Original ContributionA useful panel for the diagnosis of Hirschsprung disease in rectal biopsies: calretinin immunostaining and acetylcholinesterase histochesmistry☆,☆☆,★
Introduction
The pathological evaluation of rectal biopsies for the diagnosis of Hirschsprung disease has been a challenging issue. Typical histologic features of this congenital malformation of the enteric nervous system include the absence of ganglion cells and increased number of hypertrophic nerves in the submucosa and myenteric nerve plexuses [1], [2], [3], [4], [5], [6]. The assessment of serial hematoxylin-eosin (H&E)–stained rectal biopsies has often some difficulties, including superficial samples with limited submucosa or samples from too distal sites of the anorectum with a physiological paucity of ganglion cells and problems on the identification of ganglion cells, particularly in neonates, owing to immaturity of the enteric nervous system [7], [8], [9]. For these reasons, conventional histology has been supplemented with histochemical and immunohistochemical methods to assist the identification of ganglion cells [4], [10], [11]. Acetylcholinesterase (AChE) histochemical staining has been used as an ancillary method in the evaluation of rectal biopsies for the diagnosis of Hirschsprung disease [4], [10], [12], [13], [14]. The presence of this enzyme in cholinergic nerve fibers stained in dark brown to black, within the muscularis mucosa and the lamina propria, has been considered diagnostic for Hirschsprung disease [4], [5], [10]. The main advantage of this method is that superficial biopsies are adequate to reveal the activity of the enzyme, confirming the diagnosis of Hirschsprung disease [4], [5], [10]. However, this histochemical method requires frozen tissue samples, and the findings may be difficult to interpret, leading to false-positive and false-negative results [15], [16], [17]. In this context, over the past years, a series of immunohistochemical markers such as S-100 protein and peripherin, neuron-specific enolase, and glial fibrillary acidic protein have been proposed to aid in the diagnosis of Hirschsprung disease [18], [19], [20]. However, none of these markers have shown significant advantage over conventional histopathologic analysis performed by an experienced examiner [5], [11].
Barshack et al [21], [22] have found that calretinin, a 29-kd calcium binding protein, which plays an important role in the organization and functioning of the central nervous system was not expressed in aganglionic segments, whereas in normal colon, both ganglion cells and nerve fibers were immunostained by calretinin [21], [22]. Other retrospective studies have shown similar results suggesting the usefulness of this immunohistochemical marker in the evaluation of rectal biopsies for the diagnosis of Hirschsprung disease [23], [24], [25].
Therefore, we aimed to investigate prospectively the diagnostic accuracy and applicability of calretinin immunostaining, as compared with AChE histochemistry and conventional histology (H&E), in the evaluation of rectal biopsies from patients under investigation for Hirschsprung disease.
Section snippets
Study population
A prospective study was conducted from the monitoring of children under investigation for Hirschsprung disease, at Botucatu Medical School Hospital, São Paulo State University, São Paulo, southern Brazil, in a 2-year period, from January 2010 to January 2012. A total of 83 patients were initially investigated with anorectal manometry and/or barium enema as screening tests. Of them, 43 patients had results suggestive of Hirschsprung disease and were submitted to rectal biopsies.
Patient characteristics
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. They were 27 boys (62.8%) and 16 girls (37.2%). According to the age distribution, they presented a median of 35 (1/195) months. The distribution of patients, according to the age groups, is shown in Fig. 1.
The diagnosis of Hirschsprung disease was performed on rectal biopsies in 13 (30.2%) of 43 patients. They were 9 boys (69.2%) and
Discussion
In the present study, we have investigated, in a prospective series of cases, the applicability and diagnostic accuracy of calretinin immunostaining as compared with AChE histochemistry and conventional histopathology (H&E), in the evaluation of rectal biopsies from 43 patients under investigation for Hirschsprung disease. The final diagnosis was performed in 13 patients who were submitted to surgical treatment. The histopathologic analysis of surgical specimens confirmed the diagnosis of
References (34)
- et al.
Hirschsprung's disease and idiopathic megacolon
Lancet
(1949) Practical pathology and genetic of Hirschsprung's disease
Semin Pediatr Surg
(2009)- et al.
The histopathology of gastrointestinal motility disorders in children
Semin Pediatr Surg
(2009) - et al.
Ganglion cell distribution in the normal rectum and anal canal: a basis for the diagnosis of Hirschsprung's disease by anorectal biopsy
J Pediatr Surg
(1968) - et al.
Experience with rectal suction biopsy in the diagnosis of Hirschsprung's disease
J Pediatr Surg
(1969) - et al.
Acetylcholinesterase activity in suction biopsies of the rectum in the diagnosis of Hirschsprung's disease
J Pediatr Surg
(1972) - et al.
Problems and advantages of acetylcholinesterase histochemistry of rectal suction biopsies in the diagnosis of Hirschsprung's disease
J Pediatr Surg
(1990) - et al.
Utilization of peripherin and S-100 immunohistochemistry in the diagnosis of Hirschsprung disease
Mod Pathol
(2010) - et al.
Distribution and localization of glial fibrillary acidic protein in colons affected by Hirschsprung's disease
J Pediatr Surg
(1989) - et al.
Calcium-binding proteins in the nervous system
Trends Neurosci
(1992)
Calretinin immunostaining as an adjunct in the diagnosis of Hirschsprung disease
Ann Diagn Pathol
Calretinin immunohistochemistry: a simple and efficient tool to diagnose Hirschsprung's disease
Mod Pathol
A rectal suction biopsy tube for use in the diagnosis of Hirschsprung's disease
J Pediatr Surg
Transanal endorectal pull-through for Hirschsprung's disease
J Pediatr Surg
Controversies concerning diagnostic guidelines for anomalies of the enteric nervous system: a report from the Fourth International Symposium on Hirschsprung's disease and related neurocristopathies
J Pediatr Surg
Calretinin and calbidin-D28k immunoreactivity in the human gastrointestinal tract
Gastroenterology
Functional intestinal obstruction on congenital neurogenic basis in infancy
Am J Dis Child
Cited by (25)
Advances and Pitfalls in the Diagnosis of Hirschsprung Disease
2020, Surgical Pathology ClinicsDiagnostic value of rectal suction biopsies using calretinin immunohistochemical staining in Hirschsprung's disease
2016, Journal of Pediatric SurgeryCitation Excerpt :With clinical follow-up for 6 months of patients with positive CIS only, thus far no false-negative cases have been identified. In the literature, in cases where the standard technique (both AChE and H&E staining) was used, the percentage of cases with inconclusive or equivocal results is not negligible [7,11]. Our study therefore supports the use of CIS as the primary method for the diagnosis of HD.
Diagnostic Pathology: Gastrointestinal
2015, Diagnostic Pathology: GastrointestinalCalretinin immunohistochemistry for the diagnosis of Hirschprung disease in rectal biopsies
2015, Pathology Research and PracticeCitation Excerpt :In the guideline prepared by the Gastro 2009 International Working Group, of which Kapur was a member, the phrases “aganglionic calretinin immunohistochemical pattern” and “normal calretinin immunohistochemical pattern” were added to the report format for rectal biopsies, however they mentioned that re-biopsy is required in the absence of ganglion cells, in spite of calretinin positivity, as there was, as yet, insufficient data [2]. There are a few other articles about the use of calretinin staining in HD in the English literature [8,17–24]. Calretinin is a calcium binding protein and it plays an important role in the organization and function of the CNS.
Early Detection and Intervention for Hirschsprung’s Disease: A Key to Successful Outcomes
2024, Clinical Medicine Insights: Case Reports
- ☆
This study is part of a PhD thesis in pathology prepared by Pedro Lourenção and presented at Botucatu Medical School, University of São Paulo State.
- ☆☆
The study did not receive support or funding from any organization.
- ★
The authors declare no conflicts of interest.