Citation: Hawez, T.; Graneli, C.; Erlöv,T.; Gottberg, E.; Munoz Mitev, R.; Hagelsteen, K.; Evertsson, M.; Jansson, T.; Cinthio, M.; Stenström, P. Ultra-High Frequency Ultrasound Imaging of Bowel Wall in Hirschsprung’s Disease—Correlation and Agreement Analyses of Histoanatomy. Diagnostics 2023, 13, 1388. https://doi.org/10.3390/ diagnostics13081388 Academic Editor: Takuji Tanaka Received: 9 February 2023 Revised: 2 April 2023 Accepted: 4 April 2023 Published: 11 April 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). diagnostics Article Ultra-High Frequency Ultrasound Imaging of Bowel Wall in Hirschsprung’s Disease—Correlation and Agreement Analyses of Histoanatomy Tebin Hawez 1 , Christina Graneli 1 , Tobias Erlöv 2 , Emilia Gottberg 3 , Rodrigo Munoz Mitev 3 , Kristine Hagelsteen 1 , Maria Evertsson 4 , Tomas Jansson 4 , Magnus Cinthio 2 and Pernilla Stenström 1, * 1 Department of Pediatric Surgery, Children’s Hospital, Skåne University Hospital Lund, Lund University, 221 85 Lund, Sweden 2 Department of Biomedical Engineering, The Faculty of Engineering, Lund University, 223 63 Lund, Sweden 3 Department of Clinical Genetics and Pathology, Skåne University Hospital, Lund University, 222 42 Lund, Sweden 4 Department of Biomedical Engineering, Skåne University Hospital Lund, The Faculty of Engineering, Lund University, 221 00 Lund, Sweden * Correspondence: pernilla.stenstrom@med.lu.se Abstract: Hirschsprung’s disease (HD) is characterized by aganglionosis in the bowel wall, requiring resection. Ultra-high frequency ultrasound (UHFUS) imaging of the bowel wall has been suggested to be an instantaneous method of deciding resection length. The aim of this study was to validate UHFUS imaging of the bowel wall in children with HD by exploring the correlation and systematic differences between UHFUS and histopathology. Resected fresh bowel specimens of children 0–1 years old, operated on for rectosigmoid aganglionosis at a national HD center 2018–2021, were examined ex vivo with UHFUS center frequency 50 MHz. Aganglionosis and ganglionosis were confirmed by histopathological staining and immunohistochemistry. Histoanatomical layers of bowel wall in histopathological and UHFUS images, respectively, were outlined using MATLAB programs. Both histopathological and UHFUS images were available for 19 aganglionic and 18 ganglionic specimens. The thickness of muscularis interna correlated positively between histopathology and UHFUS in both aganglionosis (R = 0.651, p = 0.003) and ganglionosis (R = 0.534, p = 0.023). The muscularis interna was systematically thicker in histopathology than in UHFUS images in both aganglionosis (0.499 vs. 0.309 mm; p < 0.001) and ganglionosis (0.644 versus 0.556 mm; p = 0.003). Significant correlations and systematic differences between histopathological and UHFUS images support the hypothesis that UHFUS reproduces the histoanatomy of the bowel wall in HD accurately. Keywords: Hirschsprung’s disease; bowel wall; ultra-high frequency ultrasound; histopathology; children 1. Introduction Hirschsprung’s disease is a congenital bowel motility disorder characterized by agan- glionosis in the bowel wall. Inhibiting relaxation, aganglionosis leads to a life-threatening functional obstruction [1–3]. Treatment of Hirschsprung’s disease is by surgery, including resection of the aganglionic and transition zone segments, followed by the establishment of bowel continuity [4,5]. Aganglionosis always stretches in an oral direction from the anus. In the majority of patients, only the rectosigmoid colon is affected, but aganglionosis can also extend over a longer distance [6]. When deciding upon the length of bowel to be resected, intraoperative fresh frozen biopsies are required to confirm the level of gangliono- sis. Acknowledged clinical problems are that the frozen biopsy method is a rather blunt technique, and that resection of a too short or a too long bowel segment can cause severe postoperative problems [7,8]. Additionally, waiting times for frozen biopsy analyses during Diagnostics 2023, 13, 1388. https://doi.org/10.3390/diagnostics13081388 https://www.mdpi.com/journal/diagnostics