Image Processing and Analysis of Mucosal Calretinin Staining to Define the Transition Zone in Hirschsprung Disease: A Pilot Study

Najjar, Saleh; Ahn, Sangtae; Kasago, Israel; Zuo, Chunlai; Umrau, Kavita; Ainechi, Sanaz; Whyte, Christine; Sheehan, Christine E.; Homan, Suzanne M.; Lee, Hwajeong

doi:10.1055/s-0037-1618594

Cited by 10 publications

(17 citation statements)

References 25 publications

(37 reference statements)

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“…Aganglionosis should be excluded firstly because it is the most frequent congenital cause of constipation. We chose calretinin as a marker of mature/immature ganglion cells because it is believed to be the most reliable staining [5, 6, 8–13]. It can be particularly useful for less experienced pathologists.…”

Section: Discussionmentioning

confidence: 99%

Pathogenesis of chronic constipation in a Polish group of paediatric patients – an attempt to create the optimal histopathological diagnostic protocol

Szymańska

Markiewicz-Kijewska

Pyźlak

et al. 2019

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IntroductionHistopathological diagnosis of chronic constipation in children is difficult and time-consuming because the aetiology of the problem is heterogenous.AimTo create the optimal immunohistochemical (IHC) and histological diagnostic protocol using novel antibodies and assessing precisely their patterns.Material and methodsTwenty-eight paediatric patients were enrolled to the study. The study group consisted of the following: 9 patients with confirmed Hirschsprung’s disease (HD), 11 patients with desmosis of the colon (DC) (3) or with chronic constipation of unknown aetiology (3), and eight children operated on due to other problems. Retrospective analysis of full-thickness material from the large intestine was performed. In each specimen the number of ganglion cells was estimated per square millimetre as well as the number of submucosal and intramuscular ganglion cells per ganglion. The following IHC and histological stains were also performed: calretinin, CD117, picrosirius, and trichrome gomori. Patterns (nuclear vs. cytoplasmic vs. membranous) and intensity (strong vs. faint) of the stainings were analysed.ResultsThere was no statistically significant difference between groups while comparing the intensity and pattern of each staining, except HD (no staining due to lack of ganglion cells), p > 0.001. Calretinin was positive in each patient with ganglion cells; however, it did not unequivocally stain all cells identified in routine haematoxylin and eosin staining.ConclusionsCalretinin is helpful in identifying ganglion cells; however, it cannot replace an experienced paediatric pathologist. In children with chronic constipation it is worth obtaining a full thickness intestinal biopsy in order to perform additional histological and immunohistochemical stains starting with picrosirius red.

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Section: Discussionmentioning

confidence: 99%

Pathogenesis of chronic constipation in a Polish group of paediatric patients – an attempt to create the optimal histopathological diagnostic protocol

Szymańska

Markiewicz-Kijewska

Pyźlak

et al. 2019

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“…Pixel count (PC), defined as the percentage of calretinin-stained pixels in the mucosa, was calculated for each image using IPA similar to that in the previous study. 1 In this study, we attempted to exclude mast cells, which are usually round, from segmented calretinin-stained pixels as follows. For each connected cluster of calretinin-stained pixels, the ratio of the squared circumference and the area for the cluster was used to exclude potential mast cells (note that the ratio represents the degree of roundness).…”

Section: Hd and Tca Casesmentioning

confidence: 99%

“…5 We compared mucosal innervation of TZ in TCA and colorectal HD by quantifying calretinin-positive mucosal nerve fibers using image processing and analysis (IPA). 1 Although the proximal boundary of TZ was not defined by IPA, mucosal calretinin staining gradually increased in the distal TZ toward the possible proximal TZ/distal NZ of colorectal HD. 1 We hypothesize that the gradient of calretinin-positive mucosal nerve fibers in TZ of TCA may fall within the range of those in conventional colorectal HD.…”

Section: Introductionmentioning

confidence: 97%

“…1 Although the proximal boundary of TZ was not defined by IPA, mucosal calretinin staining gradually increased in the distal TZ toward the possible proximal TZ/distal NZ of colorectal HD. 1 We hypothesize that the gradient of calretinin-positive mucosal nerve fibers in TZ of TCA may fall within the range of those in conventional colorectal HD. Assuming that the length of TZ correlates with the gradient, we speculate the length of TZ in TCA is similar to that of conventional colorectal HD.…”

Section: Introductionmentioning

confidence: 97%

“…Its most probable etiology is an incomplete migration of neuroblasts from the neural crest to the colon as well as inadequate differentiation of neuroblasts to ganglion cells. 1 HD can be classified into 3 clinical groups based on the length of the aganglionic segment: ultra-short segment (limited to the distal 1 to 2 cm of the rectum), short segment (rectum and distal sigmoid), and long segment (extends proximal to the sigmoid, up to the splenic flexure). 2,3 Total colonic aganglionosis (TCA) is defined as aganglionosis involving the entire colorectum and up to 50 cm of the distal ileum.…”

Section: Introductionmentioning

confidence: 99%

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Transition Zone in Total Colonic Aganglionosis and Colorectal Hirschsprung’s Disease Shows a Similar Trend of Mucosal Innervation: Image Processing and Analysis Study

et al. 2019

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The aganglionic segment of bowel in Hirschsprung’s disease (HD) varies in length. It is not clear whether total colonic aganglionosis (TCA) merely represents a long form of HD or a different phenotype of the disease. Animal model studies suggest that TCA may have a longer transition zone (TZ) than conventional colorectal HD. We compared mucosal innervation of TZ in 2 TCA cases and 10 conventional colorectal HD cases by quantifying calretinin-positive mucosal nerve fibers using image processing and analysis. One TCA was associated with esophageal atresia-tracheoesophageal fistula, the other with trisomy 21. The gradients of calretinin-stained pixel count increase per distance from the beginning of TZ (slope) for TCA were not significantly different from those for the conventional HD group. Given this observation, it is speculated that the length of TZ in TCA may fall within the range of and may not be much longer than conventional colorectal HD.

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Increasing Trend of Calretinin-Positive Mucosal Innervation from Aganglionic Zone toward Transition Zone in Hirschsprung's Disease

et al. 2021

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Introduction Diagnosis of very short-segment Hirschsprung's disease (vsHD) by rectal suction biopsy is challenging as its aganglionic zone (AZ) overlaps with physiologic hypoganglionic zone and calretinin-positive mucosal nerves may extend from the transition zone (TZ) into AZ. We studied whether an increasing trend/gradient of calretinin-positive mucosal nerves along the distance from AZ toward TZ aids in diagnosis of HD. Materials and Methods In this study, 46 rectal suction biopsies from non-HD and HD, and 15 pull-through specimens from short-segment HD were evaluated by mucosal calretinin immunostain (CI) and image processing and analysis (IPA) to measure pixel count (PC, the percentage of calretinin stained pixels in the mucosa). Consecutive longitudinal sections of proximal AZ toward distal TZ in HD pull-through specimens were utilized as a vsHD surrogate model. First, we studied variability of mucosal CI in non-HD biopsies along the distance from dentate line. Second, we determined a cutoff point of mucosal CI by IPA that separated non-HD versus HD and applied this cutoff to longitudinal sections from proximal AZ to distal TZ segments in HD pull-through specimens. Third, we studied whether an increasing trend of mucosal CI was universally observed in HD pull-through. Results Our findings included a significant variability in PC along the biopsy distance in non-HD cases. Positive mucosal CI was found in proximal AZ in 6 (43%) of 14 HD pull-through, among which 1 case lacked submucosal nerve hypertrophy in the proximal AZ. All 14 HD pull-through cases showed an increasing trend/gradient of PC from AZ toward TZ. Conclusion Based on our findings, the presence or absence of mucosal CI positivity and submucosal nerve hypertrophy may not reliably diagnose vsHD in rectal suction biopsy. While we acknowledge that the density of mucosal innervation in variable contexts and anatomical locations is unknown and yet to be explored, our study suggests that an increasing trend of positive mucosal CI from AZ toward TZ by IPA might prove to be a useful tool for the diagnosis of vsHD in the future.

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Image Processing and Analysis of Mucosal Calretinin Staining to Define the Transition Zone in Hirschsprung Disease: A Pilot Study

Cited by 10 publications

References 25 publications

Pathogenesis of chronic constipation in a Polish group of paediatric patients – an attempt to create the optimal histopathological diagnostic protocol

Pathogenesis of chronic constipation in a Polish group of paediatric patients – an attempt to create the optimal histopathological diagnostic protocol

Transition Zone in Total Colonic Aganglionosis and Colorectal Hirschsprung’s Disease Shows a Similar Trend of Mucosal Innervation: Image Processing and Analysis Study

Increasing Trend of Calretinin-Positive Mucosal Innervation from Aganglionic Zone toward Transition Zone in Hirschsprung's Disease

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