frozen section. Here, a definite diagnosis can be made on a single section by demonstrating specific patterns of increased AChE activity. [11] On the other hand, if the biopsy is fixed in formalin, AChE staining cannot be performed and the absence of this robust diagnostic corroboration substantially limits the diagnostic workup in suspect HD. In this situation, one is required to examine 60-80 serial sections to confirm aganglionosis. This laborious exercise based on a negative finding is both time consuming and fraught with uncertainty. We faced this dilemma often when mucosal biopsies were received in formalin and have felt the need for a marker that could be help identify ganglion cells/nerve patterns in formalin fixed mucosal biopsies with certainty. This study aims to evaluate the feasibility and utility of calretinin as an immune marker in the diagnosis of HD and compares it with the current standard-AChE.

Calretinin, a 29 kD calcium binding protein is expressed primarily in neurons of central and peripheral nervous system and as a rich network in the ENS. This protein is involved in the transport of calcium, and when stained, is seen as thin fibrillary network in the mucosa as an indirect evidence of ganglionosis. [17],[18] The distinct granular network of fibers is seen in the MM and between the crypts mimicking the tree trunk mucosal pattern of staining of AChE in HD. Due to excess neuro-excitability and neuro-exhaustibility in HD, there is excess cytoplasmic calcium with loss of calretinin [19] and hence no stainable fibers with IHC. This is reflected as a "silent mucosa" in rectal biopsies of HD. Thus, negative staining with calretinin indicates HD and positive staining for calretinin indicates normal innervation. In other words, normal innervation is AChE negative and calretinin positive, while HD is AChE positive and calretinin negative.

In this comparative study, the results of calretinin IHC were comparable with rapid AChE except for one false-positive case (one pathologist) and fewer or no equivocal findings. Considering that both the pathologists have been routinely reporting AChE and H and E stained rectal biopsies for the last 10-14 years and had not reported calretinin on rectal mucosal biopsies earlier, the results are impressive. Their assessment was strictly based on the controls (full-thickness biopsies from HD) and individual positive and negative controls commercially provided with every batch of IHC staining.

We highlight certain advantages of calretinin IHC over AChE in the diagnosis of HD.

  1. Frozen section facility: AChE histochemistry being a very specialized technical stain imposes a tangible burden on pathology laboratories as it is used only for diagnosis of HD. It requires technical expertise and cryostat facilities. Though our staining technique is modified to cater to a general pathology laboratory of a developing country, [11] it adds a significant work load. In contrast, Calretinin is available as one of the diagnostic markers in most laboratories with IHC facility.
  2. Fresh tissue: Calretinin IHC can be performed on formalin fixed paraffin embedded mucosal biopsy that has been used earlier for conventional H and E/tissue remains after frozen H and E/AChE staining. Unlike rapid AChE, it does not require fresh and frozen tissue.
  3. Adjunct to AChE staining: Since calretinin IHC can be performed on the paraffin sections of frozen remains after AChE on fresh tissue, an occasional difficulty in the interpretation with AChE can be resolved by a consensus opinion of the information obtained from all three stains - H and E, AChE and calretinin on a single fresh mucosal biopsy, provided the biopsy is handled with care.
  4. Age: Calretinin stains well across all ages. Unlike the occasional problems with AChE in neonates and premature subjects, the distinct positive staining with calretinin is consistently noted in all. Some neonates with HD exhibit an "equivocal pattern" of increased AChE activity; this delicate pattern may be easily missed by the reporting pathologist unless one is aware of the entity and has an eye for it. A silent mucosa with no stainable fibers with both AChE and calretinin in a neonate is diagnostic of HD.
  5. Depth of biopsy: While mucosa with MM is adequate for calretinin immunostain, except for AChE - pattern A where the superficial mucosa may be adequate for characterization, the interpretation of other patterns (pattern B, equivocal pattern) requires the presence of submucosa in the biopsy.
  6. Level of biopsy: Calretinin stained satisfactorily even on low level biopsies and provided the diagnostic information in four of our cases. Importantly, this may obviate the need for a repeat biopsy with the conventional low level biopsies when one reports only on H and E stained section.
  7. Long segment disease: The increased AChE activity with typical patterns of staining in HD is restricted to the mucosa distal to the splenic flexure; it dies down proximally in the aganglionic segment in LScHD/TCA as also in the appendix in TCA. [20] In contrast, we have noted calretinin positive fibers in the entire length of colon, appendix and small intestine when they are ganglionic. This unique finding has not been highlighted in literature before and confers a decisive advantage in evaluating the colon/appendix in LScHD and TCA.
  8. Calretinin is a great help to identify quadrants of abnormal innervation in doughnuts when they show no fibers and thus, prove them to be from transition zone. This is definitely advantageous over AChE where the latter fails to stain tissues when they are from proximal colon and ileum as shown in our case.
  9. AChE staining requires the mixing of reagents that are toxic to human body [12] while calretinin involves minimal or no handling of toxic material.

However, there are certain disadvantages with calretenin IHC too:

  1. Processing time: Since calretinin needs heat antigen retrieval for immunostaining, the formalin fixation and processing of the tissue requires a minimum of 24 hours. Unlike AChE stain, it cannot be employed on fresh tissues for intra-operative diagnosis of aganglionosis on rectal biopsy or subsequent leveling to guide a single stage pull through. AChE staining currently requires approximately 40 min for staining a frozen section on the rectal mucosa.
  2. Need for stringent control: As the negative calretinin immunostaining is a positive (diagnostic) finding in HD, it is imperative that a well-standardized and validated IHC staining protocols must be in place in order to differentiate from negative calretinin results from technical errors. This is partially overcome by running a ganglionated biopsy as a concurrent positive control in the batch as well as identifying staining of the mast cells (inbuilt positive control) in the test section.
  3. Viability of biopsy: Viability of tissue is a prerequisite to study calretinin fibers. This is illustrated by the false-negative staining of calretinin in the mucosa of ganglionic bowel noted with early ischemic necrosis. This is in contrast to AChE, which highlights fibers in aganglionosis even when mucosa shows autolytic changes at morphology.
  4. Principle of staining: The aim of AChE staining is to highlight the increased parasympathetic activity seen in HD due to the absence of ganglion cells. However, calretinin staining is not based on enzyme pathology or on the primary pathogenetic mechanism, but aims at highlighting the normal calcium ion transport process in nerve processes when ganglion cells are present. It does not stain the submucosal hypertrophic nerve bundles seen in HD. However, calretinin is found to stain faintly nerve bundles in the submucosa in occasional cases of Hirschprung disease when tissue is in proximity to transition zone. [7],[21]


Calretinin is a reliable single immune marker in ruling out the diagnosis of HD by positive granular staining of nerve fibers in the mucosa and submucosa of formalin fixed rectal mucosal biopsies in NHD. The improvised rapid AChE histochemistry remains indispensable to confirm HD on fresh biopsies by demonstrating increased activity in submucosa and mucosa, thus, facilitating surgical decisions based on conclusive intra-operative diagnosis. Calretinin staining is technically less demanding and easier to interpret than AChE. Like AChE, it also reduces the necessity to study multiple serial sections for "ganglion cells" in minute rectal biopsies by routine H and E staining. In addition, it could obviate the need for repeat biopsies in low level or "superficial" rectal biopsies. Employing both stains - one positive (AChE) and one negative (calretinin) could, thus, maximize the accuracy of diagnosis in HD.

   Acknowledgments Top

The authors thank Mainak Deb, Ashley D'Cruz, S. Ramesh, Anand Alladi and Santosh Kurbet and their respective Departments of Pediatric Surgery for clinical material; Sudhir Krishna and Sweta Srivastava for help and guidance; technical staff of Department of Pathology, St. John's Medical College for laboratory assistance and Dr. Nachiket Shankar for statistical assistance.

   References Top

Goldberg EL. An epidemiological study of Hirschsprung's disease. Int J Epidemiol 1984;13:479-85.  Back to cited text no. 1
Suita S, Taguchi T, Ieiri S, Nakatsuji T. Hirschsprung's disease in Japan: Analysis of 3852 patients based on a nationwide survey in 30 years. J Pediatr Surg 2005;40:197-201.  Back to cited text no. 2
Amiel J, Lyonnet S. Hirschsprung disease, associated syndromes, and genetics: A review. J Med Genet 2001;38:729-39.  Back to cited text no. 3
Kini U, Babu MK, Yadav L, Mohanty S, Divya P, Saleem KM, et al. Role of syanptophysin in leveling circumferential full thickness (doughnut) bowel. Indian J Pathol Microbiol 2012;55:s10.  Back to cited text no. 4
Barshack I, Fridman E, Goldberg I, Chowers Y, Kopolovic J. The loss of calretinin expression indicates aganglionosis in Hirschsprung's disease. J Clin Pathol 2004;57:712-6.  Back to cited text no. 5
Kapur RP, Reed RC, Finn LS, Patterson K, Johanson J, Rutledge JC. Calretinin immunohistochemistry versus acetylcholinesterase histochemistry in the evaluation of suction rectal biopsies for Hirschsprung Disease. Pediatr Dev Pathol 2009;12:6-15.  Back to cited text no. 6
Guinard-Samuel V, Bonnard A, De Lagausie P, Philippe-Chomette P, Alberti C, El Ghoneimi A, et al. Calretinin immunohistochemistry: A simple and efficient tool to diagnose Hirschsprung disease. Mod Pathol 2009;22:1379-84.  Back to cited text no. 7
de Arruda Lourenção PL, Takegawa BK, Ortolan EV, Terra SA, Rodrigues MA. A useful panel for the diagnosis of Hirschsprung disease in rectal biopsies: Calretinin immunostaining and acetylcholinesterase histochesmistry. Ann Diagn Pathol 2013;17:352-6.  Back to cited text no. 8
Holland SK, Ramalingam P, Podolsky RH, Reid-Nicholson MD, Lee JR. Calretinin immunostaining as an adjunct in the diagnosis of Hirschsprung disease. Ann Diagn Pathol 2011;15:323-8.  Back to cited text no. 9
Gonzalo DH, Plesec T. Hirschsprung disease and use of calretinin in inadequate rectal suction biopsies. Arch Pathol Lab Med 2013;137:1099-102.  Back to cited text no. 10
Babu MK, Kini U, Das K, Alladi A, D'Cruz AJ. A modified technique for the diagnosis of Hirschsprung disease from rectal biopsies. Natl Med J India 2003;16:245-8.  Back to cited text no. 11
Kobayashi H, Miyahara K, Kusafuka J, Yamataka A, Lane GJ, Sueyoshi N, et al. A new rapid acetylcholinesterase staining kit for diagnosing Hirschsprung's disease. Pediatr Surg Int 2007;23:505-8.  Back to cited text no. 12
Kini U, Das K, Babu MK, Mohanty S, Divya P, Saleem KM. Role of rapid modified acetylcholinesterase histochemistry in the diagnosis of Hirschsprung disease. Indian J Pathol Microbiol 2010;53:s127.  Back to cited text no. 13
Swenson O, Sherman JO, Fisher JH, Cohen E. The treatment and postoperative complications of congenital megacolon: A 25 year followup. Ann Surg 1975;182:266-73.  Back to cited text no. 14
Martucciello G, Pini Prato A, Puri P, Holschneider AM, Meier-Ruge W, Jasonni V, et al. Controversies concerning diagnostic guidelines for anomalies of the enteric nervous system: A report from the fourth International Symposium on Hirschsprung's disease and related neurocristopathies. J Pediatr Surg 2005;40:1527-31.  Back to cited text no. 15
Kapur RP. Can we stop looking? Immunohistochemistry and the diagnosis of Hirschsprung disease. Am J Clin Pathol 2006;126:9-12.  Back to cited text no. 16
Baimbridge KG, Celio MR, Rogers JH. Calcium-binding proteins in the nervous system. Trends Neurosci 1992;15:303-8.  Back to cited text no. 17
Weidmann S, Schrödl F, Neuhuber W, Brehmer A. Quantitative estimation of putative primary afferent neurons in the myenteric plexus of human small intestine. Histochem Cell Biol 2007;128:399-407.  Back to cited text no. 18
Jande SS, Maler L, Lawson DE. Immunocytochemical mapping of vitamin D-dependent calcium binding proteins in brain. Nature 1981;294:765-7.  Back to cited text no. 19
Meier-Ruge W, Bruder E. Disturbed peristalsis of the gut. Immaturity of the enteric nervous system. Pathobiology 2005;72:3-6.  Back to cited text no. 20
Volpe A, Alaggio R, Midrio P, Iaria L, Gamba P. Calretinin, ß-tubulin immunohistochemistry, and submucosal nerve trunks morphology in Hirschsprung disease: Possible applications in clinical practice. J Pediatr Gastroenterol Nutr 2013;57:780-7.  Back to cited text no. 21

Correspondence Address:
Usha Kini
Department of Pathology, St. John's Medical College, St. John's National Academy of Health Sciences, Bengaluru - 560 034, Karnataka
Login to access the Email id

Source of Support: St. John’s Research Society, Bangalore and National Centre for Biological Sciences, Bangalore, Conflict of Interest: None

DOI: 10.4103/0377-4929.138717

Rights and Permissions


  [Figure 1], [Figure 2], [Figure 3]

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]

This article has been cited by
1 To assess calretinin immunohistochemistry in Hirschsprung’s disease and allied disorders
Sharada Rane, Pooja U Patil, Dasmit Singh Khokar
Indian Journal of Pathology and Oncology. 2020; 7(3): 408
[Pubmed] | [DOI]
2 Comparison of Haematoxylin and Eosin Staining and Calretinin Immunohistochemistry in Clinically Suspected Hirschsprung Disease to Evaluate the Diagnostic Utility of Calretinin in Hirschsprung Disease
Keerthi C.P, Prema N.S
Journal of Evolution of Medical and Dental Sciences. 2019; 8(49): 3702
[Pubmed] | [DOI]
3 Hirschsprung Disease — Current Diagnosis and Management
Kanishka Das,Suravi Mohanty
The Indian Journal of Pediatrics. 2017; 84(8): 618
[Pubmed] | [DOI]
4 Appendicular Biopsy in Total Colonic Aganglionosis
Suravi Mohanty,Usha Kini,Kanishka Das,Divya Puttegowda,Lokendra Yadav,Manjally Kunjipapu Babu,Kiran Mahadevappa,Prasanna Kumar,Shubha Attibele Mahadevaiah,Mainak Deb
Pediatric and Developmental Pathology. 2017; 20(4): 277
[Pubmed] | [DOI]
5 Rectal biopsy for Hirschsprung’s disease: a review of techniques, pathology, and complications
Eleanor Dorothy Muise,Robert Anthony Cowles
World Journal of Pediatrics. 2016; 12(2): 135
[Pubmed] | [DOI]
6 Diagnostic value of rectal suction biopsies using calretinin immunohistochemical staining in Hirschsprungæs disease
Viet Quoc Tran,Kim Thien Lam,Dinh Quang Truong,Minh Hoang Dang,Thao Thi Phuong Doan,Valerie Segers,Marilyn West Butler,Annie Robert,Philippe Goyens,Henri Steyaert
Journal of Pediatric Surgery. 2016; 51(12): 2005
[Pubmed] | [DOI]
7 How Reliable Are Diagnostic Methods of Hirschsprung Disease?
Hanbaro Kim,Dae Yeon Kim,Seong Chul Kim,Jung-Man Namgoong,Ji-Hee Hwang
Journal of the Korean Association of Pediatric Surgeons. 2014; 20(2): 33
[Pubmed] | [DOI]


    Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
    Email Alert *
    Add to My List *
* Registration required (free)  

    Materials and Me...
    Article Figures
    Article Tables

 Article Access Statistics
    PDF Downloaded421    
    Comments [Add]    
    Cited by others 7    

Recommend this journal