Reevaluation of concurrent acetylcholinesterase and hematoxylin and eosin staining for Hirschsprung's disease

Pediatr Int. 2021 Sep;63(9):1095-1102. doi: 10.1111/ped.14596. Epub 2021 Jul 29.


Background: Acetylcholinesterase (AChE) histochemistry has been widely performed for the histopathological diagnosis of Hirschsprung's disease (HD). However, we occasionally come across diagnostic difficulties. We conducted concurrent AChE histochemistry and hematoxylin and eosin (HE) staining to validate the ancillary value of this technique.

Methods: Of 177 patients diagnosed using AChE histochemistry from January 2014 to December 2016, 90 patients underwent formalin-fixed paraffin-embedded HE staining. The histopathological findings and diagnostic abilities were investigated and compared retrospectively.

Results: The sensitivity, specificity, accuracy, and kappa index of AChE histochemistry and HE staining were 94.1%, 100%, 98.9%, and 0.964 and 76.5%, 84.9%, 83.3%, and 0.530, respectively. The specificity, accuracy and kappa index of AChE histochemistry were significantly higher than those of HE staining (P < 0.001, <0.001, and <0.05). Hematoxylin and eosin staining supported the suspected diagnosis of total colon aganglionosis at the initial biopsy; furthermore, HE staining helped confirm the distinct shape of ganglion cells and hypertrophic nerve bundles.

Conclusion: We re-confirmed that AChE histochemistry is an excellent method for diagnosing HD. Although the diagnostic ability of HE staining is limited, it has acceptable utility as an ancillary method. Thus, AChE staining is a useful test and it should be performed together with HE staining.

Keywords: Hirschsprung’s disease; acetylcholinesterase histochemistry; hematoxylin and eosin staining; rectal mucosal punch biopsy.

MeSH terms

  • Acetylcholinesterase*
  • Adolescent
  • Adult
  • Aged
  • Biopsy
  • Child
  • Child, Preschool
  • Eosine Yellowish-(YS)
  • Female
  • Hematoxylin
  • Hirschsprung Disease* / diagnosis
  • Humans
  • Infant
  • Infant, Newborn
  • Male
  • Middle Aged
  • Rectum
  • Retrospective Studies
  • Staining and Labeling
  • Young Adult


  • Acetylcholinesterase
  • Eosine Yellowish-(YS)
  • Hematoxylin