[Histonet] Need a chemist's opinion on formalin fixation reversal

[Due, Brice]

Hi, I have a related tidbit for you. I am not a chemist, but I just recently
read deep on HIER for an ISH protocol we started. Several sources (DAKO manual I
think, and a couple rigorous papers I found) suggested that Heat Induced Epitope
Rerieval (HIER) on Formalin Fixed Paraffin Embedded (FFPE) tissue sections
serves (in part) to accelerate what washing at room temp could in theory do
alone. In other words, washing long enough can remove the bound formaldehyde
and/or reverse other FFPE fixation effects. It was estimated that washing alone
could take months to achieve what HIER does in 30-60min. Sorry I don't have
references handy, but if you want them, bug me and I'll dig through my folders
sometime. 

-brice
Neuropathology Lab
Brigham & Women's Hospital
Boston


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-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu]On Behalf Of Luis
Chiriboga
Sent: Tuesday, June 07, 2005 2:21 PM
To: Andrea T. Hooper; Patsy Ruegg; histonet <@t> lists.utsouthwestern.edu
Subject: RE: [Histonet] Need a chemist's opinion on formalin fixation
reversal


Hey Andrea
I don't know if the exact chemical mechanism is known. Most likely, the
hydroxymethyl adducts and early cross links (lysine and arginine
predominately) are unstable and easily reversed by h20(and alcohol?)  There
was a really nice 2 part article published in JOH 2001 by Eltoum IE. et al
(unfortunately I don't have those issue because if it's not nailed down,
somebody steals it !) I believe Jerry Fredenburgh was a co-author (yup, he
presented at NYSHS) so you may want to contact him......let me know
L


-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu]On Behalf Of Andrea T.
Hooper
Sent: Monday, June 06, 2005 5:54 PM
To: Patsy Ruegg; histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Need a chemist's opinion on formalin fixation
reversal


Can one of the chemists on board please address the issue of washing
to reverse formalin fixation? (sorry if you have done so already but
I must have missed it).

This is a new concept to me and makes me very very nervous about some
of my routines in the lab as often times I can only occasionally
process and have samples in the fridge for sometime after fixation
yet before processing ... yikes!

Thank you thank you!
Andrea



>I have been asked to elaborate on my reference to washing tissues to
reverse
>the effects of formalin fixation iliminating the possible need for AR.
>Here goes:
>
>
>I found the reference to Washing in a book called Introduction to
>Immunocytochemistry 3rd Edition  J.M. Polak and S. Van Noorden pg. 24-
3.8.2
>"The simplest form of reversing the effects of formalin is to wash the
>tissue well before processing, but this is not usually possible in
>histopathology laboratories, where rapid turnowver of specimens is
>required."
>
>I have not tested this extensively but have done a little, especially on
>over fixed samples, the data is yet to be confirmed but I am looking into
it
>as we speak.  I will keep you all posted.
>
>Patsy

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