RE: [Histonet] Formalin Fixation times for IHC samples

From:"Morken, Tim - Labvision"

I thought Dr Leong converted all you in Oz to microwave fixation!

Tim Morken

-----Original Message-----
[] On Behalf Of Bill Sinai
Sent: Wednesday, February 23, 2005 3:09 PM
To: 'Barry Madigan'; histonet (E-mail)
Subject: RE: [Histonet] Formalin Fixation times for IHC samples

Hi Barry,

I couldn't agree more.  We are a major centre for NSW Breast Screen Western
and The Breast Cancer Institute and the surgeons perform their operations on
Wednesdays and Thursdays then have a meeting Monday and Tuesday and expect
the results including Immunohistochemistry at these meetings.  Fixation for
most specimens is minimal at best and the results show poorly on
Immunohistochemistry.  As you said incomplete fixation means sections
looking tatty or falling off.  The positive controls, usually collected
after the specimen has fixed for at least another 24 hours, always have
great morphology and staining.  We find it is very much surgeon driven as
they want results for meetings for discussion about course of treatment and
for discusion with patients.  Many do not understand the importance of good
fixation to the diagnostic process. Automation (Tissue Processors, Staining
Machines, Automated coverslippers and automated Immunohistochemistry) helps
a little with TAT but these are only some facets of the total process which
begins with fixation.  Microwave fixation helps but is not the answer to all
the problems.  Until someone discovers a revolutionary new fixation method I
will still stick to "good old formaldehyde".  I have tried several other
formaldehyde substitutes over many years but the pathologists still prefer
the known artefacts produced by formaldehyde.

Bill Sinai
Laboratory Manager
Tissue Pathology, ICPMR
Westmead NSW 2145
Ph 02 9845 7774

-----Original Message-----
[]On Behalf Of Barry Madigan
Sent: Thursday, 24 February 2005 9:42 AM
Cc: 'Katri Tuomala'
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 of
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 in
the way specimens are fixed or processed by these referring laboratories.
Both lymph nodes and breast tissue are the major specimens affected by under
fixation. With lymph nodes the peripheral staining is excellent but the
morphology of 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 on 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 cost.
How many other laboratories experience this problem?

Barry Madigan
Brisbane, Queensland, Australia

-----Original Message-----
[] On Behalf Of Katri
Sent: Thursday, 17 February 2005 12:23 PM
To: Anne C Lewin;
Subject: Re: [Histonet] Formalin Fixation times for IHC samples

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 24

hours) can be a bigger problem with certain antibodies like Her-2, ER and PR

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 antigens

with weaker retrievals (proteolytic enzymes). The heat over 60C was actually

banned from any immunohistochemistry protocols 20 years ago.
My two cents worth...


Katri Tuomala
Hamilton, Ontario, Canada

----- Original Message -----
From: "Anne C Lewin" 
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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