[Histonet] 37% formalin or penfix and false negative ER/PR stains

Rene J Buesa rjbuesa <@t> yahoo.com
Wed Oct 12 09:48:35 CDT 2005

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 <eckjm <@t> yahoo.com> 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.


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