Role of calretinin immunohistochemical stain in evaluation of Hirschsprung disease: an institutional experience

Abstract

Background: The use of calretinin immunostain (IHC) in the evaluation of rectal suction biopsies for Hirschsprung disease (HD) has been reported by Kapur et al. and others. The first goal of this article is to report our institutional experience with the use of calretinin in specimens for evaluation of HD. The second goal is to describe the pattern of expression of calretinin in the junction of ganglionic-to-aganglionic segment of pull through specimens of patients with a previous diagnosis of HD on suction rectal biopsy.

Material and methods: Three pathologists at University of Texas at Houston evaluated 28 rectal biopsy specimens from 2010-2011. The patients' age ranged from 15 days to 8 years. Twenty-three cases were suction biopsies, and five were rectal full thickness biopsies. Hematoxylin-eosin (H&E) stain was performed on at least 80 levels for the suction biopsy specimens. Calretinin immunohistochemical stain was performed on levels 40-42 in all cases, with adequate controls. The H&E slides of nine pull through specimens with a diagnosis of HD on a suction rectal biopsy that was evaluated in this study, were evaluated. Calretinin IHC was performed on the slide(s) showing the junction of aganglionic-to-normal rectum, along with adequate controls.

Results: The presence of ganglion cells consistently correlated with calretinin-positive thin nerve fibrils in the lamina propria, muscularis mucosae and superficial submucosa. These nerve fibrils were absent in the aganglionic segments of bowel and in the areas without ganglion cells from the junction of normal with diseased rectum. Calretinin was strongly expressed in the submucosal and subserosal nerve trunks in the ganglionic segment. It had faint expression in the thick nerve trunks from the areas without ganglion cells 1.6-2.5 cm proximal to the normal rectum. No calretinin expression was seen in the nerve trunks in the rest of the aganglionic segment.

Conclusion: The pattern of expression of calretinin in rectal suction biopsies in HD and normal rectum coincide with the ones previously described in the literature. Calretinin IHC offered additional diagnostic value in the specimens with inadequate amount of submucosa and rarely seen ganglion cells. The pattern of expression of calretinin in HD pull-through specimens correlates with the rectal biopsy ones. Faint positivity of the thick submucosal and subserosal nerves in the absence of ganglion cells and calretinin positive nerve fibrils, is characteristic of the junction of the aganglionic-to-normal rectum. We are the first ones to document this finding.

Keywords: Hirschsprung; calretinin; pull-through specimen.

Figure 1

A: Calretinin IHC x400: Section shows rectal biopsy lamina propria with calretinin positive thin, linear, granular nerve fibrils, in a non-HD case. Figure 1A inset: Calretinin IHC x400: The submucosa of the same case shows positive ganglion cells and expression of calretinin in the nerve trunks. B: Calretinin IHC x400: In another non-HD case, lamina propria shows linear nerve fibrils with a granular pattern of reactivity on calretinin IHC. C: Calretinin IHC x40: Suction rectal biopsy specimen with suboptimal amount of submucosa and very few ganglion cells seen at cutting through the block. There are a few submucosal cells that express calretinin, possibly mast cells. D: Calretinin IHC x400: This is a section of the same case represented in Figure 1C. Even if the amount of submucosa was suboptimal, and the ganglion cells were difficult to find when cutting through the block, the lamina propria shows the linear, granular pattern of reactivity that was mentioned above. E: Calretinin IHC, x400: Section shows the lamina propria of an HD specimen. No nerve fibrils are present in the lamina propria. There were no nerve fibrils present in the superficial submucosa and muscularis mucosae. There is non-specific pale stain in the crypts. F: Calretinin IHC, x400: In a limited number of suction rectal biopsies with a diagnosis of HD, the submucosal hypertrophied nerve trunks showed axonal expression of calretinin in a faint, granular pattern. This finding correlated with the junction of aganglionic-to-ganglionic segment of bowel in pull-through specimens, and extended 1.5-2.5 cm proximal to the aganglionic segment.