[Histonet] Ventana BenchMark IP stainer

[Sebree Linda A.]

Karen,
We find that once you go to mild CC1 or CC2, you may have problems with tissue morphology and/or tissue loss yet plain CC1 or CC2 may not be long enough retrieval time.  Sometimes if we need more HIER but don't want to extend our time to the mild setting we'll add a weak protease (P2 or P3) for a short time to enhance the retrieval effect without further tissue damage. Also, the reason you're probably getting worse results with CC1 is that it is the harsher of the two buffers (EDTA) at a higher pH than CC2.  We generally do not oven dry slides going on the Benchmark; just make sure they are well air-dried.  So maybe your slides are just being exposed to too much heat between the oven and deparaffinization on the instrument.  So try backing off on a few things that may be damaging your tissues and see if that helps.  Feel free to contact us with specific questions as we have dealt with a number of the same issues.

Linda A. Sebree, HT(ASCP)
University of Wisconsin Hospital & Clinics
IHC/ISH Laboratory
A4/204-2472
600 Highland Ave.
Madison, WI 53792
(608)265-6596
FAX: (608)262-7174


-----Original Message-----
From: Bauer, Karen [mailto:Bauer.Karen <@t> mayo.edu]
Sent: Thursday, October 09, 2003 1:38 PM
To: Histonet (E-mail)
Subject: [Histonet] Ventana BenchMark IP stainer


Hi,

One of our Pathologists is unhappy with some of the results from our
BenchMark IP stainer.  Not all, but some tissues fall off and there are
stains where the tissue seems to be "burnt" or washed out.  We realize that
fixation has a lot to do with the way a stain can turn out, and I've told
him this many times.  I'm sure that everyone has had to deal with "Hurry and
get this on the Processor tonight" and then the next morning we are supposed
to make everything cut, stain, and look perfect.  This isn't always the
case.  There will be well fixed tissue that will look and stain the same
way.  

We were having this problem with our Ventana Nexes stainer, but after some
micron and heating time adjustments, things started looking good again.  Now
that we've upgraded to the BenchMark, it's like we are starting from scratch
again.  We've had it since July of this year and our Pathologist would
really like our Nexes back.  The thought of changing all of our IP
procedures again does not sound appealing to me.

Right now, this is the way we cut and prepare our slides for IP staining on
the BenchMark:  Cut sections at 4 to 5 microns and place on a positive and
negative control slide. (We use the charged slides with the red control box
on them.)  We let them air dry for 30 minutes and then we place them in a 60
degree C oven for 30 minutes to 1 hour.  We then place them on the BenchMark
for staining.  

The stains that we are having the most troubles with are the ones using the
CC1.  I've checked our buffers and the pH is fine.  We're having difficulty
with our Pathway Her 2 also.  This uses the CC2 and it ends up looking
washed out.  If we keep it at the mild CC2, there's staining but it's light.
Standard CC2 looks like the tissue is "eaten up or burnt out", and Extended
CC2 makes the tissue pretty much fall off.  This only happens to the pt.
tissue.  Our control tissue seems to stain fine. (Pt. tissue and control
tissue are on the same slide.)

I've talked to our Ventana Rep and their technical personnel and no one has
given me any ideas that we haven't tried already.  Can anybody give me some
hints or solutions to these problems?  Has anyone else out there had these
problems?  

Thanks!!

Karen L. Bauer
Histology Department
Luther Hospital
715-838-3205



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