Cytometrical image analysis for immunohistochemical hormone receptor status in breast carcinomas

A cytometrical image analyzing method for nuclear protein was established using WinROOF, a commercially available, inexpensive software, to determine the status of both estrogen and progesterone receptors. Immunohistochemical evaluation of estrogen receptors (ER) and progesterone receptors (PR) was performed with the anti-ER (clone 1D5) and the anti-PR (clone PgR636), respectively, combined with dextran polymer reagent EnVision+, all of which are approved in vitro diagnostics in Japan. The immunostained results were captured as digital images in Windows, and then analyzed in WinROOF with macroinstructions for analyzing each captured area either immunolabeled with chromogen or counterstained with hematoxylin. This image analysis method graded the immunostained nuclei of carcinoma cells based on staining intensities, and calculated the labeling index (LI) for both ER and PR. Furthermore, the LI correlated highly with the results from a histology score (HSCORE) when 20 breast carcinomas were quantified. Regarding ER, when 20% in the LI was considered as the cut-off point for positive, the positivity of ER in computer-assisted analysis was 75% (15 of 20 cases), and was completely concordant with that of HSCORE-based analysis. These results indicate that the cytometrical image analysis-based quantification could be appropriately applied to the objective determination of the immunohistochemical status of both ER and PR.