[Histonet] overfixation with formalin

Sun Nov 4 17:29:46 CST 2012

I would have explained this as poorly fixed tissue with the mushiness due to alcohol fixation from the dehydrating alcohols.

Alcohol shrinks the tissue (as it removes the water from the cells), making it very difficult to infiltrate with xylene and the wax giving a mushy centre. 
This is also common if tissue samples are too thick (where they are squeezing out of the cassette holes).

Formalin forms crosslinks with cell constituents (proteins etc) resulting in a "scaffold-like" rigidity of the cells. 
This allows easier movement of processing solutions in and out of the cells with little collapse of the cells.

Regards 
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
Laboratory Manager & Senior Scientist 
Tel: 612 9845 3306 
Fax: 612 9845 3318 
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Gudrun Lang
Sent: Sunday, 4 November 2012 7:03 PM
To: Charles.Scouten <@t> LeicaBiosystems.com
Cc: Histonet <@t> lists.utsouthwestern.edu
Subject: AW: [Histonet] overfixation with formalin

This margin effect was especially seen in needle biopsies. In 2 mm cores.
But he showed also a lymphnode slide, I would estimate 0,5-1 cm, with a margin-effect, that causes bad cutting in the center (loosened aereas). In this context he said, that a lymphnode sitting in higher concentrated formaldehyde over the weekend isn't properly fixed in the center because of the hardened surface.

I'm not with his opinion.

Gudrun

Von: Charles.Scouten <@t> LeicaBiosystems.com
[mailto:Charles.Scouten <@t> LeicaBiosystems.com]
Gesendet: Samstag, 03. November 2012 23:20
An: gu.lang <@t> gmx.at; histonet-bounces <@t> lists.utsouthwestern.edu
Betreff: Re: [Histonet] overfixation with formalin

Is this due to the well known effects that formaldehyde penetrates tissues very slowly, at a rate of about 18mm/25 hours.  And that autolysis (tissue
breakdown) begins to occur  immediately with extraction.   If so, then in
your tissue the center decayed before the fixative reached it.  How large was the starting piece of tissue, how far was the center negative area from any direct exposure to formaldehyde?  Was it precut in slices less then a mm thick, before being dropped in fixative? 

Cordially, 

Charles W. Scouten, Ph.D
Product Specialist
Leica Biosystems Richmond, Inc.
5205 Route 12
P.O. Box 528
Richmond, IL 60071
United States of America
Telephone 630 964 0501
facsimile +1 630 964 0576
 <http://www.myneurolab.com/> www.MyNeuroLab.com  <http://www.leica-microsystems.com/> www.leicabiosystems.com 

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From:        "Gudrun Lang" <gu.lang <@t> gmx.at> 
To:        <histonet <@t> lists.utsouthwestern.edu> 
Date:        11/03/2012 01:42 PM 
Subject:        [Histonet] overfixation with formalin 
Sent by:        histonet-bounces <@t> lists.utsouthwestern.edu 

  _____  

Hi histonetters!

I'm just attending a histo-course, where the teacher told us his opinion about overfixation.

For him overfixation takes place in any formaldehyde solution with a concentration above 5%. This should cause the margin-artefact, that leads to false-positive IHC at the margins of the tissue and to false-negative results in the center. The higher concetrated fixative should harden and shrink the surface, so it cant be penetrated any more by the fixative.

I told him about the publication of Cecil Fox, who saw shrinkage only in solutions with formaldehyde concentration above 30% (I think) and said, that the methanol-part is responsible for that.

I believe, that these margin-artefacts are due to drying at the time of biopsy or an effect of the needle-shot itself. (But believing is no
evidence)

In our lab we use 8% formaldehyde as standard fixative, buffered with low-molar phosphatebuffer. There are no complains from the doctors about margins.

Please help me with the histonet-wisdom. What's your opinion? 

Bye

Gudrun Lang

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