Immunohistochemical evaluation of neuronal dysfunction in paediatric patients with Hirschsprung’s disease and allied disorder

Yashika Bhatia, Sunita Singh, Kamal Nain Rattan, Padam Parmar, Megha Ralli, Rajeev Sen


Background: Neonatal bowel obstruction may result due to defect in the intestine wall which may be classified as neuropathic, myopathic or idiopathic types according to the pathological changes observed. The present study was conducted between September 2014 to December 2015 with the aim to study histomorphological changes and evaluate the role of various IHC markers (calretinin, S-100, CD117) in Hirschsprung’s disease (HD) to assess neuronal dysfunction in these patients.

Methods: Thirty cases with clinical suspicion of HD were included in our study. The tissue sections were processed and wax blocks were prepared. Histopathological diagnosis was established on routine H and E. Representative sections were further subjected to IHC staining with calretinin, CD117 and S-100 protein. A descriptive study was carried out. Chi-square was used with P-value less than 0.05 accepted as statistically significant.

Results: Out of 30 cases with clinical suspicion of HD, 13 cases were diagnosed as HD, 10 as Non-HD motility disorder whereas 7 were without any definitive diagnosis. All the cases were subjected to IHC staining using calretinin. Out of 13 cases diagnosed as HD, 1 case showed presence of ganglion cell using calretinin. All 7 equivocal cases were accurately diagnosed by calretinin. Thus 12 cases were confirmed HD while 18 were diagnosed as Non HD motility disorder. On statistical analysis, sensitivity (92.3%) of calretinin was lower than specificity (100%). Nerve bundle hypertrophy was observed in 11 cases of HD and 9 cases of Non-HD motility disorder using S-100 as an IHC marker. CD117 was used to demonstrate altered density and distribution of ICCs was statistically significant in cases of Non-HD motility disorder.

Conclusions: IHC is being widely used as a reliable adjunctive test in evaluation of motility disorders of bowel. In view of its ease and reproducibility, it can be routinely used, avoiding need for repeated biopsies, and delay in treatment.


Calretinin, CD117, Equivocal, Hirschsprung’s disease, S100

Full Text:



Francine R, Pascale S, Aline H. Congenital anomalies: Prevalence and risk factors. Univers J Public Health. 2014;2:58-63.

Sharma R. Birth defects in India: Hidden truth, need for urgent attention. Ind J Hum Genet. 2013;19:125-9.

Saiyad SS, Jadav HR. Study of congenital malformations in central nervous system and gastro-intestinal tract. Natl J Med Res. 2012;2:121-3.

Sarkar S, Patra C, Dasgupta MK, Nayek K, Karmakar PR. Prevalence of congenital anomalies in neonates and associated risk factors in a tertiary care hospital in Eastern India. J Clin Neonatol. 2013;2:131-4.

Komborozos VA, Skrekas GJ. Neuronal intestinal dysplasia: An entity of chronic intestinal pseudo-obstruction. Ann Gastroenterol. 2000;13:95-7.

De Lorijn F, Kremer LC, Reitsma JB, Benninga MA. Diagnostic tests in Hirschsprung disease: A systematic review. J Pediatr Gastroenterol Nutr. 2006;42:496-505.

De Lorijn F, Reitsma JB, Voskuijl WP, Aronson DC, Ten Kate FJ, Smets AM, et al. Diagnosis of Hirschsprung's disease: A prospective, comparative accuracy study of common tests. J Pediatr. 2005;146:787-92.

Holschneider AM, Steinweig I. Functional diagnosis. In: Holschneider AM, Puri P, editors. Hirschsprung´s Disease and Allied Disorders. 3rd ed. New York: Springer;2008:152-84.

Wieczorek TJ, Krane JF. Diagnostic utility of calretinin immunohistochemistry in cytologic cell block preparations. Cancer. 2000;90:312-9.

Walters JR, Bishop AE, Facer P, Lawson EM, Rogers JH, Polak JM. Calretinin and calbindin-D28k immunoreactivity in the human gastrointestinal tract. Gastroenterol. 1993;104:1381-9.

Blair PJ, Rhee PL, Sanders KM, Ward SM. The significance of interstitial cells in neurogastroenterology. J Neurogastroenterol Motil. 2014;20:294-317.

Gfroerer S, Rolle U. Interstitial cells of Cajal in the normal human gut and in Hirschsprung disease. Pediatr Surg Int. 2013;29:889-97.

Rolle U, Piotrowska AP, Nemeth L, Puri P. Altered distribution of interstitial cells of Cajal in Hirschsprung’s disease. Arch Pathol Lab Med. 2002;126:928-93.

Bancroft JD, Layton C. The hematoxylins and Eosin. In: Suvarna SK, Layton C, Bancroft JD, editors. Theory and Practice of Histological Techniques. 7th ed. New York: Churchill Livingstone;2012:173-186.

Jackson P, Blythe D. Immunohistochemical techniques. In: Bancroft JD, Gamble M, editors. Theory and Practice of Histological Techniques. 7th ed. New York: Churchill Livingstone;2012.

Becheanu G, Manuc M, Dumbrav M, Herlea V, Hortopan M. The evaluation of interstitial Cajal cells distribution in non-tumoral colon disorders. Rom J Morphol Embryol. 2008;49:351-3.

Samuel VG, Bonnard A, Lagausie PD, Chomette PP, Alberti C, Ghoneimi AE, et al. Calretinin immunohistochemistry: A simple and efficient tool to diagnose Hirschsprung’s disease. Mod Pathol. 2009;22:1379-84.

De la Torre L, Santos K. Hirschsprung disease. Evaluation of calretinin and S-100 as ancillary methods for the diagnosis of aganglionosis in rectal biopsies. Acta Pediatr Mex. 2012;33:246-51.

Hiradfar M, Sharifi N, Khajedaluee M, Zabolinejad N, Jamshidi S. Calretinin Immunohistochemistery: An aid in the diagnosis of Hirschsprung’s Disease. Iran J Basic Med Sci. 2012;15:1053-9.

Kannaiyan L, Madabhushi S, Malleboyina R, Are NK, Reddy KR, Rao B. Calretinin immunohistochemistry: A new cost-effective and easy method for diagnosis of Hirschsprung's disease. J Indian Assoc Pediatr Surg. 2013;18:66-8.

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.

Anbardar MH, Geramizadeh B, Foroutan HR. Evaluation of calretinin as a new marker in the diagnosis of Hirschsprung Disease. Iran J Pediatr. 2015;25:367.

Zuikova V, Franckevica I, Strumfa I, Melderis I. Immunohistochemical diagnosis of Hirschsprung’s Disease and allied disorders. Acta Chirurgica Latviensis. 2015;15:50-7.

Jain D, Moussa K, Tandon M, Culpepper J, Proctor D. Role of interstitial cells of Cajal in motility disorders of the bowel. Am J Gastroenterol. 2003;98:618-24.

Streutker CJ, Huizinga JD, Campbell F, Ho J, Riddell RH. Loss of CD117 (c-kit) and CD34-positive ICC and associated CD34-positive fibroblasts defines a subpopulation of chronic intestinal pseudo-obstruction. Am J Surg Pathol. 2003;27:228-35.

Anatol T, Mohammed W, Rao C. Interstitial cells of Cajal and intestinal function in Trinidadian children. West Indian Med J. 2008;57:393-7.

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.

Geramizadeh B, Akbarzadeh E, Izadi B, Foroutan H, Heidari T. Immunohistochemical study of enteric nervous system in Hirschsprung's disease and intestinal neuronal dysplasia. Histol Histopathol. 2013;28:345-51.

Puri P, Montedonico S. Hirschsprung’s disease: clinical features. In: Holschneider AM, Puri P, editors. Hirschsprung´s disease and allied disorders. 3rd ed. New York: Springer. 2008:107-114.