DECALCIFICATION
Presented by: Madhura Shekatkar.
INTRODUCTION
To study the histological structure, the tissue should be appropriately prepared
for microscopic examination.
Tissue specimen must be thin enough to permit the passage of light & should
be one cell thickness for detailed morphology.
Decalcification is a routine procedure with the purpose of making a calcified
tissue compatible with the embedding media for cutting micro slides and
subsequent staining.
DEFINITION
IT IS THE PROCESS OF REMOVING INORGANIC CALCIUM(CONTENT)
OF THE BONE OR TISSUE(TOOTH) BEFORE PROCESSING THE
SPECIMEN AFTER FIXATION.
AIM
To obtain
Presence
Resultsofinsatisfactory
calcium
torn andsalts
paraffin section of calcified
prevent
raggedthesections
preparation
and
tissue.
damage
Theofaverage
goodtosections
cutting edge
thicknessbyis 4-
ofroutine
microtome
methods.
6 µm knife.
3
CHOICE OF DECALCIFYING AGENT
Urgency of the Degree of
case mineralization
Extent of Staining
investigation technique
IDEAL DECALCIFYING AGENT
Complete removal of calcium.
Minimal damage to cells and tissues.
Non impairment of subsequent staining.
Reasonable speed.
Enamel: 96% minerals (crystalline calcium phosphate)
COMPOSITION
90-95% Elastic collagen fibers called ossein.
Dentin: 70% inorganic material Ground substance: Calcium phosphate (hydroxylapetite) which gives bone its rigidity.
Cementum: 45% inorganic material.
TOOTH BONE
METHODS
Acid decalcification
Electrolytic decalcification
Chelating Agents
Ion exchange resins
DECALCIFYING AGENTS
• 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
STRONG MINERAL 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.
Nitric acid Perenyi’s fluid Hydrochloric acid Von Ebner’s solution
Sodium chloride saturated
10% nitric acid 40ml
soln. 50ml
5% in distilled water 0.5% chromic acid 30ml 5-10% in distilled water Distilled water 42ml
Absolute alcohol 30ml
Hydrochloric acid 8ml
Fix the selected block for 2-3 days A traditional decalcifier that Formalin should be washed
in buffered neutral formalin decalcifies more slowly than from specimen before
Change the nitric acid solution aqueous nitric acid.
daily until bubbles cease to evolve Quite rapid in action, exceeding
placing in HCl to avoid the
from the tissues end-point will impair staining. formation of bis- Rapid in action, exceeding
Wash in 3 changes of 90% Mix shortly before use. chloromethyl ether (a end-point will impair
alcohol. Chromic acid must be collected carcinogen). staining.
Dehydrate , clear in xylene or for proper disposal. Rapid in action, exceeding
benzene and embed in paraffin. Its used specially for small
Rapid in action, exceeding end- specimen which are not densely end-point will impair
point will impair staining. calcified. staining.
WEAK ACIDS
Formic acid Evans and Krajian Kristensen Gooding and Stewart
Formic acid 5-
Formic acid 18ml
Formic acid 25ml 25ml
Sodium formate
10% in distilled Sodium citrate 10g 40%formaldehyde
3.5g
water Distilled water 5ml
Distilled water
75ml Distilled water
82ml
75ml
A simple effective decalcifier.
Formic acid is the only acid used
A formic acid
extensively as a decalcifier.
An effective formic
Formic acid can be used as a
decalcifier with
simple 10% aqueous solution or
A simple effective acid decalcifier
combined with formalin or with a
decalcifier. added formalin,
buffer. buffered with
Although it is slower than the claimed to fix and
strong acid agents it is much formate
gentler in action and less likely to decalcify.
interfere with nuclear staining.
Aceti
c acid
They cause tissue
swelling and thus
not used alone as
primary
decalcifiers but
are found as
components of
Carnoy’s or
Bouin’s fixatives.
Picric
acid
OTHER
OTHER DECALCIFYING
DECALCIFYING FLUIDS
FLUIDS
Absolute alcohol 73ml
Distilled water
Chloroform
10ml
10ml
Formal saline (10%) 95ml
Glacial acetic acid 3ml
Hydrochloric acid 4ml Trichloroacetic acid 5gm
JENKINS TRICHLORO
FLUID ACETIC ACID
The process of decalcification is slow hence these solution cannot be used for dense bone or big bony
pieces.
Used for simple biopsies.
ACID DECALCIFICATION
• Principle: Acid releases calcium from its chemical combinations
with phosphates and carbonates in bone through ionic exchange
giving soluble calcium salt.
• Most commonly used method.
• Various acid solutions can be used either alone or in combination
with a neutralizer.
• The neutralizer helps prevent the cells from swelling.
For best results gauze- wrapped
bone/tooth should be suspended at
the centre of the fluid.
CHELATING AGENTS
• Organic chelating agents absorb metal ions. Ethylene di amine tetra acetic acid (EDTA) is the most commonly
used chelating agent for decalcification.
• EDTA can bind calcium to form a non – ionized soluble complex.
• It works best for cancer bone. This is the best way to decalcify bone marrow biopsies as it best preserves
cytological details.
• Marrow glycogen is preserved.
• Care should be taken when the specimen contains cartilage because over exposure can remove proteoglycans
and weaken staining.
• This method makes uses of EDTA Solution.
• EDTA Solution: Tissue doesn’t harden after EDTA
decalcification.
EDTA 5.5 gm
Formalin 100 ml Easier to cut by microtome.
Distilled water 900 ml
ELECTROLYTIC DECALCIFICATION
ION EXCHANGE RESINS
• Ammonium salts of sulfonated polystyrene resin are used in this
method. The salt is layered at the bottom of the container and the
fluid – containing formic acid is filled. Mineral acid should not be
present in the decalcifying fluid. Only complete decalcification can
be determined by X rays.
The benefits of this method are :
• Faster decalcification
• Well preserved tissue structures
• Longer use of resin
TECHNIQUE
Selection of tissue
Fixation
Decalcification
Acid neutralization
Thorough washing
SELECTION OF TISSUE
BONE / TEETH:
• Large specimen: Fine toothed bone saw or hack saw is
used to slice the section.
• Small specimen: Diamond impregnated cutting disc is
used to lice the section.
( Slices should not exceed 4-5mm in thickness)
EFFECTS OF DECALCIFICATION ON TISSUE
Nitric acid:
Crumbling of tissue EDTA:
Tissue becomes Formic acid:
Teeth decalcified with
indistinct when Minimal soft-tissue
neutral EDTA
observed under shrinkage and minimal
microscope responses the best to
loss of tissue
Soft-tissue attachment microtome knife
and soft-tissue shrinkage
FACTORS AFFECTING THE RATE OF DECALCIFICATION
Concentration of decalcifying agent:
●
Large volume of the fluid compared with the volume of tissue- 20 to 1 is recommended to avoid total depletion of the acid or chelator by their reaction with calcium.
●
Fluid should be changed several times during the decalcification process .
Temperature:
●
Increased temperature accelerates decalcification but also increases the damaging effects of acids
on tissue. 18 degree Celsius- 30 degree Celsius is acceptable.
Agitation:
●
Gentle agitation may increase the rate slightly by influencing fluid
exchange within as well as around tissues.
Suspension:
●
Fresh decalcifier should have ready access to all surfaces of the specimen. Enhance diffusion
and penetration into the specimen and facilitate solution, ionization and removal of calcium.
SURFACE DECALCIFICATION
• When partly decalcified bone/ unsuspected mineral
deposits in tissue are found during paraffin sectioning
surface decalcification is done.
• After finding the calcified area, the exposed surface in
the paraffin block is placed face side down in 5% HCL for
1 hour or 10% formic acid for 15mins to 1 hour.
• Then the block is rinsed well to remove the corrosive
acids and re-sectioned.
END POINT DECALCIFICATION
Requires manipulation like bending, probing, trimming of the
●
Physical test
specimen.
Calcium oxalate test: Appearance of turbidity indicates presence of
●
Chemical Test
calcium.
Acid reacts with calcium carbonate in bone to produce carbon dioxide,
●
Bubble test seen as a layer of bubbles on the bone surface.
Radiography ●
Decrease in the radio-opacity.
NEUTRALIZATION OF ACIDS
Saturated lithium 5-10% aqueous
carbonate sodium bicarbonate
solution solution
Culling
(1974)
Tap water 2 changes of
rinsing 70% alcohol for
PROCESSING OF DECALCIFIED BONE
• Small biospy specimens : little dense bone : routine
method.
• Larger blocks : quantities of bone : double-embedding
procedure.
DOUBLE EMBEDDING PROCEDURE
• Combine the advantages of paraffin and nitro-cellulose as embedding
media. Suitable for blocks up to 5 mm thick which may contain cortical
bone and soft tissue.
70% alcohol and mercuric chloride-formalin 8 hours
90% alcohol 8 hours
Absolute alcohol (industrial alcohol 99) 24 hours
Alcohol-ether (equal parts of diethyl ether and 8 hours
industrial alcohol 99)
1% celloidin in alcohol-ether 16 hours
Chloroform 16 hours
Paraffin wax 16 hours
MICROTOMY
• Small bone samples and biopsies usually section well with knife angles set for routine soft tissue
microtomy.
• Slight adjustment of a knife angle can be attempted if dense cortical bone sectioning is not working
with the routine soft tissue knife angle, and can be increased or decreased at a microtomist’s
discretion.
• Knives must be changed frequently, sometimes after cutting one ribbon or a few sections of cortical
bone. When sectioning any bone sample, a sharp knife is necessary in order to get flat, uncompressed,
wrinkle-free sections.
• Hard tissues cut more easily if cooled by a melting ice block to allow water penetration into the tissue
surface. Extensive soaking causes visible tissue swelling way from the block face and, even though the
tissue cuts more easily, the sections fall apart on the water bath.
• An optimal thickness for bone sections is the same as that for soft tissues, 4–5 μm, and cut routinely
from adequately processed blocks. Bone marrow biopsies should be cut at 2–3 μm for marrow cell
identification, and sliding microtome sections may vary from approximately 5 to 8 μm.
STAINING METHODS FOR DECALCIFIED TISSUE
• Most routine soft tissue staining methods can be used without modification for staining
decalcified bone sections.
• Acid decalcification, particularly when prolonged or used with a heat producing
method, e.g. microwave, sonication, or electrolytic, can adversely affect the H&E and
some special stains.
• When the temperature exceeds 37°C during decalcification, Giemsa staining may be
too pink and the historical Feulgen stain for DNA will be negative because of excessive
protein hydrolysis.
• Staining is successful after EDTA treatment, but the slower decalcification rate usually
rules it out in favor of faster acid methods.
• H&E is still the primary stain used for most final diagnoses with the aid of special stains.
ARTEFACTS
UNDER Inability to section.
●
●
Incomplete infiltration of paraffin.
●
Staining characteristics lost.
DECALCIFICATION
●
Bone dust.
●
SOLUTION: Surface decal, Re- decal.
OVER ●
●
Nuclear detail lost or severely compromised.
Disruption of cell membrane and cytologic properties.
●
Loss of glycogen.
DECALCIFICATION ●
Swelling of tissue, Especially collagen.
Artefacts of over Bone dust
decalcification
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.
• 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 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.
• 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
ULTRASONIC ENERGIZATION IN DECALCIFICATION
Decalcification of bone specimens of 2-
5mm thickness can be achieved in 5 hours
or less when the decalcifying fluids are
agitated by ultrasonic energization.
MICROWAVE DECALCIFICATION
• Microwave assisted decalcification saves from 10X to
100X of the time required by routine methods.
• The use of dilute acids (i.e. nitric or formic) in place of
EDTA which accelerate the process. The solution should
be changed after each cycle.
• Temperature restriction between 42-45o C for best
results.
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.
