[Histonet] Re: Weight Loss/Weight Gain decalcification endpoint test

Mon Aug 11 16:35:09 CDT 2014

Trevor and Jennifer, 

This method originally came from Mawhinney et al.  Control of rapid nitric
acid decalcification J Clin Pathol 1984 37:1409-1415 and was cited by Cathy
Sanderson (Mayton)in a publication using EDTA, found in Biotechnic and
Histochemistry.  Mawhinney acutally did a chemical test on final acid change
to see that calcium was not present, but we never had to do that.   If you
end up with a bit of residual calcium in block, I would surface decalcify at
microtomy.   I used it for years when we downsized and gave away our
FAXITRON.   Radiography is still the most accurate, and if you had either a
micro CT or digital FAXITRON available, it would be a better test.     I
used Cathy's method and will be happy to send the original publication for
anyone's files/future referencing.   A chemical test for EDTA is a pain to
do if a FAXITRON is not available.   

I have put the full method below, with a bit more detail.   

WEIGHT LOSS/WEIGHT GAIN ENDPOINT TEST

This is a method of choice for EDTA decalcification although it was
originally used by Mawhinney et al for testing nitric acid decalcification.

Many samples can be decalcified together in one container, i.e. 25 mouse
femurs in 1 liter of 10% formic acid.   If all the samples are the same
size, i.e. mouse femurs, tibia, paws, choose several as representative
samples and test only those to save time.   Always suspend bones in the
decalcifier. 

Requires a balance that reads in milligrams to 3 places for greatest
accuracy.  Specimen must be blotted free of fluid for accurate weighing each
time you weigh the sample.  We suspend bones in nylon specimen bags for easy
removal to weigh.  Bags can be marked with pencil too. 

Technique:

1.  Rinse NBF off bone, blot with paper towel, WEIGH BONE, RECORD BEGINNING
WEIGHT.  Suspend bone in acid or EDTA decalcifier.  During acid
decalcification CO2 bubbles are given off, so stir during decalcification to
release bubbles or small samples will float.   EDTA does not create CO2
bubbles, only acids.  Large bones can be started at end of day in acid
decalcifier and sit overnight with testing the next morning.   

2. After 4 to 5 hours in acid or overnight in EDTA, remove bone, rinse with
water, BLOT, weigh.  RECORD WEIGHT.  If bone shows loss of weight, change
acid decalcifier to refresh acid.  Return samples to resume decalcification,
and repeat as many times as necessary.  EDTA should be changed but not as
often as acid.   Always use a large volume of decalcifier i.e. 20:1 or more.
Remove bone from specimen bag, and place in weighing boat to protect balance
from acids/EDTA. 

3.  When bone begins to GAIN WEIGHT, the bone is decalcified.  Once calcium
is removed, water is taken on and the weight increases.  This water does not
affect the bone.  

4.  Rinse bone with running tap water for an hour or longer to remove these
decalcifiers. Either store in 70% alcohol or process.    Store endpoint
tested decalcified bones in 70% alcohol while waiting for other samples to
finish decalcifying and mass processing run.  

Reminders:  For EDTA, one can suspend bones and check every day for accuracy
but bones can be left in the EDTA over a weekend or several days without
damage as long as the bones were well fixed. Acid decalcified bones cannot
be left over a weekend, remove from acid, put in NBF to stop
decalcification. Bones should be endpoint tested before stopping
decalcification so you can resume decalcification on the next working day.
Rinse off NBF briefly before resuming decalcification.   Do not overexpose
bones to acids or you will damage antigens and nuclear staining.   

Enjoy the method, as it truly is fast and easy.

Gayle M. Callis

HTL/HT/MT(ASCP) 

____________________________________________________________________________
_____

Ha Wow...that's almost too easy. Thank you for this!

Trevor Jordan Wait

University of Texas Health Science Center, San Antonio

Class of 2017 MD Candidate

Abilene Christian University Class of 2013 Graduate

B.S.  Biochemistry

________________________________

From: Jennifer MacDonald <
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mtsac.edu>

Sent: Monday, August 11, 2014 2:33 PM

To: Wait, Trevor Jordan

Cc:  <http://lists.utsouthwestern.edu/mailman/listinfo/histonet> histonet
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Subject: Re: [Histonet] Weight Loss/Weight Gain Decal

I believe this was originally from Patsy Ruegg

Decalcification End Point: Weight Loss, Weight Gain

1.        Blot sample to remove excess fixative

2.        Weigh bone in mg, record as beginning weight

3.        Next day, rinse bone, blot and weigh bone daily, record weight.
Change decalcifying solution to refresh acid OR EDTA. Return bone to fresh
decalcifying solution.

4.        When bone begins to GAIN weight, remove from decalcifying
solution, rinse and process.

        YOU MUST WEIGH IN MILLIGRAMS FOR ACCURACY

Once calcium is totally removed (bone loses weight as this happens), water
replaces the calcium and weight begins to go up.  This is the point at which
calcium should be totally gone.  The original method used a chemical test at
the end to insure no calcium was in the decalcifying solution.  If you do
this, you cannot stir the solution during decalcification.  Be sure to
suspend bone in the solution to insure all sides of bone are in contact with
decalcification solution.

From:        "Wait, Trevor Jordan" <
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livemail.uthscsa.edu>

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histonet <@t> lists.utsouthwestern.edu" <
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Date:        08/11/2014 12:29 PM

Subject:        [Histonet] Weight Loss/Weight Gain Decal

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________________________________

Hello all! I'm currently doing some decalcification and was curious if
anyone had some particular advice about the weight loss/weight gain method.
I understand that when the decalcification process is complete, the tissue
block will begin to increase in weight. However, I'm confused when I should
record the weight for the block once they have been taken out of the EDTA
solution. You see, for the times I weighed the blocks before... the weights
were a little skewed because there were differing amounts of solution on the
blocks while they were sitting on the balance. I just want to standardize my
protocol a little more so that I can be sure the block is actually gaining
weight due to the calcium loss rather than just extra solution sitting on
the outside of the block. Would letting the blocks sit out of solution for
about 30 minutes before being weighed help with the matter? I know that the
blocks take on water once they are completely decalcified so I'm not sure
how much this will affect that.

Trevor Jordan Wait

University of Texas Health Science Center, San Antonio

Class of 2017 MD Candidate

Abilene Christian University Class of 2013 Graduate

B.S.  Biochemistry

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