Introduction
Bone is a specialised connective tissue which forms the basis of the skeleton and, as such, its functions are numerous and complex. One of these functions, although not necessarily the most important, is to protect the internal organs from damage which could result from the physical trauma of everyday life. In combination with the associated musculature, the bones of the skeleton also provide a means of physical support, locomotion and related movement. One further role is that of a reservoir for a multitude of inorganic ions (including calcium) which are subsequently recruited by various physiological systems. It could be argued that this latter function may be the most important, because in conditions of calcium deprivation, it is the mass of the skeleton which is sacrificed at the expense of the other functions. Bone is never static: the cells of the skeleton act continuously to maintain the physical structure of bone by a process known as remodelling. These cells are also responsible for the maintenance of plasma calcium homeostasis.

The physical hardness which is a unique characteristic of bone poses particular problems requiring specialised laboratory procedures to achieve a high standard of tissue section preparation. For instance, before attempting conventional histological methods, it is generally necessary to "soften" bone (and other calcified tissue), by removing the mineralised component. This procedure requires special treatment, and the time it adds to the overall tissue processing cycle will inevitably cause some delays in the assessment of diagnostic specimens. For this reason, it is essential to optimise the laboratory procedures for handling such tissues. In addition there are specific techniques which enable the preparation of sections from calcified tissue, without the need to remove the mineralised phase. Apart from bone many other kinds of mineralised specimens may be encountered since virtually any normally soft tissue can become calcified as a result of a disease process.

Anatomy of a bone
Two types of bone are observed in the normal, mature human skeleton (Figs. 1, 2). Although macroscopically and microscopically different, the two forms are identical in their chemical composition. Cortical (also known as compact) bone is found along the shafts of the long bones (femur, tibia, radius, ulna) and is the principal component of the flat bones (skull and ribs). It has an extremely dense physical structure arranged around Haversian systems and Volkmann's canals which are responsible for providing cellular nutrition. Because of its strength cortical bone plays a significant role in the support of the body weight and in the protection of internal organs. Approximately 80% of skeletal mass is cortical bone. On the other hand, cancellous (also known as trabecular or spongy) bone is considerably finer and more delicate in appearance. Its physical arrangement of broad plates connected by thin struts provides for maximum support but with a minimum of raw material. Cancellous bone is found principally in the vertebrae of the spinal column and at the epiphyses of the long bones.

Figure 3 shows the arrangement of cortical and trabecular bone in the proximal femur, where the outer dense cortical bone of the femoral shaft encases the finer trabecular structure of the cancellous bone. The trabeculae have adopted a preferential alignment along the direction of principal mechanical forces. The passage of materials to and from cells which reside within the bony system occurs mainly by diffusion through the trabecular elements which, in the normal situation are usually about 150 µm thick. The metabolic activity of cancellous bone is thought to be considerably higher than that of cortical bone, with the result that diseases, which are caused by aberrations of metabolism, are invariably seen in the cancellous compartment first.

Interspersed between the trabeculae of cancellous bone is the haematopoietic marrow compartment which consists of blood sinusoids and adipocytes as well as the different haematological cell types. In view of the clear mesenchymal envelope which surrounds the bony elements, this compartment is considered to belong to a micro-environment which is structurally and physiologically distinct from the bone.

Composition of bone tissue
Bone is a composite of organic and inorganic phases. Water accounts for approximately 20% of the wet weight of bone whilst about 75% of the dry weight is organic material.

COLLAGEN
Type 1 collagen (consisting of two a 1 chains and one a 2 chain) represents approximately 90% of the organic composition of all bone tissue. Deposition of collagen in the form of osteoid is the initial event in the bone formation process. There are small amounts of other collagen types in normal bone, but these are present as only a small fraction of the total content.¹ In the normal adult skeleton, collagen is found in layers approximately 3 µm thick on the surface of pre-existing bone, immediately adjacent to the marrow. This lamellar arrangement is normal only after the first few years of life: before this, all newly-formed bone appears as a woven matrix in which collagen fibres are deposited in an irregular mosaic pattern, without any preferential orientation (Fig. 4). While this situation is normal for infants, it clearly indicates in the adult that bone has been laid down in a hurried and disorderly fashion. In the adult skeleton, it is not uncommon to find foci of woven bone at sites of fracture repair for example, but more extensive amounts are indicative of abnormal growth and raise the suspicion of some underlying pathological change.

NON-COLLAGENOUS PROTEIN
There are several non-collagenous proteins found in bone, all of which are thought to be synthesised by specific bone-forming cells known as osteoblasts (see later). Osteocalcin (bone g -carboxyglutamic acid, BGP) constitutes up to 2% of vertebrate bone protein.^2-3 The three g -carboxyglutamic acid residues give this macromolecule of molecular weight 5,800, a strong affinity for hydroxyapatite (see following section).⁴ Much of the BGP produced finds its way into the general circulation and is therefore a reliable indicator of osteoblast activity. Another abundant bone protein also synthesised by osteoblasts is osteonectin, which has a molecular weight of around 32,000. It has a strong affinity for both hydroxyapatite and collagen. Osteonectin is also found in platelets. Other less abundant non-collagenous bone proteins include osteopontin (or sialoprotein¹), some small proteoglycans, bone morphogenetic protein and bone-derived growth factors.

Mineralization of bone
The hard nature and rigidity of bone result from the incorporation of mineral into the osteoid matrix template. This inorganic component, known as hydroxyapatite is a crystalline substance which comprises calcium, phosphate and hydroxyl ions [Ca₁₀(PO₄)₆(OH)₂]. Small amounts magnesium, fluoride, carbonate, citrate and potassium as well as other ions are also found in mineralised bone. The skeleton is a conveniently accessible source of calcium and other ions, with approximately 98% of the total calcium, 85% of the phosphorus and around 50% of the sodium and magnesium occurring in bones. The exact mechanism of bone matrix mineralisation is highly complex. An extensive review of current concepts is given elsewhere.⁵

Bone cells
The cells directly associated with bone tissue are relatively few in number and probably derive from the same lineage as other more familiar cells in the bone marrow cavity. There are three distinctly recognisable forms of bone cells, osteoblasts, osteocytes and osteoclasts: each has a specific function in the highly ordered sequence of formation, maintenance and removal of bone mineral.

Osteoblasts actively synthesise many proteins during their developmental and maturation stages: the principal role of these cells is to secrete the matrix framework upon which bone is built. In the active state they appear in histological sections as plump, cuboidal cells lying in a palisade arrangement closely apposed to the bone surface. Active osteoblasts are seen most often in histological sections adjacent to a new osteoid seam. They are rarely seen next to a bare bone surface, since the time interval between cell activation and matrix formation is relatively short. They have eccentric nuclei with prominent Golgi apparatus, and also show abundant rough endoplasmic reticulum and secretory granules. Gap junctions connect the cells with each other and possibly with the more mature osteocytes which lie within the bone structure. When they are less active, osteoblasts take on a more fusiform appearance and are morphologically similar to fibroblasts lining the bone surface. It is believed that smaller, apparently inactive osteoblasts may still be engaged in bone synthesis, but at a much slower rate. The functional life span of an osteoblast can range between 3 and 18 months with an average of 5-6 months.⁶ Osteoblasts are rich in alkaline phosphatase and osteocalcin, both of which may have a role in the mineralisation of the osteoid matrix.

As the collagen framework becomes mineralised, some osteoblasts become trapped in the newly-formed matrix. These remaining cells, now known as osteocytes, reside in the bone matrix until that portion of bone dies, or is degraded during its normal life cycle. Osteocytes are abundant and easily recognisable being arranged in a relatively ordered fashion, in both cortical and lamellar bone. They provide the bone tissue around them (often deep beneath the surface) with a supply of nutrients from the marrow cavity, by way of cytoplasmic communicating channels known as canaliculi. These channels are never more than a few micrometers apart and represent an effective signalling system. Osteocytes are found in peri-cellular lacunae, small independent regions surrounding each cell. These cells are not capable of division and are lost when the bone in which they reside is degraded - the lifespan of an osteocyte thus being dictated by the lifespan of the bone. The arrangement of these cells with their cytoplasmic communications suggests that they may well be responsible for detecting mechanical strain and organising bone cell function as necessary.

Mineralised bone tissue is degraded by specialised giant cells known as osteoclasts, which originate in the bone marrow (possibly from the circulating monocyte). In thin H&E stained sections they appear plump and have abundant foamy eosinophilic cytoplasm. These cells are commonly multinucleate probably as a consequence of the union of several smaller cells. In addition osteoclasts contain prominent Golgi apparatus, abundant mitochondria and numerous lysosomes. Osteoclasts are activated in response to low levels of plasma calcium and their reactions to chemical signals such as parathyroid hormone1, dihydroxyvitamin D and calcitonin are well characterised. Osteoblasts may also be involved in the activation process.⁷ The hallmark of the osteoclast is the high level of lysosomal tartrate-resistant acid phosphatase which when released, dissolves the mineralised bone matrix and releases calcium (and other) ions into the circulation (osteoclasts are not active along a non-mineralised bone surface). A characteristic feature of osteoclasts is their close proximity to an eroded bone surface (Howship's lacuna). Electron microscopic studies reveal a distinctive ruffled cell border in the region where these cells are in contact with bone matrix. This may act to increase the surface contact through which they resorb bone. The lifespan of an osteoclast is thought to be in the range of 4 to 6 weeks.

Indications for bone biopsy
Invariably, samples of bone are taken only after extensive non-invasive clinical examinations which might include conventional plain radiography, photon absorptiometry and computed tomography. Additional laboratory investigations such as the chemical analysis of serum and urine can also provide valuable insight into skeletal metabolic disorders.

Types of specimens
Bone biopsy specimens vary from relatively small "chips" or a needle trephine to full resections of major bones or whole limb amputations. It is advisable, for the expedient treatment of larger specimens, to refer to the clinical radiographs if possible so that the area of interest can be isolated from the bulk of the specimen.

Curettings and small biopsies of bone often arrive at the laboratory in a minimal amount of fixative. To avoid the histological artefacts which result from delayed fixation, a volume ratio of about 20:1 should be established as soon as possible. These samples should be fixed for a minimum of 12-16 hours (or longer if possible), depending upon the density of the tissue. Larger specimens which may include ribs, whole or parts of digits and resected femoral heads are frequently encountered and should be trimmed with a saw to a more manageable size before further processing. Ideally, as with most pathological specimens, a macroscopic description should be made in the first instance. If the specimen is particularly interesting or unusual it should be photographed to maintain a permanent record of its original appearance before a representative portion is selected for processing.

Specimen hndling
As previously mentioned clinical notes and radiographs are useful references to guide in the selection of blocks. When a sample of bone-containing tumour is received it is essential to include all of the resection margins in the blocks taken to ensure that there has been complete excision of the tumour. Similarly, blocks from specimens submitted for investigation of a fracture, must include tissue from the fracture site to determine whether the cause was trauma or less obvious pathology such as osteoporosis or tumour infiltration.

Amputated limbs which may appear at first glance to be difficult to handle due to their size are frequently the simplest to manage. Rarely is it the case that anything other than the bone is of pathological interest, and unless there is tumour or other disease process which involves the adjacent soft tissue, the muscle and skin can be stripped away and discarded. If it is not possible to deal with the specimen immediately, to avoid prefixation artefacts it should be kept in its original state in a refrigerator or cold room and preferably wrapped thoroughly to avoid desiccation. The specimen should be X-rayed as soon as possible to determine the areas of pathological involvement and then cut into more manageable pieces using either a bandsaw or handsaw. These smaller samples can then be fixed for the standard time and processed in the usual manner.

Radiography
If X-rays of bone samples are required it is possible to provide them quickly using a small X-ray machine (Fig. 5). It is more convenient than taking the material to a radiology department although this is still necessary for specimens which exceed 30 cm in diameter due to the internal size restriction of laboratory machines. Radiographs are useful not only for determining the extent of mineralisation in a bone sample, but also for the demonstration of small deposits of calcification in other tissues, as well as identifying foreign bodies, such as prostheses and fragments of glass and metal in soft tissue samples.

With radiological film appropriate for laboratory diagnostic work (such as Kodak Min-R™) small bone biopsies and thin slices of tissue are adequately exposed after approximately 18 seconds at 60 kV. Film can be developed in a commercial automatic processing machine. Fine detail radiography for structural analysis is possible and can be optimised by selecting the appropriate film type and the level and length of exposure.

Fixation
Bandsaws and small hacksaws are essential tools for dividing bone samples to minimise the dimensions of the block at the outset. In the case of an amputation specimen, skin and surrounding soft tissue impair penetration of the fixative, and should be removed as soon as possible.

The choice of fixative will depend upon exactly what is required from the preparation. For most purposes 10% phosphate buffered formalin is adequate. Mercuric chloride containing fixatives such as Zenker's or Heidenhain's 'Susa' may give improved cellular detail following extended decalcification.⁸ While this causes no obvious harm to the tissue, it does result in deposition of mercury pigment, which must be removed before staining. Additionally, samples fixed in mercuric solutions cannot be analysed radiographically to determine the endpoint of decalcification, due to the presence of the radio-opaque metal. Fixation using 70% ethanol is essential for the demonstration of uric acid crystals and for the retention of fluorochrome labels in studies of tissue dynamics in (undecalcified) bone.

Regardless of which agent is used, the need for thorough fixation before decalcification cannot be over-emphasised. The duration of fixation depends to a large extent on the size and density of the tissue block: a specimen consisting of dense cortical bone requires longer in fixative than one which is composed principally of more porous cancellous bone. Samples should always be cut into smaller blocks in order to facilitate more complete and rapid penetration of the fixative. (This action also has the advantage of accelerating subsequent decalcification and processing). As a general rule, it is advisable to restrict blocks to a thickness of approximately 3 mm, and under no circumstances should they be thicker than 5 mm for routine diagnostic work. Fixation is best achieved by ensuring that all faces of the block are equally exposed to the fluid. This can be done either by resting the sample on a soft layer of cotton wool or gauze or tying it with suture or string and suspending in the fixative. A freshly cut slice of bone must not be allowed to lie flat on the base of the container.

Decalcification
Essentially, bone is submitted for the definitive diagnosis of tumour or other condition such as arthritis, traumatic fracture or osteomyelitis. In these cases, it is necessary to examine the tissue for relevant histological changes by clearly demonstrating the arrangement of cells in the bone or within the bone marrow cavity. It is generally not necessary to demonstrate the extent to which the bone is mineralised. Therefore, most bone is subjected to a process of softening known as decalcification. This term implies removal of calcium only but in this sense is not correct since calcium is only one of several constitutive minerals which are extracted. A more accurate description would be "demineralisation", however, calcium is by far the most abundant mineral in bone and the word "decalcification" is well established in the literature of bone histology.

Selection of blocks
The importance of careful selection of tissue blocks from a specimen has already been emphasised and the same rationale applies with decalcification. For example, if radiographs indicate that a tumour involves regions of both cortical and cancellous bone, it would be prudent to sample the cancellous component initially, since this will decalcify sooner. A diagnosis may well be made from these blocks while the cortical bone is still undergoing decalcification. Likewise, if an excised femoral head is submitted for examination following an acute sub-capital fracture, blocks must be taken from around the site of fracture. In addition, blocks should also be taken from other (apparently unaffected) regions to determine if there is any pathology associated with the fracture.

Choice of declcifying agents
Traditionally decalcifying agents are divided into two groups - mineral acids and chelating agents.⁹

MINERAL ACIDS
Weak acids
Examples of this group are acetic and picric acids. Occasionally these components may be incorporated into the chemical formulae of certain fixatives (including Bouin's and Carnoy's fluid), so that they have the simultaneous roles of fixation and decalcification. Weak acid decalcifiers are relatively slower and more gentle than most others and are best used when only a small amount of mineralisation is known or suspected to be present.

Strong acids
Examples in this group include nitric, hydrochloric and sulphuric acids which are most often used alone in aqueous solution at concentrations up to 10%. They are faster in their action than those of the previous group but need to be monitored closely as they carry an increased risk of tissue damage due to hydrolysis of proteins (which can result in "maceration" or the dissolution of the soft tissue components, with possible complete loss of histological detail). It is generally recommended that strong acids are used only if very rapid decalcification (within 48 hours) is required. It should be noted that the yellow staining of tissue which results from prolonged decalcification with nitric acid solutions may detract from the macroscopic appearance but does not affect the histological examination since the colour leaches from the specimen during processing.

The most noticeable effect of acid decalcification is the impairment of staining properties. In particular, nucleic acids stain poorly with haematoxylin and other cationic dyes and the cytoplasm risks being over-stained by the briefest exposure to anionic dyes such as eosin. This effect can be of diagnostic significance if nuclei which have failed to stain through excessive decalcification are falsely interpreted as being non viable. For this reason, even the most fundamental of staining procedures must be performed carefully following acid decalcification.

CHELATING AGENTS
The other major type of decalcifying substance is the chelating agent. The most common example is ethylenediaminetetraacetic acid (EDTA), in the form of the disodium salt, [CH₂N(CH₂COOH)CH₂COONa].2H₂O. EDTA binds Ca++ from the outer shell of the hydroxyapatite crystal. As these calcium ions are chelated, the physical dimensions of the crystal are reduced, and demineralisation of the tissue sample proceeds. EDTA is used in aqueous solution close to saturation (approximately 14%).

Preparation Of EDTA For Decalcification
REAGENTS REQUIRED
1 Solution A
Sodium di-hydrogen orthophosphate 31.2 g
Distilled water 1 l
2 Solution B
Di-sodium hydrogen orthophosphate (anhydrous) 28.4 g
Distilled water 1 l
3 EDTA (di-sodium salt) 140 g

METHOD
1 Add 280 ml of solution A to 720 ml of solution B and make up to 2 l.
2 Add EDTA.

Decalcification using chelating agents occurs at approximately neutral pH, thereby avoiding the undesirable effects on tissue morphology and staining which are seen following uncontrolled acid decalcification. The process is considerably slower than acid decalcification but does have advantages where time is not as critical as the optimal preservation of cellular morphology and detail.

Chelating agents are frequently used in combination with other chemicals, particularly mineral acids, in solutions which are designed to maximise the favourable characteristics of both components and to minimise exposure of the sample to the decalcifying solution. Thus the ideal decalcifying fluid is one which combines the speed of acid decalcification with the moderation of a chelating agent. Most proprietary solutions contain a mineral acid at a concentration of approximately 10%, in combination with a chelating agent. The chelating agent acts to prevent saturation with calcium ions and to extend the active life of the solution. Thus working solutions of decalcifying fluid should be changed at least daily to ensure maximum effect. It is also considered appropriate to maintain a fluid to tissue volume ratio of at least 50:1 so that the active constituents of the decalcifying fluid are not depleted, and to ensure that the reaction occurs as rapidly as possible.

SURFACE DECALCIFICATION
Surface decalcification is achieved by exposing the cut surface of a trimmed block to decalcifying fluid for a short period (approximately 15 minutes) before sectioning. The advantages are that, as only the surface layer of the tissue is treated, the process is completed in a relatively short time and artefacts due to over-decalcification can be avoided. The block should be washed thoroughly before sectioning to ensure that the acid does not carry across to the microtome knife and destroy the cutting edge. This form of decalcification is best suited to material containing small spicules of mineralisation.

OTHER METHODS OF DECALCIFICATION
Ion exchange resins, electrolysis and high frequency sound waves have in the past been advocated as methods for decalcification but are now rarely applied. Ion exchange resins (available commercially) can be used in conjunction with mineral acids to remove calcium ions and prolong the working life of the solution. The resins can be regenerated by washing in acid to remove the calcium ions followed by thorough rinsing in water. Given that it is common practice to change decalcifying solutions daily, the expense and effort involved with the use of ion exchange resins may not be considered worthwhile.

Another process which relies on the dissolution of calcium ions at low pH is the citric acid/citrate buffer (pH 4.5) method. This can also be used to distinguish between uric acid crystals (in gout) and calcium deposits (in chondrocalcinosis or pseudo-gout) in tissue sections.

Factors which determine the rate of decalcification
There are several factors relating to strong acid/chelating agent mixtures which can be varied to either increase or decrease the rate at which specimens are decalcified.⁹

CONCENTRATIONS OF ACID
As the concentration of the acid component increases, the speed of decalcification also increases. However, there is a parallel increase in the degree of tissue damage from hydrolysis of proteins. Conversely, decalcification is slowed with a reduction in the acid concentration. This effect varies between particular species of acids but, in general, mineral acids are most effective at a concentration of around 10%.

REACTION TEMPERATURE
An increase in temperature accelerates decalcification and a decrease slows it down. However if the temperature is too high, the tissue can be damaged and if it is too low, decalcification will not proceed at a satisfactory rate. A suitable temperature range is 20-25°C. If necessary (for example if processing cannot be completed promptly) specimens can be stored at 4°C to prevent over-decalcification.

AGITATION
Mechanical agitation probably has minimal effect on promoting the chemical exchange between bone and fluid unless the specimens are kept on a rolling device such as a blood tube mixer.

SUSPENSION
It is critical to ensure that all surfaces of the tissue receive adequate exposure to the decalcifying fluid, especially if flat slices of tissue are to be treated. In order to achieve this it may be necessary to suspend the tissue in the fluid or place some absorbent material on the bottom of the specimen container.

REDUCED PRESSURE
Contrary to a number of claims, reducing the pressure has not proven to have a significant effect on the rate of decalcification.

Detecting the end-point of decalcification
It is important to remove specimens from decalcifying fluid as soon as they are decalcified to avoid damage from over-exposure.

PHYSICAL METHOD
Probing the tissue with needles or other instruments is a crude practice and should not be seriously considered due to the risk of permanent tissue damage. If specimens are sufficiently large, it may be possible to bend them gently to gain some idea of the amount of mineral which remains in the tissue.

CHEMICAL METHOD
This is a relatively cheap and easy procedure and especially useful if X-ray facilities are not available. It is the only method which is suitable for use following mercuric chloride fixation since the metallic deposition renders the tissue radio-opaque.

Chemical Detection Of The Endpoint Of Decalcification
REAGENTS REQUIRED
1 Concentrated sodium hydroxide
2 Ammonium oxalate (saturated solution)

METHOD
1 Take a sample of decalcifying fluid from the specimen container and neutralise it with concentrated sodium hydroxide.
2 Add an equal volume ammonium oxalate and allow to stand for up to 30 minutes.

RESULT
The fine white precipitate which forms is calcium oxalate, indicating the presence of free calcium ions in the fluid.

TECHNICAL NOTES
1 It is necessary to neutralise the solution since calcium oxalate remains in solution up to pH 5.0.
2 This method does not indicate if there is any mineral remaining in the tissue at the time of the test and the decalcifying fluid should be changed and checked until free calcium ions cannot be detected. Clearly this procedure carries a risk of the specimen being over-decalcified.

RADIOGRAPHY
This is the method of choice for determining the extent of mineralisation in a sample. At the completion of decalcification residual acid in the tissue should be neutralised before further processing. Immersion in a 6% aqueous solution of sodium sulphate or 1% aqueous sodium bicarbonate for a few hours will suffice although washing in running (alkaline) tap water for a similar period will have the same effect. Decalcified tissue can be stored in 70% ethanol.

Processing and embedding
Paraffin wax is a suitable support medium for bone which has been decalcified (Table 1). Small pieces of tissue can be embedded using plastic cassette systems, but larger blocks may need to be hand-processed and embedded in other ways (for instance on wooden blocks) for sectioning on larger microtomes.

Double embedding
Double embedding introduces a plastic such as celloidin into the tissue before infiltration with paraffin wax. It is generally used for embedding tissues of mixed consistency or extremely delicate samples such as stapes which are susceptible to distortion during cutting.

Manual processing
Occasionally it is necessary to process blocks which are larger in both overall area and thickness, than can be managed using regular block processing. In such cases a glass jar or bottle with a sealable lid is used and the dehydrating and clearing fluids are changed manually. Vacuum processing, as in automated processing schedules, is essential with large tissue slices. Tissue blocks can be embedded in wax using rubber moulds or rigid angle irons, and then melted onto wooden or metal backs ready for sectioning.

Paraffin section microtomy
Most bone blocks can be cut using a standard bench-top rotary microtome but larger specimens may require the stronger and more stable base sledge microtome. Most problems in sectioning are due to residual mineral foci remaining in the tissue. Decalcified bone blocks cut much more easily if cooled on wet ice for a little longer than other tissue. Sections are normally 5 µm or less in thickness although difficult tissue may need to be cut slightly thicker.

Occasionally, a block will present problems despite apparent adequate decalcification. In this instance, brief surface decalcification after trimming or a tissue softening agent may be helpful.

Disposable microtome blades used for routine sectioning of paraffin-embedded soft tissue are quite adequate for cutting decalcified bone. Trimming the block prior to sectioning however, should be done using a solid knife as disposable blades do not have sufficient rigidity. Paraffin sections should be collected from the water bath directly onto adhesive coated microscope slides.

Staining methods
Bone sections are commonly stained by H&E and, depending upon components to be demonstrated, may also be treated with haematoxylin and van Gieson, alcian blue for acidic mucosubstances or PAS.

Alizarin Red S For Sites Of Calcium Deposition¹⁰
Sodium alizarin sulphonate is a bidentate chelating agent for calcium ions when used at the appropriate pH.

SECTION PREPARATION
Formalin fixation is suitable (avoid acid-containing fixatives). Decalcified tissues give a negative result. Sections are cut at 3 to 4 µm. Always include a known positive control.

REAGENTS REQUIRED
1 2% aqueous solution of Alizarin Red S (CI 58005)
Adjust pH 4.1-4.3 with dilute ammonium hydroxide using a glass electrode pH meter. The solution should be a deep iodine colour and keeps well.
2 Acetone, xylene

METHOD
1 Dewax and bring sections to 50% alcohol.
2 Rinse briefly with distilled water.
3 Cover the sections with the stain and leave for 20 seconds.
4 Shake off excess stain, blot dry.
5 Treat immediately with acetone for 20 seconds.
6 Treat with 50:50 acetone:xylene for 20 seconds.
7 Treat with xylene for 20 seconds.
8 Mount in an appropriate medium.

RESULTS (Fig 6)
Calcium sites orange/red
Background faint pink

TECHNICAL NOTES
1 Step 3 - examine the staining by light microscopy. Development of the orange-red staining and should not appear diffuse.
2 Step 5 - avoid alcohol and prolonged exposure to dehydration, since this may decolourise the end result.

DISTINCTION BETWEEN URATES AND CALCIUM IN TISSUE SECTIONS
It is important to discriminate between these two entities, which appear similar histologically, to make the diagnosis of chondrocalcinosis (pseudo-gout). The von Kossa silver nitrate stain is used with pre-incubations of citrate buffer, pH 4.5 (to remove calcium) and/or aqueous lithium carbonate (to remove urates).

Identifying Urates And Calcium In Tissue Sections
REAGENTS REQUIRED
1 Citrate buffer
0.2 mol/l disodium hydrogen phosphate 9.09 ml
0.1 mol/l citric acid 10.91 ml
Adjust pH to 4.5
2 Aqueous (saturated) lithium carbonate