In the day-to-day work of a histotech, we are always troubleshooting--from processing to embedding, microtomy, special stains, or immunohistochemistry (IHC). We are constantly trying to fix or improve on something. I want to tell you about some tricks I have learned over the last 20 years performing immunohistochemistry.
At the beginning of my career, most of the work was manual. We had some machines to facilitate the work, but it was mostly done manually. I know some of you still process IHC manually, and for you, my best advice is to follow your recipe from beginning to end without skipping any steps. It does not matter what company you are using to get your reagents. That company will provide you with kit instructions. After optimization and validation of your protocols, make sure to follow them. More often than not, skipping steps trigger problems that lead to negative results. If you find yourself having issues, I recommend making a checklist of your protocol to help you remember your steps. Then, if you are positive that all the steps were followed correctly, check to make sure all your solutions were done properly. If you continue to have the same negative results and cannot identify the issue, go through your checklist again, and make changes to only one step for each run; that way, it will help you identify the problem. Controlled changes will teach you where to look next time it happens.
Now, when we are working with automation, there are other things to pay attention to that spark questions to ask yourself., For example, “Is your IHC machine working properly? Is your machine set properly?” Sometimes, depending on the error, you will get clues to your issue. The most important thing to pay attention are the details. For example, why was the solution not dispensed when your slides have a positive stain but no counterstain? Sometimes something as simple as the cap in one of the reagents not having been removed or the safety lock not being removed would be the first place to look, followed by verifying if the dispenser was empty/leaking or dry, causing the machine to generate the same error.
The same concept will apply to your antibodies or detection system. If a stain that was working before suddenly stops working, I usually check my dispensers first. One of the first things I do before starting my runs is check my reagents to make sure they are in good condition (not empty, not leaking, or dry) before placing them in the machine. If you get used to doing that, you know that when something is wrong, your reagents are not the problem, and you will need to look for another issue. But, let’s say, you are not sure about your reagent; then, the best thing to do is repeat the process; if everything is good, then the reagents are not the problem. In that case, it is time to take a closer look at other components of your machine, which would most likely indicate the need to call your service technician.
Another common issue in the IHC area is losing your sample from the slide. This is something that happens during manual and/or automated IHC processing. First, we need to make sure the tissue was adequately handled, well fixed, and processed correctly. Most of the time, the tissue is lost due to improper fixation. Underfixed tissue or delayed fixation will generate weak staining, no staining, or background staining while overfixation will give you weak staining. In the case of overfixation, antigenicity can be restored with a more extended pretreatment protocol. Just remember to make a validation for any new protocol. Also, remember to make sure your tissue is well immersed in formalin. If the formalin volume is low, the tissue will fix on the outside, but it will not penetrate the inside properly, generating weak or variable staining, good in the outside margin but weak or variable in the insides. But let’s say the tissue is good and processed well; then we need to look at the slides; we may have a bad slide lot number, and the positive charge is lost, or the slides are old (check expiration date). Another possibility is that the tissue was cut on a regular slide instead of a plus charge slide. You will think these points are obvious, but when looking for issues, sometimes we forget simple details.
For example, some of those details are the baking of slides, the time and the temperature will vary depending on your lab protocol, but most of us do 30 to 60 minutes at 60 degrees. Ensure the water is not under your section that it is well-dried; if not, your tissue will likely be lost during pre-treatment.
Another common issue is the preparation of your buffer. I remember on one occasion we were losing the counterstain from all the slides; after checking my dispenser and making sure the machine was working properly, we checked the pH of the tap water. We discovered it was very acidic. When doing the final rinsing of the slides, the tap water was taking out most of the counterstain. We changed the water for a while until the problem was resolved, and returned to normal.
Another issue I have encountered was with the deparaffinization process. Some samples exhibited incomplete removal of paraffin. When troubleshooting the issue, we discovered that the preparation/dilution of the deparaffinization solution was incorrect. It turns out the deparaffinization solution was ready to use, but we processed it as if it needed to be diluted (when it didn’t). In this case, as part of the troubleshooting process, we verified that the oven temperature was at the correct setting (paraffin melting point). Also, we verified that the heat pads the machine uses to heat the slides in the deparaffinization process were working. If your deparaffinization process is incorrect/deficient, you will get weak or no staining because your epitopes will not be fully exposed for the primary antibody to attach.
At the end of the IHC process, take into consideration if you are using a non-permanent chromogen, like AEC, because this type of chromogen is soluble in alcohols, and if they are exposed for a long time to alcohols, you can also get weak staining. A good recommendation is to avoid alcohols and put slides directly in the oven to dry off before coverslipping. I have covered only a few troubleshooting examples with tips on how to overcome them. Feel free to add your own tips, tricks, and comments related to IHC processing.
References
Cell Marque. (2023, March 19). Cell Marque. Retrieved from Cell Marque. Website: https://www.cellmarque.com/troubleshooting/
Written By: Elba Vidal, BS, MBA, HTL(ASCP), QIHC (ASCP)
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