Vectra multispectral slide analysis system

Multispectral Analysis of dual-Color IHC/eosin stained samples were imaged using the PerkinElmer’s Vectra® multispectral slide analysis system. For each stained mammary section, multispectral images of the entire slide were captured manually for each 20X field containing ductal structures. For quantification of the DAB staining, the multispectral images were reviewed and analyzed using inForm® Tissue Finder software. A pattern recognition algorithm was used for processing as follows: (1) trainable tissue segmentation to segment ductal epithelial cells and (2) segmentation of the DAB-positive cells within this population from eosin-stained nuclei. Automatic quantification of these populations was used to calculate percent positive cells. For pERK quantification, automated cell segmentation of the pERK-positive tissue category was used to locate the subcellular compartments and scored by binning the spectrally unmixed DAB signal into four categories depending on the staining intensity (0+, 1+, 2+, and 3+), providing data in percent. The H-score, which ranges from 0 to 300, was calculated using the following formula: [1 × (% cells 1+) + 2 × (% cells 2+) + 3 × (% cells 3+)]. Thus, H-score measures staining intensity as well as percentage of positive cells in a given cellular compartment.