[Histonet] Attention Gudrun Lang Re EDTA method for bone marrow biopsies

Gayle Callis gayle.callis at bresnan.net
Thu Aug 31 11:01:43 CDT 2017


Dear Gudrun,

 

EDTA will always be slower than a buffered formic acid and there are caveats
about using it for some enzyme methods for bone. 

 

This EDTA method which is better than most but is avoid pH that is too low.
This EDTA method is made with tetra-sodium EDTA (Fisher BP 121-500 which has
a very high molecular weight).  

The pH is adjusted DOWN to pH of your IHC buffers.   I have a review of
decalcification which explains how EDTA decalcifies bone as a function of
pH.   The correct EDTA with a high molecular weight information is also
attached.  The method was developed by a bone guru. Webb Gee,  many years
ago, and has remained a favorite of mine and others for many years.  You
might get by decalcifying in 24 hours with the higher concentration of EDTA
but there is no true way to speed it up.    We used acetic acid to adjust
the pH down since this EDTA is very alkaline but is highly soluble.  HCL can
be used to adjust the pH.  I liked pH 7.6 which alone, speed up the rate a
bit, and this is the same pH as TRIS buffered saline.   I made up my EDTA in
Dulbecco's PBS, or any PBS will work.   You decalcify at RT.   EDTA is not
affected by temperature since it is a chelator.    A trick to help you is
cut off the cortical bone plug at some point during decalcification since
pathologists should be interested in only the trabecular bone with marrow
components.  This will speed up decal too. 

 

METHOD: 14% EDTA Tetrasodium DECALCIFICATION (Webb Jee, Stain Technology) 

 

140 g EDTA, tetrasodium salt in 800 ml distilled water.  Dulbeccos PBS
(DPBS) is preferred over distilled water.  Starting pH is approx. 11, very
alkaline and could damage alkaline sensitive protein linkages.  Hence using
a pH close to your IHC buffers is desirable. 

 

Adjust with glacial acetic acid (or HCL) using pH meter, mechanical stirrer
and continuous readout on pH meter.  It takes approximately 18 ml glacial
acetic acid for every liter of 14% EDTA (tetrasodium salt).    Since one
adds a large volume of glacial acetic acid at beginning, start out with
suggested gm of EDTA in less buffer,i.e. 800 ml, adjust the pH down.  When
pH 7.4- 7.6 reached, bring final volume to 1 liter using the solvent i.e.
water, DPBS or PBS.   This is basically a titration technique with
continuous readout on a pH meter.  

 

Suspend bone in decalcifying solution and stir or rock gently.  

 

This decalcifying method is NOT used for articular or other cartilage work
since EDTA extracts proteoglycans and will change the tinctorial quality of
cartilage stains i.e. Toluidine Blue, and Safranin O/Fast green so these
appear weaker.   It can extract proteoglycans needed for IHC.  Samples can
be left in EDTA solution over a weekend, but change frequently to replenish
active decalcifying agent EDTA.   Use a sufficient volume, 20:1 for adequate
decalcification.  

 

I have a colleague using this EDTA method for murine bones, and she loves it
even for doing decalcified bone frozen sections.  

 

Contact me privately and I will send a publication explaining how EDTA works
chemically as a function of pH, Webb Jee publication and the weight
loss/weight gain decalcification endpoint method.   

 

Take care, and stay in touch.  

 

Gayle 

 

 

You wrote: 

 

I would be happy about some input about decalcification protocols with EDTA
of trephine bone marrow biopsies.

 

recommended duration of fixation?  Complete fixation.  NBF totally fixes in
24 h but you may not be able to wait that long.  

 

recommended duration of decalcification? Maybe 24 h, less is debatable.
EDTA will always be slow since it is a chelation process.  There is a weight
loss/weight gain decalcification end point test, highly advisable, to know
when sample is decalcified.  This requires a balance which weighs in mg, or
you can use xray method.  A chemical method for EDTA is painfully tedious.  

 

strategies for speed-up of decal? Use higher concentration 14% EDTA,
tetrasodium at pH 7.6 with some gentle agitation.

 

recommended EDTA-solution formula?  Provided above it 14% Tetrasodium EDTA,
pH 7.6.  This is highly alkaline EDTA and the pH is adjusted down with
acetic or HCl.  

 

Hopefully some experienced histotechs can share their knowledge with me.

 

thanks in advance

 

Gudrun

 

 



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