[Histonet] Re: Histonet Digest, Vol 102, Issue 26

[Kuhnla, Melissa]

Hello,
I also use Ventana Ultra instruments.  We have had our air share of
tissue adhesion issues as well. We currently have most of it under
control by doing the following:


1. We switched from standard plus slides to Fisherbrand Excel Adhesion
slides (cat #22-034-985). I believe they are meant for automated
staining, solutions of varying temp and pH, etc.  We did see an
improvement!!
2. We at all time handle our slides with gloves on.  We dip and dry
controls upside-down.  I was very clear to us that dipping and handling
the slide while cutting the control section altered the charge of the
rest of the slide.  As a rule of thumb...if we need to repeat things due
to tissue loss, we run it on a slide by itself and run a separate
control.  If a focus of tumor is very small or tissue is minimal to
begin with, a pathologist may request separate controls right from the
start.
3. We also altered some of our processing schedules.  At first we were
loosing a lot of small biopsies and FNAs.  We shortened our processing
and now they adhere fine!! 
4. We discontinued the use of recycled Xylene in the processor as well.

BTW, We routinely cut at four microns, dry vertical for 15 minutes and
bake at 60 for 90-120 minutes


We have this issue under such a microscope here that we know it will be
impossible to get rid of this all together.  Breast cases (and sometimes
skin) remain to be the only culprits.  These tissues just by nature may
always be a problem.  We now are contemplating running batched controls
for ER/PR cases so all those cases are on their own slides.
Hope this helps.
Melissa
-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of
Madeleine Huey
Sent: Wednesday, May 23, 2012 12:29 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Re: Histonet Digest, Vol 102, Issue 26

>> I'm a Histotechnologist working in the Regional Hospital in Barrie,
> ON Canada. We are using the Ventana Ultra for our Immunohistochemistry
> (IHC). Since the end of February, we have been having issues with some
> tissues lifting off our positive (marked with +) charged slides. It
> seems to be mostly with the fatty and/or larger sections. We now dry
> our slides for one hour at room temperature (R.T.) and an additional
> hour at 60 degrees C. We cut our IHC sections at 4 um. Since we have
> tried 2 different types of + slides and will be trying another type of
> charged slide (from Newcomer this time) I was wondering if anyone has
> any other suggestions?
>> I also have another question regarding a QC (quality control) issue.
> We use a multi-tissue control that is applied to the top of all our
> test slides for IHC. One of our paths commented that there is some
> positive staining in the smooth muscle nuclei of thenormal bowel when
> we are testing for Progesterone (PR). We are using a Heat Induce
> Epitope Retrieval (HEIR) of 36 minutes with CC1 (Ventana's proprietary
> buffer @ pH of 8.0-8.5) and a primary antibody incubation time of 16
> minutes with PR clone 1E2. (Ventana instrumentation provides
> pre-diluted antibodies and the user adjusts the concentration of the
> antibody by adjusting the time the primary antibody is incubated with
> the tissue).
>> I am concerned about the implications of this staining and I have
> not been able to find a reference to this kind of unusual staining
> pattern. The bowel tissue that we are using as QC is from a 62 year
> old female patient. I was wondering if anyone has had any experience
> with this kind of staining and /or any references that I could use.
>>
>> Thanking you in advance,
>> I look forward to your input,
>> Nancy Cloughley-Gray MLT

Nancy,

Your problem is very common, due to fatty tissue is difficult to
process.

Here's my suggestion if you want to salvage your fatty tissue blocks
(presumably the fatty tissues are well fixed);

1) melt down the fatty tissue block & re-process

2) cut & put on charged slides (Fisher Scientific "Ultra Stick"
charged slides work the best)

3) bake slide for 30 min @ 60c

4) do antigen retrieval with Biocare's Diva for 3 min in Pascal or
Biocare pressure cooker (very important; do NOT use Triology & Cell
Marque pressure cooker).

5) do your ihc as usual

Three important elements; (1) well process tissue, (2) Biocare Diva AR
buffer, & (3) Pascal pressure cooker.

Keep us posted what's your outcome.

Madeleine Huey BS, HTL/QIHC (ASCP)
Supervisor - Pathology (histology & IPOX)
madeleine_h <@t> elcaminohospital.org

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