College of American Pathologists
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Innovations In Pathology

December 2003
Seth L. Haber, MD

Handling bones and other calcified tissue

Izak B. Dimenstein, MD, PhD, HT (ASCP), of Loyola University Medical Center, Maywood, Ill., is a frequent and valued contributor to this column. Now, this month, he shares his expertise in meeting the challenge that bones and other calcified tissue present at the surgical pathology grossing table. If you would like to learn more of Dr. Dimenstein’s innovations, you can write to him at You might also consult his Web site at Although the site is a work in progress, he’ll post his manuscript on bones and enough other pearls to make it worth checking regularly.

Safety (first!)

Bones are the most hazardous specimens we handle because of the bone dust and the danger of injuries from the instruments, solutions, and sharp bone fragments. Like many inanimate objects, the tools necessary to hold and cut bones tend to be spiteful, stubborn, and vindictive, and have a perverse ability to attack the unwary user-often with painful results. So be careful, and wear at least one cut-resistant glove at all times.

The pathologist should wear cut-resistant gloves, made of Lycra, polyethylene-wire, Spectra, or chain mail, and a double layer of latex gloves. Cut-resistant gloves are much less expensive in sporting goods, industrial safety, or hardware stores than in scientific supply houses. Or check the Web. A face shield is mandatory, particularly if an electric saw is used.

Universal safety precautions should be followed, of course; there should be adequate room, laminar exhaustion of the air over the table, and a nearby supply of running water.

Dissection and fixation It’s all summed up in five steps:

  1. Orient and dissect the specimen.
  2. Fix the attached soft tissue.
  3. Cut the bone or calcified tissue.
  4. Complete fixation of the bone and other tissue.
  5. Decalcify as needed.

To hold an extremity in position while you dissect it, cut a couple of semicircles out of opposite ends of the rim of a gallon-size (or larger) plastic container, and fit the limb into them. You might add water for increased stability.

Important soft tissue, such as tumor, that is attached to the bone should be fixed before it is cut. Then cut the bone and fix the cut section again. Cutting into the bone will enable the fixative to penetrate the periosteum and cortical bone more quickly.


The purpose of decalcifying bone is to make the bone suitable for cutting on the microtome. The bone should never be placed in decalcifying solution until it is completely fixed. Some commercial decalcifying solutions are said to save time by mixing the decalcifying solution, formic acid, with the fixative, formalin. Even the dilute mineral acids ruin unfixed tissues. Keeping the tissue in decalcifying solution for too long is worse than insufficient decalcification. The histotechnologist can always decalcify the surface of the "faced" block, if necessary, but over-decalcification is irreparable.

The degree of decalcification can be judged by the flexibility of the specimen. It can also be judged adequate if your fingernail leaves an indentation. The deeper bone can be checked by probing with a dissecting needle.

At the Kaiser Permanente Medical Center, Santa Clara, Calif., we use RDx, a proprietary potassium permanganate solution, like those the plumbers use to de-scale pipes. They are not harmfully acidic, and they work quickly on completely fixed tissue. We also increase the speed and efficiency of any decalcifying solution by adding to the container (in granular or liquid form) about 20 cc of an ion exchange chelating resin, like Winthrop’s WIN-3000, Geary’s Sequestrene, EDTA, or EGTA. These resins bind the calcium out of the solution and shift the Henderson-Hasselbach equation in your favor. They are available inexpensively from most chemical and reagent supply sources, and, since so little is used and they are reusable, a bottle of it will last a long time. I prefer WIN-3000 because that’s what Nancy E. Warner, MD, taught me about 40 years ago, but I’m not sure any one of them has great advantages over the others.

After decalcification, wash the tissue in warm water for at least 30 minutes.

Preliminary orientation

The specimen should be oriented, clinically and anatomically, as to its position in the body. Margins and important structures should be identified and marked.

All soft tissue incisions should be made carefully in the areas of intended bone cuts, while holding the bone down on some support.

Marking margins on surgical specimens of bone and soft tissue

If you want to know all about the advantages and disadvantages of the various methods and media of marking tissue, please refer to pages 120-122 in Innovations in Pathology: The Best of Thirty Years. There, I discuss a score of liquid and dry marking materials, from laundry bluing to tattoo powders, how to best apply them, and how to fix the margin marking materials to the tissue. You can consult or download The Best of Thirty Years free (access is restricted to CAP members) at → Pathologists → CAP Press → Online Books. (Members must be logged on to the Web site to download.) For those who are not CAP members or who don’t have access to the Web, direct your request for the information to Melody Myers at the CAP (847-832-7368 or 800-323-4040 ext. 7368).

Saws and other instruments

When I think of bone, I think of saws. First, the band saw: fuggetaboudit. A band saw is more of an affectation than a tool-except for butchers or carpenters. It takes up too much space; is difficult to impossible to keep clean, much less sterilize; and takes a lot of hands-on training, supervision, and practice to even approach competence. When the band saw attacks, the injuries can be debilitating and ugly.

Stryker oscillating saws are much safer. A handheld circular saw is unnecessarily hazardous; stick to the oscillating saws, with a variety of changeable blades. You probably don’t need anything as large as the Stryker saws used by orthopods; the size used by maxillofacial surgeons is just right. Don’t forget the bone dust collectors that are designed for these saws. Also, you might want to consider diamond saws, like the Bueler Isomet Low Speed Saw, which does not force bone dust into the interstices at the surfaces or send clouds of it into the air.

Most of the bone saw dust that oscillating saws introduce into the face of the specimen can be removed with a soft brush. It’s a gloriously productive recycling of an old toothbrush.

Generally, a handsaw, also called a hacksaw, which takes a 10-inch blade, belongs at every cutting table. Although blades with 14 teeth per inch cut faster, those with 32 TPI are better for our purposes. Such saws are inexpensive and easy to clean, and the blades are easy to change and dispose of.

When I buy a handsaw, generally at a hardware store, I pay particular attention to the length of the pegs that hold the blades-the longer the better. It should be at least 5 mm long. That way, I can mount two blades together, spaced by small washers, which are generally 1 mm to 1.5 mm thick, to cut perfectly spaced parallel specimens out of the bone.

Similarly, you might try epoxying two thin scalpel handles together so that you can mount two blades parallel to each other. This makes it easier to cut thin soft tissue specimens with parallel surfaces.

You will also want a fret saw (AKA coping saw or jigsaw) for smaller, irregular, and more delicate specimens. Most of them take 6-inch blades, but you get to choose the thickness and teeth per inch of the blades and the depth of the saw’s throat. Five minutes with the clerk who sells tools at the local Home Depot will teach you a lot more than I know about this. Incidentally, spend time looking over the saws, clamps, and other small hand tools. You’ll make serendipitous discoveries, gain confidence in choosing the best tools for you, and save a small fortune by buying for far less than you could from the scientific instruments companies.

The remainder of this tutorial on overcoming the problems presented by bones and other calcified materials at the grossing table will conclude in the March 2004 "Innovations in Pathology" column. We will also discuss other tools you will want, vices every pathologist needs, and immobilizing the specimen for cutting.

Dr. Haber is emeritus chief of the Department of Pathology, The Kaiser Permanente Medical Center, Santa Clara, Calif., and clinical professor of pathology at Stanford University School of Medicine. He can be reached at 1375 Pitman Avenue, Palo Alto, CA 94301; phone 650-321-3441; e-mail; and fax 650-321-6773.