There are a number of options available when the histologist is required to produce sections from bone or other calcified specimens. In choosing a technique and processing method consideration must be given to the type of investigation being carried out. For example if a metabolic bone disease is being investigated and it is necessary to differentiate mineralized bone from osteoid, or if morphometric measurements are required, it may be necessary to retain and demonstrate the mineral content by producing sections of “un-decalcified” bone. As mineralized bone is such a hard material there are a limited range of techniques available to produce sections from it. After fixation it can be directly sawn into thin wafers and then ground using abrasive surfaces to produce thin “ground” sections (see Figure 1). It is also possible to prepare bone specimens by infiltrating them with acrylic or epoxy resins which, when polymerized, have a hardness equivalent to that of mineralized bone. You can then produce ground sections from the infiltrated specimens or section them directly using a heavy-duty microtome (such as the Leica SM2500) and a tungsten-carbide or diamond knife (see Figure 2). Frozen sections of mineralized cancellous bone are another possibility.^1-3

Bone consists of cells (osteocytes) surrounded by a calcified matrix containing Type 1 collagen fibres. In the matrix calcium is in the form of hydroxyapatite crystals [Ca₁₀(PO₄)₆(OH)₂] which are deposited between the fibrous elements. These crystals are dissolved out during the process of decalcification which, if performed correctly, leaves cohesive tissue with the physical characteristics of dense fibrous connective tissue.

There are two types of mature bone. Cortical or compact bone forms the shafts of long bones and the major parts of the flat bones of the skull and has a very dense structure based on an arrangement of cylindrical structures called osteons (Figure 3). Cancellous, trabecular or spongy bone has a much more delicate arrangement consisting of thin partitions (trabeculae) connecting bony plates between which is found the bone marrow. It is located in the vertebra and at the epiphyses of long bones (Figure 4). Both compact and cancellous bone develop in layers or lamelle in which the collagen fibres are structurally orientated and show a characteristic picture under polarized-light microscopy (see Figure 5). ^{2-3, 5}

In order to protect the cellular and fibrous elements of bone from damage caused by the acids used as decalcifying agents, it is particularly important to thoroughly fix these specimens prior to decalcification. ^2-3 Poorly-fixed specimens become macerated during decalcification and stain poorly afterwards. This is very noticeable in areas containing bone marrow. It is therefore common practice for laboratories to extend fixation times for bone specimens before commencing decalcification. It is important to provide ready access for the fixative to penetrate the bone, so skin and soft tissue should be removed from large specimens if practicable. Bone specimens should be sawn into thin slices as soon as possible to enhance fixation and an adequate volume of fixative provided. High-quality fine tooth saws should be used to prepare bone slices. Coarse saws can cause considerable mechanical damage and force bone fragments into the soft tissues present in the specimen (see Figure 6).

Buffered formalin is a satisfactory fixative for bone but where the preservation of bone marrow is important some laboratories will use alternatives such as one of the Zinc formalin mixtures, B5, formol acetic alcohol (Davidson’s fixative), or Bouin.

Strong acids such as hydrochloric or nitric acid at concentrations up to 10% are the most rapid in action but if used for an excessive time will rapidly cause a loss of nuclear staining and can macerate tissues. It is important that an appropriate end-point test is used to minimise exposure of the specimens to these agents. Generally proprietary decalcifiers that are claimed to be rapid in action are based on strong acids, most commonly hydrochloric acid, and should be used conservatively with attention to the provided instructions if good results are to be obtained. For example Surgipath’s Decalcifier II®  is rapid in action and contains hydrochloric acid. Figure 7 demonstrates the consequences of prolonged treatment with a mineral acid decalcifier beyond the appropriate end-point.

Table 1 lists several of the more common mineral acid-based decalcifiers. Standard textbooks of histotechnology should be consulted for a more comprehensive listing.

Table 1: Mineral acid decalcifiers

If high-quality results are to be obtained it is important to determine the point at which all the calcium has been removed, because, from this point on, tissue damage seems to occur at an increasing rate. Over-decalcification, particularly with the strong acid decalcifiers, spoils the staining of basophilic elements such as cell nuclei and in some circumstances can cause maceration of the softer tissue elements. On the other hand specimens that are incompletely decalcified may be difficult or impossible to section.

The best method, particularly with large specimens such as femoral heads, is to X-ray the specimen. A good-quality X-ray will clearly reveal tiny residual calcium deposits and allow further treatment if required. It is an excellent method for following the process of decalcification of large specimens such as femoral heads (see Figure 8). A simple chemical test can be applied when some acid decalcifiers are used (particularly formic acid). Ammonium oxalate solution is added to a sample of the final change of decalcified that has been neutralized with ammonium hydroxide.¹ If calcium is present a precipitate of calcium oxalate will form indicating that decalcification is probably incomplete and a longer time in decalcified is required. Of course this test is best done on a relatively recent change of decalcifier (exposed to the tissue for say, one hour only). Physical tests require manipulation, bending probing or trimming of the specimen to “feel” for remaining calcified areas. Whilst this method may be successful in experienced hands it is generally considered to be unreliable. Mechanical damage can occur during bending or probing and small deposits of calcium can easily be missed.⁷ A method of determining the endpoint by carefully weighing the specimen after rinsing and blotting has also been described. This may be an effective method for large specimens.¹⁰

If you believe the decalcification end-point is close and you wish to slow the process down so as to avoid over-decalcification and consequent tissue damage, as might be the case when your laboratory is unoccupied during a weekend, specimens can be removed from decalcifier, rinsed and placed back into formalin (important if hydrochloric acid is being used). Decalcification can then be resumed when convenient.¹⁰ An alternative is to refrigerate the specimen at 4˚C in its decalcifier to slow down the process.¹

This is a method of dealing with small unexpected deposits of calcium that may be encountered in paraffin blocks (see Figure 10). Normally it is after trimming the block in the microtome to expose the specimen that the calcium is discovered. It is important at this stage to try to avoid disrupting the block surface extensively. After the tissue has been exposed the block can be removed from the microtome and placed face down in an acid decalcifier for 15 – 60 minutes. This surface treatment will allow the decalcifier to penetrate a small distance into the block and dissolve the calcium. The block can then be thoroughly rinsed in water to remove residual acid, chilled and sectioned. Careful realignment of the block will be required because the decalcifier will penetrate a very small distance into the block allowing only a couple of sections to be taken.^{1, 11}