[Histonet] Formalin Fixation times for IHC samples

Bill Sinai bills <@t> icpmr.wsahs.nsw.gov.au
Wed Feb 23 17:08:30 CST 2005

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

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

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu]On Behalf Of Barry
Sent: Thursday, 24 February 2005 9:42 AM
To: Histonet <@t> lists.utsouthwestern.edu
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
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

Try to convince Pathologists that fixation is the problem is just about
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-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Katri
Sent: Thursday, 17 February 2005 12:23 PM
To: Anne C Lewin; Histonet <@t> lists.utsouthwestern.edu
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" <anne.lewin <@t> bms.com>
To: <Histonet <@t> lists.utsouthwestern.edu>
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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