[Histonet] Re. Decalcification with formic acid sodium citrate

[Merissa]

Thank you, Gayle! This is exactly what I was looking for and we are willing to make this in house. We are trying just for if acid and water, but the buffering salt should be added. I will try de calcifying mouse knees next week with this protocol. Thank you for the reference, I appreciate your help!

Sincerely,
Merissa



> On Jul 25, 2015, at 10:24 AM, Gayle Callis via Histonet <histonet at lists.utsouthwestern.edu> wrote:
> 
> 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
> your preference. 
> 
> 
> 
> Gayle M. Callis 
> 
> HTL/HT/MT(ASCP)         
> 
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> 
> Written by Tim and Merissa:   
> 
> 
> 
> 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. 
> 
> 
> 
> 
> 
> Tim Morken
> 
> Pathology Site Manager, Parnassus 
> 
> Supervisor, Electron Microscopy/Neuromuscular Special Studies
> 
> Department of Pathology
> 
> UC San Francisco Medical Center
> 
> 
> 
> -----Original Message-----
> 
> From: M.O. via Histonet [mailto:
> <http://lists.utsouthwestern.edu/mailman/listinfo/histonet> histonet at
> lists.utsouthwestern.edu] 
> 
> Sent: Wednesday, July 22, 2015 1:24 PM
> 
> To:  <http://lists.utsouthwestern.edu/mailman/listinfo/histonet> histonet at
> lists.utsouthwestern.edu
> 
> Subject: [Histonet] understanding reagents in decalcifier; making it
> in-house
> 
> 
> 
> Hello Histonet
> 
> 
> 
> 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 %
> 
>     Water                      77-80
> 
> Formic acid                 21-23
> 
>    Fluorad                       >1
> 
> 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,
> 
> Merissa
> 
> 
> 
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