[Histonet] Re. Decalcification with formic acid sodium citrate
gayle.callis at bresnan.net
Sat Jul 25 12:24:19 CDT 2015
Merissa and Tim,
This formic acid decalcifying solution is basically the classic Evans and
Krajian fluid (Sheehan and Hrapchak, Theory and Practice of
Histotechnology, 2nd edition, P.92). Shandon has added other ingredients
for some reason, and has kept those concentrations proprietary. You really
don't need to add a surfactant or PVP emulsifier when making up this
decalcifying agent. Simply use the classic recipe for successful
decalcification. This is also referred to as buffered formic acid and in
some publications an "acidic buffer". It is excellent if IHC is needed and
less damaging, obviously, than a strong mineral HCL acid decalcifiers.
Sodium citrate crystals (a buffering salt) 10 g
90% formic acid stock 25 ml
Distilled water 75 ml
One can calculate the concentration of formic acid i.e. approx. 4.5% since
is it made from 90% formic acid stock.
Don't bother with the surfactants or PVP.
Enjoy an excellent in house formic acid decalcifying solution. I also
suggest you read Sheehan and Hrapchak textbook chapter on bone as a way to
familiarize yourself with decalcifiying solutions that manufacturers now
supply with some modifications. Some manufacturers will refer to these
methods but probably prefer not to do this since they want you to buy their
commercial product that is obviously a time saver with elimination of having
to store stock acid solutions. The classic methods made in house are
excellent if you have time to make them up. Formic acid with sodium
formate is another popular buffered formic acid. I suggest you look for
another source/manufacturer of the your favorite decalcifier in question as
more than one company will make it. Decal Corp, recently sold to Stat Lab,
could also be the source as Shandon isn't the only game in town. Others
are Newcomer Supply, Poly Scientific. Not having to make it up may remain
Gayle M. Callis
Written by Tim and Merissa:
Water 77-80 solvent
Formic acid 21-23 active ingredient
Fluorad >1 surfactant - a
wetting agent to make the solution wet the bone more easily
Sodium citrate >1 emulsifier , buffer
Polyvinyl pyrrolidone >1 emulsifier
They say less than one percent of the last three, but you really have no
idea whether that is 1%, .1% or .01%. It could be any of those.
But all those surfactants and emulsifiers are meant to keep the solution
viable for long periods on the shelf. When you make it fresh you don't
really need them.
You can either buy a different decalcifier, or make your own. Making your
own with just the water and acid will work just fine.
Pathology Site Manager, Parnassus
Supervisor, Electron Microscopy/Neuromuscular Special Studies
Department of Pathology
UC San Francisco Medical Center
From: M.O. via Histonet [mailto:
<http://lists.utsouthwestern.edu/mailman/listinfo/histonet> histonet at
Sent: Wednesday, July 22, 2015 1:24 PM
To: <http://lists.utsouthwestern.edu/mailman/listinfo/histonet> histonet at
Subject: [Histonet] understanding reagents in decalcifier; making it
The supplier for our decalcifier, TBD-2 from Shandon, is having issues with
getting the product out and we will not be receiving it for at least another
month. Our samples are piling up and I don't know what I should do, but
maybe I can make the decalcifier in-house. I am wondering if I can make my
own based on the reagents they listed and their percentages and if certain
reagents are not actually necessary.
The samples we typically decalcify are mouse knees (decal time = 2 days),
mouse spines (3 days), human bone slabs about 7mm in thickness (7-12 days).
Fixation is in zinc buffered formalin, then decalcification, then 70% EtOH.
Our choice to use TBD-2 is due to the gentle decalcification for IHC and we
get GREAT results.
Composition of Shandon TBD-2 Decalcifier:
Component Weight %
Formic acid 21-23
Sodium citrate >1
Polyvinyl pyrrolidone >1
If you have any input on what reagents I should use and the percentages for
making a decalcifier myself, it would be much appreciated!
Thank you for you help,
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