[Histonet] Re: EDTA

Mon Feb 22 13:03:10 CST 2010

For many years doing IHC on mouse bones, I have used 10% EDTA, just pHed up to where it goes into solution, with great results for frozen section immunofluorescence. It takes 5 days. For FFPE, we use Richard Allan Decal solution which is HCl and EDTA. Also works great and only takes 1 hour for murine bones. I don't use the acid decal on frozen sections as I find the autofluorescence is too much and I have better antigen preservation with the EDTA for IF.

Andrea Hooper

--- On Fri, 2/19/10, gayle callis <gayle.callis <@t> bresnan.net> wrote:

From: gayle callis <gayle.callis <@t> bresnan.net>
Subject: RE: [Histonet] Re: EDTA
To: "'Rittman, Barry R'" <Barry.R.Rittman <@t> uth.tmc.edu>, "'Histonet'" <histonet <@t> lists.utsouthwestern.edu>
Date: Friday, February 19, 2010, 11:32 PM

We use EDTA tetra-sodium salt, pH 7.6 but the pH is adjusted down with
glacial acetic acid using continuous stirring and a pH electrode in the
solution (titration with the acid). This is a recipe from a Dr. Webster
(Webb) S. S. Jee publication. 

We like this since this 14% EDTA dissolves immediately in water or PBS
compared disodium EDTA, and at a higher concentration = more molecules of
EDTA available.  

We are very careful to NOT use this 14% tetrasodium EDTA at it original pH
of around 10 or more.  

Decalcification is still very slow, but a recent bone project required EDTA
decalcification (so we thought!) to protect antigens never stained before in
our lab.  After trying both the EDTA and 10% formic acid, we found the
antigen to be very robust after formic acid decalcification too. Lesson
learned, don't put off a pilot study to test IHC after acid decalcification.
The formic acid would have been faster and cheaper.    

Gayle Callis 

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Rittman,
Barry R
Sent: Friday, February 19, 2010 3:20 PM
To: 'Histonet'
Subject: RE: [Histonet] Re: EDTA

I agree with Gayle and Rene
I would not even use a mixture of the two.
Formic acid demineralization will work fine, the theory of using EDTA with
it makes no sense.
EDTA demineralization usually uses the disodium salt. EDTA by itself is not
very soluble but its sodium salts are.
It can be present as mono-, di-, tri- or tetra-sodium salts depending on the
pH. At pH around 11 there are 4 groups that can be replaced with for example
calcium.
Usually the disodium salt of EDTA is used as the tri and tetrasodium salts
are only present in very alkaline solutions that tend to cause tissue
maceration.
At acidic pH the EDTA will only work slowly and have one group replaceable
as the monosodium EDTA.
There are several good formulae for using EDTA and formic acid in separate
formulae, depending on your specific needs..
Barry

________________________________________
From: histonet-bounces <@t> lists.utsouthwestern.edu
[histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of gayle callis
[gayle.callis <@t> bresnan.net]
Sent: Friday, February 19, 2010 4:03 PM
To: 'Histonet'
Subject: [Histonet] Re: EDTA

You wrote:

I am looking into the various decals on the market and have found one that
in addition to formic acid, has EDTA in the mix.  I have never worked with
EDTA so would appre ciate any help in your comments on the use of EDTA in
decalcification methods for bone marrow and routine specimens.

****************************************************************************
****************************************************************************
**

The purpose of EDTA in an acid decalcifier is probably NOT performing the
actual removal of calcium (or very little)  from the bone but chelating
calcium ionized from bone by the acid that then (the calcium) settles to the
bottom of the container.  I doubt very much that you will be dealing with
EDTA but with formic acid decalcification.   People who have developed acid
decalcifying solutions, with either formic acid or hydrochloric acid tout
that the bone section looks very good, better that if EDTA is not present in
the solution.  I never tested this claim in our bone studies, but it might
be something to try, an acid solution containing EDTA and one without EDTA
(keeping the same kind of acid in the study).

The acid is going to ionize the calcium from bone at a faster rate than the
EDTA can chelate the calcium.  But most importantly, EDTA does not work in a
low pH environment.  The chemistry of how EDTA acts at different pHs is well
documented by chemists, and found in book chapters on EDTA.  My physical
chemist spouse supplied me with such a chapter.  The pH of formic acid is
what, pH 3 or so, and if so, EDTA only begins to chelate calcium around pH
4, and when fully protonated at pH 8, decalcifies faster at that pH than at
pH 4, or below pH7.  The working pH for most EDTA solutions is commonly 7 to
7.6, but when the pH becomes more alkaline ( and 8 is going into alkaline
range)  then alkaline sensitive protein linkages can be damaged.

EDTA, correctly written out by Rene Buesa e.g. ethylene diaminatetraacetiic
acid comes in several molecular weights depending on whether is it EDTA
without attached sodium, EDTA disodium and EDTA tetrasodium.  Tetrasodium is
very soluble in water or PBS, but has a very alkaline pH that requires
adjustment down to pH 7 to 7.6 or that high pH will damage alkaline
sensitive protein linkages.  EDTA and disodium EDTA are not as soluble,
usually no more than a 10% solution, requiring heat or addition of sodium
hydroxide in order to dissolve.  EDTA is also expensive,  not affected by
heat up to 60C but only if the bone is totally fixed with NBF.  I would not
advise using 60C during lengthy EDTA decalcification, and possible damage to
heat labile antigens.  There are publications on using EDTA to decalcify
fresh bone samples, then snap freeze, cut frozen sections, then acetone fix
the sections for murine CD markers compromised by both NBF and acid
decalcification.

EDTA mixtures are found in Histonet Archives,  histotechnology books and on
websites e.g. IHCworld, as a chelating agent, it is a very slow decalcifier.
More advantages of EDTA are - no damage to antigens, nucleic acid staining,
nor other tissue components.  It does affect the enzyme histochemical stain
for alkaline phosphatase, and magnesium ions (chelated by EDTA) must be
replaced in the staining solution.  Most clinical laboratories prefer to use
formic acid or hydrochloric acid decalcifying solutions instead of EDTA when
rapid diagnosis is needed.

If you try EDTA alone, rinse the bone well (after decalcification) with
running tap water since residual, excess EDTA in tissue precipitates in
presence of alcohol, and the ppt makes the tissue difficult to section.
Endpoint determinations for complete removal of calcium with EDTA cannot be
done chemically, although there is a weight gain/weight loss method that we
use routinely that works or use an Xray machine, Faxitron.  You can buy EDTA
mixtures from Poly Scientific and some other suppliers, or make it up
yourself.

Otherwise, try the formic acid with EDTA, it may work very well for you
while not sacrificing speed for diagnosis. Formic acid will be less damaging
to antigens than hydrochloric acid, and is a popular decalcifier because it
is gentler than HCl.

Good luck

Gayle M. Callis

HTL/HT/MT(ASCP)

Bozeman MT 59715

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