Re: [Histonet] 37% formalin or penfix and false negative ER/PR stains
From:
Rene J Buesa (by way of histonet)
37% formalin (formaldehyde or methanal) is the "pure" substance. Being a
GASS formalin combines with water up to a concentration of 37%; there is no
stronger solution.
The regular "10% formalin" is a dilution 1:10 of the 37% and, therefore, it
is in reality a 3.7% solution of the natural gass methanal.
Having said that, the difficulty with fixation with formalin on IHC resides
in the fact that formalin fixes by cross-linking proteins (this is the
nature of the epitopes) and HIER (Heat Induced Epitope Retrieval) is aimed
at "un-crosslinking" at "undoing" the mess that formalin causes.
Now, since formalin is an aldehyde, it can oxidize with the oxygen in the
air, and when that happens it transforms into an acid (mathanoic or formic
acid in this case), and this is why the "10%" formalin has to be
neutralized with salts that will act as a buffer and maintain the pH of the
"10%" formalin within "neutral" (non acid) levels.
The problems you are having with breast tissue will NOT be solved by
increasing the formalin concentration; by doing so you will create a
stronger crosslinkage that will not be reversed during a "normal" HIER and
this could explain why ER/PR detection does not take place.
The problem will be solved with neutral buffered "10%" formal (NBF) acting
MORE time (the 24 hours you said are OK) and on THINNER breast sections.
Also, if you want to be sure that your ER/PR protocol is working
correctly, do not use breast as your positive control; use CERVIX which is
better, more stable, and consistent.
I would avoid alcoholic formalin (not necessary and of unknown effect on
epitopes).
Check you processing protocol; if the tissue is well fixed and the
processing is incorrect you will end with poorly infiltrated tissue that
will cause meny problems with the detection (specially if your sections are
not used for IHC immediately). There is not such a thing as "reprocessing";
once the tissue is processed improperly it cannot be "resurrected".
Hope this will help!
Rene J.
john eckman wrote:
Can anyone comment on what effect using 37% formalin to fix breast tissue
has on ER/PR immuno stains? Same question regarding alcoholic formalin use.
We work with pathology residents and have never ending problems with under
fixed breast tissue. Some have suggested using 37% formalin or an alcoholic
formalin. We're concerned about these fixatives altering immuno stains,
although some in our department have used 37% formalin elsewhere without
stain issues.
Our breast sections usually fix in 10% buffered formalin for 6 to 24 hours
before processing and we still get underprocessed/underfixed blocks in
histology. These blocks are then sent through the tissue processor again
(new Themo processor). Could the re-processing alter our ER/PR stains as
well?
Appreciate any comments.
John
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