Hirschsprung Disease and Use of Calretinin in Inadequate Rectal Suction Biopsies

From the Department of Pathology and Laboratory Medicine, Cleveland Clinic, Cleveland, Ohio.
Archives of pathology & laboratory medicine (Impact Factor: 2.84). 08/2013; 137(8):1099-102. DOI: 10.5858/arpa.2012-0220-OA
Source: PubMed


Context.-Up to 17% of all rectal suction biopsies performed in the evaluation of Hirschsprung disease are considered inadequate. In most instances, inadequate biopsies contain too little submucosa or are taken within the anal transition zone. Objectives.-To examine the utility of calretinin stain in the workup of inadequate biopsies for patients with clinical suspicion of Hirschsprung disease. Design.-A retrospective analysis was conducted of all rectal suction biopsies performed in the evaluation of Hirschsprung disease during the previous 12 years that were considered "inadequate." Seventeen cases were identified, and Hirschsprung disease status was determined by clinical or surgical follow-up. Immunohistochemistry for calretinin was performed for all cases containing columnar mucosa, which were evaluated without knowledge of clinical course. Results.-All 12 patients without Hirschsprung disease had calretinin-positive nerve fibers in the lamina propria or muscularis mucosae, and all 5 patients with Hirschsprung disease had no calretinin staining of nerves. Conclusions.-In this retrospective series, calretinin immunohistochemistry correctly predicted outcome in all instances. Although the gold standard for the diagnosis of Hirschsprung disease in rectal suction biopsies remains the evaluation of ganglion cells in a hematoxylin-eosin staining with sufficient submucosa, calretinin immunohistochemistry is quite helpful in triaging further workup based on clinical suspicion.

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    ABSTRACT: Background: Acetylcholinesterase (AChE) histochemistry on rectal mucosal biopsies accurately diagnoses Hirschsprung disease (HD), but is not widely employed as it requires special tissue handling and pathologist expertise. Calretinin immunohistochemistry (IHC) has been reported to be comparable to AChE staining with the loss of expression correlating with aganglionosis. Aim: The aim was to evaluate calretinin IHC as a primary diagnostic tool in comparison to the improvised rapid AChE technique in the diagnosis of HD. Materials and Methods: A total of 74 rectal biopsies (18 fresh frozen - 18 cases, 56 formalin fixed - 33 cases) from 51 cases of suspect HD were evaluated with hematoxylin and eosin/AChE/Calretinin. Ten biopsies each from ganglionated and aganglionated segments served as positive and negative controls. Ileal (3), appendiceal (3) and ring bowel (2) biopsies were also included. Two pathologists blinded to the clinical details evaluated the histomorphology with AChE and calretinin. Observations were statistically analyzed and Cohen's k coefficient employed to assess agreement between two pathologists and calretinin and the AChE. Results: The study confirmed HD in 26 and non-HD in 25 cases. There were 7 neonates, 5 low level biopsies and 14 "inadequate" biopsies. The results of calretinin were comparable with AChE with a statistically significant measure of agreement of k = 0.973 between the two. One false-positive case of HD was noted with calretinin. The advantages and disadvantages of calretinin versus AChE are discussed. Conclusion: Calretinin is a reliable single immune marker for ruling out HD by its specific positive mucosal staining of formalin fixed rectal biopsy. The improvised AChE staining remains indispensable to confirm HD on fresh biopsies and thus, along with calretinin IHC maximizes the diagnostic accuracy of HD in difficult cases.
    Indian Journal of Pathology and Microbiology 07/2014; 57(3):369-375. DOI:10.4103/0377-4929.138717 · 0.47 Impact Factor
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    ABSTRACT: Purpose: The diagnosis of Hirschsprung's disease (HD) remains challenging. The identification of ganglion cells is difficult and acetycholine esterase (AChE) staining can be subject to a great variability, particularly in the neonatal period (<8 weeks). Nerve trunks greater than 40 µm are considered to be predictive for HD. The aim of this study was to evaluate the usefulness of measuring nerve trunk size in the newborn with HD. Methods: Out of 292 biopsies 69 could be reanalyzed by three independent researchers. 40 µm was used as cutoff point for nerve trunk size. They were subdivided into three groups: (a) diagnosis of HD certain at the first biopsy, (b) no HD and (c) diagnosis of HD remains doubtful and re-biopsy taken. Results: In 87 % of group A nerve trunk size was ≥ 40 µm (SD 13.8). In 84 % of group B trunk size was < 40 µm (SD 16.2). In group C only 60 % of the patients showed a positive correlation between final diagnosis and nerve trunk size. Conclusion: Using 40 µm as the cutoff point gave 13 % false-negative and 16 % false-positive cases. Measurement of the nerve trunk in the neonatal period does not seem to be a reliable method for detecting HD.
    Pediatric Surgery International 07/2014; 30(8). DOI:10.1007/s00383-014-3541-7 · 1.00 Impact Factor
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    ABSTRACT: AimsDiagnosis of Hirschsprung's disease is based on aganglionosis and so far the presence of an increase in acetylcholinesterase-positive hypertrophic nerve fibres in the submucosa. However, acetylcholinesterase staining is laborious and requires a skilled technician. The aim of this study was to find a method to reliably diagnose Hirschsprung's disease using an immunohistochemical panel of recently proposed markers with diagnostic potential.Methods and results69 specimens from 37 patients were evaluated. Map2 and calretinin were shown to reliably stain ganglia in the submucosal and myenteric plexus of normal tissue. By contrast, reduced ganglia staining was observed in patients with Hirschsprung's disease. Glut1 and S100 were used to evaluate the number and thickness of nerve fibres. The gain of Glut1 and S100 was in contrast to the loss of calretinin and MAP2. Hypertrophic submucosal nerve fibres in Hirschsprung's disease develop a perineurium with a ring-like Glut1 staining similar in size and intensity to that observed in deeper subserosal tissueConclusions The diagnosis of Hirschsprung's disease using immunohistochemical panels could be as accurate as with conventional cryo-techniques. In particular, the comprehensive combination of markers for ganglia and hypertrophic nerve fibres highlighting a prominent perineurium in Hirschsprung's disease could be an alternative method.This article is protected by copyright. All rights reserved.
    Histopathology 08/2014; 66(6). DOI:10.1111/his.12527 · 3.45 Impact Factor
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