[Histonet] Losing tissue on IHC slides

[Clare Thornton]

I have a couple of things.  First off, are the sections coming off the slides entirely, or does it have a "chewed up" appearance?  If the sections are coming completely off, I would agree with the others that water is the issue.  Also, make sure you are cutting no thicker than 4 microns.  We had repeated a test twice due to sections falling off, and the tech who cut them has a tendency to cut on 5 microns.  I cut them the third time at 4 microns, and let the sections really flatten out on the water bath for a minute.  Then baked them well, and third time was the charm.  The sections remained on.  As far as the "double dipping" of precut control slides go, I spoke directly with a representative from Erie (the makers of all superfrost plus slides) and was told you could dip those slides a hundred times in water, it will not affect the charge on the slides.  We've never worried about double dipping and it has never caused us a problem.  If the sections have more of a chewed up appearance, then it's probably a combination of factors:  fatty tissue, not well fixed, strong antigen retrieval.  We've done something for years now that seems to help.  After baking, we "post fix" some of our IHC slides in formalin for about 15 minutes.  Rinse in tap water then put on stainer as usual.  This seems to help when certain antibodies like to chew up the tissue.  We don't do this will all antibodies, mostly breast markers and those with strong antigen retrieval.  I hope this helps!  Good luck.


Clare J. Thornton, HTL(ASCP)
Assistant Histology Supervisor
Dahl-Chase Diagnostic Services
417 State Street, Suite 540
Bangor, ME 04401
cthornton <@t> dahlchase.com
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From: histonet-bounces <@t> lists.utsouthwestern.edu [histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Deloris Carter [delsuec <@t> gmail.com]
Sent: Friday, July 26, 2013 5:09 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Losing tissue on IHC slides

Hi all,
I'm looking for some troubleshooting help.  In the last couple of months
we've been having an increasing trend in losing tissue from our IHC
slides.  We use a Ventana Benchmark XT.  PM is done on it quarterly.  The
problem seems to be with the patient tissue, not the control tissue.  We
use Fisherbrand Superfrost Plus slides, picking up the control by dipping
the top end of the slide to avoid "double dipping".  We use tap water in
the waterbaths which I know could cause an issue, but that's the way it's
always been done here, so there's been no change to that part of the
procedure. (We did try using DI water, but it didn't make any difference.
Tissue still fell off of slides.) We do use DI water to mix the bulk
reagents.The control slides may sometimes have been cut for a longer than
recommended time, but that part of the procedure hasn't changed either,
same process as always.  If we run separate slides for control and patient
tissue, we get much better results, but still have some tissue loss.  I
can't say it's all of our tests, but it is a steadily worsening problem.
I'm going through tons of reagent, and I can't seem to nail down the
problem.  Ventana rep said to try DI water, don't double dip, try adhesive
slides (which didn't really make any difference that we could tell).  I
would appreciate any suggestions
Deloris Carter
Shawnee Mission Medical Center
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