Abstract
Immunohistochemistry is a powerful technique that is widely used in biomedical research and clinics; it allows one to determine the expression levels of some proteins of interest in tissue samples using color intensity due to the expression of biomarkers with specific antibodies. As such, immunohistochemical images are complex and their features are difficult to quantify. Recently, we proposed a novel method, including a first separation stage based on non-negative matrix factorization (NMF), that achieved good results. However, this method was highly dependent on the parameters that control sparseness and non-negativity, as well as on algorithm initialization. Furthermore, the previously proposed method required a reference image as a starting point for the NMF algorithm. In the present work, we propose a new, simpler and more robust method for the automated, unsupervised scoring of immunohistochemical images based on bright field. Our work is focused on images from tumor tissues marked with blue (nuclei) and brown (protein of interest) stains. The new proposed method represents a simpler approach that, on the one hand, avoids the use of NMF in the separation stage and, on the other hand, circumvents the need for a control image. This new approach determines the subspace spanned by the two colors of interest using principal component analysis (PCA) with dimension reduction. This subspace is a two-dimensional space, allowing for color vector determination by considering the point density peaks. A new scoring stage is also developed in our method that, again, avoids reference images, making the procedure more robust and less dependent on parameters. Semi-quantitative image scoring experiments using five categories exhibit promising and consistent results when compared to manual scoring carried out by experts.