16 Bone
Normal bone
Bone collagen
Bone cells
There are three types of bone cell other than the marrow cells belonging to the hemopoietic system.
Techniques for analyzing bone
• Decalcified bone for frozen, paraffin, and transmission electron microscopy (TEM).
• Mineralized bone for frozen, plastic (microtomed or sawn/ground sections), transmission or scanning EM samples.
Fine-detail specimen radiography
Radiographs of bone slabs, blocks, or fragments are useful for four main purposes:
1. To examine the nature and extent of a lesion.
2. To provide a diagram of a lesion prior to block selection for processing.
3. To check progress of decalcification, i.e. decalcification endpoint test.
4. To confirm the presence of foreign materials, e.g. prosthetic devices, metal or glass fragments implanted by trauma.
An example of manual exposure requirements is as follows:
• bone slab 3–5 mm thick on polyethylene sheet (moisture barrier)
• Kodak X-OMAT 2, Ready Pak (Kodak Ltd)
• film-to-source distance (FTSD), 50 cm (lowest shelf level)
Decalcification
Decalcifying agents
As noted previously there are two major types of decalcifying agent, i.e. acids and chelating agents, although Gray (1954) lists over 50 different mixtures. Many of these mixtures were developed for special purposes with one used as a fixing and dehydrating agent. Other mixtures contain reagents, e.g. buffer salts, chromic acid, formalin, or ethanol, intended to counteract the undesirable swelling effects that acids have on tissues. Many popular mixtures used today are from the original formulae developed many years ago (Evans & Krajian 1930; Kristensen 1948; Clayden 1952). For most practical purposes, today’s laboratories seem to prefer simpler solutions for routine work. Provided the bone is totally fixed and treated with a decalcifier suitable for removal of the amount of mineral present, the simple mixtures work as well or better than more complex mixtures.
Strong inorganic acids, e.g. nitric, hydrochloric
Strong acids are used for needle and small biopsy specimens to permit rapid diagnosis within 24 hours or less. They can be used for large or heavily mineralized cortical bone specimens with decalcification progress carefully monitored by a decalcification endpoint test (Callis & Sterchi 1998). The following is a list of strong acid decalcifying solutions. The formulae and preparations are available in Bancroft & Gamble, sixth ed. (2008).
Weak, organic acids, e.g. formic, acetic, picric
Of these, formic is the only weak acid used extensively as a primary decalcifier. Acetic and picric acids cause tissue swelling and are not used alone as decalcifiers but are found as components in Carnoy’s, Bouin’s, and Zenker’s fixatives. These fixatives will act as incidental, although weak, decalcifiers and could be used in urgent cases with only minimal calcification. Formic acid solutions can be aqueous (5–10%), buffered or combined with formalin. The formalin–10% formic acid mixture simultaneously fixes and decalcifies, and is recommended for very small bone pieces or needle biopsies. However, it is still advisable to have complete fixation before any acid decalcifier is used. The salts, sodium formate (Kristensen 1948) or sodium citrate (Evans & Krajian 1930), are added to formic acid solutions making ‘acidic’ buffers. Buffering is used to counteract the injurious effects of the acid. However, in addition to low 4–5% formic acid concentration, increased time is needed for complete decalcification. Formic acid is gentler and slower than HCl or nitric acids, and is suitable for most routine surgical specimens, particularly when immunohistochemical staining is needed. Formic acid can still damage tissue, antigens, and enzyme staining, and should be endpoint tested. Decalcification is usually complete in 1–10 days, depending on the size, type of bone, and acid concentration. Dense cortical or large bones have been effectively decalcified with 15% aqueous formic acid and a 4% hydrochloric acid–4% formic acid mixture (Callis & Sterchi 1998). The following is a list of weak acid decalcifying solutions. The formulae and preparations are available in Bancroft & Gamble, sixth ed. (2008).
Factors influencing the rate of decalcification
Temperature
The optimal temperature for acid decalcification has not been determined, although Smith (1962) suggested 25°C as the standard temperature, but in practice a room temperature (RT) range of 18–30°C is acceptable. Conversely, lower temperature decreases reaction rates and Wallington (1972) suggested that tissues not completely decalcified at the end of a working week could be left in acid at 4°C over a weekend. This practice may result in ‘over-decalcification’ of tissues, even with formic acid. A better recommendation is to interrupt decalcification by briefly rinsing acid off bone, immersing it in NBF, removing from fixative, rinsing off the fixative, and resuming decalcification on the next working day. Microwave, sonication, and electrolytic methods produce heat, and must be carefully monitored to prevent excessive temperatures that damage tissue (Callis & Sterchi 1998).
Agitation
The effect of agitation on decalcification remains controversial even though it is generally accepted that mechanical agitation influences fluid exchange within as well as around tissues with other reagents. Therefore, it would be a logical assumption that agitation speeds up decalcification, and studies have been done which attempt to confirm this theory. Russell (1963) used a tissue processor motor rotating at one revolution per minute and reported the decalcification period was reduced from 5 days to 1 day. Others, including Clayden (1952), Brain (1966), and Drury and Wallington (1980), repeated or performed similar experiments and failed to find any time reduction. The sonication method vigorously agitates both specimen and fluid, and one study noted cellular debris found on the floor of a container after sonication could possibly be important tissue shaken from the specimen (Callis & Sterchi 1998). Gentle fluid agitation is achieved by low-speed rotation, rocking, stirring, or bubbling air into the solution. Even though findings from various studies are unresolved, agitation is a matter of preference and not harmful as long as tissue components remain intact.
Completion of decalcification
Decalcification endpoint test
There are several methods for testing the completion of decalcification, with two considered to be the most reliable. These are specimen radiography, using an X-ray unit and the chemical method to test acids and EDTA solutions. Another method first used to test nitric acid is a weight loss, weight gain procedure that provides relatively good, quick results with all acids and EDTA (Mawhinney et al. 1984; Sanderson et al. 1995). Although still used, ‘physical’ tests are considered inaccurate and damaging to tissues. Probing, ‘needling’, slicing, bending, or squeezing tissue can create artifacts, e.g. needle tracks, disrupt soft tumor from bone, or cause false-positive microfractures of fine trabeculae, a potential misdiagnosis. The ‘bubble’ test is subjective and dependent on worker interpretation.
Calcium oxalate test (Clayden 1952)
Method
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