Rectal suction biopsy (RSB) is a gold standard for diagnosing Hirschsprung disease (HD). Calretinin staining of RSB is increasingly used by experienced pathologists due to non-complex examination and comparable diagnostic accuracy with acetylcholinesterase (AChE). However, the diagnostic accuracy of calretinin examined by unexperienced pathologists remains to be elucidated. Therefore, we aim to compare diagnostic accuracy of calretinin with AChE on RSB for diagnosing HD when examined by unexperienced pathologists. We prospectively analyzed sections from RSB stained with AChE + HE and calretinin. Blinded examination was done by five unexperienced pathologists (pathology residents) and three experienced pathologists (senior pediatric gastro-enterology pathologists) assessing for the presence of HD. Cases for the study included ones proven to be HD on resection specimens and cases without HD. Diagnostic accuracy was determined calculating area under the curve (AUC), sensitivity, specificity, likelihood ratio, and posttest probability. Fleiss’ kappa analysis was performed to assess interobserver agreement between reviewers. Eleven of 18 included patients (61%) were diagnosed with HD. Comparing the diagnostic accuracy of unexperienced pathologists, calretinin versus AChE + HE showed sensitivity of 80.0% versus 74.5% and specificity of 100% versus 65.4%, AUC of 0.87 (0.78–0.96) versus 0.59 (0.45–0.72). Unexperienced pathologists showed substantial agreement with calretinin (kappa 0.72 [0.61–0.84]) and fair agreement with AChE + HE (kappa 0.34 [0.23–0.44]). We found calretinin having higher diagnostic accuracy in diagnosing HD compared to AChE + HE when examined by unexperienced pathologists. Therefore, we recommend to use calretinin as the standard technique for staining RSB in diagnosing HD.
Original languageEnglish
Pages (from-to)245-252
Number of pages8
JournalVirchows Archiv
Issue number2
Early online date5 May 2022
Publication statusPublished - Aug 2022


  • Acetylcholinesterase
  • Calretinin
  • Diagnostic accuracy
  • Hematoxylin–eosin
  • Hirschsprung disease
  • Pathologist
  • Rectal suction biopsy

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