[Histonet] IHC edge effect

[Jeffrey Silverman]

Hi Katelin, 
 
It would be helpful to view some images- can you post them on Histonet? 
Just to be sure: You say you have intense peripheral non-specific staining and do not see the expected immunoreactivity in the center of the tissues, or do you see expected reactivity centrally but of weaker intensity? 
Don't know what more to tell you but some questions come to mind...
..
Formalin fixed tissues?  Any microwave  processing or fixation going on? Antigen retrieval? SMA, in my experience, does not require antigen retrieval. 
 
Are you staining manually or on an instrument?  If instrument, which one? Is it calibrated and PM'ed to deliver the proper amount of reagent? The progressive involvement and the problem's new appearance in other antibodies suggests maybe a deteriorating problem with an instrument's function. 
 
 If manual, do you drag reagent well past the edges of the tssue to more than cover each section?  Is only one person doing the stains? ie could it be a problem with someone's technique? 
 
How do you dry your sections? Immediately after cutting place in a 60 degree C oven. Be sure it is  no more than that, hotter than 60 deg C will damage sections and immunoreactivity. Or do you  air dry first and then oven?  This might allow lifting of the periphery with subsequent entrapment of reagent under the edges that might not be removed with interstep washing. 
 Just some more ideas. 
 
Jeff Silverman HT HTL QIHC (ASCP)
Pathologists' Assistant
Southside Hospital - NSLIJ Health System

--- On Wed, 1/13/10, Katelin Lester <histology <@t> medsurgpath.com> wrote:


From: Katelin Lester <histology <@t> medsurgpath.com>
Subject: Re: [Histonet] Fw: Waste paraffin and edge effect
To: "Jeffrey Silverman" <pathmaster <@t> yahoo.com>
Date: Wednesday, January 13, 2010, 12:33 PM


Hi Jeffrey,
I'm curious about what you said about the edges of the tissue having more
fixation than the rest of the tissue. I'm curious because that would make
sense with some of the uterus sections we are using for our control for
SMA, but I'm not sure how that would work for the patient tissue as well.
Every SMA I run, control and patient, any area of the slide shows this
dark edge staining with minimal staining in the center, as well as
nonspecific. We are also seeing it in CK AE1/AE3 and our CD117 has started
demonstrating it on previously perfect controls.  Any other ideas or
suggestions?
Katelin Lester, HTL(ASCP)
MedSurg Pathology Associates, Inc.
(503)443-2157
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> Hello everyone,
>  
> Our system's  hazardous chemical waste consultants changed our method of
> disposal- we had been solidifying the paraffin in a hood and then red
> bagging it, ie treating it as regulated medical (biohazard) waste.
>  
> But, due to it's contamination with xylene, the very reason for our
> disposing of it, the paraffin is really hazardous chemical waste. Believe
> it or not, EPA regs prohibit most hospitals from treating their chemical
> waste and the consultants worried that solidifying it under the hood was
> considered "treating" the waste.  Yeah right!!   Anyway, the blocks of
> solidified paraffin are now dumped in it's own specially labelled drum and
> has become a part of our hazardous waste stream, manifested like the
> xylene and dye waste. I'd be careful about putting hazardous chemicals
> into regulated medical waste.
>  
> Interestingly, our system recently inquired if formalin in discarded
> surgicals needs to be decanted before disposal- many hospitals are doing
> just that, but most of those, but not all, recycle formalin (yuk!). Our
> disposal facility informed us that as long as the containers are plastic,
> ie are flammamble, they can be incinerated with the formalin and there is
> no need to decant.  Glory be!!
>  
> In IHC slides, edge effect, or more intense, sometimes nonspecific,
> staining at the periphery of the tissue, can be caused  by more intense
> fixation of a block at the periphery, drying out of the edges of a block
> before fiation,  and/or by some degree of drying of antibodies and
> detection chemistry reagents during staining- this cause is more common in
> manually stained slides rather than those stained on automated stainers.  
>  
> Also electrocautery used during the excision can also cause intense
> staining at the edges of specimens.
>  
> Jeffrey S. Silverman HT HTL QIHC (ASCP)
> Southside Hospital- NSLIJ Health System
> Pathologists' Assistant and Laboratory Safety Officer
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