Acetylcholinesterase staining for the pathological diagnosis of Hirschsprung’s disease


Hirschsprung’s disease (HD) is a congenital disease manifesting various degrees of functional bowel obstruction caused by the absence of enteric ganglion cells, which are usually absent in the colonic segment of the HD patient. Because the aganglionic segment of HD always includes the rectum, pathological diagnosis can be made using a rectal sample. HD should be diagnosed as early as possible because serious complications, such as acute enterocolitis or toxic megacolon, can develop without a definitive diagnosis and appropriate treatment. In the mid-1900s, HD was diagnosed by HE staining of specimens obtained by full-thickness biopsy. Since then, the combination of rectal mucosal biopsy and rubeanic acid-amplificated AChE staining has been brought about by the following milestones: the discovery that the submucosal plexus and the intermuscular plexus had the same level of nerve migration; the findings of research on acetylcholine (ACh) and acetylcholinesterase (AChE) in the intestinal tract; and the establishment of a rubeanic acid amplification method. Consequently, the diagnostic rate of HD improved dramatically in the 1980s. This review outlines the history of diagnostic methods for HD, the roles of ACh and AChE in the intestine, and the method of AChE staining.

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Hirschsprung’s disease






Enteric nerve system


Rectal mucosal biopsy


Full-thickness biopsy


  1. 1.

    Haricharan RN, Georgeson KE. Hirschsprung disease. Semin Pediatr Surg. 2008;17(4):266–75.

    Article  Google Scholar 

  2. 2.

    Taguchi T, Obata S, Ieiri S. Current status of Hirschsprung’s disease: based on a nationwide survey of Japan. Pediatr Surg Int. 2017;33:497–504.

    Article  Google Scholar 

  3. 3.

    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.

    Article  Google Scholar 

  4. 4.

    Dobbins WO 3rd, Bill AH Jr. Diagnosis of Hirschsprung’s disease excluded by rectal suction biopsy. N Engl J Med. 1965;272:990–3.

    Article  Google Scholar 

  5. 5.

    Swenson O, Fisher JH. Hirschsprung’s disease in the newborn. Arch Surg. 1959;79:987–93.

    CAS  Article  Google Scholar 

  6. 6.

    Gherardi GJ. Pathology of the ganglionic-aganglionic junction in congenital megacolon. Arch Pathol. 1960;69:520–3.

    CAS  PubMed  Google Scholar 

  7. 7.

    Shandling A. new technique in the diagnosis of Hirschsprung’s disease. Can J Surg. 1961;4:298–305.

    CAS  PubMed  Google Scholar 

  8. 8.

    Hirose R, Hirata Y, Yamada T, Kawana T, Taguchi T, Suita S. The simple technique of rectal mucosal biopsy for the diagnosis of Hirschsprung’s disease. J Pediatr Surg. 1993;28:942–4.

    CAS  Article  Google Scholar 

  9. 9.

    Nakao M, Suita S, Taguchi T, Hirose R, Shima Y. Fourteen-year experience of acetylcholinesterase staining for rectal mucosal biopsy in neonatal Hirschsprung’s disease. J Pediatr Surg. 2001;36:1357–63.

    CAS  Article  Google Scholar 

  10. 10.

    Budianto IR, Obata S, Kinoshita Y, Yoshimaru K, Yanagi Y, Miyata J, et al. Reevaluation of acetylcholinesterase staining for the diagnosis of Hirschsprung disease and allied disorders. J Pediatr Gastroenterol Nutr. 2015;60:606–12.

    CAS  Article  Google Scholar 

  11. 11.

    Yoshimaru K, Kinoshita Y, Yanagi Y, Obata S, Jimbo T, Iwanaka T, et al. The evaluation of rectal mucosal punch biopsy in the diagnosis of Hirschsprung’s disease: a thirty-year experience of 954 patients. Pediatr Surg Int. 2017;33:173–9.

    Article  Google Scholar 

  12. 12.

    Muise ED, Cowles RA. Rectal biopsy for Hirschsprung’s disease: a review of techniques, pathology, and complications. World J Pediatr. 2016;12:135–41.

    Article  Google Scholar 

  13. 13.

    Challa VR, Moran JR, Turner CS, Lyerly AD. Histologic diagnosis of Hirschsprung’s disease. The value of concurrent hematoxylin and eosin and cholinesterase staining of rectal biopsies. Am J Clin Pathol. 1987;88:324–8.

    CAS  Article  Google Scholar 

  14. 14.

    Kamijo K, Hiatt RB, Koelle GB. Congenital megacolon; a comparison of the spastic and hypertrophied segments with respect to Cholinesterase activities and sensitivities to acetylcholine. DFP Barium Ion Gastroenterol. 1953;24:173–85.

    CAS  Google Scholar 

  15. 15.

    Karnovsky MJ, Roots LA. “Direct-coloring” thiocholine method for cholinesterases. J Histochem Cytochem. 1964;12:219–21.

    CAS  Article  Google Scholar 

  16. 16.

    Meier-Ruge W. Das megacolon seine diagnose und pathophysiologie. Virchows Arch Path Anat. 1968;344:67–85.

    CAS  Article  Google Scholar 

  17. 17.

    Meier-Ruge W, Lutterbeck PM, Herzog B, Morger R, Moser R, Schärli A. Acetylcholinesterase activity in suction biopsies of the rectum in the diagnosis of Hirschsprung’s disease. J Pediatr Surg. 1972;7:11–7.

    CAS  Article  Google Scholar 

  18. 18.

    Nakamura T, Torigoe K. A new method for enhancing contrast of Hatchett’s brown at the sites of acetylcholinesterase activity by Rubeanic acid. Acta Histochem Cytochem. 1981;14:67.

    Article  Google Scholar 

  19. 19.

    Goto S, Ikeda K, Toyohara T. An improved staining technique for acetylcholinesterase activity using Rubeanic acid in the diagnosis of Hirschsprung’s disease. Jpn J Surg. 1984;14:135–8.

    CAS  Article  Google Scholar 

  20. 20.

    Goto S, Ikeda K, Toyohara T. Histochemical diagnosis of HIrschsprung’s disease by an improved acetylcholinesterase staining technique using rubeanic acid (in Japanese). Jpn J Pediatr Surg. 1984;20:717–22.

    Google Scholar 

  21. 21.

    Friedmacher F, Puri P. Current practice patterns of rectal suction biopsy in the diagnostic work-up of Hirschsprung’s disease: results from an international survey. Pediatr Surg Int. 2016;32:717–22.

    Article  Google Scholar 

  22. 22.

    Zani A, Hall NJ, Rahman A, Morini F, Pini Prato A, Friedmacher F, et al. European paediatric surgeons’ association survey on the management of Hirschsprung Disease. Eur J Pediatr Surg. 2017;27:96–101.

    Article  Google Scholar 

  23. 23.

    Jiang M, Li K, Li S, Yang L, Yang D, Zhang X, et al. Calretinin, S100 and protein gene product 9.5 immunostaining of rectal suction biopsies in the diagnosis of Hirschsprung’ disease. Am J Transl Res. 2016;8:3159–68.

    CAS  PubMed  PubMed Central  Google Scholar 

  24. 24.

    Tran VQ, Lam KT, Truong DQ, Dang MH, Doan TT, Segers V, et al. Diagnostic value of rectal suction biopsies using calretinin immunohistochemical staining in Hirschsprung’s disease. J Pediatr Surg. 2016;51:2005–9.

    Article  Google Scholar 

  25. 25.

    Inestrosa NC, Alvarez A, Pérez CA, Moreno RD, Vicente M, Linker C, et al. Acetylcholinesterase accelerates assembly of amyloid-beta-peptides into Alzheimer’s fibrils: possible role of the peripheral site of the enzyme. Neuron. 1996;16:881–91.

    CAS  Article  Google Scholar 

  26. 26.

    Moore SW, Johnson G. Acetylcholinesterase in Hirschsprung’s disease. Pediatr Surg Int. 2005;21:255–63.

    CAS  Article  Google Scholar 

  27. 27.

    Rectal YK, Biopsy M. In: Taguchi T, Matsufuji H, Ieiri S, editors. Hirschsprung’s disease and the allied disorders—status Quo and future prospects of treatment. Singapore: Springer Nature; 2019. p. 75–83.

    Google Scholar 

  28. 28.

    Ikawa H, Yokoyama J, Morikawa Y, Hayashi A, Katsumata K. A quantitative study of acetylcholine in Hirschsprung’s disease. J Pediatr Surg. 1980;15:48–52.

    CAS  Article  Google Scholar 

  29. 29.

    Ikeda K. Basic and clinic of Hirschspring’s disease. Herusu Shuppan, Co. Inc.; 1989. p. 38–40 (In Japanese).

  30. 30.

    Layer PG, Kaulich S. Cranial nerve growth in birds is preceded by cholinesterase expression during neural crest cell migration and the formation of an HNK-1 scaffold. Cell Tissue Res. 1991;265:393–407.

    CAS  Article  Google Scholar 

  31. 31.

    Lazar M, Vigny M. Modulation of the distribution of acetylcholinesterase molecular forms in a murine neuroblastoma x sympathetic ganglion cell hybrid cell line. J Neurochem. 1980;35:1067–79.

    CAS  Article  Google Scholar 

  32. 32.

    Garrett JR, Howard ER, Nixon HH. Histochemical diagnosis of Hirschsprung’s disease. Lancet. 1969;2:436.

    CAS  Article  Google Scholar 

  33. 33.

    Kurer MH, Lawson JO, Pambakian H. Suction biopsy in Hirschsprung’s disease. Arch Dis Child. 1986;61:83–4.

    CAS  Article  Google Scholar 

  34. 34.

    Morris MI, Soglio DB, Ouimet A, Aspirot A, Patey N. A study of calretinin in Hirschsprung pathology, particularly in total colonic aganglionosis. J Pediatr Surg. 2013;48:1037–43.

    Article  Google Scholar 

  35. 35.

    Touloukian RJ. Acquired aganglionic megacolon in a premature infant: report of a case. Pediatrics. 1975;56:459–62.

    CAS  PubMed  Google Scholar 

  36. 36.

    Bjørn N, Rasmussen L, Qvist N, Detlefsen S, Ellebæk MB. Full-thickness rectal biopsy in children suspicious for Hirschsprung’s disease is safe and yields a low number of insufficient biopsies. J Pediatr Surg. 2018;53:1942–4.

    Article  Google Scholar 

  37. 37.

    Meier-Ruge WA, Holschneider AM. Histopathologic observations of anorectal abnormalities in anal atresia. Pediatr Surg Int. 2000;16:2–7.

    CAS  Article  Google Scholar 

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This study was supported in part by Grants from The Ministry of Health, Grants-in-Aid for Scientific Research (18K16260). We thank Rie Funatsu and Tomoko Yamazaki of the Department of Pediatric Surgery, Reproductive and Developmental Medicine, Faculty of Medical Sciences, Kyushu University, for their excellent assistance.

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Correspondence to Toshiharu Matsuura.

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Yoshimaru, K., Yanagi, Y., Obata, S. et al. Acetylcholinesterase staining for the pathological diagnosis of Hirschsprung’s disease. Surg Today (2020).

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  • Hirschsprung’s disease
  • Acetylcholinesterase staining
  • Rectal biopsy