X-ray microtomography is a novel method for accurate evaluation of small-bowel mucosal morphology and surface area

Abstract

The often poorly orientated small-bowel mucosal biopsies taken for the diagnostics of celiac disease and other intestinal disorders are prone to misinterpretation. Furthermore, conventional histopathology has suboptimal sensitivity for early histopathological changes observed in short-term challenge studies. X-ray microtomography (micro-CT) is a promising new method for accurate imaging of human-derived biological samples. Here, we report that micro-CT could be utilized to create virtual reconstructions of endoscopically obtained intestinal biopsies. The formed digital 3D images enabled selection of always optimal cutting angles for accurate measurement of the mucosal damage and revealed diagnostic lesions in cases interpreted as normal with conventional histomorphometry. We also demonstrate that computer-assisted point cloud analysis can be used to calculate biologically meaningful surface areas of the biopsies in different stages of mucosal damage with excellent replicability and correlation with other disease parameters. We expect the improved diagnostic accuracy and capability to measure the surface areas to provide a powerful tool for the diagnostics of intestinal diseases and for future clinical and pharmaceutical trials.

Figure 1

Comparison of conventional histology and micro-CT imaging of the small-bowel mucosal biopsies. After sampling, the specimen is fixed with formalin and dehydrated with increasing ethanol series. Next, in conventional histology (left route), the biopsy is embedded in paraffin via xylene and then cut into thin sections, stained with H&E, and evaluated under light microscopy. The assessment is performed either applying grouped classification or by quantitative determination of the villous height-crypt depth ratio. Correct orientation of the biopsy is a prerequisite for reliable measurements. This is accomplished by recutting the sections until acceptable readouts are obtained, assuming that the biopsy size is sufficient for this. Micro-CT imaging (right route) starts by placing fixed and ethanol-dehydrated biopsies with or without paraffin embedding into I₂E solution to increase the intrinsically low soft tissue contrast. Subsequently, mechanically stabilized biopsy is scanned using X-ray source and detector, and the acquired transmission images are reconstructed to 3D model. The model is freely orientable for precise morphometric measurements and also enables measurement of the mucosal surface areas.

Figure 2

Examples of 3D biopsy reconstructions and digitally cut sections from an untreated celiac disease patient diagnosed in routine practice (A), from a seropositive individual with duodenal morphology interpreted as normal (potential celiac disease, B), and from a non-celiac control subject with negative serology and normal villi (C). The image scaling is uniform throughout the figure. The 3D model allows free virtual orientation and rapid setting of an optimal cutting angle for precise digital morphometry. Although the biopsy in Panel B was originally interpreted as normal, possibly due to incorrect orientation or patchy mucosal lesion, the abnormal surface structure with widened and blunted villi can be readily seen in the 3D reconstruction; particularly when comparing with the longer villi in (C). The orientated digital section on the right side of the (B) confirms the decreased villous-crypt ratio characteristic of celiac disease. (C) Also demonstrates the variability in the villous morphology even in healthy mucosa. The structural differences seen in the middle of the image (right side of the panel) is only random variation that has no diagnostic significance.

Figure 3

Duodenal biopsies evaluated in routine practice with conventional histology (photos on the left), orientated sections investigated by quantitative histomorphometry (middle photos) and digital cuttings obtained via reconstructed micro-CT models (photos on the right). In patients 1–6 biopsies were also taken after approximately one year on a gluten-free diet (GFD). Patients 1–3 had received a celiac disease diagnosis, subjects 4–6 had positive celiac disease serology but were originally considered to have normal villi (potential celiac disease) and subjects 7–8 exemplify non-celiac controls with negative serology and normal histology. Numbering of the patients and samples corresponds Table 1.

Figure 4

Demonstration of the surface area measurement from the digital 3D models of the small-bowel mucosal biopsies by computer-assisted point cloud analysis (Avizo software; Thermo Fisher Scientific, Waltham, MA, USA). The image scaling is uniform throughout the figure. Panel (A) represents morphologically normal mucosa from a non-celiac individual and Panel (B) advanced celiac disease with almost flat mucosa. For the measurements, a rectangle of pre-defined size is aligned with the mucosa and the surface area is calculated within these boundaries. It is possible to use different rectangular side lengths and various measurement shapes. Use of equal image resolution and noise filtering is required for consistent results. The surface area in Panel (A) was 5.973 mm² and in Panel (B) 1.548 mm² when applying a measurement rectangle with an area of 1.000 mm². Black bar = 1.0 mm.