Re: [Histonet] Immuno slide problem

From:Todd Sherman

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I have not used the Ventana Nexus IHC or any slides of the type you
describe, but I have a question for your QC follow-up. What happens when
you use only positive control sections on the slide, i.e. a row of
positive sections only? Do you observe the same unequal distribution of
positive staining in a similar (or for that matter, different) pattern?

If I understand you correctly, you observed the following:
(Hopefully this schematic will resolve properly in your email reader. It
is in ASCII format so you should be able to cut, copy, and paste this
graphic representation on your computers notepad editor.)

~  Label    +Control     Specimen L    Specimen R
|       | +Con LCA + |     LCA +        LCA +      |
|       |            |                             |
|       | +Con L26 + |     L26 -        L26 +      |
|       |            |                             |
|       | +Con CD3 + |     CD3 -        CD3 +      |
~                  ==                           ====
~                ====                       ========
~              ======                   ============
~            ========               ================
~          ==========           ====================

~          Red "Box"           Specimen Area
~         Hematoxylin+          Hematoxylin+

+ indicates positive immunodetection
- - indicates negative immunodetection
= indicates levels of polarized IHC staining via histogram (more bars
equates to higher intensity)

It would seem to me, as you have alluded, that the red barrier material
is acting as a sort of hydrophobic barrier (like a PAP pen) or a
physical one whereby a slightly tilted slide tray might cause pooling of
liquid volumes. The red "raised" barrier could impede the natural flow
of liquid to the far end (label terminal vs label proximal) of the slide
and allow the polar staining pattern that you see.

If you mount a row of positive control sections on the same slide,
immunostain as before, and observe the same pattern, it implicates the
slide. If you change slides (i.e. sans red barrier), mount the same row
of positive control sections, and observe only positive staining near
the terminal end (opposite label), then that implicates the
immunostainer or possibly the tray. It might be out of balance or tilted.

Since I'm not familiar with your staining procedure, this is just
troubleshooting. Incubation times for immunoreagents are typically much
longer than for hematoxylin, so the sections will stain much more
readily for hematoxylin. It wouldn't surprise me that hematoxylin
staining works while IHC detection doesn't work because of the nature of
the chemical reactions and thermodynamics that are occurring. An
adequate volume of reagent must coat the entire section at all times, as
you know, so a bi-polar pooling of reagents exacerbated by that "red
barrier" could explain your staining patterns.

Let us know what happens with all positive controls on a slide and let
the experts consult.

Hope this helps,

Todd Sherman
HistoSoft Corporation
"Biology in a new form..."
Member Services:

From:	"Anissa Choi"

We run our immuno on Ventana Nexus IHC. We place a positive control
section on every slide together with patient 's tissue section. We use
charged slides with a red edge square which is right next to the barcode
label. The square is labelled "control" and that is where we place the
positive control section. We had a case of a punch skin bx on which the
pathologist requested LCA, L26 and CD3. Because of the small size of
specimen, 2 sections were picked up on each slide. There was no problem
with LCA, the positive control and the 2 skin sections stained strongly.
For L26, the control worked but only 1 skin section ,which was at the
bottom end of the slide, was immunostained strongly  for L26.  The other
skin section in the middle and lying next to the positive control  was
completely negative for L26 but nuclei were stained with hematoxylin.
The same staining pattern was seen in CD3-----the skin section in the
middle did not get stained while the control and the other section
worked just fine. Ventana suggested us to switch to other kind of
slides, saying some stuff from the red edge may get onto the slides and
cause staining problem. We requested a maintenance check on the stainer
and Venatana could not find anything wrong with the machine. We recut
the section using same kind of slides and repeated L26 and CD3. We got
normal result with both skin sections stained. We also re-run the
origional problem slides on Ventana but they turned out the same, the
skin section in the middle still not stained.

I am convinced  that there is something on the slides that is blocking
the staining but I am puzzled by the fact that the nuclei are stained. I
would like to get some comments or opinions on this. I also hope to hear
from people using the same type of slides to see if they have any
staining problems.

Thanks in advance for your inputs.
Anissa Choi
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