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Automated Signal Counting for SISH, Dual CISH, & RNA ISH Steven Hashagen CEO, Indica Labs, Inc. http://indicalab.com Outline • • • • • • • • • • • • • • • • Indica Labs Overview ISH Tool Overview ISH Algorithm Tuning ISH Algorithm Outputs SISH Example Dual CISH Example Dako Dual CISH Example Ventana RNA ISH Example Affymetrix ViewRNA Example Failure Modes Product Demonstration Future Enhancements Other Tools – Steatosis Quantification Other Tools – Muscle Fiber Quantification Other Tools - Axon Quantification Questions & Discussion http://indicalab.com Indica Labs, Inc. Overview Indica Labs is a privately held corporation based in Albuquerque, New Mexico. We specialize in building image analysis software for the Aperio ScanScope platform. Our products integrate seamlessly into ImageScope and Spectrum and compatible with a number of other standard and proprietary image formats. We are the only company worldwide, whose sole focus is on Image Analysis for digital pathology. http://indicalab.com ISH Algorithm Overview • Single tool that can be configured to quantify SISH, CISH, Dual ISH & RNA ISH signals on a wide range of tissue types and reagent kits. INTENDED FOR RESEARCH USE ONLY. • Integrates seamlessly into Aperio ImageScope and Aperio Spectrum software. • User never leaves the Aperio environment, and all generated data and images remain part of the Aperio environment. • Works on a variety of other file formats including Aperio, Hamamatsu, Zeiss, Bioimagene, TIFF, JPG photomicrographs. http://indicalab.com ISH Algorithm Tuning – Single Probe Stain Separation Separate nuclear stain from positive signal stain RNA ISH stain separation Affymetrix ViewRNA stain separation TIP: Use ImageScope eyedropper plugin to simplify stain selection. FREE download from indicalab.com. http://indicalab.com ISH Algorithm Tuning – Dual Probe Stain Separation Separate nuclear stain, probe 1 stain, and probe 2 stain Nuclear Stain Probe 1 (HER2) Probe 2 (Chr17) http://indicalab.com ISH Algorithm Tuning – Cell Detection Cell Radius parameter controls how far the cell boundary extends beyond the nucleus. This allows you to restrict signal detection to within the nucleus, within the nucleus and cytoplasm, or anywhere. Original Image Radius = 0 µm Spots are detected within the nucleus only. Radius = 7.9 µm Radius = ∞ Spots are detected within the nucleus and the surrounding cytoplasm. All spots are counted including spots from tangentially cut cells who’s nuclei aren’t present. http://indicalab.com ISH Algorithm Tuning – Spot Counting Manual Scoring recommendations in the Roche SISH Interpretation guide. Clusters are categorized as either small or large. Digital Scoring allows for clusters to be accurately sized and counted according to size. Individual signals are colored orange, clustered signals are colored red. http://indicalab.com ISH Algorithm Outputs • • • • • • • • • Total Signals Total Cells Avg Signals Per Cell Total Cell Area Total Signal Area Average Signal Optical Density Signals Per Cell Histogram Probe 1 / Probe 2 ratio Probe 1 / Probe 2 ratio per cell histogram http://indicalab.com SISH Example Software detects nuclei (blue) individual signals (yellow) and clustered signals (red) http://indicalab.com Ventana Dual CISH Example Ventana INFORM HER2 Dual ISH Stain Separation Markup Image Results http://indicalab.com Dako Dual CISH Example Dako kit based on Dako DuoCISH Stain Separation Markup Image Results http://indicalab.com RNA ISH Example Algorithm detects nuclei (blue), cytoplasmic regions (yellow), and RNA signals (red). http://indicalab.com Affymetrix ViewRNA Example In this example the algorithm is tuned for the Affymetrix ViewRNA stain colors. It detects nuclei (blue), cytoplasmic regions (yellow), and RNA signals (red). http://indicalab.com Failure Modes All image analysis software has failure modes. This software can perform sub optimally when any of the following are true: • Nuclei are not well defined • Positive signals bleed • Probe 1 and Probe 2 signals are difficult to distinguish http://indicalab.com Product Demonstration http://indicalab.com ISH - Future Enhancements More histograms Support FISH Support Radio-Probe ISH Feature to exclude cells with dual ISH ratios that are too low or too high • Report standard deviations, of probe 1, probe 2, and probe 1:2 ratio among cells • Export all raw data for each cell • • • • http://indicalab.com Other Tools – Muscle Fiber Quantification Algorithm uses muscle fiber basement membranes to detect individual muscle fibers and reports: • Total Fiber Count • Average Fiber Area • Average Fiber Perimeter • Average Fiber Min, Max, and Median diameter http://indicalab.com Other Tools – Steatosis Quantification H&E - detecting lipid droplets in fatty liver. Configured for PAS and Oil Red-O. Note, nuclear size and shape can be used to distinguish hepatocytes from other nuclei. Algorithm reports: • total number of nuclei • Total number of vacuoles • Avg vacuoles per cell • Avg vacuole, area, diameter, perimeter • Vacuole diameter histogram http://indicalab.com Other Tools – Axon Quantification In this example, software measures axons and myelin in human optic nerve This tool reports: • Total Axon Count • Total Axon Area • Total Myelin Area • Average Myelin Thickness • Average Axon diameter • Axon Diameter Histogram http://indicalab.com Thank You Dr. Gene Hung, ISIS Pharmaceuticals Will Casavan, Aperio Technologies Questions and Comments? http://indicalab.com
