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Effectiveness of calretinin immunohistochemistry with digital morphometry in mapping of different segments of Hirschsprung disease

Abou Gabal, Hoda H.; Osman, Wesam M.; Abd El Atti, Rasha M.

Egyptian Journal of Pathology: July 2016 - Volume 36 - Issue 1 - p 9–18
doi: 10.1097/01.XEJ.0000482435.40584.bd
ORIGINAL ARTICLES
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Background Definitive diagnosis of Hirschsprung disease (HD) is exclusively based on histological examination of rectal biopsies. Among the immunohistochemical markers, calretinin appears to be an encouraging marker for HD diagnosis. Calretinin expression needs to be more elaborated in the transition zone.

Aim This work evaluates the usefulness of calretinin immunostaining with morphometric assessment of the nerve caliber in the diagnostic workup of HD, essentially the transitional zone.

Materials and methods A retrospective study including 25 HD patients and 10 control full-thickness colonic biopsies was conducted. Different sections representing each segment were evaluated by calretinin immunostaining and digital morphometry.

Results A highly significant difference was demonstrated between aganglionic, transitional, and ganglionic areas as regards submucosal and myenteric nerve fiber caliber (P=0.001). There was overlap of nerve caliber in the transitional zone and the ganglionic segment. The cutoff value for submucosal and myenteric nerve caliber to delineate the transitional zone from the ganglionic area was ≥34.5 and ≥28.4 μm, respectively, and that from the aganglionic area was ≤62.25 and ≤80 μm, respectively. Calretinin immunostaining of ganglion cells and nerve fibers disclosed highly significant differences between aganglionic and ganglionic segments and between aganglionic and transitional segments (P=0.001), whereas there was no significant difference between transitional and ganglionic segments in submucosal and myenteric innervations by calretinin staining (P>0.05).

Conclusion Morphometric analysis with calretinin immunohistochemical study is a better method for precisely characterizing the transitional zone and easily delimiting the proximal margin of the transitional zone, which is considered a critical issue in surgery.

Department of Pathology, Faculty of Medicine, Ain Shams University, Cairo, Egypt

Correspondence to Hoda H. Abou Gabal, MD, Department of Pathology, Faculty of Medicine, Ain Shams University, 11566 Cairo, Egypt Fax: +202 26847824; e-mail: abougabalhoda@yahoo.com

Received November 15, 2015

Accepted November 30, 2015

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Introduction

Hirschsprung disease (HD) is a congenital malformation of the enteric nervous system in which the obligate diagnostic feature is absence of intrinsic ganglion cells in the submucosal and myenteric intestinal plexus from the distal rectum and a variable length of contiguous bowel. HD affects 1 : 5000 liveborns and is considered in differential diagnoses of patients with severe and/or chronic constipation at any age, especially the pediatric age (Kapur, 2006).

HD is thought to be related to the mutations of multiple genes, including the ret proto-oncogene that affects the development and craniocaudal migration of ganglion cells in the colon by 12 weeks of gestation; they are also implicated in early ganglion cell death (Uesaka et al., 2008). Decreased expression of Ca2+ channel receptors was also implied in the pathogenesis (Piotrowska et al., 2003). In ∼70% of cases, the aganglionic segment involves the rectum and the sigmoid colon only (classical HD), whereas in 20% of cases the aganglionic segment involves the more proximal bowel, with the total colon being aganglionic in 8–10% (Kacar et al., 2012).

A definite diagnosis of HD is exclusively based on histological examination of rectal biopsies in which the typical histological features of HD include the absence of ganglion cells and increased numbers of hypertrophic nerves (Barshack et al., 2004; Martucciello et al., 2005; De Lorijn et al., 2006). Standard histopathology obtained from the rectal suction biopsy, which is increasingly becoming the procedure of choice for obtaining specimens for the initial diagnosis of HD, demands the examination of numerous serial sections (up to 100) (Guinard-Samuel et al., 2009; Rahman et al., 2010), which is time-consuming. In addition, obstacles are encountered in certain circumstances when the biopsy is either too superficial with inadequate submucosa or too distal with physiological hypoganglionosis or when there is difficulty in identifying the ganglion cells unequivocally, especially in neonates (Guinard-Samuel et al., 2009; Hiradfar et al., 2012).

As a result, a number of ancillary methods have been introduced. Ease of diagnosis has been improved with the use of acetylcholinesterase histochemistry (ACHE staining), which has been widely adopted as an ancillary technique (Meier-Ruge and Bruder, 2008). However, drawbacks such as the need for frozen material, related technical and interpretive difficulties, and lack of sensibility in neonates are encountered (Guinard-Samuel et al., 2009).

Several immunohistochemical markers such as S-100 protein, neurone-specific enolase 14, or glial fibrillar acid have been tried, but none have been shown to be superior to acetylcholinesterase or to exceed the accuracy of hematoxylin and eosin (HE) morphology (Martucciello et al., 2005; Kapur, 2006; Guinard-Samuel et al., 2009). Among the different immunohistochemical markers, calretinin appears to be an encouraging marker for the diagnosis of HD (Barshack et al., 2004; Guinard-Samuel et al., 2009; Alexandrescu et al., 2013). Calretinin is a vitamin D-dependent calcium-binding protein involved in calcium signaling, which has an important role in the organization and functioning of the central nervous system (Baimbridge et al., 1992; Barshack et al., 2004).

The transitional zone features altered density and nonuniform distribution of enteric neurons (Volpe et al., 2013). Accordingly, the calretinin expression needs to be more elaborated in the transition zone to aid in its delineation as the recognition of the discordant, subtle histologic features in this zone needs an experienced pathologist. The persistence of a residual transitional zone is a possible cause of postoperative dysmotility, with subsequent revisional pull-through operation manifesting disappointing results (Ghose et al., 2000; Langer, 2004; Kapur and Kennedy, 2013).

This study evaluates the putative usefulness of calretinin immunostaining on surgical specimens of HD in conjunction with morphometric assessment of the nerve caliber in the diagnostic workup of HD with special emphasis on their role in characterizing the transitional zone.

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Materials and methods

This is a retrospective study of 25 patients with confirmed histopathologic diagnosis of HD who underwent surgical colonic resection (pull-through specimens). Cases were retrieved from pathology labs of Ain Shams University Hospitals, Cairo, Egypt, during the period from January 2013 until January 2015. The control group included 10 colonic specimens of full-thickness biopsies in which the diagnosis of HD was excluded (matched for age and sex) and colonic surgical margins of the resected tumors. Clinical data were obtained from the patients’ medical records and included age and sex. The study was carried out with full local ethical approval.

The pull-through specimens were previously sampled as per the following protocol. They were opened longitudinally. For each case at least one or two representative blocks containing multiple longitudinal full-thickness sections across the circumference – from ganglionic, transitional, and aganglionic segments – were submitted. Additional blocks were taken when available with the orientation maintained from proximal to distal. In addition, a cross-section of the proximal and distal margin was submitted en face for each specimen. The transitional zone was defined as the segment between the contracted aganglionic segment and the normal or dilated ganglionated bowel, as designated by the surgeon (Barshack et al., 2004; Knowels et al., 2009; Volpe et al., 2013). The formalin-fixed paraffin-embedded blocks and the 4-μm-thick HE-stained serial sections were retrieved from the entire specimens of all the cases.

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Histopathological and morphometric analysis

The retrieved HE-stained serial sections and the extra deeper serial sections of each case from the different segments were carefully reviewed for the presence or absence of ganglion cells in the submucosa and myenteric plexus. Morphometric assessment of nerve trunk caliber in the submucosa and myenteric plexus was carried out. In each examined slide, five well-representative fields were evaluated at magnification ×200, and the nerve trunk caliber was measured by estimating the maximal nerve thickness perpendicular to the long axis (Kapur, 2014) from a digital image using a computerized image analysis system [an Olympus microscope (Cx51) equipped with an Olympus camera using SIS software (Olympus, Tokyo, Japan)].

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Calretinin immunohistochemical staining and interpretation

For all specimens, 4-µm-thick sections of paraffin-embedded tissue were prepared on positively charged slides and subjected to calretinin immunohistochemical staining. The paraffin sections were deparaffinized in xylene and rehydrated through graded ethanol solutions to distilled water. Antigen retrieval was performed using high-temperature treatment in citrate buffer for 15 min in a microwave oven; the sections were then left to cool for 20 min. Endogenous peroxidase activity was blocked by adding 2–3 drops of 3% H2O2 in methanol for 5 min. Protein blocking was carried out using protein blocking serum for 10 min. Thereafter, the slides were incubated for 1 h with the primary antibody at room temperature using the mouse monoclonal antibodies against calretinin (Calret 1; Dako, Glostrup, Denmark) followed by rinsing in PBS (pH=7.6). This was followed by the secondary biotin-conjugated antibody for 30 min and finally peroxidase-conjugated streptavidin for another 20 min. Diaminobenzidine tetrachloride (freshly prepared) was added for 25 min, then counterstained in Harris hematoxylin, followed by dehydration, clearing, and mounting. Negative controls were obtained by excluding the primary antibody and replacing it with a nonimmune antibody. Positive calretinin reaction in the mast cells served as the internal positive control.

Calretinin immunostaining was scored as either positive or negative (Barshack et al., 2004; Guinard-Samuel et al., 2009). Calretinin immunoreactivity and pattern of staining for ganglion cells and also nerve fibers in different layers of the bowel, lamina propria, submucosa, and muscularis propria were evaluated separately for positivity for calretinin.

The histopathological evaluation, morphometric analysis, and results of the immunohistochemical staining were assessed independently by each author and then a consensus was reached with a multiheaded microscope.

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Statistical analysis

The collected data were revised, coded, tabulated, and entered into a PC using the Statistical Package for Social Science (SPSS 15.0.1 for Windows, 2001; SPSS Inc., Chicago, Illinois, USA). Quantitative nonparametric variables are expressed as mean and SD. Qualitative variables are expressed as frequencies and percentages. The analysis of variance test was used to compare a continuous variable between more than two study groups. The χ2-test and Fisher’s exact test were used to examine the relationship between categorical variables. The receiver operating characteristic curve was used to evaluate the sensitivity and specificity of nerve caliber in the prediction of the transitional zone. P-values less than 0.05 were considered statistically significant.

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Results

This retrospective study included 25 patients with a confirmed diagnosis of HD. Among the 25 studied patients, 17 (68%) were male and eight (32%) were female, with a proportion of 2.1/1. Their ages ranged from 0.25 to 120 months, with a median of 9 months. Among the 10 control cases, five (50%) were male and five (50%) were female, with a proportion of 1/1. Their ages ranged from 4 to 60 months, with a median of 15 months.

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Hematoxylin and eosin results

Histopathological examination

Analysis of HE-stained serial sections of the distal segment of HD patients (25 cases) revealed the absence of ganglion cells in both the submucosa and the muscularis propria in all cases. The histologic evaluation of the control cases (10 cases) and the proximal segment of the HD patients (25 cases) showed the presence of ganglion cells in the nerve plexus in all cases. The transition segment featured sparse ganglion cells in 11 cases and absence of ganglion cells in four cases, and 10 cases were inconclusive for the presence of ganglion cells.

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Morphometric analysis

In the HE-stained sections, no significant difference was demonstrated between the control group and ganglionic segment as regards submucosal and myenteric nerve fiber caliber (P>0.05) (data not tabulated). On the other hand a highly significant difference was demonstrated between aganglionic, transitional, and ganglionic areas as regards submucosal and myenteric nerve fiber caliber (P=0.001). The nerve caliber was significantly decreased from the distal aganglionic segment to the transitional zone to the proximal ganglionic segment (Figs 1–3). There was overlap of nerve caliber measurements in the transitional zone with those found in the ganglionic segment. The maximum diameter of submucosal nerve fiber was 112.5, 61.5, and 39 μm in the aganglionic, transitional, and ganglionic areas, respectively, whereas the maximum diameter of the myenteric plexus was 135, 70, and 25.5 μm in the aganglionic, transitional, and ganglionic zones, respectively (Table 1).

Fig. 1

Fig. 1

Fig. 2

Fig. 2

Fig. 3

Fig. 3

Table 1

Table 1

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Analysis of variance

Nerve fiber calibers ≥50 and ≥59 μm in the submucosal and myenteric nerve plexus, respectively, were considered to be highly significant differentiating cutoff values between aganglionic and ganglionic areas (data not tabulated). Further, the best discriminating cutoff value for the submucosal and myenteric nerve caliber to delineate the transitional zone from the ganglionic area was ≥34.5 and ≥28.4 μm, respectively, and that from the aganglionic area was ≤62.25 and ≤80 μm, respectively (Tables 2 and 3, Figs 4 and 5).

Table 2

Table 2

Table 3

Table 3

Fig. 4

Fig. 4

Fig. 5

Fig. 5

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Immunohistochemical results for calretinin

Control group and ganglionic segments of patients with HD: Calretinin immunostaining in nerve fibers and ganglion cells showed no significant differences between the control group and ganglionic segments of patients with HD (P>0.05) (data not tabulated). There was granular nuclear and cytoplasmic staining of ganglion cells in the submucosa and the muscularis propria layers in both the control group and the ganglionic bowel areas (100%) (Fig. 6). Also, calretinin was expressed in a linear granular nonhomogenous pattern in the lamina propria nerve fibrils and in the submucosal and muscularis propria nerve plexus without skip areas (Fig. 6). Only one case showed negative immunostaining in the lamina propria (92.3%).

Fig. 6

Fig. 6

In the aganglionic segments: Calretinin was negative at all levels in all cases (100%). No ganglion cells were demonstrated by calretinin. Only a few cells, mostly mast cells, expressed calretinin in a homogenous nongranular pattern (Fig. 7).

Fig. 7

Fig. 7

In the transitional zone: Calretinin immunopositivity was demonstrated in ganglion cells in 18/25 cases. There was either linear or punctate immunostaining in the lamina propria nerve fibrils and in the submucosal and muscularis propria nerve trunks with or without skip areas in 19/25, 21/25, and 21/25 cases, respectively (Fig. 8).

Fig. 8

Fig. 8

Calretinin immunostaining of ganglion cells and submucosa, myenteric, and lamina propria innervations disclosed a highly significant difference between aganglionic and ganglionic segments and between aganglionic and transitional segments (P=0.001), whereas no significant differences were identified between transitional and ganglionic segments in terms of submucosal and myenteric innervations by calretinin (P>0.05) (Table 4).

Table 4

Table 4

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Discussion

HD is a common congenital intestinal disorder with absence of ganglion cells in the colonic wall. The diagnosis is based on clinical data, imaging studies, and histopathological features (Kapur, 2009; Anbardar et al., 2015). Despite the seeming anatomic simplicity of this condition, HE-based diagnosis of HD can be a stressful experience, especially in suction rectal biopsies (Kapur, 2006; Guinard-Samuel et al., 2009).

The reliability of distinguishing between the presence or absence of ganglion cells can be complicated by the fact that submucosal ganglions are relatively sparse and widely distributed, and thus adequate sampling is critical (Kapur, 2006). The presence of immature ganglion cells that are confused with endothelial cells and plasma cells is often cited as a difficulty associated with HE-based diagnosis (Kapur, 2006; Kacar et al., 2012).

As a consequence, a search has continued for new simple, less time-consuming, and reliable diagnostic methods, concentrating on immunohistochemical studies (Kapur, 2006; Burtelow and Longacre, 2009). Several immunohistochemical markers have been introduced, but the most promising one is calretinin (Barshack et al., 2004; Guinard-Samuel et al., 2009; Kacar et al., 2012). Calretinin is a vitamin D-dependent calcium-binding protein involved in the physiological buffering of excess cytosolic calcium ions, calcium transport, and protection against calcium overload. In its absence, there is an accumulation of excess cytosolic calcium ions, causing hyperexcitability and often neurodegeneration (Baimbridge et al., 1992; Barshack et al., 2004).

Calretinin immunostaining was first tested by Barshack et al. (2004) (cal1) in HD patients; calretinin was not expressed in aganglionic segments of HD, whereas in ganglionic HD segments and in normal colon both ganglion cells and nerve fibers were immunopositive for calretinin. Immunohistochemical staining for calretinin was found to show perfect concordance when compared with the gold standard (HE-stained section) (Guinard-Samuel et al., 2009) and to be highly sensitive and specific in diagnosing HD. Calretinin showed 100% specificity and more than 90% sensitivity in determining aganglionic segments in HD patients (Anbardar et al., 2015) (cal6). Also, calretinin is more accurate than acetyl cholinesterase and can be an alternative for it in evaluating either full-thickness or suction rectal biopsies of HD (Guinard-Samuel et al., 2009; Kapur et al., 2009; Kacar et al., 2012; Małdyk et al., 2014). Calretinin immunostaining has binary pattern of interpretation (either positive or negative), making the diagnostic criteria of HD more distinctive and reproducible (Barshack et al., 2004; Guinard-Samuel et al., 2009; Anbardar et al., 2015).

The present study further emphasizes the previous results as regards the beneficial role of calretinin immunohistochemistry in the diagnostic workup of HD. A highly significant difference between ganglionic and aganglionic segments was detected as regards calretinin immunoreactivity. There was intense granular nuclear and cytoplasmic staining of the ganglion cells. Immunoreactive nerve fibers in almost all the layers (lamina propria, submucosa, and muscularis propria) featured a linear granular heterogenous staining pattern in the ganglionic segment with a pattern of expression similar to that of the control group. Similar to Kacar et al. (2012), a continuous pattern of calretinin staining without skip area in the lamina propria and submucosa was demonstrated in the present work, a useful feature that would overcome problems associated with small superficial biopsies. Only one case showed negative calretinin immunoreactivity in the lamina propria similar to that seen by Hiradfar et al. (2012) and Anbardar et al. (2015), which shows the importance special attention being paid to the submucosal layer in rectal suction biopsies. However, Alexandrescu et al. (2013) together with Gonzalo and Plesec (2013) have postulated the usefulness of calretinin immunostaining in inadequate rectal suction biopsies with insufficient submucosa depending on interpretation of the lamina propria.

As regards the aganglionic segment, the current study demonstrated absence of ganglion cells together with negative calretinin reactivity in the nerve fibers at all the bowel layers in all the cases, similar to the study by Barshack et al. (2004). The presence of calretinin immunopositivity in a few non-neuronal cells, mostly mast cells, added another dimension for calretinin usage, by acting as a positive internal control (Anbardar et al., 2015). Other studies additionally extrapolated a faint calretinin staining restricted to some large nerve fibers either in the submucosa or in the muscularis propria in the absence of ganglion cells and without the staining of lamina propria nerve fibrils (Barshack et al., 2004; Alexandrescu et al., 2013; Volpe et al., 2013), or even faint staining at the mucosal level in the study by Kapur (2014). The authors attributed such unexpected faint positivity in the aganglionic segment to the beginning of the transition zone in the short-segment HD leading to a possible diagnostic pitfall and false-negative diagnosis.

This work further supports the emerging evidence that calretinin immunostaining is a valuable ancillary technique for determining aganglionosis, reducing the need for either repeated biopsies, full-thickness biopsies, or serial sectioning of blocks. However, owing to the heterogenous nature of HD, the use of calretinin immunohistochemistry in particular settings such as transitional zone assessment remains to be uncovered.

Incomplete resection of the transitional zone between the ganglionic and the aganglionic bowel in HD is a putative cause of postoperative functional consequences, with symptoms of constipation, diarrhea, and incontinence (Ghose et al., 2000; Kapur and Kennedy, 2012). Hence, determination of the transitional zone is crucial from the surgical point of view.

Few studies have investigated the calretinin expression in the transitional zone; Barshack et al. (2004) showed a broad spectrum of calretinin immunohistochemical patterns with calretinin positivity in most of the ganglions (10/13) and nerve fibers (12/13), with partial focal staining. In a study by Volpe et al. (2013), a staining gradient between aganglionic and ganglionic segments in 13/17 cases ranged from negative/faint to strongly positive.

The present study was not dissimilar from the above-mentioned studies, as calretinin immunopositivity was exhibited in ganglion cells (which were sparse) in 18/25 cases. Linear or punctate immunostaining in the lamina propria nerve fibrils and submucosal and muscularis propria nerve trunks with or without skip lesions was identified. The statistical analysis disclosed a highly significant difference between aganglionic and transitional segments. Thus, absent calretinin staining strongly supports the diagnosis of the aganglionic segment. In contrast, no significant differences were identified between transitional and ganglionic segments regarding the submucosal, myenteric, and lamina propria calretinin expression, in contrast to the observations of Volpe et al. (2013), who appreciated significant differences in calretinin visual scores between transitional and proximal ganglionic segments of HD. Such a discrepancy was attributed to the use of different methodologies. This work categorized calretinin immunohistochemical results as positive or negative regardless of the staining intensity in order to reduce the subjectivity and render the evaluation simple.

In our study only some ganglion cells were immunoreactive for calretinin, whereas relatively more nerve fibers demonstrated positivity. This could be explained by the possibility that those positive nerve fibers represented connections to the proximal calretinin-positive ganglion cells (Barshack et al., 2004). The presence of calretinin-positive nerve fibers alone is not sufficient to consider the examined bowel as normally innervated, especially in the presence of skip areas along with the absence of ganglion cells in areas suspicious to be the transitional zone. Another scenario that could be encountered in the transitional zone is the presence of weak/strong calretinin expression in nerve fibers even in the presence of normally innervated ganglion cells. It has not yet been clearly settled whether the presence of normal ganglion cells is sufficient evidence of good innervation as the complete functional activity of the enteric nervous system may begin some centimeters more proximally to that expected by observing normally innervated ganglion cells. This may further support resecting a longer tract of colon, which may avoid the risk of including the transitional area in the anastomosis (White and Langer, 2000; Kapur, 2009; Volpe et al., 2013).

Thus, as seen in our results, calretinin immunostaining confers relative limitations in delineating the transitional zone from the ganglionic segment; therefore, morphometric analysis is strongly necessary to determine the nerve hypertrophy to delineate the transitional zone.

The current work revealed significant differences between aganglionic, transitional, and ganglionic segments as regards the mean submucosal and myenteric nerve fiber calibers. The nerve caliber was significantly decreased from the distal aganglionic segment to the transitional zone to the proximal ganglionic segment. This could constitute a useful morphologic feature for distinguishing the transition area as suggested by Coe et al. (2012), Kapur and Kennedy (2012), and Volpe et al. (2013).

In the ganglionic segment, the maximum diameter of the submucosal nerve fiber was 39 μm, with a mean of 29.08 μm, compared with the studies by Monforte-Munoz et al. (1998), Bandyopadhyay et al. (2000), Yutaka Kakita et al. (2000), and Volpe et al. (2013), who found the maximum diameter to be 32, 42.84, 50, and 45.8 μm, respectively. In the aganglionic segment, submucosal thicker nerves dominated, with the maximum diameter reaching 112.5 μm, with a mean of 85.88 μm. Similar to our results, Bandyopadhyay et al. (2000) showed the maximum nerve trunk to be 120 μm. Yutaka Kakita et al. (2000) detected maximum diameter measurements of 150 and 100 μm in the resection and biopsy specimens, respectively, whereas the maximum diameter was 84.6 μm in another study by Volpe et al. (2013). Morphometric studies by Monforte-Munoz et al. (1998) and Kapur (2014) reported that submucosal nerve caliber greater than 40 μm aids in the detection of the aganglionic segment, whereas Bandyopadhyay et al. (2000) indicated a value greater than 45 μm. The present work implied a submucosal nerve caliber of at least 50 μm to be a differentiating cutoff value between the ganglionic and aganglionic segments, in agreement with Yutaka Kakita et al. (2000).

As regards the transitional zone in the current work, there was overlap of nerve caliber measurements with those found in the ganglionic segment. But the thicker nerve fibers were encountered in the transitional zone at which the maximum diameter was 61.5 μm with a mean of 48.48 μm, more or less similar to other studies by Yutaka Kakita et al. (2000) and Volpe et al. (2013), with maximum diameters of 60 μm in most cases and 64.55 μm, respectively. Owing to such recorded overlap between ganglionic and transitional segments, introducing the discriminating cutoff values to possibly map the transitional zone from ganglionic and aganglionic segments is essential and one should not rely only on cutoff values between ganglionic and aganglionic segments. Thus, in this study, the receiver operating characteristic curve disclosed that nerve fiber caliber of at least 34.5 μm in the submucosa was a highly significant differentiating cutoff value between transitional and ganglionic areas. Moreover, the best discriminating cutoff value for the submucosa to delineate the transitional zone from the aganglionic area was 62.25 μm or less.

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Conclusion

Morphometric analysis along with calretinin immunohistochemistry should be adopted to precisely characterize the transitional zone and easily delimit the proximal margin of the transitional zone, which is considered a critical surgical issue.

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Acknowledgements

Conflicts of interest

There are no conflicts of interest.

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