Working in a histology laboratory provides many opportunities and challenges. One such challenge, for example, is to formulate procedures for the processing of hard tissues. Specimens of bone, fingernail and toenail present potential problems that even the most experienced histologist may have trouble solving. Solutions to problems related to hard tissues are explored.

Bone Specimens: Back to the Basics

Bone specimens may arrive at your laboratory for a number of reasons. Iliac crest biopsies are done for metabolic bone studies on the patient. These specimens may be processed in a special mineralized "non-decalcified" manner and are beyond the scope of our subject here. However, other bone biopsies may be done to determine if the patient has leukemia or other bone cancer. Additionally, bone specimens may be taken from oral sites for the determination of inflammatory disease and/or bone tumors.

Adult human bones come in two types. Cortical, compact bone forms the shafts of long bones and surfaces of flat bones, while trabecular bone is located in the marrow cavities of long bones. Contrary to popular belief, bones are not static, simple support structures of the skeleton. Bones are living, dynamic centers of growth and regeneration and are constantly being remodeled.

Cells called osteoblasts are located on the surfaces of growing bone and function to lay down new bone matrix. This matrix is composed of hydroxyapatite, whose composition is one-third calcium. This is what makes bone so strong (and hard). When osteoblasts become trapped within the matrix they are making, they become osteocytes and remain in small cavities, or lacunae, which stay connected to one another through a system of spaces called canaliculi.

Osteoclasts are bone cells that "digest" the hydroxyapatite to release calcium into the blood stream. This ensures a constant calcium level for proper metabolism. These are giant multinucleated cells seen individually or in clusters on bone surfaces. These bone cells are shown in Fig. 1.

Bone may be processed in histology in one of two ways:

• Mineralized or non-decalcified sections may be prepared by processing the bone tissue into methacrylate plastic.
• The more common way to process bone tissue is to decalcify the bone after fixation and prior to processing. This allows the bone to be routinely processed into paraffin-embedded blocks for subsequent microtomy. The choice of decalcifying agent will depend on exactly what structures need to be seen in the final microscope slide.

Click the image avobe and see the additional Figures that accompany this story.

Chelating agents such as ethylene-diamine-tetracetic acid (EDTA) is the gentlest decalcifying agent and may be used in research projects that require the best histology possible and/or the ability to perform enzyme histochemistry. The drawback of using chelating agents is that they are very slow acting, requiring weeks of time to complete the decalcification process.

More commonly, acid decalcifying agents are used. Strong decalcifiers that contain nitric and/or hydrochloric acid are fast acting and can decalcify bone specimens in a matter of hours. However, the time must be rigorously controlled to prevent a degradation of histology and staining quality.

Weak acid decalcifiers are usually preferable because their use results in superior histology. Bouin's fixative contains picric acid, and Carnoy's fixative contains acetic acid, which can also decalcify bone. However, the primary weak acid decalcifiers usually contain formic acid. If the formic acid is made up in 10 perecnt formalin, the tissue will continue to fix as it decalcifies. This should be straight 10 percent formalin made in water, not buffered formalin. The solution must be acidic to work; any buffers added will negate this effect.

Regardless of whether you choose a strong or weak acid decalcifier, some basic principles must be followed to ensure the highest quality bone slides possible:

• The bone specimen must be completely fixed in formalin prior to decalcification. Without proper fixation, the resulting decalcification step may have a deleterious effect on the histology of the specimen.
• Once fixed, the specimen (usually contained within a tissue processing cassette) should be immersed in a container of acid decalcifier of a volume equal to at least 100x the volume of the specimen.
• Once immersed, the specimen should be agitated in the solution. This can be accomplished by either placing the container on a rotary shaker or inserting a stir bar in the solution and placing on a stir plate. This will move the decalcifier around the specimen and enhance the removal of the calcium.

It is important to check the bone specimen daily. If it is not decalcified, the decalcifier solution should be removed and replaced with fresh solution. As the acid in the solution removes the calcium from the bone, the calcium binds irreversibly to the acid in the solution, thereby stopping its ability to continue to bind more calcium. The solution must continue to be changed daily to accomplish complete decalcification. After 24 hours in the acid decalcifier, the specimen should be removed and checked for decalcification progress. This can be done physically by using a blade or needle to attempt to penetrate the bone, or it can be done biochemically (see Procedure 1 at www.advanceweb.com/mlp).

Either way, once the bone is soft enough, it should be cut into 2 mm slices to increase surface area and make decalcification more efficient. Once decalcification is complete, the specimen should be rinsed in running tap water for 2-3 hours to remove traces of decalcifying reagent.

The tissue is now ready to be routinely processed. Once the slides are cut, they should be picked up on gelatin coated slides, as described below for nail specimens. This procedure results in optimal slides, as shown in Fig. 2.

Nail Specimens

The challenges for bone and nail specimens are two-fold: soften the tissue enough to allow sectioning and ensure adherence of the sections to the slides as they undergo routine and special stains procedures, including immunohistochemistry (IHC).

Specimens of fingernails and toenails are composed of a protein called keratin, a combination of different molecular weight keratin proteins densely packed together. The result is a very strong, tough and hard structure.

Fingernail or toenail specimens may be received in the laboratory for histology. Usually, the clinician suspects a fungal infection. If this is the case, the specimen will be routinely processed into paraffin for hematoxylin and eosin (H&E) staining.

Additionally, periodic acid Schiff's stain and/or Gomori's methenamine silver stain will be done to detect the presence of fungi.

In some cases, a malignant melanoma may be suspected.

Thus, the nail with underlying nail bed must be routinely processed into paraffin for H&E staining and other special stains that may include Fontana-Masson and/or IHC stains (Figs. 3 and 4).

The nail must first be fixed thoroughly in formaldehyde. After fixation, the nail should be held overnight in nail softening solution, which will soften and continue to fix the nail.

Other keratin softeners may be employed. Some histologists use 0.5 percent sodium hydroxide; however, if the nail is left for too long in this solution, it may dissolve.

The following day, the specimen may be processed routinely into a paraffin block. After cutting, the sections should be picked up on gelatin coated slides.

This ensures that the sections will adhere to the slide during H&E, PAS and immunohistochemical staining. To save time, multiple unstained slides may be cut and saved at the time of initial microtomy.

Procedures 2 and 3 summarize these protocols (www.advanceweb.com/mlp).

The methods described here result in high-quality microscope slides of decalcified bone and nail specimens on a consistent basis.

Clifford M. Chapman is technical director, Strata Pathology Services Inc., Lexington, MA.