decalcified section
TRANSCRIPT
DECALCIFIED SECTION
By Dr.G.M.Kailash
kumar
content Definition Criteria for decalcification Structure of bone & tooth Fixation Decalcifying agents – Overview Decalcifying agents – Strong acids Decalcifying agents – Weak acids Decalcifying agents – Chelating agents Factors influencing the rate of decalcification Other techniques for increasing the efficiency of decalcification Determining the end-point of decalcification Treatment following decalcification and prior to processing Surface decalcification Conclusion
DefinitionDecalcification is the process of removing
inorganic calcium ( mineral) content of the bone /tissue before processing the specimen after fixation
Purpose of decalcification
To remove calcium salts from bone or other calcified tissue while preserving the cellular and organic components. Without the removal of these salts microtomy is virtually impossible using standard microtomy procedures resulting in torn, compressed, and ragged sections
•Examination of bone marrow and for the diagnosis of tumors, infections or for other purposes
•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)
Structure of bone
Bone consists of cells (osteocytes) surrounded by a calcified matrix containing Type 1 collagen fibres. The matrix calcium is in the form of hydroxyapatite crystals [Ca10(PO4)6(OH)2] which are deposited between the fibrous elements
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 structural arrangement of cylindrical structures called osteons
•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
Structural composition of teethEnamel : 95-98% inorganic 1-2% organic 1-4% waterDentine : 70% inorganic
20% organic 10% water
Cementum: 45-50% inorganic 50-55% organic & water
• To protect the cellular and fibrous elements of specimen from damage
•extend fixation times for bone specimens before commencing decalcification.
•Buffered formalin is a satisfactory fixative for bone
•For the preservation of bone marrow alternatives such as Zinc formalin mixtures, B5, formalin acetic alcohol (Davidson’s fixative), or Bouin
Fixation
Decalcifying agents TypesStrong mineral acidWeaker organic acidChelating agent
Decalcifying agents – Strong acids
Strong acids - hydrochloric or nitric acid at concentrations up to 10% are the most rapid in action.- excessive time -will cause a loss of nuclear staining and can macerate tissues.
It is important that an appropriate end-point test is used to minimize exposure of the specimens to these agents.
Following Figure demonstrates the consequences of prolonged treatment with a mineral acid decalcifier beyond the appropriate end-point.
Decalcifier Formula Comment
Nitric acid 5-10% in distilled water Rapid in action, exceeding end-point will impair staining.
Perenyi’s fluid (1882) 10% nitric acid 40ml
0.5% chromic acid 30ml
Absolute ethanol 30ml
A traditional decalcifier that decalcifies more slowly than aqueous nitric acid. Quite rapid in action, exceeding end-point will impair staining.
Hydrochloric acid 5-10% in distilled water Formalin should be washed from specimen before placing in HCl to avoid the formation of bis-chloromethyl ether (a carcinogen). Rapid in action, exceeding end-point will impair staining.
Decalcifying agents – Weak acids
widely used weak acid is formic acid Formic acid can be used as a simple 10% aqueous
solution or combined with formalin or with a buffer. it is slower in action and gentler than the strong
acid and less likely to interfere with nuclear staining.
It also contains formalin and is claimed to fix as well as decalcify and be gentle in action.
Trichloracetic acid (TCA) have also been used. Picric acid, as a component of some fixatives has
weak decalcifying properties.
Decalcifier Formula Comment
Formic acid 5-10% in distilled water A simple effective decalcifier.
Formic acid + formalin
Formic acid 5-10 -%Formaldehyde 37-40%
Both fixing and decalcification
Buffered formic acid 20% sodium citrateFormic acid
Reduce adverse effectProlonged duration
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. The process is very slow & gentle
This reagent is not suitable for urgent specimens But appropriate for research applications where
very high quality morphology is required It is used at a concentration of approximately 14% as
a neutralized solution. The rate of action 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.
Decalcifier
Formula
Comment
Neutral EDTA EDTA disodium salt 250g
Distilled water 1750ml
Acts slowly but causes little tissue damage. Conventional stains are largely unaffected.
Factors influencing the rate of decalcification
Concentration TemperaturesAgitationFluid access
Efficiency of decalcificationSonication used with EDTA has been used to accelerate
decalcification.During the process the temperature must be carefully controlled.
Microwave treatment has been used with hydrochloric acid decalcifiers but the raised temperature may damage morphology and cause staining artefacts.
Ion-exchange resins have been incorporated into some decalcification
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.
Determination of end point of decalcification Over Decalcification cause 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, for large specimens such as femoral heads, is to X-ray the specimen
A simple chemical test -Ammonium oxalate solution is added to a sample of the final change of decalcified that has been neutralized with ammonium hydroxide.
Physical tests - bending probing or trimming of the specimen to “feel” for remaining calcified areas. Mechanical damage can occur during bending or probing
Bubble test: Acid+ ca carbonate to form co2 bubble
weighing the specimen after rinsing and blotting is an effective method for large specimens.
• If the decalcification end-point is close then specimens can be removed from decalcifier, rinsed and placed back into formalin. Decalcification can be resumed when convenient.
• An alternative is to refrigerate the specimen at 4˚C in its decalcifier to slow down the process
Treatment following decalcification and prior to processing
Various methods for neutralizing residual acid decalcifier before processing .
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
Surface decalcification
This is a method of dealing with small unexpected deposits of calcium that may be encountered in paraffin blocks
after trimming the block in the microtome if the calcium is discovered. the block can be removed from the microtome and placed in an acid decalcifier for 15 – 60 minutes. This 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.
Conclusion
Decalcification is a straightforward process but to be successful requires:
A careful preliminary assessment of the specimenThorough fixationPreparation of slices of reasonable thickness for
fixation and processingThe choice of a suitable decalcifier with adequate
volume, changed regularlyA careful determination of the endpointThorough processing using a suitable schedule.