[Histonet] Image analysis sample size



I have a question regarding image analysis and sample size (microscopic fields). Our lab has been estimating proliferation indices in IHC stained (BrdU and Ki-67) rat mammary gland and mammary tumors by evaluating ten 400x microscopic fields chosen at random, e.g. green dots placed on the tissue section prior to review under the scope in order to prevent selection bias toward heavily stained "hot spot" areas. We're using a CAS-200 imaging system that measures total nuclear area and DAB percent positive area above a specified threshold. The problem is that specimens can have a large range/variance depending upon the areas chosen for analysis and sampling 10 fields may not be enough. I'm thinking the best way to compensate for this problem would be to increase the number of fields until the variance stabilizes. Does anyone have an algorithm like an Excel macro or something similar that addresses this problem, i.e. will estimate how many more fields to count beyond 10 based on current variance? Thanks in advance.


John N. McGinley
Cancer Prevention Laboratory
Colorado State University

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