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4. An Introduction to Decalcification

An Introduction to Decalcification
Geoffrey Rolls, BAppSc, FAIMS
Decalcification describes the technique for removing minerals from bone or other calcified
tissue so that good-quality paraffin sections can be prepared that will preserve all the essential
microscopic elements. Decalcification is carried out after the specimen has been thoroughly
fixed and prior to routine processing to paraffin. In this paper, the basic structure of bone is
described and the technical options for preparing sections are outlined. The procedure for
decalcification and the successful monitoring of the process is discussed and some popular
options for the choice of reagents are provided.

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Introduction

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 undecalcified bone. As
mineralized bone is such a hard material, there is a limited range of techniques available to
produce sections from it. After fixation, it can be directly sawed 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

Figure 1: An unstained ground section of compact bone. A number of osteons (Haversian

systems) are cut transversely. The osteons consist of concentric layers of bone (lamellae)
surrounding a central Haversian canal containing blood vessels. Within the lamellae are
lacunae (spaces) normally containing osteocytes. In dried ground sections such as this, they
appear as black structures due to particles of abrasive and air contained within them.
 Figure 2: An undecalcified section of cancellous bone (Von Kossa). The bone was fixed in
formalin and processed and embedded in epoxy resin for sectioning. Calcified bone is black,
and a rim of osteoid on the surface of the trabeculae is stained blue, as are the components of
the bone marrow. Note that despite the support of polymerized resin, in this case, the
calcified matrix has cracked during preparation of the section.
Much more commonly, bone and other calcified specimens are decalcified (demineralised)
following fixation and processed using a standard method to produce paraffin sections.
Decalcified sections are used for the examination of bone marrow and for the diagnosis of
tumours, infections, or for other purposes. The specimens may be in the form of iliac crest
trephines, or bone pieces removed at operation (such as femoral heads) or dissected from
amputation specimens. Fine detail radiographs are often used to assist in the selection of
appropriate bone specimens for processing. Apart from bone, other tissues may undergo
calcification associated with degenerative processes such as necrosis (dystrophic
calcification), or it may occur in the walls of blood vessels or in kidney, lung, or elsewhere
(metastatic calcification).4 If the calcified areas in tissue specimens are substantial, it may be
impossible to obtain decent sections without first decalcifying the specimen. Another
possibility is to apply “surface decalcification” to a paraffin block to allow sections to be
obtained where the presence of calcium was not anticipated when the specimen was
processed.

The decalcification procedure is relatively straightforward and is extensively discussed in

standard texts of histological technique. 1-3 If high quality results are to be obtained however,
some points are worthy of emphasis.
Structure of bone

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 lamellae in which the collagen fibres are structurally orientated and show a
characteristic picture under polarized-light microscopy (see Figure 5).2-3, 5

Figure 3: A transverse section from a long bone optimally decalcified using formic acid
(H&E). Numerous osteons with peripheral cement lines are shown. Well-stained osteocyte
nuclei are present indicating that the decalcification endpoint was not exceeded.
 Figure 4: A decalcified section of cancellous bone (pink) and hyaline cartilage (blue) from
the epiphysis of a long bone (H&E). The delicate trabeculae of the bone are well preserved,
as is the fine structure of the bone marrow and associated adipocytes. The
basophilic/acidophilic balance of the stain is well maintained indicating that the formic acid
decalcification was optimal.
 Figure 5: A decalcified section of compact bone from the shaft of a long bone (H&E). The
section is photographed under polarized light to demonstrate the concentric lamellae forming
the osteons. The birefringence is due to the orientation of collagen fibres in the bone matrix
which differs between successive layers.
It is important to carefully consider the nature of any bone specimen received for processing
because the relative amounts of cortical and cancellous bone are important and will determine
the time required for decalcification and processing. For example, the iliac crest trephine
specimen consists of a rim of cortical bone overlying a cylinder of cancellous bone. The
cortical bone will be the last to give up its calcium salts during decalcification, and unless it is
completely decalcified, difficulty will be experienced in obtaining decent sections.
Fixation of bone

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.

Figure 6: A trephine specimen in which bone fragments have been forced into the marrow
spaces during preparation (arrows). This is an un-decalcified resin section stained with Von
Kossa’s method to demonstrate calcium.
Decalcifying agents – Overview

There are three main types of decalcifying agents:

 Those based on strong mineral acids

 Those based on weaker organic acids
 Those composed of chelating agents.

For convenience, most laboratories choose from the many proprietary reagents available.
Potential users of these products should consult the relevant MSDS to determine the active
component present if it is not clearly stated in the technical information provided.
Decalcifying agents – Strong acids

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

Decalcifier Formula Comment

Nitric acid6 5% in distilled water Rapid in action
impair staining
Perenyi’s fluid6 (1882) 10% nitric acid 40ml A traditional d
slowly than aq
0.5% chromic acid 30ml action, exceedi
staining.
Absolute alcohol 30ml
Hydrochloric acid 3
5-10% in distilled water Formalin shou
before placing
of bis-chlorom
Rapid in action
impair staining
Von Ebner’s solution6 Sodium chloride saturated soln. 50ml Rapid in action
impair staining
Distilled water 42ml

Hydrochloric acid 8ml

Figure 7: A section of decalcified cancellous bone (H&E). This specimen was decalcified
with a hydrochloric acid decalcifier for an excessive time without using an appropriate
endpoint test. Although the tissue elements are cohesive, the staining is very poor showing a
complete absence of nuclear staining together with strong, poorly-differentiated eosin.
Decalcifying agents – Weak acids

Weak acids such as formic acid are popular and are widely used for decalcification. Formic
acid can be used as a simple 10% aqueous solution or combined with formalin or with a
buffer. Although it is slower than the strong acid agents, it is much gentler in action and less
likely to interfere with nuclear staining. 1, 7 An example of a proprietary decalcifier based on
formic acid is Surgipath’s Decalcifier I®. It also contains formalin and is claimed to fix as
well as decalcify and be gentle in action. Other acids such as trichloracetic acid (TCA) have
also been used. Picric acid, as a component of some fixatives, has weak decalcifying
properties.

Table 2: Weak acid decalcifiers

Decalcifier Formula Comment

Formic acid8 10% in distilled water A simple effec
Evans and Krajian8 Formic acid 25ml An effective fo
with citrate.
Sodium citrate 10g

Distilled water 75ml

Kristensen 8
Formic acid 18ml An effective fo
with formate
Sodium formate 3.5g

Distilled water 82ml

Gooding and Stewart 8
Formic acid 5-25ml A formic acid
claimed to fix
40%formaldehyde 5ml

Distilled water 75ml

Decalcifying agents – Chelating agents

Chelating agents such as ethylenediaminetetracetic acid (EDTA) work by capturing the

calcium ions from the surface of the apatite crystal, slowly reducing its size. Because the
process is very slow but very gentle (weeks may be required depending on the size of the
specimen), this reagent is not suitable for urgent specimens but more appropriate for research
applications where very high quality morphology is required or particular molecular elements
must be preserved for techniques such as IHC , FISH or PCR.9 It is used at a concentration of
approximately 14% as a neutralized solution.1,10 The rate at which EDTA will decalcify is pH
dependent. It is generally used at pH7.0. It works more rapidly at pH10, but some tissue
elements can be damaged at alkaline pH. 10

Table 3: Chelating agents

Decalcifier Formula Comment

Neutral EDTA1 EDTA disodium salt 250g Acts slowly bu
Conventional s
 Distilled water 1750ml

Bring to pH 7.0 by adding sodium hydroxide

(about 25g will be needed).
Factors influencing the rate of decalcification

Concentration.
The concentration of active agent will affect the rate at which calcium is removed. Published
formulations for decalcifying solutions strike a balance between speed and degree of tissue
damage. It must be remembered that the concentration of active agent will be depleted as it
combines with calcium and so it is wise to use a large volume of decalcifier and renew it
several times during the decalcification process.

Temperature.
Increased temperature will speed up the decalcification rate but will also increase the rate of
tissue damage so must be employed with great care.

Agitation.
Gentle agitation may increase the rate slightly.1

Fluid access.
As with fixation, fresh decalcifier should have ready access to all surfaces of the specimen.
This will enhance diffusion and penetration into the specimen and facilitate solution,
ionization, and removal of calcium.
Other techniques for increasing the efficiency of decalcification

Sonication used with EDTA has been successfully used to accelerate decalcification of
trephine specimens for subsequent molecular analysis. During the process, the temperature
must be carefully controlled.9 Microwave treatment has been used with hydrochloric acid
decalcifiers, but the raised temperature may damage morphology and cause staining
artefacts.10Ion-exchange resins have been incorporated into some decalcification protocols.
They are added to the container holding the decalcifier and take up the ionized calcium
maintaining the effectiveness of the acid. If acid decalcifiers are used in adequate volumes
and replaced regularly, the use of such resins is probably unnecessary.3 Electrolytic
decalcification in which the bone is placed in acid decalcifier and attached to an electrode
through which current is applied is a technique that has not found wide acceptance because of
the potential to cause heat damage to the specimen.3
Determining the end-point of decalcification

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.1If 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

Figure 8: An X-ray series following the process of decalcification of a femoral head with
formic acid/citrate decalcifier. The radiographs were produced using a Hewlett-Packard
Faxitron® and allow the process to be accurately followed and the endpoint to be properly
identified.
 Figure 9: A decalcified paraffin section of fibula (H&E). Note the size of the specimen. It
was decalcified with a formic acid agent using a chemical endpoint test then processed to
paraffin using an extended schedule. The 13 y/o male sustained a pathologic fracture of his
right fibula after a fall. X-ray showed bony enlargement of the mid-shaft of the (R) fibula.
Pathologic diagnosis was bone cyst with pathologic fracture. The section was prepared in the
bone laboratory of the Armed Forces Institute of Pathology (USA) in 1983.
Treatment following decalcification and prior to processing

Various methods for neutralizing residual acid decalcifier before processing have been
published, including extensive washing in tap water or the application of alkaline solutions.
Generally a short, effective wash in tap water should be sufficient as any remaining acid will
be removed during processing.1 It is important to remove the bulk of the decalcifier to avoid
contaminating the processing reagents and the processor with acid.
Choosing a suitable schedule for decalcified bone or other decalcified tissues

Once the mineral has been removed a standard processing schedule can be used. It must be
borne in mind that despite complete decalcification, bone, particularly compact bone, will
contain dense areas that require thorough processing. It is better to use a schedule which is
too long than too short. Your choice will depend on the nature and size of the specimen.
Application of vacuum during wax infiltration should improve the quality of the finished
blocks.
Surface decalcification

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
 Figure 10: A section from a granuloma specimen in which unanticipated calcium deposits
were encountered (blue). Sections of better quality could have been prepared if surface
decalcification had been applied to this block.
Conclusion

Decalcification is a straightforward process but to be successful requires:

 A careful preliminary assessment of the specimen

 Thorough fixation
 Preparation of slices of reasonable thickness for
fixation and processing
 The choice of a suitable decalcifier with adequate
volume, changed regularly
 A careful determination of the endpoint
 Thorough processing using a suitable schedule.

References
1. Page KM. Bone. In Bancroft JD and Stevens A
eds. Theory and Practice of Histological
Techniques. New York: Churchill Livingstone,
1996.
2. Moore RJ. Bone. In Woods AE and Ellis RC
eds. Laboratory histopathology. New York:
Churchill Livingstone, 1994;7.2-10.
 3. Carson FL. Histotechnology. 2nd ed. Chicago:
ASCP Press, 2007.
4. Vardaxis NJ. Pathology for the health sciences.
Melbourne: Macmillan Australia Pty Ltd, 1997.
5. Young B, Heath JW. Wheater's Functional
Histology. 4th ed. Edinburgh: Churchill
Livingstone, 2000.
6. Clayden EC. Practical section cutting and
staining. Edinburgh: Churchill Livingstone,
1971.
7. Skinner RA, Hickmon SG, Lumpkin CK,
Aronson J, Nicholas RW. Decalcified Bone:
Twenty Years of Successful Specimen
Management. The Journal of
Histotechnology 1997;20;267-277.
8. Wallington EA. Histological Methods for Bone.
London: Butterworths, 1972.
9. Reineke T, Jenni B, Abdou MT et al. Ultrasonic
Decalcification Offers New Perspectives for
Rapid FISH , DNA, and RT_PCR Ananlysis in
Bone Marrow Trephines Am J Surgical
Pathology 2006;30.
10. Callis G, Sterchi D. Decalcification of Bone:
Literature Review and Practical Study of
Various Decalcifying Agents, Methods, and
Their Effects on Bone Histology. The Journal of
Histotechnology 1998;21;49-58.
11. Rolls GO. Difficult Blocks and Reprocessing.
Leica Microsystems, 2011.

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About the presenter

Geoffrey Rolls, BAppSc, FAIMS
Geoffrey Rolls is a Histology Consultant with decades of experience in the field. He is a
former Senior Lecturer in histopathology in the Department of Laboratory Medicine, RMIT
University in Melbourne, Australia.

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