decalcification questions/RDO

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From:Gayle Callis <> (by way of histonet)
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The chemical endpoint test is a bit subjective.  If the ppt is slight,
calcium oxalate, this indicates that endpoint is near, and not a great
deal of calcium is present in the bone, or being ionized out of bone
by the acid (hydochloric in your RDO).  To be accurate on endpoint, the
bone should be taken out of the solution you just tested then rinsed
with tap water to remove any calcium ions residing on the surface as a
result of the acid ionizing it from bone.  This calcium could contaminate
the next change of fresh decalcifier.  The rinsed bone should be placed in
fresh decalcifier in order to have a more accurate endpoint test.  The bone
can be decalcified for another 5 hours (near endpoint) retested. If near
the endpoint, a clear test solution can be allowed to stand for 30 min,
just insure good ppt of any calcium present in used decalcifying solution.
A good way to read this test, put a black paper behind the tube with good
lighting. Have series of tubes with heavy, moderate, light and blank (neg)
calcium results to compare the test solution, old med tech trick for reading
turbid solutions.  Standardizes your tests with all your techs.

The proprietary black or dark coloration of RDO could skew results, but I
know people who diluted RDO 1:1, rather than use it full
strength, get excellent decalcification results, and use chemical
endpoint test, even did IHC on the bone.  Dilution with water did not
slow down the decalcification that much, bone staining was not compromised,
and may have permitted easier visual chemical endpoint test, less black
stuff. Guess it depends on how much white ppt you have in a brownish

An interesting way to think about decalcification is that it
results in a pH change of acid decalcifying  solution.  The pH will change
from low pH to higher pH.  I always wanted to do an endpoint test that
used pH meter to determine when acid is depleted, and needed
changing. Could be coupled with chemical endpoint test to see just when
endpoint is reached, a double whammy would be the more sensitive xray
endpoint test with each chemical test and pH reading, even more correlation.

Cortical bone is just denser, more of it by comparison to delicate trabecular
bone, hence more calcium in cortical. If your pathologist is kind, this
tiny cortical
plug could be cut off, it is THE  bone marrow that interests them.  Pins
and razors are poor choices for testing bone decalcification.  Have
done this in past, bone was never totally decalcified, finally realized
my feel for grittiness was poor, nerve endings just not sensitive
enough to detect very fine calcium deposits left in bone.  Pins also leave
bad ski tracks in bone!  That is why one probably ends up having to
surface decal. If you decalcify long enough to remove CA from cortical bone,
you may overexpose the marrow cells to acid, compromising the staining and
IHC.  A good test for any fine deposits of calcium in the cortical bone,
is stain one of the sections (do not surface decal!), do H&E, look for
blue calcium deposits.  Time in decalcifier could be increased, but will
it ruin those cells???

What a long whew! of info!  TGIF!  Guess bone remains a challenge for me.

Gayle Callis

A cold water soak often helps soften the block enough to get a good
section, be sure to use fresh sharp edge.

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