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-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of nap...@siscom.net Sent: Sunday, November 29, 2009 11:54 AM To: histonet@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 _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet __________ Information from ESET Smart Security, version of virus signature database 4647 (20091129) __________ The message was checked by ESET Smart Security. http://www.eset.com __________ Information from ESET Smart Security, version of virus signature database 4647 (20091129) __________ The message was checked by ESET Smart Security. http://www.eset.com _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet