Bone is a specialized connective tissue that offers protection to internal organs and works with the musculature to provide physical support and movement. Bone has a hard, rigid nature due to the inclusion of minerals in its osteoid matrix. This crystalline component of the bone, known as hydroxyapatite, serves as a reservoir for a multitude of inorganic ions that are subsequently utilized by various physiological systems. It is composed of calcium, phosphate and hydroxyl ions along with small amounts of magnesium, fluoride, carbonate, citrate, potassium and several other ions.
Although some labs process mineralized bone for resin embedding, most routine labs demineralize, or “decalcify”, bone specimens to make them soft enough to section in paraffin. Bone specimens can be particularly challenging because they are often submitted with the surrounding cartilage and soft tissue that can be easily damaged when exposed to many of the common decalcification solutions. Decalcification is time consuming, which delays diagnostic reporting, but procedures can be expedited with microwave exposure. Monitoring of individual specimens is required.
Specimens submitted for decalcification should be cut into small pieces to expose the marrow, since that is normally the area of interest. The selection of the decalcification method and agent should be made after determining their effects on the subsequent diagnostic techniques that will be used on the specimen. There are two commonly used methods of decalcification: acid methods and chelating methods.
Calcium salts dissolve and then ionize when exposed to acids. The acids used for decalcification are either inorganic acids (hydrochloric and nitric) or organic acids (formic and acetic). Formic acid is very commonly used because it is fairly slow and gentle to the tissue. Staining is usually very strong, even after prolonged exposure. The inorganic acids remove calcium faster and, when the end point is carefully controlled, are more compatible with immunohistochemistry procedures.
The calcium ions that have been removed can saturate the solution around the specimen and prevent further decalcification if the solution is not agitated and changed regularly. Vacuum will facilitate infiltration and remove carbon dioxide bubbles that form on the specimen surfaces.
EDF™, Enhanced Decalcification Formulation, (CAT# SL85-32) is a combination formic acid/formalin solution designed to enhance fixation and gently decalcify bone specimens. Nuclear staining is excellent, even after prolonged exposures. EDF™ is available in 32 oz. bottles.
Chelating agents are organic compounds that are capable of binding with certain metals. They are typically very slow acting and gentle, making them good fixatives for electron microscopy and immunohistochemistry studies. Ethylenediaminetetraacetic acid (EDTA) is the most common chelating agent used for decalcification. It binds with calcium ions and gradually depletes the crystal size of the outer layer of the hydroxyapatite crystal. Care should be taken when specimens contain cartilage because overexposure can remove proteoglycans and weaken staining.
RDF™, Rapid Decalcification Formulation, (CAT# D1210) is a mixture of hydrochloric acid, ethylenediaminetetraacetic acid (EDTA), disodium dihyrate and water. RDF™ completely removes and binds all calcium in specimens, providing excellent sectioning and staining characteristics. It is available in 32 oz. bottles and 1 gallon containers.
Tips for Proper Decalcification
The first rule: Specimens must be fixed before exposure to an acid solution. If a combination fixative/decalcifying solution is used, the specimen should be at least partially fixed first.
The second rule: Specimens must be washed in running water before and after exposure to acid solutions, especially hydrochloric acid. The combination of formalin and hydrochloric acid can create the formation of bis-chloromethyl ether which is a known carcinogen.
- Suspend the specimen so it is not in contact with any of the surfaces of the container. This allows exposure to all of the specimen surfaces and allows the precipitated calcium salts to sink to the bottom of the container.
- Use either x-ray or chemical end-point determination techniques. Do not use probes, needles, scalpels or bending. This will cause physical damage to the specimen.
- Small specimens should not be left in the solution overnight. If decalcification is incomplete, wash it in water and return it to fixative. Wash again before returning to the decalcification solution to complete the process.
- Consider extending processing times for large specimens.
- Embed decalcified bone specimens in “hard” paraffin for additional support during microtomy.
- Embed the harder cortical bone so that it is the last surface to be sectioned and at an angle so that the knife does not contact the entire surface at once.
- Use a sharp knife or well supported disposable blade. A heavy duty blade might be necessary.
- Soak difficult specimens briefly in ice water.
- If decalcification was incomplete, surface decalcification techniques may be used. Be certain to rinse the block before it is placed in the microtome.
- Remember that bone has all of the same tissue elements as any other tissue. In order to demonstrate them effectively the specimen must be well fixed and carefully monitored.