[Histonet] bone fixation and decal question

Sun Nov 29 15:13:28 CST 2009

The only way you can do a frozen section on undecalcified bone is using a
tungsten carbide knife($12200 to $1500 per knife and need reconditioning)
but you need a universal knife holder in your cryostat. Even then, doing an
frozen on undecalcified fresh bone is difficult, it tends to crumble if not
held together is some way.  Instrumedics Cryojane (sold by Leica
Microsystems) can solve this problem as you would get the margins of an
intact section (soft tissue and attached bone) you need at the time of Mohs
surgery. There would be no waiting for fixation, decalcification or
cryoprotection into the next day.  Cryojane sectioning does not require
fixation or decalcification. There is a wonderful demonstration of Cryojane
on http://www.alphelys.com/site/us/pHGP_CoupesCongelees.htm

There is another frozen section tape method available now, and you stain the
section while it is on the tape, then cover slip the tape (section side
down) onto a slide.  Go to this website  http://section-lab.jp and read
about the - Kawamoto Cryofilm method.  It is less expensive than Cryojane,
but does NOT transfer the section onto a slide surface - the section stays
on the tape.  However, transferring to the slide surface is something you
may not need to do anyway since diagnosis can be made very quickly on day of
Mohs surgery.   This part of the Kawamoto website has references with all
pdf's downloadable,  http://section-lab.jp/English/Referece.htm and his
original publication is there along with many others on how this Cryofilm
technology is used. I am not sure if permanent mounting medias can be used
with Cryofilm.  

Your assessment of the problem was right on.  If the Surgipath Decal I is
the same kind of fixative/decalcifier as is Calrite (a combination
formalin/formic acid mixture) then this is a better option that protects
tissue integrity with fixation at the same time as decalcifying the bone
fragment. HCl or any acid used without fixing the tissue first should NEVER
be done - this will macerate the tissue and bone, destroying cellular and
tissue morphology. The staining will be horrible.  HCl does not have
fixative qualities, and any enzymes causing autolysis are certainly
destroyed by the acid along with everything else in tissue - without
possibility of rescuing the tissue.  The practice of HCl alone is not only
flawed but potentially a legal issue if the tissue/cells are destroyed by
acid without using fixation first - it could be considered mishandling of a
tissue sample so diagnosis isn't possible.  These labs need to do some
reading on working with bone fixation, decalcification, etc. and to order a
good histotechnology textbook!    

You should cryoprotect fixed/decalcified sample with 20 to 30% sucrose
before freezing or large ice crystal freezing artifact damages tissue
morphology, often making tissue unrecognizable. Cryoprotection can be
speeded up by vacuuming during cryoprotection plus exchanging the
fixative/decalcifier should rinse the residual acid from tissue.  Residual
acid can damage staining and also damage metal parts in cryostat. 

The other option is being able to raise the fibrous periosteum without
taking bone fragments - there are surgical instruments designed for doing
this. Question is:  is basal cell cancer also found in the bone? and that
means taking the bone fragment too.   

Good luck

Gayle Callis
HTL/HT/MT(ASCP)  

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of
napoli <@t> siscom.net
Sent: Sunday, November 29, 2009 11:54 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] bone fixation and decal question

Question regarding fresh tissue, in this case bone.
Scenario:

 Mohs surgeon removes layers of skin on a patient's scalp,
eventually discovering the basal cell carcinoma extends to
the periosteum and so a bit of bone is taken and tested to
ensure the margins are free of cancer. The only problem is
that since it is bone, in order to cut it is needs to be
decalcified. 

In order to decalcify this tissue in order to cut it either
in cryostat or on regular microtome, it of course must be
fixed, correct? Reason being is I have heard of Mohs
dermatologic surgeons taking bones frags, letting their
histotech put the fresh bone in hydrochloric acid solution
for decal overnight and then cutting it in the morning on
the cryostat. This seems flawed to me as the acid would seem
to destroy architecture and critical proteins? Isnt this
practice flawed as it doesnt allow fixation? Does the
hydrochloric solution have fixative properties? Does decal
halt autolysis? Also, is there any real reason why formalin
fixed tissues cannot be mounted in OCT and cut frozen? Would
it hurt the frozen section for the specimen to be placed
into one of the decals i have seen that are both fixative
and decal? (Like Surgipath Decal I)It is said to work
reasonably quickly and the bone would be thin. Seems like
this would be the thourough way of getting quick
intraoperative results with a bit of bone on cryo.

Any comments? thanks in advance

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