BACKGROUND: In prognosis and therapeutics of adrenal cortical carcinoma (ACC), the selection of the most active areas in proliferative rate (hotspots) within a slide and objective quantification of immunohistochemical Ki67 Labelling Index (LI) are of critical importance. In addition to intratumoral heterogeneity in proliferative rate i.e. levels of Ki67 expression within a given ACC, lack of uniformity and reproducibility in the method of quantification of Ki67 LI may confound an accurate assessment of Ki67 LI.RESULTS: We have implemented an open source toolset, Automated Selection of Hotspots (ASH), for automated hotspot detection and quantification of Ki67 LI. ASH utilizes NanoZoomer Digital Pathology Image (NDPI) splitter to convert the specific NDPI format digital slide scanned from the Hamamatsu instrument into a conventional tiff or jpeg format image for automated segmentation and adaptive step finding hotspots detection algorithm. Quantitative hotspot ranking is provided by the functionality from the open source application ImmunoRatio as part of the ASH protocol. The output is a ranked set of hotspots with concomitant quantitative values based on whole slide ranking.CONCLUSION: We have implemented an open source automated detection quantitative ranking of hotspots to support histopathologists in selecting the 'hottest' hotspot areas in adrenocortical carcinoma. To provide wider community easy access to ASH we implemented a Galaxy virtual machine (VM) of ASH which is available from .VIRTUAL SLIDES: The virtual slide(s) for this article can be found here:,
Diagnostic pathology
Department of Bioinformatics

Lu, H., Papathomas, T., van Zessen, D., Palli, D., de Krijger, R., van der Spek, P., … Stubbs, A. (2014). Automated Selection of Hotspots (ASH): enhanced automated segmentation and adaptive step finding for Ki67 hotspot detection in adrenal cortical cancer. Diagnostic pathology, 9. doi:10.1186/s13000-014-0216-6