Is nerve bundle hypertrophy a reliable criteria for diagnosing Hirschsprung disease? A case control study using Calretinin as an adjunct tool for confirming Hirschsprung disease.

Objective: To determine the frequency of nerve bundle hypertrophy in aganglionic segments in relation to the site of biopsy, along with the assessment of value of Calretinin immunostaining in the diagnosis of suspected cases of Hirschsprung disease. Study Design: Cross Sectional study. Setting: Department of Pathology, Pakistan Institute of Medical Sciences (PIMS) Islamabad. Period: September 2018 to March 2019. Material & Methods: After routine tissue processing colonic biopsies were examined for presence of ganglion cells and hypertrophic nerve presence or absence. Diagnosis of Hirschsprung disease was based on the absence of ganglion cells in submucosal and myenteric plexus, the presence or absence of hypertrophic nerves (more than 4 nerves >30 µm thick/×200 field or more than 2 nerves >40 µm thick/×200 field) was also noted in all cases of Hirschsprung disease (aganglionic segments). Calretinin immunostaining was applied to all the cases and controls and findings were recorded as positive or negative staining. Statistical Analysis: Data was analyzed using SPSS version 23. Qualitative data was calculated as frequencies and percentages. Pearson Chi square test was used to establish the association of nerve bundle hypertrophy with the site of biopsy. Results: Total biopsies were 60; 30 each from ganglionic and aganglionic segments. Calretinin sensitivity in our study was 90%, specificity 83.3%. In 30 cases of aganglionosis hypertrophic nerves were present in 13(21.7%) and they were absent in 17 (28.3%). No significant association (p value= 0.447) was seen in nerve bundle hypertrophy and site of biopsy. Conclusion: Calretinin immunohistochemistry can be used as a reliable ancillary technique in the diagnosis of HD. Aganglionosis may not always be associated with submucosal nerve hypertrophy which alone should not be used as a criteria for HD diagnosis but instead adjunct methods like Calretinin immunostaining must be utilized to confirm presence or absence of ganglion cells. There is no association of nerve hypertrophy with site of biopsy.