[Histonet] IHC question

From:"Orr, Rebecca"

Hi Karen,
My experienced guess would be 2 things.
I am suggesting this based on my experiences in the field
troubleshooting just this type of issue, so I'm not really an expert.

But,  I would consider these things:
1) The CD 3 and CD 20 epitope sites may not need as much formalin
fixation to stain appropriately. There may be some "wiggle room"  in the
titer of these antibodies.  What is your pretreatment for all 3?  Are
they all the same?  In my lab I run concentrates and my CD3 works best
with Tris HIER and the CD 20 works best with citrate HIER.  Back in the
day, LCA didn't even NEED HEIR!  
2)You may have simply over done the HIER and caused a prozone effect.

Because the LCA control worked that shows me that the Antibody itself is
working appropriately. These immunogens are developed separately so even
though LCA will stain the same sites as CD3 and CD20, they aren't "the
This may not be a fact but comes more from my experience. 
I'm sure you could make the LCA work if you compensated for the variable
that changed, in this case the formalin/processing time.  
Either lighten up on the hier or if you can, extend the incubation time.
Or Change the concentration.

Sometimes errors are made and you have make a change to the standardized
protocol to compensate.

Hope this helps

Becky Orr CLA,HT(ASCP)
Assistant Manager, Anatomic Pathology
Evanston Northwestern Healthcare
-----Original Message-----

Message: 23
Date: Fri, 7 Apr 2006 11:57:23 -0500
From: "Bauer, Karen" 
Subject: [Histonet] IHC Question...

Content-Type: text/plain;	charset="iso-8859-1"

Hi to all. 
I recently stained a lymph node case and it has brought up an
interesting question that I can't explain to our Pathologist.  I'm
hoping that someone out there can give me some suggestions.  First of
all, a little background...
The lymph node was processed, by accident, on a "fast run" on our VIP
Tissue Tek.  The processing run was complete in about 3 and a half
hours.  (We thought there were just small biopsies on the run, but the
lymph node cassette was probably thrown in the wrong basket by mistake.)
Anyway, the lymph node tissue didn't turn out all that bad, but the
fatty tissue did not process adequately (no surprise there!!).  Since
the lymph node tissue cut fairly well, the block was not run back and
re-processed.  H&E's were turned in and stains were ordered.  
Here's my problem:  The CD3 and CD20 stained well and the lymph node
tissue and controls looked as they should, but the LCA on the patient
tissue did not stain at all.  We put the patient tissue on a control
slide, so the control and tissue are stained the same all the way
through the run.  The LCA control looked beautiful, but the lymph node
tissue had no staining.  How can you have positive staining for CD3 and
CD20, but no staining for LCA on a lymph node?  
Since we use a tonsil for our control, we re-ran the LCA with the tonsil
control, a different lymph node case and then the same lymph node case
that we were staining.  Again, the control looked great, but the lymph
node did not stain again.  The different lymph node case that was also
run turned out just fine, so we knew it was tissue specific.  We thought
maybe this lack of staining was due to the insufficient processing, but
then why did the CD3 and CD20 turn out so well?  Shouldn't they have no
staining also?
I'm perplexed and was wondering if anyone else out there has had this
happen to them.
As always, thank you.
Karen Bauer HT(ASCP)
Histology Supervisor
Luther Hospital
Eau Claire, WI
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