Fwd: RE: [Histonet] Immuno's on Thin Prep specimens

From:Rene J Buesa

Rene J Buesa  wrote:  Date: Fri, 28 Sep 2007 07:21:26 -0700 (PDT)
From: Rene J Buesa 
Subject: RE: [Histonet] Immuno's on Thin Prep specimens
To: Shawn Leslie 

  My ABSOLUTE involuntary mistake. You are right, formalin crosslinkage is the one "targeted" by HIER, to undo what formalin did to the epitope reactivity.
  But let me tell you, there is something also HIER is intended to: in 1980 JB Matthews points out in "Influence of clearing agent on immunohistochemical staining of paraffin-emebedde tissue" that the omnipresent XYLENE causes: 
  "Many laboratories use xylene as their routine clearing agent, which has been shown to have an extracting quality on cell cytoplasm as seen ultrastructuraly and to decrease immunoreactivity" (sic.). 
  This author further states: "The results indicate that xylene processing is deleterious to the immunoreactivity of tissue antigens and that the choice of clearing agent may be an important factor determining the ability to stain non-trypsinised sections of formalin-fixed, paraffin-embedde tissue".
  This author was working in a pre-HIER period and used trypsine instead.
  So, BOTH formalin AND xylene reduce the epitope reactivity. When I totally eliminated xylene from my tissue processing protocols I noticed an intensity increase in the IHC reactions of the order of 1+ step in intensity.
  This author did this research at the Immunology Lab., Dept. of Oral Pathology, The Dental School, St. Chad's Queensway, Birminham B4 6NN, UK
  Thank you for pointing out my "slip of the mind/fingers".
  René J.

Shawn Leslie  wrote:
  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-----
From: histonet-bounces@lists.utsouthwestern.edu
[mailto:histonet-bounces@lists.utsouthwestern.edu] On Behalf Of Rene
J Buesa
Sent: Friday, September 28, 2007 7:49 AM
To: AWeiss@shorememorial.org; histonet@lists.utsouthwestern.edu 
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.

AWeiss@shorememorial.org 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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