[Histonet] Nuclear artefact II

[Lee & Peggy Wenk]

>From what I've read about nuclear bubbling, it all starts with inadequate
fixation. Anything else we do to the tissue just makes it worse. Nothing we
can do will ever improve poorly fixed tissue.

If there are not enough cross-links of formalin (or any other fixative) due
to underfixation, then these few cross-links can be easily broken later on.
This causes the proteins and DNA being easily moved around into new
locations.

So some of the things that can move the proteins and DNA are:
- processing (going through alcohols and xylene will warp these under
cross-linked proteins/DNA even more)
- too much heat in processing (alcohols, xylene, paraffin)
- too long of time in hot paraffin
- too much heat during slide drying

The reason we see this artifact mostly with formalin fixed tissue is two
fold:
1) Formalin needs 1-2 days to completely fix 3 mm thick tissue sections. In
other words, we need that amount of time to create enough cross-links so
that nuclear bubbling and other protein deformities do not occur later with
processing, heat, etc.
2) Formalin is a lousy nuclear fixative. Mercury and Zinc are much better
fixatives for nuclei. And tend to fix faster than formalin. So B5, Zenker,
Zinc-Formalin are less likely to show nuclear bubbling. However, the worst
case of nuclear bubbling I ever saw (orphan annie eyes) was in bone marrow
biopsies that were underfixed in zinc formalin (tech didn't want to wait the
usual amount of time, so only let them fix about half the time required).

So, increasing formalin fixative by one hour is NOT going to correct this
problem. It's going to take several hours increase in time in formalin
before some decrease in nuclear bubbling is seen. So leave the tissue on the
processor in the formalin as long as possible at the beginning of the cycle,
NOT in the hot paraffin at the end of the cycle.

After that, watch the temperatures during processing (keeping at 37 degrees
C would be better than 42 degrees, for example for the alcohols/xylene; keep
paraffin as low as possible (2 degrees above melting point)). Keep the
temperature on the slide dryer down too. 60 degrees C for 30 minutes will be
better than 80 degrees C for 15 minutes.

And tell the people grossing that they MUST cut thin sections (thickness of
a nickle = 2 mm), and NOT overstuff the cassettes. These will not allow
formalin to flow around the tissue. So the tissue won't get fixed! So no
cross-links, so no protein/DNA stabilization. Ergo, another reason for
nuclear bubbling!

Peggy A. Wenk, HTL(ASCP)SLS
William Beaumont Hospital
Royal Oak, MI 48073

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Gale, Nadia
Sent: Thursday, June 28, 2007 12:05 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Nuclear artefact II

I've looked at everyone's suggestions/comments and am sad to report that I
cannot get rid of the nuclear "bubble" artefact from our slides.
 
I have tried
 
- decreasing water bath temp
- increasing/decreasing drying oven time before H&E staining
- varying fixation times in formalin
 
I am currently trying
 
- decreasing drying oven temperature
- changing the "wrap" that I use for the biopsies when grossing
- looking at our wax times on the processor
 
I agree with Bob that it's extremely frustrating that our community cannot
seem to agree on a common cause for this (what I am now
realizing) common artefact.
 
I'll keep you posted with my results.
 
Thank you so much for all of your ideas and help with this, I am most
grateful.
 
 
Sincerely,
Nadia Gale
Histotechnologist
Centre for Translational and Applied Genomics (CTAG) 3427-600 West 10th
Avenue Vancouver, BC V5Z 4E6 tel 604-877-6000 ext 2426 fax 604-877-6089
ngale <@t> bccancer.bc.ca _______________________________________________
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