Re: [Histonet] Formalin Fixation times for IHC samples

From:"Bryan Hewlett"


You asked >>How many other laboratories experience this problem?

The majority of histopathology labs in Canada and the US!

For over 25 years, some of us have been on a crusade to correct this.
The result,
When shown the undeniable evidence, as you have done, the IHC technologists
get it immediately but few others choose to listen.

And I thought that we were in the era of 'evidence-based' medicine!!!

Bryan Hewlett
Quality Management Program-Laboratory Services
Ontario, Canada.

----- Original Message -----
From: "Barry Madigan" 
Cc: "'Katri Tuomala'" 
Sent: Wednesday, February 23, 2005 5:42 PM
Subject: RE: [Histonet] Formalin Fixation times for IHC samples

> I find the major impediment to good quality Immunohistochemistry is turn
> around times, which has been a buzz word here in Australia for a number of
> years. The result of this management tool has led to poorly fixed and
> processed tissue samples that are unable to withstand the harsh treatment
> heat retrieval.
> Thanks to Murphy's Law, we usually find that the most vital block for
> diagnosis is always the one that is poorly preserved.
> We receive material for Immunohistochemistry from a large number of
> hospitals in Queensland (Australia). Unfortunately there is no uniformity
> the way specimens are fixed or processed by these referring laboratories.
> Both lymph nodes and breast tissue are the major specimens affected by
> fixation.
> With lymph nodes the peripheral staining is excellent but the morphology
> the centre is compromised.
> The central structures of these specimens have not been strengthened by
> fixation and as a result the morphology is damaged by heat retrieval.
> With breast specimens there is an increase risk of tissue lifting from the
> slide.
> Try to convince Pathologists that fixation is the problem is just about
> impossible.
> We place control material, which has received at least 24 hours fixation
> the same slide. The control material is always perfect and the Pathologist
> always concurs but questions why the test morphology is damaged.
> It's great to get pathology reports out quickly but at what diagnostic
> How many other laboratories experience this problem?
> Barry Madigan
> Brisbane, Queensland, Australia
> -----Original Message-----
> From:
> [] On Behalf Of Katri
> Tuomala
> Sent: Thursday, 17 February 2005 12:23 PM
> To: Anne C Lewin;
> Subject: Re: [Histonet] Formalin Fixation times for IHC samples
> Anne,
> I think you are doing well with your protocol. Although I don't think
> "overfixation" is as a big problem any more with the advent of different
> HIER protocols with various buffers being used. Under fixation (less than
> hours) can be a bigger problem with certain antibodies like Her-2, ER and
> to mention few. This concept of over fixation is a remnant from the time
> prior to HIER, when we all had a problem trying to demonstrate many
> with weaker retrievals (proteolytic enzymes). The heat over 60C was
> banned from any immunohistochemistry protocols 20 years ago.
> My two cents worth...
> Katri
> Katri Tuomala
> Hamilton, Ontario, Canada
> ----- Original Message -----
> From: "Anne C Lewin" 
> To: 
> Sent: Tuesday, February 15, 2005 10:28 AM
> Subject: [Histonet] Formalin Fixation times for IHC samples
> > Could I get a general concensus from Histoland about the amount of time
> > needed to fix a sample for IHC without over-fixing?  My training/journal
> > searches  have led  me to believe that the longer a sample is in
> > formalin, the stronger the protiens are cross-linked, and the more
> > difficult it is to break those bonds with antigen retreival and get your
> > antibody to the target.  I generally tell the scientists in my
> > department to fix overnight (most samples vary from about the size of a
> > pea to a lima bean), and then to transfer to 70% Ethanol for me to
> > process for paraffin.  The total time in fixative tends to be around 24
> > hours.  Morphology has always been great, and my IHC's work well.  This
> > question is mainly for my information, since I have used the same tissue
> > prep protocol for a few years now, I want to make sure I am keeping up
> > with current opinions.
> > Thanks!
> > -Anne
> >
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