RE: [Histonet] Microwave Processing

From:"Steven E. Slap"

Hi Terry & HistoNetters

Let me take a stab at this, although I am not a 
chemist (Dick Dapson, where are you when I really 
need you?)...

Microwave stabilization uses heat to harden the 
tissue through a process of coagulation of the 
proteins in the tissue.  Kok & Boon liken this to 
poaching an egg.  Alcoholic "fixation" also 
hardens the tissue by coagulation of the 
proteins, but also removes the water (the higher 
the percentage of alcohol, the more the hardening 
effect).  The use of alcohol as the primary 
fixative results in a characteristic shrinkage 
within the tissue.  Aldehydes "fix" the tissue 
through cross-linking of the proteins.

When I talk about microwave stabilization not 
chemically "fixing" tissue, I mean to say that 
the proteins are not cross-linked.  In Leong's 
work, the biopsies were microwave-stabilized in 
saline (hardened), then further hardened in 
graded ethanols on a conventional processor 
(normally referred to as "alcoholic fixation"), 
but still not cross-linked.  With larger tissues, 
I would worry about shrinkage with this method, 
but Leong seemed to have success with it on 
biopsies.  I still don't think they would look 
quite the same as formalin-fixed specimens.

I'm sorry if I didn't make myself clearer.

To muddy the waters further, some labs carry out 
microwave stabilization of gross tissue specimens 
in formalin at 50C, rather than in saline at 
65C, but this still isn't fixation, because the 
formalin doesn't penetrate the tissue 
sufficiently to cross-link.

Whew.

best regards,
Steven Slap
Microwave Consultant

At 12:48 PM +0100 4/21/04, Marshall Terry Dr, 
Consultant Histopathologist wrote:
>My confusion has not been dispelled.
>
>Steven writes:
>
>"The saline will not work
>alone as a fixation step, except in the case of
>very small biopsies (as written up in an article
>by Tony Leong)- ..."
>
>In the next post, we get:
>
>"Yes, in the Leong method for biopsies, the specimens are heat
>stabilized in the microwave in saline, and not really chemically
>fixed.  They get fixed in the ethanol in a traditional processor ..."
>
>These statements are mutually incompatible.
>
>Moreover, what is the difference between "stabilisation" and "fixation".
>
>A further muddle is introduced by:
>
>"not really chemically fixed."
>
>Well, hell, are they fixed or no? Heat fixation 
>is fixation - who cares whether chemically fixed?
>
>Of course, the major problem is that mechanisms 
>of fixation are varied (with the fixative) and 
>not well understood.
>(Speak for yourself do I hear someone say?)


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