I performed an immunohistochemical study and I have estimated the percentage of expression of different tumor markers, in this case, can I estimate fold changes with respect to the control ?
Dear Settar,
Speaking strictly from the perspective of anatomical pathology / histopathology semi-quantitative/comparative analysis (approximate estimation of fold change) is a tricky subject when dealing with tissue samples from FFPE blocks. (Unless we are talking about fixed cells mounted onto slides which makes it a bit easier).
Immunohistochemical staining depends highly on the tissue you are processing and you should consider the following:
1. You must examine the same area of interest, using the same field of view every time in sample and control.
2. Your tissue must contain a sufficient amount of tumor cells (which must be distinguishable from the adjacent stroma by their size, nucleus-to-cytoplasm analogy, morphology, etc.).
3. Your antibodies must bind specifically to cancer cells and not the adjacent stroma. (if you are interested entirely in the tumor cells and not the effect on the microenvironment)
4. Your non-specific binding (noise) does not exceed a limit and that there is uniformity between sample and control slide. (there should be equal amount of non-specific binding in both control and sample sections).
5. There should be sufficient but not excessive haematoxylin counterstaining.
After considering the aforementioned there are two options and in both cases you should set your IHC Intensity Scoring (e.g. negative, mild positivity, moderate positivity, strong positivity) or follow the guidelines (if any) for each marker.
1. Your first option is to ask a histopathologist to do the assessment.
2. Your second option is to use a digital pathology image analysis program (that also requires the digitization of your slides).
You can see a comparison between both methods in the following link:
Article Comparison of Semi-Quantitative Scoring and Artificial Intel...
I really hope that was helpful,
Kind regards
Antonis Tsintarakis
I really appreciate your help ! Indeed I have already done the counting and scoring, I am now looking if I can estimate fold changes with these results or not !?
Dear Settar Amina
I am afraid the short answer to your question is: No.
I guess you would like to estimate the fold changes in the target protein concentration in the tissues. In general, this kind of quantification is not possible using ordinary indirect immunohistochemistry due to several reasons, including the binding of secondary antibodies and signal amplification systems. If you have different controls with known expression levels (e.g. cell cultures) it may be possible to estimate with digital image analysis, but otherwise, you can only estimate the changes on an ordinal scale (using different scoring systems, as you have already done).
Kind regards
Dear Christian Thomsen
I am very grateful for the insightful response.
Would I say that the estimation of fold changes is possible only after a Hybridization in Situ method, isn't it?
Dear Settar Amina
mRNA-ISH (such as RNAScope) is more quantitative that conventional immunohistochemistry because you can count dots and also it provides a more wide dynamic interval. However, that is the relative change in mRNA you are measuring, and you possibly do not know exactly how this is related to the amount of protein. In a previous study, we studied the relationship between estrogen receptor IHC and mRNA-ISH and found a non-linear correlation between mRNA (dots) and protein (H-score). Others have reported the same for PD-L1.
Kind regards
- Badges
- Science method