Calretinin immunostaining (grade 0) was negative at all levels in 19 cases. In 3 cases, a single sample from the proximity of transition zone showed a faint positive staining (grade I).
The immunostains for β-tubulin were grade I in 7 cases and grade II in 4 cases. Cytophotometric analysis showed an 89.99 mean gray value (range 55.59–164.81) with 90.9% concordance between visual score and instrumental analysis (Table 4).
The transition segment was represented by 2 subsequent samples in 9 cases and by 1 sample in 8 cases. It was characterized by a gradient of ganglion cells, from absent to normally represented Nerve trunks diameter ranged from 12.18 to 64.55 μm (mean 30.06 μm, median 27.13 μm; Table 2). Hypertrophy was evident in 7 cases, 5 of them showing also hypertrophic nerves (Table 3).
Calretinin immunostaining was completely negative (grade 0) in 2 cases and weakly positive (grade I) and strongly positive (grade II) in another 2 cases, respectively. In the remaining patients, a gradient was observed from negative/faint staining (grade 0 or grade I) to intense staining (grade II), along a single sample (5 cases) or along 2 subsequent samples (8 cases).
The immunostains for β-tubulin were grade I in 8 and II in 3 cases. Cytophotometric analysis showed a 101.95 mean gray value (range from 63.59 to 149.34) with 72.7% concordance between visual score and image analysis (Table 4).
All of the HE-stained sections were normally ganglionic. Nerve trunks diameter at proximal resection margin ranged from 8.70 to 45.33 μm (mean 23.70 μm median 23.62 μm; Table 2), hypertrophy was documented in a single trunk in 1 case, and no hyperplasia was observed (Table 3). In 5 cases, a single enlarged ganglium, containing >10 enteric neurons and exceeding 40 μm in diameter, was measured. Calretinin was positive in all patients as grade II at all levels in 14 cases and grade I/II in the 4 remaining cases.
Visual review for β-tubulin staining showed a grade I or II in 3 and 8 of 11 cases, respectively. Cytophotometric analysis showed a 96.86 mean gray value (range 61.82–155.95 with 100% concordance with image analysis (Table 4).
The 3 cases with TCA showed negative calretinin immunostaining (grade 0) in all samples from colon and ileum, except 1 case, which presented scattered positive fibres (grade I) both in the most proximal colonic and the most distal ileal samples. The transition segment was represented by ileal samples, which showed a faint calretinin staining (grade I) in 1 case and a gradient from negative (grade 0) to faint (grade I) and strong (grade II) in the other 2 cases. The proximal ganglionic segment was examined in only 2 patients and showed an intense calretinin staining (grade II) in all ileal samples.
β-Tubulin cytophotometric analysis was not performed on TCA specimens because at visual review, no staining was detected both at the level of nerve trunks and ganglion cells. Mean gray values measured from ganglionic ileum were comparable with those acquired from the ganglionic colon of patients with short-segment HD.
All specimens of the control group showed an intense calretinin immunostaining, whereas β-tubulin, performed only on 16 cases, had a mean gray value of 64.46 (range 43.57–111.01); the visual review showed grade II in 10 cases and grade I in 6. The concordance rate with image analysis was 87.5% (Table 4).
Age of patients at time of surgery ranged from 0.9 to 86.1 months (median 5.56, mean 9.10), and mean length of follow-up was 30 months. Transient constipation was the most common complication reported, affecting 10 of 22 (45.4%) patients, whereas soiling was encountered in 6 constipated patients (27.3% of all patients, 60% of constipated patients); 2 of these 6 patients developed also fecal impaction (9.1% of all patients, 20% of constipated patients). Enterocolitis occurred in 4 cases, 2 of them constipated (18.2% of all cases, 20% of constipated patients). None of the cases developed incontinence and those constipated patients who soiled improved with laxatives and enemas.
Statistical analysis was performed to verify the significance of differences of nerve trunks diameters in the 3 analyzed areas of HD specimens; association between nerve diameter and age of patients at time of surgery; differences of calretinin visual score in the 3 different innervated areas of HD specimens and in control bowel; differences of β-tubulin expression at cytophotometric analysis in the 3 innervated areas of resected HD and in control bowel; differences in immunohistochemical (calretinin based) and histological (HE based) length of the proximal (ganglionic) segment; and difference of proximal segment length between patients with postoperative intestinal dysmotility and patients with uneventful follow-up. Statistically significant results are summarized in Table 5.
The histological diagnosis of HD is often challenging, based on a negative finding, the absence of ganglion cells. Although acetylcholinesterase (AchE) staining plays a key role, the need for frozen material and the related technical difficulties represent an intrinsic limit of this diagnostic tool (26). Since its first description as a marker for normal ganglia (18), the use of calretinin in the diagnostic workup of HD has been tested in subsequent series of suction rectal biopsies and demonstrated a higher diagnostic accuracy than AchE, with fewer errors and equivocal findings. The evident positive staining in normal ganglia and intrinsic nerve fibers of the upper submucosae, muscolaris mucosae, and lamina propria, compared with its absence in aganglionic biopsies, is easily interpreted even by unaccustomed pathologists, making the diagnostic criteria more reproducible. Calretinin immunohistochemistry does not require additional frozen material, as for AchE, and can be performed on the same paraffin-embedded tissue used for HE-stained sections for direct correlation (19–22).
The present study further supports the usefulness of calretinin in the diagnostic workup of HD, showing completely negative immunostainings at almost all levels of the aganglionic segment and a strong and diffuse positive staining of ganglion cells and nerve fibers in the entire ganglionic segments, with a pattern of expression identical to normal controls. There were no relevant differences in calretinin pattern of expression between TCA and short-segment HD, and this suggests calretinin as a reliable marker of aganglionosis but not predictive of the involved length of bowel. A faint calretinin immunostaining limited to some nerve fibers, without staining of muscularis mucosae, lamina propria, nuclei, and cellular bodies of enteric neurons, was found in the most proximal part of the aganglionic segment, in 3 HD and 2 TCA samples, probably indicating the beginning of the transition zone. This is in agreement with previous reports, in which a weak calretinin staining was observed in rectal biopsies from extremely short aganglionic tract, leading to false-negative diagnoses (18,19).
The transition zone interposed between aganglionic and ganglionic segments is a critical area from a surgical point of view because it is not yet clearly established whether the presence of ganglion cells itself is a sufficient evidence of good intestinal innervation. For this reason, some authors suggest the use of additional investigations, as intraoperative rapid histoenzymatic/immunohistochemical stainings or determination of nerve hypertrophy, to obtain further anatomic information about the surgical margin (27–31). In our study, the presumptive transition zone was accurately studied with HE, calretinin, and morphometric analysis of nerve trunk diameter. Of the 17 short-segment HD cases in which the analysis could be performed, a staining gradient between aganglionic and ganglionic segment could be appreciated in 13 cases: transition from negative to strongly positive staining characterized 4 cases, a more gradual transition from faint to intense staining in the other 9. The evidence of weak calretinin expression in the transition zone, even in the presence of normal enteric neurons, and the stronger staining of the more proximal areas of colon support the hypothesis that the presence of ganglia alone is not a sufficient criterion to consider the bowel as normally innervated; the complete integrity and functional activity of the enteric nervous system may begin some centimeters more proximally to that expected by observing ganglion cells. This would further support the surgical practice of resecting a longer tract of colon than that suggested by leveling biopsies, which avoids the risk of including the transition area in the anastomosis (4,32).
The different definition of transition zone obtained by calretinin staining determines a different length of the proximal segment, moving proximally the limit between transition and normal bowel; in our cases, the mean length of proximal normal segment was 7.09 cm according to HE staining, but it was shorter, 5.33 cm, according to the calretinin staining. These observations suggest the higher sensitivity of calretinin than the histological features of aganglionosis in revealing the complex anomalies of enteric nervous system in HD.
Nerve hypertrophy, as suggested by Coe et al (11), was also investigated to understand whether it could represent a useful morphologic feature for the identification of the transition zone. A significant difference in size of nerve trunks was indeed observed at morphometric analysis; the nerve's diameter was progressively reduced from the distal aganglionic segment to the proximal ganglionic segment. This confirms the role of nerve hypertrophy in recognizing the transition zone, although the number of hypertrophied trunks rapidly reduced from aganglionic to transition segment, thus requiring an accurate examination to detect isolated and larger nerves.
Intestinal dysmotility observed in our patients with HD during the follow-up seems to be independent from the pathological features analyzed in the proximal segment: ganglion cells were fully represented, nerve hypertrophy and hyperplasia were not observed (only a single trunk exceeding 40 μm was found), and the presence of enlarged ganglia was not significantly associated with occurrence of intestinal dysfunctions. Moreover, lengths of proximal segment were comparable in all of the patients. Different from those investigated by Coe et al (11), our patients did not require further surgery: intestinal dysfunctions recorded during follow-up were controlled or solved by medical therapy, such as oral laxatives or enemas for constipation and endovenous antibiotics for enterocolitis. The intestinal dysmotility, encountered in patients who received accurate pathological analysis and correct surgical treatment, could be the result of an underlining dysfunction of enteric innervation, not detectable at histology.
In spite of promising results demonstrated by other proteins associated with cytoskeleton (23), β-tubulin expression was not a reliable marker in HD, although the reduced expression compared to the normal colon found by cytophotometry in submucosal nerve trunks at all levels in HD was detectable also at visual analysis and easily reproducible. Its use may confirm the diagnosis of HD, but it does not add information to the identification of the transition zone. Nevertheless, the reduced β-tubulin immunostaining in neural trunks of submucosal plexus suggests an alteration at inner cytosketelal structures of neural cells in HD, disclosing the possibility this may represent the effect of a failure in the dynamic control of microtubules in these axons.
The Goldberg-Shprintzen syndrome, a syndrome associated with HD that includes megacolon, facial dysmorphisms, microcephaly, and disruption of white matter tracts (33,34), is caused by a mutation kinesin–binding protein, a tubulin isotype implied in microtubular transport and dynamics. Similar to what happens in the cerebral cortex (35,36), the enteric neurons may require a precise microtubular activity to guide migration and setting of different precursors in 2 concentric layered plexi. The diffuse microtubular defect that involves the whole colon could deeply affect the specific population of neural crest cells assigned to the future aganglionic tract, being the cytoskeletal integrity, a developmental or survival requirement for that spatial-related precursor population. In proximal, although ganglionic colon, the same β-tubulin deficiency could play a role in postsurgical intestinal motility disorders as a weakening factor of bowel neuromuscular transmission.
In conclusion, our study confirms the diagnostic use of calretinin in HD, showing a specific pattern of immunostaining in differently innervated areas. Moreover, it can help to identify the transition zone before surgery. β-Tubulin seems, at the moment, to be a less reliable marker, showing only a minor intensity of staining compared with normal controls. Nonetheless, its diffusely weak staining at all levels in HD supports the hypothesis that other factors, such as cytoskeleton protein impairment, may be involved in the pathogenesis of HD and contribute to the occurrence of postsurgical intestinal dysfunction.
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Keywords:© 2013 by European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and North American Society for Pediatric Gastroenterology,
β-tubulin; calretinin; Hirschsprung; transition zone