Semi-Automated Image Analysis Methodology to Investigate Intracellular Heterogeneity in Immunohistochemical Stained Sections

The discovery of tissue heterogeneity revolutionized the existing knowledge regarding the cellular, molecular, and pathophysiological mechanisms in biomedicine. Therefore, basic science investigations were redirected to encompass observation at the classical and quantum biology levels. Various approaches have been developed to investigate and capture tissue heterogeneity; however, these approaches are costly and incompatible with all types of samples. In this paper, we propose an approach to quantify heterogeneous cellular populations through combining histology and images processing techniques. In this approach, images of immunohistochemically stained sections are processed through color binning of DAB-stained cells (in brown) and non-stained cells (in blue) to select cellular clusters expressing biomarkers of interest. Subsequently, the images were converted to a binary format through threshold modification (threshold ~ 60%) in the grey scale. The cell count was extrapolated from the binary images using the particle analysis tool in ImageJ. This approach was applied to quantify the level of progesterone receptor expression levels in a breast cancer cell line sample. The results of the proposed approach were found to closely reflect those of manual counting. Through this approach, quantitative measures can be added to qualitative observation of subcellular targets expression.