KASM Newsletter June 2015
How to study colors from whole slide images
Image analyzing algorithms extract meaningful quantitative data that are accurate and
reproducible comparing to subjective-prone manual scoring. Antibody targeting a
particular antigen reveals localization and semi-quantitative measurement of a protein of
interest on immunohistochemical (IHC) slides. Positive Pixel Count enables us to study
colors and quantify specific stains from IHC whole slide images (WSIs). Hue value, width
and color saturation threshold are three important parameters for the algorithm to study a
color on a WSI. A Hue value represents a specific color on the color wheel and can be
illustrated with a number. To study brown staining revealed by DAB, the value, 0.1,
defines brown color as a positive stain (Figure 1A). Smaller Hue width means more
restriction in defining the positive color. Continuing with our previous example, Hue width
of 0.5 specifies that slightly reddish brown to slightly greenish brown are acceptable in
defining positive pixel (Figure 1B).
Figure 1. Color wheels. A) Hue value of brown color can be represented by 0.1. B) Hue width defines
the acceptable range of positive stain between red and green on the color wheel. Saturation
represents the purity of brown color that are to be analyzed by Positive Pixel Count.
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KASM Newsletter June 2015
Figure 2. Mouse tissue stained with Ki-67.
A) Brown stains revealed by DAB reaction
yields localization and quantity of Ki-67 in
the tissue. B) Positive Pixel Count shows
positivity of brown color in yellow, orange
and red presenting weak, medium and
strong positives. Ki-67 positive pixels in
the selected area of a tissue section are
calculated and could be exported to Excel
files for further statistical analysis.
Saturation represents the purity of the positive color and saturation less than a
threshold value will not be reported. Positive pixel count will analyze the selected area on
WSIs and reveal positive stain in three levels of red while representing negative stains in
blue (Figure 2). Positivity of brown stain on a WSI is quantified as total number of the
positive pixel divided by total number of pixels, and is a meaningful value to compare with
other slides from a set of experiment. Our custom WSI and image analysis services
have helped our clients to effectively manage their studies in reaching significant
milestones. How could we help to translate your pixels into success-leading
decisions?
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