Techniques for digital histological morphometry of the pineal gland

The pineal gland is a small photo-neuro-endocrine organ. This study used human post-mortem pineal glands to microscopically assess immunohistochemical marker intensity and percentage of positivity using known and novel digital techniques. An experimental non-inferiority study has been performed on 72 pineal glands harvested from post-mortem examinations. The glands have been stained with glial fibrillary acidic protein (GFAP), synaptophysin (SYN), neuron-specific enolase (NSE), and neurofilament (NF). Slides were digitally scanned. Morphometric data were obtained using optical analysis, CaseViewer, ImageJ, and MorphoRGB Strong and statistically significant correlations were found and plotted using Bland-Altman diagrams between the two image analysis software in the case of mean percentage and intensity of GFAP, NSE, NF, and SYN. Software such as SlideViewer and ImageJ, with our novel software MorphoRGB were used to perform histological morphometry of the pineal gland. Digital morphometry of a small organ such as the pineal gland is easy to do by using whole slide imaging (WSI) and digital image analysis software, with potential use in clinical settings. MorphoRGB provides slightly more accurate data than ImageJ and is more user-friendly regarding measurements of parenchyma percentage stained by immunohistochemistry. The results show that MorphoRGB is not inferior in functionality. The described morphometric techniques have potential value in current practice, experimental small animal models and human pineal glands, or other small endocrine organs that can be fully included in a whole slide image. The software we used has applications in quantifying immunohistochemical stains. [Display omitted] •Digital morphometry with immunohistochemistry is useful in evaluating the activity of endocrine glands.•Digital pathology offers multiple morphometry tools, especially for small organs, such as the pineal gland.•Quantification of immunohistochemistry with MorphoRGB renders similar results to ImageJ.•Assessment of immunohistochemical intensity using RGB image analysis can eliminate the bias of optical microscopy.