[Histonet] overfixation with formalin

Tony Henwood (SCHN) tony.henwood <@t> health.nsw.gov.au
Sun Nov 4 17:21:25 CST 2012

Hi Gudrun,
Over-fixation is likely to decrease IPX staining at the edges (since it would require harsher antigen retrieval).

I agree with you. Many of the edge affects with IPX and several special stains (including H&E and Modified Orcein (for HBSAg etc) on Liver bx) are more probably dur to air-drying of the biopsies prior to fixation (similar seen on cautery specimens) or compression due to the needle as you suggested.

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 5:41 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] overfixation with formalin

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


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? 



Gudrun Lang


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