Positive pixel count, deprecated as of 0.2.0
There are several ways to measure areas, the most popular of which is Analyze->Region identification->Positive pixel count (experimental).
Basically, a selected annotation will be divided up into positive, negative, and neither pixels. Summary measurements will be added to the parent annotation. The positive pixel and negative pixels will be detection objects, while pixels that meet neither threshold will be left blank. Here you can see the red (positive) pixels, blue (negative/hematoxylin pixels), and some empty space that was below the hematoxylin threshold.
Resulting measurements include:
Positive % of stained pixels: Positive/(Positive+Negative)*100
Positive % of total ROI area: Positive/(Positive+Negative+Neither)*100
The first one ignores white space/areas below the hematoxylin threshold.
0.1.2 warning: In 0.1.2, each area analyzed by the tool required at least one “negative” pixel, or else the percentage positive would error out. This problem could usually be compensated for by placing a negative value in the Hematoxylin threshold (‘Negative’), but if your whole project will revolve around this feature, I would recommend figuring out the setup of version 0.1.3 or trying out 0.2.0m#.
Downsample factor: As normal, a pure increase in pixel size. Larger values will run faster, but be less accurate.
Gaussian sigma: Blur. Increase this for smoother annotations or to increase performance due to masses of different pixel objects on screen.
Hematoxylin threshold (‘Negative’): Essentially the threshold for tissue versus whitespace.
Eosin/DAB threshold (‘Positive’): Pixels above this threshold in the second deconvolved channel will be considered Positive.
IMPORTANT: I have often flipped my positive and negative vectors and thresholds due to one specific interaction between positive and negative. If a pixel is above both the negative and positive threshold, it is considered “positive.” In cases where there is dark background in another stain that is causing problems (Masson’s trichrome, H-DAB+Red, background from black shadows, etc), I have swapped my color vectors so that Hematoxylin has the color vector for my marker of interest. That way if there is something that is very dark in the other channel, it will be treated as “negative.” More details on that and dealing with the area issues mentioned next in this thread and the links contained within it.
Positive pixel count frequently struggles over large areas, and I strongly recommend creating annotation tiles (see tiles section) before running it, and then summing the results either in a script or in a spreadsheet program after.
Were you looking for something else for your area measurement? Or would you like to review a how to generate a simple script to summarize your measurements?