Diagnostic roles of calretinin in hirschsprung disease: A comparison to neuron-specific enolase

Abstract

Background/aim: Diagnosis of Hirschsprung's disease (HD) can be hard and requires good experience, principally for pathologists who infrequently encounter the disease. However, diagnosis is not always possible with hematoxylin and eosin (H and E) because staining has limitations in the identification of immature ganglion cells in neonates and the submucosal area.

Aim: To assess the diagnostic role of calretinin immunostaining in HD in comparison to neuron-specific enolase.

Patients and methods: Formalin-fixed paraffin tissue blocks of full-thickness distal colonic and rectal biopsies for 48 patients who clinically presented with symptoms suspicious for HD were collected for the period from December 2012 to January 2016. All biopsies were already studied by routine H and E histopathological examination for the presence or absence of ganglion cells. Further confirmation of ganglion cells and nerve fibers was performed by immunohistochemical study for neuron-specific enolase and calretinin, respectively, in a private pathology laboratory.

Results: According to the histopathological assessment, cases with absent ganglionic cells were considered to be HD, which comprised 40 cases out of the total 48 cases. The mean age for HD cases was 19.43 months. The male-to-female ratio in HD cases was 2.34:1. All HD cases showed negative expression of calretinin in small nerve fibers of the lamina propria, musularis mucosae, and submucosa, and negative expression of neuron-specific enolase in ganglionic cells. The sensitivity, specificity, positive predictive value, and negative predictive values for both the markers in the confirmation of diagnosis of HD were all 100%.

Conclusion: Calretinin immunostaining, similar to that of neuron-specific enolase, is a highly sensitive and specific diagnostic aid to histopathological examination in suspected HD.

Figure 1

UltraView Universal DAB Detection Kit Reaction

Figure 2

Hirschsprung disease; absence of myenteric plexus (Auerbach's plexus) ganglion cells along the interface between the two layers muscularis layer of colon (arrows). Hematoxylin and eosin stain, ×10

Figure 3

Normally occurring myenteric plexus (Auerbach's plexus) ganglion cells between the two layers muscularis layer of colon; large pyramidal shaped cells with large vesicular nucleus and prominent nucleolus (arrows). Hematoxylin and eosin stain, ×40

Figure 4

Hirschsprung disease showing negative immunohistochemical staining of neuron specific enolase and calretinin (same case) with absence of myenteric plexus (Auerbach's plexus) ganglion cells along the interface between the two layers muscularis layer of colon (arrows). (a) Neuron specific enolase (×10), (b) calretinin (×10)

Figure 5

Hirschsprung disease with complete absence of staining of calretinin in small nerve fibers of lamina propria and submucosa layers. (a) power ×10; (b) power ×40

Figure 6

Neuron specific enolase: normal staining pattern for myenteric plexus (Auerbach's plexus) ganglion cells between the two layers muscularis layer of colon; strong brown cytoplasmic stain for a group of ganglion cells (arrows), ×40

Figure 7

Calretinin: normal presence and distribution in lamina propria and submucosa layers of small nerve fibers showing strong brown cytoplasmic staining pattern for calretinin (arrows). (a) power ×10; (b) power ×40

Figure 8

Normal staining pattern of calretinin for myenteric plexus (Auerbach's plexus) ganglion cells between the two layers muscularis layer of colon; strong brown cytoplasmic stain for two groups of ganglion cells (arrows); ×10