AR-V7 protein expression in the UW CSPC cohort was determined for each case by a pathologist (L.D. True) as described above. AR-V7 protein expression in the UW CRPC cohort was determined using automated digital scoring as follows: TMA slides were scanned with an Aperio ScanScope (Leica Biosystems) at ×40 (0.25 μm/pixel). Using Aperio ImageScope software, the AR-V7–stained TMA slides were annotated to create regions of interest (ROIs) for analysis. Quantitative image analysis of the annotated ROIs was performed using Aperio Brightfield Image Analysis Toolbox software (Leica Biosystems). The analysis data for each TMA spot were extracted into Microsoft Excel for further analysis. The quantitative analysis data for each TMA spot included total numbers and percentages of nuclei (positive and negative), average positive intensity, average positive OD, and area of analysis. The intensity is a measurement of the light transmission, or brightness, of the positive staining in the nuclei and is logarithmically related to the OD. The OD is a measurement of absorbance and is linearly related to the amount of staining present. Automated scores for AR-V7 protein expression were reviewed and confirmed by a pathologist (L.D. True), and have been shown to correlate highly with manual scoring (Supplemental Figure 9 and refs. 84 (link)–86 (link)).
Aperio brightfield image analysis toolbox software
Manufactured by Leica
The Aperio Brightfield Image Analysis Toolbox software is a digital pathology solution developed by Leica. It is designed to provide automated image analysis capabilities for brightfield microscopy slides. The software offers tools for image acquisition, processing, and quantitative analysis of various histological and cytological samples.
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Lab products found in correlation
2 protocols using aperio brightfield image analysis toolbox software
1
Automated Quantification of AR-V7 Protein Expression
2
Quantitative Analysis of pTau and pTDP-43 Immunostaining
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As previously described (6 (link)), an Aperio ScanScope AT2 (Leica Biosystems Pathology Imaging, Vista, CA) at 20x (0.5 microns/pixel) was used to scan the sections immunostained for pTau and phosphorylated TDP‐43 (pTDP‐43). Regions of interest (ROI) on each tissue section were highlighted for analysis using Aperio ImageScope software (Leica Biosystems Pathology Imaging, Vista, CA), and quantitative image analysis of the annotated ROIs was performed (Aperio Brightfield Image Analysis Toolbox software [Leica Biosystems Pathology Imaging, Vista, CA]). The calibrated red‐green‐blue (RGB) color vectors for the immunohistochemical stain components were measured and input as parameters into the Color Deconvolution Area Analysis algorithm (pTau+ and pTDP‐43+ stained slides) and Immunohistochemistry (IHC) Nuclear Quantification algorithm (pTDP‐43+ stained slides) (Figure 1 ).
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