For quantitative assessment of IHC staining, when do we measure/calculate " optical density", "area %" and "% of positive cells "?
Thanks alot David for making it clear and simple. I have been discussing that with my colleagues and we were confused. Thanks for the article too.
As Mr. Meyerholz mentioned - interpretation of IHC staining depends on What IHC marker you study and Where (tissue)? I wrote a small review about the scoring approaches in IHC.
http://www.diagnosticpathology.org/content/9/1/221
Dear Amal
I agree with David, it depends on what you want to represent from your immuno results. Immunohistohemsitry is a semi-quantitative technique and the main objective is to show the cellular localization and expression of a specific protein. Quantification of the protein expression is performed according to the pattern of expression. For instant, if the protein is localized in the cytoplasm of all cells from the different study groups so there is no need to count the percentage of cells and measuring the intensity of of brown staining is sufficient. The larger the number of groups the wider the scaling system you use (e.g. you can use +, ++, +++ if comparing between 2-4 groups but a scoring system 0-1 negative, 2-4 weak positive etc is used for 5 or more groups).
In case you have localization in different compartments of your tissue (e.g. cytoplasmic & nuclear or membrane) then you can compare each compartment separately. You apply the number of positive cells (%) in case where only specific cells are expressing the molecule (specific immune cells or specific cells e.g. goblet cells) then you need to count the number of positive cells in 5 random sections from each sample to obtain the mean +/- SD to measure whether there is a significant difference between the groups.
Hope this is useful.
Wishes,
Bassem
Thanks alot Fedchenko and Bassem for your help and the attached article. It's useful indeed. Thanks again
how do we assest area % and % of positive cells In immunohistochemistry staining? can we calculate with software image? and any free software to download?
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