[Histonet] Re: Decalcification

gayle callis gayle.callis <@t> bresnan.net
Wed Aug 4 14:19:25 CDT 2010

Dorothy Webb wrote: 


I would appreciate any feedback on what all are using in your
decalcification process.  We get a lot of large bones in and the past 2-3
months have noticed a huge problem in our microtomy process with these
samples.  We have been grossing the bones in and leaving the sample in the
cassette in 10% formalin for 24 hours befoere placing in decal for up to 8
hours and still having the inner portion of the sample look underprocessed
and crunchy!  Any suggestions would be appreciated!






Large bones need to fix longer than 24 hours unless you cut bone into
smaller slabs or representative samples e.g. size that fits into a cassette.
If the samples are NOT cut open or slabbed to allow good fixation as soon as
you receive in the lab, you will have to still allow time for fixation.  Any
residual pinkness/reddish color means unfixed.  Whole bones take a long time
to fix in NBF, so bisect, open, cut slabs.   Decalcification can be done
with buffered formic acid, ranging from commercial 4.5% formic concentration
up to 20% formic acid.  Bones must be TOTALLY FIXED and must be to be
protected from effects of acid decalcification, then decalcification can
proceed.  We simply fix  longer and then decalcify in 10% to 15% aqueous
formic acid, do the weight loss/weight gain endpoint determination for bone
sectioning  without problems. 


The other problem is processing and what is probably happening is the bone
is actually under processed, and you need to use a longer processing
schedule.  That is difficult in a hospital setting, so make sure the samples
are smaller if your processing shortened.  Also, poor dehydration, clearing
and most important - paraffin infiltration.  Opaque appearance and poor
microtomy are indicators of 1) poor fixation 2) incomplete decalcification
3)poor dehydration/clearing and paraffin infiltration or ALL of these.  You
can solve the poor  paraffin infiltration by leaving the bone in paraffin
longer, preferably with under a vacuum.   We have a heated vacuum oven to do
this,  but usually have automated bone processing schedules that are longer
than a routine schedules for soft tissues.   The latter may not be feasible
for you in your hospital.    


I also suggest you look into the commercially available kit for endpoint
determination from Poly Sciences IF you don't want to make up the solutions
for this in house. This method is commonly found in histotechnology text
books but I will send the protocol privately IF you want it.   The weight
loss/weight gain method is what we use now since we no longer have an xray
machine, aka FAXITRON.  The chemical test is accurate.  


You could use a rapid decalcifying solution (HCl, commerical solutions are
fine, just read the MSDS to know the acid concentration), but use the
endpoint test - that will save you a lot of grief by preventing overexposure
to acid ("overdecalcification").   


Here is a very quick decalcification endpoint test as long as you have a
balance that weighs in milligrams.   This is a published method and
excellent for EDTA decalcifcation since there is NO good chemical test for
EDTA.  If you don't have a balance, then do the chemical endpoint test. 



ADVANTAGE:  One can decalcify  many samples in one container, fast,

DISADVANTAGES:  Requires a balance that reads in milligrams.   Specimen must
be blotted free of water, for accurate weighing each time you weigh the



1.Rinse NBF off bone, blot with paper towel, WEIGH BONE and  RECORD
BEGINNING WEIGHT.  Suspend bone in acid or EDTA decalcifier, agitate.  Large
bones can be started at end of day, sit overnight in  decalcifier.
Hydrochloric acid decalcifiers are very fast, formic acid is slower/gentler.

2.After 4 to 5 hours in strong acid or overnight in formic acid or EDTA,
remove bone, rinse with water, BLOT, weigh.  RECORD WEIGHT.  If bone shows
loss of weight, change decalcifying solution,  resuspend, and repeat as many
times as necessary. EDTA should be changed but not as often as acid.   

3.When bone begins to GAIN WEIGHT, the bone is decalcified.  Once calcium is
removed, water is taken on and the weight increases. 

4.Rinse bone with running tap water for a several  hours, and process.  

For EDTA, one can suspend bones and check every day for accuracy but bones
can be left in the EDTA over a weekend.  Acid decalcified bones cannot be
left over a weekend, remove from acid, put in NBF, then resume
decalcification on next working day. 


Gayle M. Callis


Bozeman MT 



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