World Journal of Pathology Volume No 7

Research Open Access

Primary Diagnosis of Hirschsprung Disease – Calretinin Immunohistochemistry in Rectal Suction Biopsies, with Emphasis on Diagnostic Pitfalls

Kok Hing Lim, Wei Keat Wan, Tony Kiat Hon Lim, Alwin Hwai Liang Loh, Shireen A Nah and Kenneth Tou En Chang
World Journal of Pathology 2014, 3:3



Calretinin immunohistochemistry is an adjunctive diagnostic technique in the primary diagnosis of Hirschsprung disease (HD). Rectal suction biopsies from non-HD patients show calretinin immunoreactivity of small mucosal nerves. Those from HD-patients show absent immunolabeling of these nerves.

Study Design and Materials and Methods

This is a prospective study of rectal suction biopsies taken from 99 patients necessitating investigation for HD in our institution during which calretinin immunohistochemistry was routinely performed. At the end of the study period, all calretinin-stained sections were also subjected to a blinded review by 3 external reviewers.


Of the 27 patients with HD, two false negative results were obtained: one related to technical overstaining and the other to punctate immunoreactivity of deep submucosal hypertrophied nerves. Of the 72 non-HD patients, three false positive results were obtained, all relating to diminished immunoreactivity of previously frozen biopsy specimens. In terms of the blinded slide review, 2 reviewers correctly reported 100 out of 101 biopsies (from 99 patients) while 1 reviewer correctly reported 99 out of 101 biopsies. All discordant findings by the reviewers were the result of examining the sections at low (x40 or x100) magnification only and misinterpreting positive calretinin staining as being absent.


Calretinin immunostaining is a reliable ancillary technique in the investigation of HD, if its potential pitfalls (described both by ourselves and previously published studies on this topic) are noted. On this basis, we attempt to formulate a protocol incorporating routine calretinin immunohistochemistry for the diagnosis or exclusion of HD.


Hirschsprung disease, aganglionosis, calretinin immunohistochemistry

Download PDF    Full Text