Calretinin immunohistochemistry: a simple and efficient tool to diagnose Hirschsprung disease

Abstract

Diagnosis of Hirschsprung disease (HD) is quite entirely based on the histopathological analysis of suction rectal biopsies. This hematoxylin and eosin approach has some limitations, despite the help of acetylcholinesterase staining. The aim of this study was to assess the diagnostic value of calretinin immunochemistry as a simple and reliable method in the diagnosis of HD. A total of 131 initial rectal biopsies carried out for suspicion of HD in children were retrieved, and calretinin immunohistochemistry was carried out on paraffin-embedded biopsies. Diagnosis of HD was made when no staining was observed. The results were statistically analyzed in comparison with our standard method (histology and acetylcholinesterase staining). 130 biopsies were accurately diagnosed on the basis of the positivity or negativity of calretinin staining. The senior pathologists diagnosed all cases of HD with no false positives. Furthermore, 12 additional cases initially considered as doubtful for HD using the standard method, were accurately diagnosed using calretinin immunohistochemistry. The false negative was a case of HD with a calretinin-positive biopsy. We also demonstrate the ease of calretinin interpretation compared with acetylcholinesterase for the junior pathologist. Calretinin immunohistochemistry overcomes most of the difficulties encountered using the combination of histology and acetylcholinesterase staining, and detects almost all cases of HD with confidence, with no false positives. Thus, we demonstrate that calretinin is superior to acetylcholinesterase to complete histology and could advantageously substitute for acetylcholinesterase. Modern Pathology ( The diagnosis of Hirschsprung disease (HD) is based on a combination of clinical features, radiological appearance of the bowel and histological features on suction rectal biopsies, 1,2 that is, aganglionosis and abnormally large nerves. In practice, a definite diagnosis of HD, leading to surgical treatment, is quite entirely based on histological results. 3,4 Several efficient approaches have been devised to assess the diagnosis of HD using a combination of standard histology and histochemistry. Standard histology obtained from rectal suction biopsies that sample mucosa and underlying submucosa, demonstrating rectal aganglionosis, requires the analysis of 100 or more histological sections to ensure good specificity, and is therefore time consuming. Moreover, difficulties in analysis may arise in several situations: (1) when the site of biopsy is too distal, because of the physiological paucity of ganglion cells; (2) when the sample is too superficial with not enough submucosa; and (3) when there is difficulty in identifying ganglion cells with confidence, particularly in neonates.For these reasons, standard histology is frequently supplemented with acetylcholinesterase histochemistry. 5,6 This technique, showing staining of extrinsic nerve fibers, provides quick results but requires frozen tissue samples. The last reports show good specificity of an abnormal ac...

Hirschsprung Disease and Use of Calretinin in Inadequate Rectal Suction Biopsies

“…6 Absence of calretinin expression was first reported in patients with HD in 2004 6 in resection specimens. Two follow-up studies 7,8 confirmed these findings in RSBs. Holland et al 9 in 2011 described a sensitivity of 100% and a specificity of 83% when classifying HD status based on calretinin stain results.…”

“…In addition, AChE activity fades after time, requiring continual replenishment of HD control tissue. Moreover, interpreting AChE patterns requires experience, and AChE staining has been associated with relatively high rates of interobserver disagreement 7,8 as well as false-positive results.…”

Utilization of peripherin and S-100 immunohistochemistry in the diagnosis of Hirschsprung disease

“…There were fewer major discrepancies and fewer equivocal interpretations. In addition, Guinard-Samuel et al 26 immunostained 131 cases of suspected Hirschsprung disease with calretinin, and also concluded that this immunohistochemical stain was superior to acetylcholinesterase in this clinical scenario.…”

Calretinin Expression in Hirschsprung’s Disease – A Potential Marker of Ganglion Cells

“…14 According to Gonzalo DH, all 12 of the patients without the Hirschsprung disease had Calretininpositive nerve fibers in lamina propria or the muscularis mucosae, and all 5 of patients with Hirschsprung disease had no staining of the nerves i.e. Calretinin-negative.…”