AW: [Histonet] Immuno's on Thin Prep specimens

From:"Gudrun Lang"

I think a part of the HIER action is really restoring: Because the
methylenbridges are linked between the proteins and therefore also between
the antigen-aminoacids. This causes a change in the structure of the protein
and this can cause the "hiding" of the epitop. Paratop doesn't recognise
epitop. After HIER the antigen-aminoacids restore there former structure.

Gudrun Lang
Biomed. Analytikerin
Akh Linz
Krankenhausstr. 9
4020 Linz

-----Ursprüngliche Nachricht-----
[] Im Auftrag von Shawn
Gesendet: Freitag, 28. September 2007 16:02
An:;; Sebree
Linda A.; Rene J Buesa
Betreff: RE: [Histonet] Immuno's on Thin Prep specimens

Just a correction Rene. You stated "HIER is designed to "restore" the
epitope reactive characteristics after being cross-linked by the
paraffin ". Actually HIER was introduced to demask the epitope after
being cross-linked (methylene bridges) by the formalin not the
paraffin.... Just wanted make that correction

Shawn Leslie
Scientific Research Manager
Anatomic Pathology
University of Florida
School of Veterinary Medicine
352-392-2235 ext 4555

>>> Rene J Buesa  9/28/2007 9:43 AM >>>
  Not to start a controversy, just to discuss the issue:
  1- antibodies are designed to reveal the presence of a given
  2- such epitopes are the same regardless of the processing protocol
or the tissues they are in, either smears, thin-preps or tissues, they
just vary in concentration and distribution among the cells;
  3- HIER is designed to "restore" the epitope reactive characteristics
after being cross-linked by the paraffin to its "original" or "natural"
  4-once the epitope has been restored it is equivalent to not having
ever being altered and will react identically whenever it is present.
  IF your usual protocol to detect the epitope in your positive tissue
control is not altered from its usual way and renders the same reaction
intensitywise on the same target cells, it should also react in the thin
prep treated identically.
  I realize that since you "recently stuck your head" on this issue and
refused to do something like I am advocating, it will be rather
difficult for you to analyze the precedent paragraphs passionlessly. but
give a chance to reasoning.
  On the other hand, I have done hundreds of IHC on thin preps and
always used FFPE tissue sections as positive controls. The positive
controls always behaved as expected and the thin preps, treated
simultaneously rendered positive or negative results, according with
their specific nature.
  The thing that you have to be consistent with is using another
identical thin prep as negaive control.
  René J.  

"Sebree Linda A."  wrote:
  I disagree Rene in terms of using a paraffin section as a positive
control. Your two specimens (thin prep & + control) are not prepared the
same. In order to get your + control to stain , you may have to employ
HIER which as you state you would not do with the thin prep. That
pretreatment of the + control makes it even "more different" than your
patient unknown. I recently stuck my neck out at work for this very same
issue. I refused to be part of running a cytospin with a FFPE staining
protocol and + control. Our lab used to run cytospin IHC protocols and
many times had to adjust the FFPE protocols to work for the cytopsins.
At that time we used frozen section tissue controls for our positives
for lack of a better alternative. These came closer to the patient
specimen than FFPE controls and actually stained identically to the

Just my experience.

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

-----Original Message-----
[] On Behalf Of Rene
J Buesa
Sent: Friday, September 28, 2007 7:49 AM
Subject: Re: [Histonet] Immuno's on Thin Prep specimens

If using thin preps for IHC, you do NOT need to do HIER because they
are acetone/air dried fixed, not formalin fixed. The rest of the IHC
protocol is the same as for a FFPE tissue section. Your positive control
refers to the epitope, so your regular positive control from a tissue
section can be used, BUT you negative control has to be a thin prep from
the same case simultaneously prepared.
René J. wrote:

Is anyone doing IHC's on cytology specimens? If you 
are can you please
send staining and fixation protocol. Also, what do you use for your
positive and negative controls? Thank you

Andrea J Weiss BST CT (ASCP)
609 653 3577 Ext 4907 
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