Re: [Histonet] neutrophils

From:Barbara Bublava

Dear Robert, Dear Gayle

trying to keep my Message short, maybee I ended up beeing too short

I did not want to test if tissue can be fixed in 70% Ethanol. I did want
to show what happens if tissue isnīt fixed enough (Dorothy mentioned
expired formalin) and wents to the processor - often starting with 70%
alcohol - or transferred, as Robert mentioned, to it for longterm storage.

I tested the "extremes"!

1.) no fixation (70% alcohol, trying to simulate starting the processor
with unfixed specimen)

2.) "half" or partial fixation (transferred to 70% before fixed
completely - which means a rather big specimen for 4 hours, fixed at the
edges, not in the center) - trying to simulate staring the processor
with partial fixed specimen. The case one doctor "produced" by insisting
on fast processing

3.) and "proper" fixation. (24 hrs - for a middle sized specimen, fixed
in the center also) - correct procedure

I did this short test to demonstrate how the incosistant staining the
doctor claimed did happen and to show how important fixation is for a
correct staining (nothing really new to us, but some doctors...)

I did choose Chloracetate Esterase Kit because it was the stain burdend,
 HE also did show differences but after all it could be interpreted.
CAE did fail comletely!

I donīt know if this is the solution for Dorothyīs problem, since I did
my little test on human tissue and do not know if the stain will work on
rodent, like other people considered.

If alcohol and not autolysis is the problem - then it could maybe help
to move the specimens to fresh formalin before processing?

I know there are impairments in my little demonstration - for example
the time specimens were left in 70% Ethanol (before moving to the
processor) differs from 0 to 24 hrs (I wanted to process, cut and stain
them together - so there can be no differences)

a lot more testing could be done, and I would do it if I had the time
for it...

but at least and for my intention the test did what it should do. The
doctor understood the overhelming importance of correct fixation.

Barbara Bublava
Medical University of Vienna

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