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

From:Rene J Buesa

  Not to start a controversy, just to discuss the issue:
  1- antibodies are designed to reveal the presence of a given epitope;
  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" condition;
  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 cytospins.

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: [] 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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