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%).
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).
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).
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).
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.
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.
Conflicts of interest
There are no conflicts of interest.
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