[Histonet] Her2 Dual ISH and breast processing

[Hoekert, W.E.J.]

I was wondering,is there any literature on this subject? i.e. the minimal required fixation time of breast tissue in order to get reliable immuno staining (Oestro, Prog and Her2neu and ISH). We immunologists are alway trying to convince our pathologists about the importance of good fixation, but the pathologist are always in a hurry to get the diagnosis out. Many mamma biopsies do not get to be fixed long enough (some are even put in the processor after a few hours of fixation). It would be helpfull if I can show them some literature to back me up.
 
Willem

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Van: histonet-bounces <@t> lists.utsouthwestern.edu namens Rene J Buesa
Verzonden: ma 10-12-2012 18:23
Aan: vtolley <@t> cox.net; histonet <@t> lists.utsouthwestern.edu
Onderwerp: Re: [Histonet] Her2 Dual ISH and breast processing



Val:
Acknowledging that you have a fixation problem is the fundamental step to solving your "staining" problems.
You state that the slices have now a consistent thickness of between 2-3 mm and that is great BUT what about the fixation time?
Thin slices is a first step but until you have the slices properly fixed, you will keep having some problems.
Even when one should never assume, I assume that you are using NBF at room temperature. With that fixative and under those temperature conditions, your 2-3 mm thick breast slices will require 4 hours to be fully penetrated; will require 24 hours to be 100% covalent bound and will require 96 hours to be completely cross-linked.
The 48 hours of maximum exposition to NBF recommended by ASCO-CAP will guarantee a 100% cross-linking of 1 mm thick slices, but your 2-3 mm slices will require 96 hours to be completely cross-linked.
Until you reach an optimum fixation you will keep having sporadic problems of your protocols (depending on greater or lower fat contents of the samples).
René J.

From: "vtolley <@t> cox.net" <vtolley <@t> cox.net>
To: histonet <@t> lists.utsouthwestern.edu
Sent: Monday, December 10, 2012 11:48 AM
Subject: [Histonet] Her2 Dual ISH and breast processing

Hello all--

My lab is having some problems with inconstant results with our Her2 Dual ISH on the Ventana Ultra.  Right now, we know we have a problem with our large breast tissue being under-fixed.  There have been many gripes to the PAs and residents about the thickness of the tissue sections and they have listened.  We are now getting breast sections that are consistently cut between 2-3mm in thickness.  However, we are still having issues with inconsistency in our dual ISH staining.  Many times, the staining is absent.

We are currently processing our breast tissue on a Tissue-Tek VIP6 processor.  Are there any labs out there that are using this processor and also running Her2 Dual ISH on the Ventana with nice, consistent results?  If so, would any of you be willing to share your processing protocol?

Thanks in advance for your help! 



Val



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