[Histonet] Soaking paraffin blocks

[Nicola Cragg]

Hi Kemlo,

The reason that we use Carnoy's fixative is that the morphology on H&E
is superior to Formalin and we perform a lot of manual histometric
analyses by light microscopy, which in particular includes the
identification of mitotic and apoptotic cells - these are much easier to
recognise in Carnoy's fixed tissue.  Also this method has a 30 year
history so I don't think I would be able to convince people to change
the fixative now!!  We do use formalin for other work, i.e. IHC.  

Nicola



-----Original Message-----
From: Kemlo Rogerson [mailto:Kemlo.Rogerson <@t> waht.swest.nhs.uk] 
Sent: 30 October 2007 15:08
To: Nicola Cragg
Cc: histonet <@t> lists.utsouthwestern.edu
Subject: RE: [Histonet] Soaking paraffin blocks

"What do you mean when you say "If the processing fluids are left on too
long, tissue goes hard" - are you referring to too much dehydration or
that the fluids have been left on the processor too long?  After a
previous discussion on Histonet, I had understood that some think it's
not possible to over dehydrate - do you not agree?  The schedule I use
is quite lengthy so I don't think it can be a case of too little
dehydration."
Nicola

Interesting point and one I've thought about before; alcohol is a
fixative as well as a dehydrating agent. If you put unfixed tissue on to
a processing machine then you logically get alcohol fixation and that
classically can make tissue 'hard'. If you put fixed tissue onto a
processing machine then I'm assuming that initial fixation 'protects'
the tissue against the effects of the alcohol. I agree you can't over
dehydrate tissue as, as they say in the Sales, 'once it's gone, its
gone'. I do think however that you can overfix tissue and I assume that
the longer you leave formalin fixed tissue (a 'soft' reversible
fixative) in alcohol then the greater effect that will have as a
fixative and then harden.

Interestingly Carnoy's is just that, an alcoholic fixative, containing
alcohol, chloroform and acetic acid (60% ethyl alcohol). It classically
is used for rapid fixation of thin pieces of tissue for relatively short
periods of time. It's said to cause considerable shrinkage, dissolves
acid soluble cell granules and pigments. It would not be the fixative of
choice for the work you do IMHO and I would suggest at least replacing
ethanol with methanol but moving to a formalin based fixative may work
better. Large blocks would have to remain in the fixative for a long
time (alcohol penetrates slowly) so by the time the middle is done, the
outside is like a brick.

You are using 'wetted ice' I think to reclaim tissue made hard by using
a rather harsh fixative known to harden tissue. 



Kemlo Rogerson
Pathology Manager
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