EpidermaQuant: Unsupervised Detection and Quantification of Epidermal Differentiation Markers on H-DAB-stained Images of Reconstructed Human Epidermis

The integrity of the reconstructed human epidermis generated in vitro can be assessed using histological analyses combined with immunohistochemical staining of keratinocyte differentiation markers. Technical differences during the preparation and capture of stained images may influence the outcome of computational methods. Due to the specific nature of the analyzed material, no annotated datasets or dedicated methods are publicly available. Using a dataset with 598 unannotated images showing cross-sections of in vitro reconstructed human epidermis stained with DAB-based immunohistochemistry reaction to visualize four different keratinocyte differentiation marker proteins (filaggrin, keratin 10, Ki67, HSPA2) and counterstained with hematoxylin, we developed an unsupervised method for the detection and quantification of immunohistochemical staining. The pipeline consists of the following steps: (i) color normalization; (ii) color deconvolution; (iii) morphological operations; (iv) automatic image rotation; and (v) clustering. The most effective combination of methods includes (i) Reinhard’s normalization; (ii) Ruifrok and Johnston color-deconvolution method; (iii) proposed image-rotation method based on boundary distribution of image intensity; and (iv) k-means clustering. The results of the work should enhance the performance of quantitative analyses of protein markers in reconstructed human epidermis samples and enable the comparison of their spatial distribution between different experimental conditions.