A Novel X-Ray 3D Histological Method for Paraffinated Prostate Samples

Prostate cancer grading currently relies on the histopathological assessment of biopsies, which are analyzed as histological slides. However, the inherent two-dimensional (2D) nature of this approach, along with the limited number of slices available for clinical analysis, increases the risk of underestimating cancerous lesions. We propose that non-destructive 3D imaging methods, such as micro-Computed Tomography, can provide a more realistic and accurate means for rapid and precise characterization of prostate malignancies, thereby minimizing diagnostic discrepancies in complex cases. In this study, we focused on the technical development of an advanced 3D X-ray micro-computed tomography method (micro-CT) for imaging formalin-fixed paraffin-embedded (FFPE) rat prostate samples. This method combines a novel paraffin-clearance protocol, the use of eosin as a contrast agent, and micro-CT imaging, followed by standard hematoxylin and eosin histology. The micro-CT imaging produced a 3D model that enabled visualization and segmentation of the prostate into two well-differentiated regions: epithelium plus stroma and lumen. The CT scan was validated with hematoxylin and eosin histology. The 3D images provided volume and area measurements from the segmented parts and displayed in fine detail the extension and continuation of the glandular luminal phase. The tissue integrity was well preserved during the process, making our protocol potentially compatible with further analytical techniques. The success of this initial approach to 3D imaging the rat prostate paves the way for the application of our method to clinical samples.