RE: sticky slides /overfixation

From:Penelope Marr <MarrP@sesahs.nsw.GOV.AU>

Hi Russ,
At last someone echoing my views!  In this day and age where we try to
process large specimens before they have been removed from the patient
(well, it seems that way), I believe that our blocks are not being processed
adequately - mainly inadequate fixation and dehydration.  All too often
those performing immuno stains assume their technique is the problem rather
than looking further afield to find the real cause of the problem.

When starting ER and PR studies some years ago the tissue constantly fell of
the slides during HIER and the staining was patchy with high levels of
background staining in many instances.  I had many arguments that the
tissues were inadequately fixed and processed.  After beating my head
against a brick wall for a long time (made for a great headache!) and
gradually tearing my hair out, I finally won (before I went bald) and got
the lab to agree to an extended processing cycle for the breasts!  We
extended the time in formalin, absolute ethanol and paraffin.  Since then my
headache has gone and I have returned to a full head of hair because there
are very few instances of tissue falling off the slides during HIER, the
background staining is greatly reduced and the specific staining is more
even.  There is still patchy staining in cases when specimens are processed
from "raw".  I am still doing battle with the pathologists to open the
specimens for fixation rather than leaving the lump whole.  

Since introducing this longer processing schedule, the only time I have had
any major lifting problems has been when the processors have missed their
weekly change of solutions.  These days I am usually the first to notice
when there is a processing problem because of the effect on the
immunoperoxidase staining.  

It is probably worth noting that on one occasion I retrieved a block from
the good old days - when we always sliced the specimens open before
overnight fixation then processed the tissue on what was then a routine
cycle (not cut down for speed) - and stained it for ER.  There was no
problem with the section lifting, staining was crisp and clear and there was
not even a hint of background staining.  

My parting question is this:  Are we really doing the patient justice by
rushing through large specimens?


Penelope Marr
St George Hospital
Gray St
Kogarah  NSW  2218

> -----Original Message-----
> Sent:	Thursday, January 18, 2001 8:01 PM
> To:
> Subject:	sticky slides /overfixation
> Here comes a blast from the past!
> I forever remember the words of Edward Brain ( Royal College of 
> Surgeons of England), That a common cause of decalcified 
> sections floating off slides during staining was incomplete 
> dehydration during processing.  I believe he was right.
> And
> I think that may also apply to immuno staining; i.e. incomplete 
> processing dehydration = loss of sections during staining.
> The concept of "overfixation" is in some ways related.
> What happens during fixation depends upon the fixing agent 
> employed.  Alcohol, for example can - and does - fix completely in 
> that it is a precipitant (alters the water balance and brings things - 
> essentially proteins - out of solution).
> If we are talking about "formalin", then the term "overfixation" is 
> much harder to comprehend for we know that the "cross-linking" 
> which formaldehyde facilitates can continue for days, nay, weeks, 
> nay, months, nay years!  Truly.  The concept of overfixation can 
> therefore be seen to be a rather arbitrary concept.
> It is likely, very likely in my opinion, that at some point the degree 
> of cross linking becomes suffieicient to hinder access of larger 
> molecules, of which antibodies are an extremely good example.  
> Therefore, fixation for immunocytochemistry needs to be optimised, 
> but that is easier said than done, for few tissues are homogenous.
> Fortunately, there is a degree of tolerance and that is what we 
> seek to establish.
> "Antigen retreival" seeks to reverse the effects of cross-linking, 
> again with a reasonable degree of tolerance, although we try very 
> hard to standardise that!
> Remember, however, the aims of fixation are to inhibit - prevent - 
> autolysis and putrefaction.  In a nut shell, that means killing bugs 
> and inactivating enzymes.  Easy peasy!
> Rip van Winkle is now going back to sleep
> Good night all!
> Russ Allison, 
> Dental School
> Cardiff
> Wales
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