Calretinin immunohistochemical staining in Hirschsprung’s disease: An institutional experience

OBJECTIVE: This study aims to evaluate the results obtained by calretinin staining on tissue samples for diagnosing Hirschsprung’s disease (HD) in a single institution, by single expert. METHODS: A retrospective evaluation was done for calretinin immunostaining in HD patients for a period of 3 years. Calretinin staining was evaluated in nerve fibers. Calretinin immunohistochemistry was considered positive if any staining was seen in nerve fibers and/or ganglion cells in the lamina propria, muscularis mucosa or submucosa. According to staining intensity, staining was classified as strong, weak or negative. The pathological diagnosis was based on presence or absence of ganglion cells (G0/G1) and nerve hypertrophy (N0/N1). Samples were classified according to the depth (presence of submucosa or intermuscular area), the type (biopsy or resection specimen) and staining intensity of calretinin (strong, weak, or negative staining). RESULTS: A total of 96 tissue samples from 56 patients were studied. Tissues were from colon (43.8%), rectum (43.8%), stoma (6.2%), ileum (3.1%) and appendix (3.1%). The pathological diagnosis was G0N0 in 14.6%, G1N0 in 54.2%, G0N1 in 25% and G1N1 in 6.2% of cases. Our materials consisted of 92 tissue biopsies and four resection specimens. Intermuscular layer was present in 87.5% of materials and 12.5% of biopsies contained submucosa. Calretinin staining was negative (C0) in 37.5% of cases, strong positive (C1) in 47.9%, and weak positive (C2) in 14.6%. When the C0 category was taken as the reference, the status of calretinin staining as C2 (weak positive) in cases with pathological diagnosis of G1N0 was found to be 37.575 times that of cases with G0N0 (OR [95% CI]: 37.575 [2.928, 482.176], p=0.006) and the status of calretinin staining as C1 (strong positive) in cases with pathologic diagnosis of G1N0 was found to be 131.401 times that of G0N0 (OR [95% CI]: 131.401 [9.263, 1864.082), p<0.001). CONCLUSION: Calretinin staining is positive whenever ganglion cells are present independent from presence of nerve hypertrophy, the depth and the site of the biopsy or staining intensity. It is negative in all aganglionic samples. Calretinin staining is a reliable ancillary test in HD diagnosis.