Histology and Image Analysis

At necropsy, the Lung/Heart, liver, and genitourinary system were collected. Tissues were processed, embedded in paraffin, and 4 – 5 μm slices of the tissues were obtained. Slides were stained with hematoxylin and eosin (H&E) stain, and were graded by a pathologist for presence of tumor tissue. Histology slides were scanned using the Aperio™ (Vista, CA) ScanScope XT with a 20x/0.8NA objective lens (200×) at a rate of 2 minutes per slide via Basler tri-linear-array. An Aperio (Vista, CA) Positive Pixel Count® v9.0 algorithm with the following thresholds: [Hue Value =.1; Hue Width =.5; Color Saturation Threshold =0.04; IWP(High) = 220; Iwp(Low)=Ip(High) = 175; Ip(low) =Isp(High) =100 Isp(Low) =0] was used to segment positive staining of various intensities. The algorithm was applied to the entire digital core image to determine the percentage of positive biomarker staining by applicable area.