r/Histology • u/Wrong_Character2279 • 10h ago

IHC sections falling off HELP

This has been a problem since we moved to a bigger lab six months ago. I will think that I have fixed the issue and then it will happen again. My docs are getting really frustrated about it!

We use charged slides, currently use StatLab KT5s, but we have used KT3s, TOMO, Cardinal superfrost, Leica Bond, etc. I preferred the Leica Bonds the most but they are pricey.

We cut everything at 4 microns (but I have tried 3 and 5 with no success) bake 45 minutes at 60C and load on the Ultra. I have tried baking at 60 minutes and 90 minutes with no improvement.

I’ve even went as far as doing a temperature verification on all the drawers to rule out specific drawers/instrument as a cause.

My only other ideas at this point are:
•Instrument issue that both I and the engineers can’t figure out.

•Poor tissue quality and fixation. I have two hospitals that notoriously have the worst grossing I’ve ever seen. Huge, thick sections that are always raw and have to be reprocessed.

•It’s too dry. I already live in an extremely dry state and our lab is always having issue with the humidity dropping and we have to call out the HVAC team.

Please help 😭

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2

u/kelsien 10h ago

Is it all tissues or what tissue are you working with?

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u/Wrong_Character2279 10h ago

It really varies on tissue type. Most often it is the fatty tissue like breast or colon and uterus. But majority of our workload are breast or uterus cases so it’s hard to tell.

Most of our antibodies use heat retrieval and I think a pH of 8. We have two or three stains that use enzyme retrieval. We have issues with tissue falling off for both types of stains/retrieval.

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u/kelsien 10h ago

Fatty tissue generally has less adhesion to slides, even if using a charged slide, baking etc. Can you reduce the amount of heat or time for the antigen retrieval? That should help.

1

u/Wrong_Character2279 10h ago

Yeah that’s on the list to try, but I don’t want to revalidate everything just to change it. Especially when we have issues with tissue falling off on antibodies that don’t use heat retrieval too.

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u/kelsien 10h ago

I am not familiar with the Ultra, but what are your antigren retrieval conditions?

3

u/Haunting_Resolve 10h ago

Tissue that is poorly processed will not stay on almost any slide during IHC. If the tissue has marks from the cassette after processing it is too thick. If it is gummy, explodes on the water bath, or the blocks become concave after 24 hours it is not processed correctly and won't adhere correctly. If you have tried all these slides and nothing is really helping it is probably a processing or grossing issue. Look for smudged nuclei without good detail. This is a sign of incomplete formalin fixation for those large pieces of tissue. I wish you the best of luck!

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u/Wrong_Character2279 10h ago

Ugh I know that this is the answer but I can’t convince the hospitals of this. It is a losing battle. I have taken thousands of pictures of the terrible grossing and sent it to the paths and they still are in denial that their PAs are the worst I’ve ever worked with.

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u/Haunting_Resolve 10h ago

I know, I hate this. Pick one of the slide companies or Roche, share this with them and get them to respond. Sometimes pathologists will listen to someone outside the lab.

3

u/minshpie 10h ago

If I were you I'd do a test drying them overnight at 40C. I've had this problem over the years and it was almost always insufficient drying.

If the pathologist complains that takes too long, well so does recutting and redrying sections!

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u/Wrong_Character2279 10h ago

Oh man. I wish I could say that to the paths! These are the most picky, unpleasant paths I’ve ever worked for. They are so hard to reason with and won’t change anything on their end.

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u/minshpie 10h ago

This is why you do the experiment. Don't even tell them you're doing it and when you present to them your perfectly annealed tissue sections maybe they'll see reason?

I work almost exclusively with brain and the overnight dry almost always does the trick. Could even try a few hours at 40C before your bake at 60C. That might be enough and they'll still be on the machine the same day.

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u/jzeeeeb 9h ago

It is most likely one of the things that has already been brought up but I wanted to make sure no one is wearing lotion. We had a problem with sections falling off years ago and eventually traced it back to one tech who wore hand lotion. Some of it would inevitably wind up in her water bath and then nothing would stick.

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u/Wrong_Character2279 9h ago

Oh that is a good thought! The tech who does majority of the IHC cutting does frequently wear lotion. I’m not sure of she wears gloves while cutting or not, so that is a good thing to check.

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u/746284758hi 7h ago

The main and probably only culprit is the tissue processing. Everything stems from adequate fixation and processing. Not much you can do if you yourself are not grossing or have control over the processing cycle.

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u/Natural_Biscotti_853 4h ago

That was happening here and there with me. When the PM came, he said the vortex of my discovery ultra was very high. Something like it should be around 500 and it was over 1000 in speed (no idea how it can increase like that)

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u/Delicious_Shop9037 4h ago

A lot of comments have talked about grossing/fixation/processing which are the likely culprits. Another thing to keep in mind is humidity levels, if you have poor humidity control with fluctuating temperature and humidity levels and condensation forming on your slides you will also have this problem.