Calretinin Immunohistochemistry Versus Acetylcholinesterase Histochemistry in Rectal Biopsies for the Diagnosis of Hirschsprung's Disease
Maria Rodrigues, Pedro Luis Lourencao, Simome A Terra, Erika V Ortolan, Bonifacio K Takegawa. Botucatu Medical School, University of São Paulo State, Unesp, Botucatu, Sao Paulo, Brazil
Background: The pathological evaluation of rectal biopsies has been the gold standard for the diagnosis of Hirschsprung's disease (HSCR) and Calretinin has been described as an ancillary tool in the diagnosis of HSCR.
Design: We aimed to investigate prospectively, the diagnostic accuracy and applicability of Calretinin as compared with Acetylcholinesterase (Ache) histochemistry, in the evaluation of rectal biopsies from patients under investigation for HSCR. A total of 83 patients were investigated with anorectal manometry and/or barium enema as screening tests. Of them, 43 patients were submitted to rectal biopsies. Frozen tissue samples were used for Ache histochemistry and paraffin-embedded sections were stained with Hematoxylin and Eosin (H&E) and Calretinin immunohistochemistry.
Results: The analysis of serial H&E stained slides did not reveal the presence of ganglion cells in 24/43 (55.8%) cases. A positive reaction for increased Ache activity, which was considered diagnostic for HSCR, was demonstrated in 13/43 (30.2%) cases, in nerve fibers strongly stained in dark brown in the muscularis mucosa and lamina propria. The disagreement between H&E and Ache occurred in 11 cases, in which the negative results for H&E were due to the absence of ganglion cells in tissue samples. Absence of immunoreactivity for Calretinin was found in 14/43 (32.6%) cases. Typical positive staining for Calretinin in non-HSCR patients was observed in 29 cases (67.4%) and consisted of granular staining in ganglion cells and intrinsic nerve fibers in the lamina propria or muscularis mucosa. The disagreement between Ache and Calretinin methods occurred in only one case, in which the negative result for Calretinin was initially performed due to the absence of ganglion cells in the samples. The results on Calretinin were in good agreement with Ache histochemistry according to the Kappa index (0,946 p< 0,001) and presented significantly higher specificity (96.7 x 63.3 p<0.002) and accuracy (97.6 x 74.4 p<0.003) when compared with H&E. The final diagnosis of Hirschsprung's disease was confirmed in 13/43 (30.2%) patients who were submitted to surgical treatment. They were 9 boys (69.2%) and 4 girls (30.8%) and in 4 cases (30.7%) the diagnosis was performed during the neonatal period.
Conclusions: These results indicate that Calretinin is an accurate and less complex diagnostic tool than Acetylcholinesterase for the diagnosis of Hirschsprung's disease in rectal biopsies.
Tuesday, March 5, 2013 1:00 PM
Poster Session IV # 119, Tuesday Afternoon