Decalcification of bone and tooth

Decalcification of bone and tooth

1. Presented by,
Dr.D.Venkatesh kumar
III year PG

2. Contents
•Introduction
•Classification of decalcifying agents
•Factors affecting the rate of decalcification
•Decalcification end tests
•Processing & Staining of decalcified bone
•Teeth decalcification
•Advanced methods
•Precautions
•Conclusion
•References

3. What is decalcification??
“Istheremovalofcalciumionsfromthe
bonethroughhistologicalprocess
therebymakingtheboneflexibleand
easyforpathologicalinvestigation”.
Bone
Teeth
Hard
tissues

4. BONE

5. lamellar bone Woven bone
Features Mature bone Woven bone
Orientation of
collagen fibres
Collagen fibres in one lamella
lies at right angles to other
lamella
Collagen fibres oriented in
different directions
Interfibrillar spaceSpace less Space is more
Deposition and
mineralization
Slow thanwoven
bone,osteocytes lesser in
number
Faster, more number of
osteocytes are present
H & Estaining Eosinophilic Basophilic

6. Choice of decalcifier
Urgency
of the
case.
Degree of
mineraliz
ation
Extent of
the
investigat
ion
Staining
technique
Be
fast
Be
goo
Do
goo
Ideal
decalcifying
agent

7. Criteria for good decalcifying agent
Complete removal
of calcium
Minimal damage to
cells and tissue
Non impairment of
subsequent
Reasonable speed

8. Classification
Decalcifying
agents
Acids
Strong
inorganic –
nitric acid,
HCLWeak
organic-
formic,
acetic, picric
acid
Chelating
agents
EDTA

9. Others
•Perenys fluid
•Formalin nitric acid
•Gooding stewarts fluid
•Trichloroacetic acid
•Ebners fluid
•Jenkins fluid
•Ion exchange resins
•Electrolytic decalcification
•Ultrasonic decalcification

10. Nitric acid & HCL
•Used as simple aqueous solutions -conc of
5-10%.
•Decalcify rapidly.
•Routinely used
•Permit rapid diagnosis.
Nitric acid-5-
10ml
Distilled water
to -100ml

11. Disadvantages of nitric acid
•Nitrous oxide -yellow color–may interfere with
subsequent staining.
•Addition of 0.1% urea obviates this color.
•Tissue left for long time –damage.
•Old nitric acid is particularly damaging & should be
replaced with fresh stock.

12. Perenyi’s fluid
Nitric acid containing…..
Decalcification time –slower than nitric acid.
Small tissue samples –which are not densely calcified
Preserves cellular details & staining -good.
often used as tissue softener
Decalcified tissues can be directly placed in 70%
alcohol.
Disposing along with Hgcl2 -Ethanol + mercuric chloride + chromic acid =

13. Disadvantage
•Routine chemical test cannot be performed to test end point
of decalcification.
•As precipitate is formed when ammonia is added to the fluid

14. Weak organic-Formic acid
•Ideal -post-mortem & research
specimen
•Also used when IHC staining is
needed.
•Excellent staining results.
•Most widely used decalcifying fluid.
•It is gentle and slower.

15. •Due to its slow action -not suitable –rapid diagnosis.
•At conc ›8% action may be rapid but cloudiness produced -
interferes with chemical test.
•X ray & chemical test are used to test for end point
Formic acid (90%) –
100ml
Formalin –50ml
Distilled water –850ml

16. Ethylene diamine tetraacetic acid (EDTA)
•Binds metallic ions -calcium &
magnesium.
•EDTA will not bind to calcium below PH 3
and
EDTA, disodium salt
–5.5g
Distilled water –90ml
40% Formaldehyde –
10ml
Hillema
n and
Lee
(1953)

17. Mechanism -very slow process.
does not damage tissue staining.
Minimizes formation of histological
artefacts.
Dense cortical bone -6-8weeks or
longer.
For small bone spicule –less than a
week.
Tissues decalcified -should not be
placed -70% alcohol
Water rinse after decalcification or
overnight storage in formal saline, NBF,
or PBS

18. Others....
Ebnersfluid :
Ideal for teeth.
Fairly rapid action.
Decalcified tissue –directly transferred to 90%
alcohol.
Nuclear staining –slightly affected.

19. Jenkins fluid
•Swelling effect of HCL counteracted by shrinkage
effect of alcohol.
•Human rib cross section are decalcified in 4-6 days
Absolute alcohol –
73ml
Distilled water –10
ml
Chloroform –10 ml
Glacial acetic acid –
3ml
HCL –4ml

20. Ion exchange resins
Principle:
•Calcium ions are removed from solution by resins which increases rate of
solubility of Ca from the tissue.
•Resin –ammonium form of sulphonatedpolystyrene
•Ammonium ions from the resins are exchanged for the calcium ions,
keeping the solutions free from calcium ions and speeding up the reaction Resins:
Cross-linkedpolystyrene
Cross-linked
polymethacrylate
Phenol-formaldehyde

21. Technique
Layer of resin about 13mm thick is spread over
bottom of the vessel
Specimen is allowed to rest on it
Decalcifying fluid about 20 times volume of resin
added.
X ray method is used to test end point

22. Advantages
•Well preserved.
•Faster decalcification.
•Elimination of the daily solution change.
•Reclaimed for further use.
•Excellent staining

23. Electrolytic decalcification
Principle : Based on attraction of positively charged calcium ions to a
negatively charged electrode.
•Calcifiedtissueisplaced-
electrolyticsolution(5%HCL&5-
10%formicacidinequalparts)
•Temperatureisraisedduring
electrolysis.
•charringofthetissue&resultsin
distortion-poornuclearstaining.
•Thereforethismethodisnot
recommended

24. Ultrasonic decalcification
•Ultrasonic waves -ultrasonic generator -metal jacket. This is
known as ultrasonic bath.
•Fluid -7.5% glacial acetic acid is placed in bath.
•Faster than conventional acid treated controls.
•Distortion and staining interference is minimal.
Thorp
e,
bellam
y&
sharp.

25. Factors influencing the rate of
decalcification
Factors
concentrat
ion
Temper
ture
Agitatio
n
Suspen
on

26. Decalcification endpoint test
Physical
methods
Chemical
methods
Radiography

27. Physical methods
•Probing, needling, slicing, bending or squeezing.
•Inaccurate and damages tissues -creates artefacts.
E.g. 1. Needle tracks
2. False positive microfracturesof fine trabeculae –a potential
misdiagnosis.

28. Chemical methods
•Most favourable, simple, reliable.
•Depends on dissolved calcium in the decalcifying fluid.
Calcium Oxalate test: (Clayden1952)
•Detection of calcium in acid solutions by precipitation of insoluble calcium
hydroxide or calcium oxalate.

29. Method
Allow solution to stand for 30min.
Add 5ml of saturated ammonium oxalate & shake well.
Add ammonium hydroxide drop by drop, Shaking after
drop, until litmus indicates solution neutral.
Add a piece of litmus paper
Take 5ml of decalcifying fluid.
Solutions:
Ammonium
hydroxide,
Saturated
aqueous
ammonium
oxalate.

30. Bubble test
•Tiny bubbles indicate –less calcium is present.
•It is subjective and unreliable.
Acids
calcium
carbon
te
carbo
n
dioxid
e

31. Radiography
•Most sensitive test -detecting calcium in bone.
•FAXITRON with a manual exposure setting of approx 1minute, 30kv, and
Kodak X-OMAT X-ray film is used.
•Possible to expose several specimens at same time.

32. Procedure
Rinse acid from
sample,
carefully place
specimen on
waterproof
polyethylene
sheet on top of
the X-ray film,
expose
to directions,
and leave until
film is developed
and examined
calcifications.

33. Metal dust particles Spicules of metal,
metallic paint, or glass
from saw blades forced deep into tissue
by a traumatic injury.
radio-opaque, sharply
delineated fragments that
never change in size,
unaffected by
decalcification,
sharply delineated but
cannot be removed
without damaging tissue
-appearing as gray specks
on the bone surface and
can be easily removed.
care must be taken
during microtomyto not
damage the knife.

34. Treatment after decalcification
•Acids -removed from tissues or neutralized.
•Chemical neutralization -saturated Lico3 or 5–10% aqu NaHco3
solution for several hours.
•Few authors -simply rinse -running tap water.
•washing in two changes of 70% alcohol for 12–18 hours -to
avoid contamination of dehydration solvents.

35. Surface decalcification
•when partially decalcified bone or unsuspected mineral deposits in soft tissue
-during paraffin sectioning.
•Done to prevent knife damage & torn tissue sections.The
exposed
tissue
surface in
a paraffin
block is
placed.
Face
in 1%
10%
formic or
proprietar
y acid
solutions.
Time: 15-
60minute
.
Rinsed to
remove
corrosive
acids, and
Resection
d.
After finding
calcification,

36. Masson’s trichrome stain
PAS
Silver impregnation
Schmorl’spicro-thionin
Weigert–van gaeson
H & E
Frost's basic fuchsin stain
Villanueva's bone stain
Villanueva's tetrachrome bone
stain
Modified Villanueva-Goldner
trichrome
A modification of Movat's
pentachrome stain
Gordon and Sweet's method for
cement lines
Phosphotungstic acid
hematoxylin
Modified Von Kossa's method
UNDECALCIFIED
BONE
STAINS
DECALCIFIED
BONE
STAINS
Staining methods

37. Staining methods
H&E:
•No modification -properly decalcified tissues.
•The routine haematoxylin staining time -
doubled.
•The acid differentiation step –shortened.
•Bluing solutions should be mild bases.

38. VON KOSSA'S METHOD:
PURPOSE:Abnormaldepositsof
calcium
PRINCIPLE:Tissuesectionsaretreated
withsilvernitratesolution,the
reducedbythestronglightand
withsilverdeposits,visualizedas
silver.
PROCEDURE:
Dehydrate, clear and mount
counterstain
Wash well in distilled water
Treat with sodium thiosulfate –5min
Wash in 3 changes of distilled water
Sivernitrate and expose to strong light -
10-60 min
Deparaffinizeand hydrate to distilled
water.
SOLUTION:
1%aqueousSilvernitrate
2.5%sodiumthiosulfate
1%saffraninOorvangieson
picrofuchsin

39. RESULTS
•Mineralized bone -black
•Osteoid -red
•Calcium salts -black
•Nuclei -red
•Cytoplasm -pink

40. Artefacts
Factor –heat
Physical test
Under
decalcification
Over
decalcification.

41. Teeth decalcification
•Teeth -same treatment as bone prior to sectioning.
•After fixation ,decalcification, processing , embed in paraffin, or cellodin to
produce thin sections.
Fixation
•Neutral buffered formalin –choice of fixative
•Adult teeth –4days fixation
•Younger teeth –24hours

42. Decalcification: Enamel is almost impossible to
preserve.
Processing:
Since the tooth consist mainly of very dense
material,
•processing times -extended similar to those
used in bone methods.

43. Study
Neutral EDTA -most considerate
& 5% nitric acid -least
considerate to the tooth structure.
Sanjai K,et al; Evaluation and comparison of decalcification agents on the
human teeth. Jomfp.2012;16(2).
EDTA
5%
nitric
acid,
formali
n–
nitrica
cid,
5%
trichlor
acetic
acid,
10%
formic
acid
Perenyi
's fluid,

44. •Application of microwave energy in histotechnology.
•Idea of using microwaves to decrease the time for decalcification of
temporal bones -rat cochleas
Maye
rs
(1970
).
Advanced methods
Microwave decalcification
Hard tissues -decalcifying agent -microwave oven -
intermittent periods -regular changes of the solution.
Decalcification significantly–from days to hours
The temperature restriction between 42-45°C for best
results
5% Formic Acid Solution

45. Procedure:
•Fix for standard times in 10% NB Formalin
•Bone biopsies -microwave-safe container with 5% Formic Acid
•Microwave for 10 minutes at 55ºC
•Repeat this procedure until desired softness is achieved
•Rinse in running tap water for at least 10 minutes
•Proceed with processing.

46. Precautions
Acids -handled carefully as concentrated acid is
hazardous.
Nitric acid: Corrosive to skin mucous
most metals,
toxic by inhalation.
Wear apron, gloves and goggles for handling
howeversmall the quantity.

47. CONCLUSION
•Techniques for the demonstration of bone and its components are possibly
more varied and difficult than for any other tissue.
•For proper diagnosis of bone lesions there should be sufficient knowledge
of decalcification procedure and decalcification agents to choose perfect
decalcification agent for faster decalcification, good staining and lesser
tissue damage.

48. References
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Livingstone Elsevier: 6th edition.
2.CullingC F A, Allison R T, Barr W T: Handbook of Histo pathological and
histochemical techniques. Butterworth & Co Ltd: 4th edition
3.Praful B. Godkar;Text book of medical laboratory technology –2
nd
edition.
4.Antonio Nanci ;TencatesOral Histology –8
th
edition
5.Medical laboratory science –J ochei , A kolhatkar.
6.Sanjai K et al. Evaluation and comparison of decalcification agents on the human

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