Image Processing and Analysis of Mucosal Calretinin Staining to Define the Transition Zone in Hirschsprung Disease: A Pilot Study
08 October 2017
06 December 2017
05 January 2018 (eFirst)
Purpose Quantification of calretinin-stained mucosal nerve fibers by image processing and analysis (IPA) may objectively define the transition zone (TZ) of Hirschsprung disease (HD). We tested the utility of IPA as an adjunctive tool in HD.
Materials and Methods Calretinin immunostain was performed on 15 HD pull-through specimens, and multiple images were captured from the proximal aganglionic zone, TZ, and probable normal zone (NZ). Pixel count (PC), defined as the percentage of brown-stained pixels in the mucosa, was quantified and plotted against distance from the rectal distal end. To validate the method, PCs from 45 images were compared with three-tiered visual scoring by five pathologists. Results were correlated against pertinent variables, which were retrieved from the clinical record.
Results The PC gradually increased in the TZ toward the proximal resection margin in 10/13 (77%) cases. The PC variation in the probable NZ and around the circumference was substantial by the coefficient of variation. The mean PC of images with a visual score of 1 was less than scores of 2 and 3 by all five (100%) pathologists (p < 0.01). One patient had possible TZ pull-through that was clinically confirmed.
Conclusion While the mucosal calretinin staining gradually increases in the TZ, for now, the boundaries of the TZ cannot be accurately defined by mucosal biopsies given the substantial variation of staining around the circumference at the same distance and in the NZ. However, the IPA technique does provide a continuous variable and warrants further utility in HD studies.
- 1 Suita S, Taguchi T, Ieiri S, Nakatsuji T. Hirschsprung's disease in Japan: analysis of 3852 patients based on a nationwide survey in 30 years. J Pediatr Surg 2005; 40 (01) 197-201
- 2 Best KE, Addor MC, Arriola L. , et al. Hirschsprung's disease prevalence in Europe: a register based study. Birth Defects Res A Clin Mol Teratol 2014; 100 (09) 695-702
- 3 Ieiri S, Suita S, Nakatsuji T, Akiyoshi J, Taguchi T. Total colonic aganglionosis with or without small bowel involvement: a 30-year retrospective nationwide survey in Japan. J Pediatr Surg 2008; 43 (12) 2226-2230
- 4 Fu M, Tam PK, Sham MH, Lui VC. Embryonic development of the ganglion plexuses and the concentric layer structure of human gut: a topographical study. Anat Embryol (Berl) 2004; 208 (01) 33-41
- 5 McKeown SJ, Stamp L, Hao MM, Young HM. Hirschsprung disease: a developmental disorder of the enteric nervous system. Wiley Interdiscip Rev Dev Biol 2013; 2 (01) 113-129
- 6 Khan AR, Vujanic GM, Huddart S. The constipated child: how likely is Hirschsprung's disease?. Pediatr Surg Int 2003; 19 (06) 439-442
- 7 De Lorijn F, Reitsma JB, Voskuijl WP. , et al. Diagnosis of Hirschsprung's disease: a prospective, comparative accuracy study of common tests. J Pediatr 2005; 146 (06) 787-792
- 8 Teitelbaum DH, Cilley RE, Sherman NJ. , et al. A decade of experience with the primary pull-through for Hirschsprung disease in the newborn period: a multicenter analysis of outcomes. Ann Surg 2000; 232 (03) 372-380
- 9 Kapur RP, Kennedy AJ. Transitional zone pull through: surgical pathology considerations. Semin Pediatr Surg 2012; 21 (04) 291-301
- 10 Kapur RP. Histology of the transition zone in Hirschsprung disease. Am J Surg Pathol 2016; 40 (12) 1637-1646
- 11 Brehmer A, Croner R, Dimmler A, Papadopoulos T, Schrödl F, Neuhuber W. Immunohistochemical characterization of putative primary afferent (sensory) myenteric neurons in human small intestine. Auton Neurosci 2004; 112 (1–2): 49-59
- 12 Kapur RP, Reed RC, Finn LS, Patterson K, Johanson J, Rutledge JC. Calretinin immunohistochemistry versus acetylcholinesterase histochemistry in the evaluation of suction rectal biopsies for Hirschsprung disease. Pediatr Dev Pathol 2009; 12 (01) 6-15
- 13 Morris MI, Soglio DB, Ouimet A, Aspirot A, Patey N. A study of calretinin in Hirschsprung pathology, particularly in total colonic aganglionosis. J Pediatr Surg 2013; 48 (05) 1037-1043
- 14 Kapur RP. Submucosal nerve diameter of greater than 40 μm is not a valid diagnostic index of transition zone pull-through. J Pediatr Surg 2016; 51 (10) 1585-1591
- 15 Coe A, Collins MH, Lawal T, Louden E, Levitt MA, Peña A. Reoperation for Hirschsprung disease: pathology of the resected problematic distal pull-through. Pediatr Dev Pathol 2012; 15 (01) 30-38
- 16 Swaminathan M, Kapur RP. Counting myenteric ganglion cells in histologic sections: an empirical approach. Hum Pathol 2010; 41 (08) 1097-1108
- 17 Gonzalo DH, Plesec T. Hirschsprung disease and use of calretinin in inadequate rectal suction biopsies. Arch Pathol Lab Med 2013; 137 (08) 1099-1102
- 18 Volpe A, Alaggio R, Midrio P, Iaria L, Gamba P. Calretinin, β-tubulin immunohistochemistry, and submucosal nerve trunks morphology in Hirschsprung disease: possible applications in clinical practice. J Pediatr Gastroenterol Nutr 2013; 57 (06) 780-787
- 19 Kovach AE, Pacheco MC. Ganglion cells are frequently present in pediatric mucosal colorectal biopsies. Pediatr Dev Pathol 2017; DOI: 10.1177/1093526617704594.
- 20 Rinner O, Gegenfurtner KR. Time course of chromatic adaptation for color appearance and discrimination. Vision Res 2000; 40 (14) 1813-1826
- 21 Byrne A, Hilbert DR. Color realism and color science. Behav Brain Sci 2003; 26 (01) 3-21
- 22 Nassar A, Cohen C, Agersborg SS. , et al. Trainable immunohistochemical HER2/neu image analysis: a multisite performance study using 260 breast tissue specimens. Arch Pathol Lab Med 2011; 135 (07) 896-902
- 23 Lohmann C, Gibney E, Cotsonis G, Lawson D, Cohen C. Progesterone receptor immunohistochemical quantitation compared with cytosolic assay: correlation with prognosis in breast cancer. Appl Immunohistochem Mol Morphol 2001; 9 (01) 49-53
- 24 Kapur RP. Calretinin-immunoreactive mucosal innervation in very short-segment Hirschsprung disease: a potentially misleading observation. Pediatr Dev Pathol 2014; 17 (01) 28-35