摘要:Background Rectal biopsy and its histopathological study with hematoxylin and eosin (HE) is the gold standard for Hirschsprung's disease (HD) diagnosis. However, there are some limitations in the diagnosis of ganglion cells in HE approach. Recently, it was reported that the utility of Calretinin is a reliable ancillary immunohistochemistry (IHC) test for HD diagnosis. We aimed to investigated Calretinin and S100 IHC staining as ancillary methods to diagnose HD. Materials and Methods: In this cross sectional study, 36 rectal biopsies taken from suspected HD patients were evaluated in pathology department of Mofid children’s Hospital. Patients ranged from 1 day to 60 months. Data were collected in a 2-year period from 2014 to 2016 in Mofid Children Hospital, Tehran, Iran. The histological study was done observing HE stained tissue sections by two pathologists and diagnoses were: twenty-four HD (aganglionic), and twelve non-Hirschsprung's (NHD) (normoganglionic) patients. Then Calretinin and S100 IHC were performed on the slides. The IHC slides were evaluated by two pathologists and the diagnostic value of Calretinin and S100 was determined in comparison with gold standard which is the presence or absence of ganglion cells in serial HE stained sections of rectal biopsies. Results The results in this study demonstrated that sensitivity, specificity, negative predictive value (NPV), and positive predictive value (PPV) for S100 were 61.9%, 93%, 93%, and 62%, respectively. Also, sensitivity, specificity, NPV, and PPV for Calretinin were all 100%. Conclusion Based on the findings it may be concluded that Calretinin Immunohistochemistry had good diagnostic value and S100 Immunohistochemistry had intermediate level diagnostic value for Hirschsprung's disease.