In order to analyze immunohistochemistry images, lately I have been using the ImageJ Macro "IHC profiler" developed by Varghese and colleagues and I have a question about the results you get from taking the "Cytoplasmic stained image" way. I don't know why sometimes you get different % of expression in every score when using the same exact image (if I am not wrong, there are like 2 different results I am getting). "Cytoplasmic" choice is quite straightforward, you don't need to adjust any threshold, so I don't know why this random answer from the Macro is happening.
When taking a look to the "numbers" in the different results obtained, I notice that in the Histogram window there is a difference in the number of counted pixels. If I am not misunderstanding this info, I think that this difference involves the Y axis of the Histogram, i.e., the number of pixels representing the intensity. Again, I don't know why this happens when using the same right image and, in case is normal, what result of both I should take into account.
Original Macro paper is here:
Varghese F, Bukhari AB, Malhotra R, De A. IHC Profiler: an open source plugin for the quantitative evaluation and automated scoring of immunohistochemistry images of human tissue samples. PLoS One. 2014 May 6;9(5):e96801. doi: 10.1371/journal.pone.0096801. PMID: 24802416; PMCID: PMC4011881.
Hello José Miguel Pardo-Sánchez ,
I also experienced the same thing in the past. Do you do an individual analysis of each image? If so, sometimes individual analyzes can produce errors like this. I also don't understand why this error can occur. However, I have a solution to avoid this error with batch processing.
Batch processing is a mechanism within ImageJ that allows users to perform multiple image analysis at once. To do so, try to follow my protocol:
1. You need to prepare the image that you will process. This means that you need to "crop to a region of interest to exclude background" on all images first.
2. After all the images are ready. Put the image in one or more folders according to your needs.
3. Click Process> batch> macro and the batch process window will appear.
4. Select the input folder containing the image you wish to analyze. Remember, you can only batch process one folder at a time! So if you are working in multiple folders, you need to do batch processing equal to the number of your folders.
5. Select the output folder where you want it. Basically, this output folder will contain the logs and histograms of the analysis of all the images in your input.
6. Set the output format according to your needs. I usually don't change the settings for "output format", "add macro code", and "file name contain".
7. Under the file name contains, there will be a large empty space. It's where you put your batch processing code. Each protocol has a different code. I have coded your protocol as the following:
run ("IHC Profiler", "mode = [Cytoplasmic Stained Image] vectors = [H DAB]");
8. After that click process, than the log and histogram window will start batch processing all the images in your input file.
This batch processing has two advantages. The first is to streamline your work on image analysis. Second, for some reason, if you use this batch processing, the error that you asked about earlier will not occur.
I hope my answer helps. I've also included my publications if you'd like to read more ( https://www.phcogj.com/article/1326 ). Contact me if you have further questions. Cheers!
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