Materials and Methods
This study was performed after the approval of Izmir Kâtip Çelebi University Faculty of Medicine Clinical Research Ethics Committee (Ethics committee date: 09.02.2017, Ethics committee decision number: 16) between 15 November 2016 and 15 March 2018 at Izmir Health Sciences University Tepecik Training and Research Hospital Pediatric Surgery Clinic. Data of 24 patients aged between 0-3 years who were hospitalized in our clinic for advanced examination and treatment with a pre-diagnosis of HD were analyzed prospectively.
Laboratory and abdominal X-Ray radiographs of the patients were evaluated. Then contrast-enhanced colon graphies and 24th hours control X-Ray graphies (retention graphy) were examined and recorded (Figure 1). Rectosigmoid index (RSI) was calculated for all patients. In the RSI evaluation, the presence of the transition zone and the presence of the barium in the intestines on the radiograph after 24 hours were evaluated and recorded. Rectosigmoid index was calculated by proportioning the diameter of the widest part of the rectum to the diameter of the sigmoid colon on barium enema graphy. If the RSI<1, it was considered in favour of HD (Figure 2)(15).
After the complaints of these patients such as abdominal distention and vomiting were treated by nasogastric decompression; SRB was performed at least 48 hours after anal dilatation or rectal irrigation procedures. The biopsy was performed at the bedside to the patients younger than 1-year-old without any anaesthesia and in the operating room with sedation (0.01 mg/kg IV midazolam) to the patients older than 1-year-old.
Suction rectal biopsies were performed with Rbi2 biopsy kit (Aus systems Pty Ltd, Allenby Gardens, Australia) (Figure 3). For histopathological diagnosis, we took two samples which are taken from 2 cm proximal of the anocutaneous line that one from the posterior wall and other from the lateral wall. Negative pressure during the procedure was adjusted to be 150 mm H2O (It was created by vacuuming 5 cc in a 10-cc injector)(14). Two fresh samples were quickly taken to the pathology laboratory, rightafter the samples were fixed in 10% formaldehyde solution, and paraffin blocks were prepared. Sections 4-5 microns thick from the paraffin blocks were stained with hematoxylin-eosin and examined under a light microscope at a magnification of 100 - 400 x (Figures 4). Ret-oncoprotein (Figure 4) and neuron-specific enolase (NSE) staining was performed by immunohistochemical (IHC) method on samples whose ganglion cells and nerve plexuses could not be selected in a routine examination. In addition, all sections were evaluated by measuring the mucosal (M) and submucosal (SM) areas in NIS Elements Ver 4.30 digital imaging program of Nikon Eclipse Ni microscope (Figure 4).