This document provides an overview of decalcification in histopathology. It discusses the need to decalcify bony tissue specimens to make them thin enough for microscopic examination. The key aspects of decalcification covered include the criteria for good decalcifying agents, factors that affect the process, common techniques and decalcifiers used, potential artifacts, and assessing the endpoint of decalcification. The techniques described aim to remove calcium from bone while minimizing damage to tissue morphology and antigenicity.

1. Decalcification in
Histopathology
Mrs Naa-Anyima Boateng

2. Overview
• Introduction
• Biopsy
• Criteria for good decalcifying agent
• Factors affecting decalcification
• Technique
• Artefacts
• Recent Advances
• References

3. Introduction
To study the histological structure, the tissue
should be appropriately prepared for
microscopic examination.
Tissue specimens must be thin enough to
permit the passage of light & should be one cell
thickness for detailed morphology.

4. Boney Section
• Average thickness is 4-6 µm.
• To section the hard mineralised
tissue decalcification is
necessary
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.

5. Biopsies
A biopsy is a procedure
performed to remove tissue or
cells from the body for
examination under a
microscope.
A bone biopsy is a procedure in
which a small sample of bone is
taken from the body and looked
at under a microscope for
cancer, infection, or other bone
disorders.

6. Decalcification
•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.

7. Criteria of a good decalcified Tissue
Sample
Complete
removal of
calcium
Absence of
damage to
tissue cells or
fibres
Non
impairment of
subsequent
staining
technique
Reasonable
speed of
decalcification

8. Factors
Affecting
Decalcification
• 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
Concentration of decalcifying agent
• Increased temperature accelerates
decalcification but also increases the damaging
effects of acids on tissue. 18º C -30º C is
acceptable.
Temperature

9. Factors
Affecting
Decalcification
II
• Agitation may increase the rate slightly
by influencing fluid exchange within as
well as around tissues.
Agitation Gentle
• 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.
Suspension

10. Technique
• Selection of tissue
• Fixation
• Decalcification
• Acid neutralization
• Thorough washing
The technique
of
decalcification is
divided into the
following stages:

11. Specimen
Selection
• BONE / TEETH- Fine toothed bone saw
or hack saw(large sp)
• Geological cutting machine fitted with
a diamond impregnated cutting disc
(small sp)
• Slices not exceed 4-5mm in thickness.

12. Fixation
• As a routine fixative, formal-saline is
preferred but bone marrow is best fixed
in Zenker formol.
• For tooth specimens , 15% formic acid
is mostly preferred
• For electron microscopy –
Gluteraldehyde
• Some fine preparations of bone have
been produced following immersion in
Mullers fluid followed by decalcification
in 3% formic acid – formalin.

13. Fixation II
Tissue damage during acid
decalcification is four times greater
when the tissue is unfixed.

14. Decalcification Step
• Decalcification is the process of removing inorganic calcium
(mineral) content of the bone /tissue before processing the
specimen after fixation.
• Choice of decalcifying agent influenced by:
1. Urgency of the case
2. Degree of mineralization
3. Extent of investigation
4. Staining technique required

15. Decalcification II
Routine decalcification
methods include:
Acids
Ion exchange resins
Electrical ionization
Histochemical
methods
Buffer mixtures
Chelating agents
Surface decalcification

16. Acid Decalcification
Principle
Acid releases calcium from its chemical combinations with phosphates and
carbonates in bone through ionic exchange giving soluble calcium salt.
Two types
• Strong Inorganic Acids
• Weak Organic Acids

17. Strong Acids
Are Inorganic Acid, eg: Nitric
Acid, Hydrochloric Acid
Recommended
Concentration - 5-10%
They decalcify rapidly by
dissolving calcium.

18. Mineral Acid Decalcifiers
Decalifier Formula
Nitric Acid 5% in dH₂0
Perenyi’s Fluid (1882) 40 ml of 10% Nitric Acid
30ml of 0.5% Chromic Acid
30ml Absolute Alcohol
Hydrochloric Acid 5% in dH₂0

19. Nitric Acid
• Nitric acid 5-10ml
• Distilled water 100ml
1. Fix the selected block of bone for 2-3 days in buffered neutral
formalin.
2. Place a mixture of 95ml distilled water and 5ml of nitric acid.
3. Change nitric acid solutions daily until bubbles cease to evolve from
the tissues(1-3 days, depending on the size and consistency of the
bone block)
4. Wash in 3 changes of 90% alcohol.
5. Dehydrate, clear in xylene or benzene and embed in paraffin

20. Nitric Acid II
•Formation of nitrous acid checked temporarily
by addition of 0.1% urea to the conc. nitric acid
It’s the fastest decalcifier, but end point must be
carefully watched .
•Yellow discolouration owing to formation of
nitrous acid, this accelerates decalcification but
also stains and damage tissues

21. Nitric Acid Pros and Cons
Advantages Disadvantages
Rapid in Action Tissue left for too long causes damage to tissue
Gives Better Nuclear staining Urea is added to remove yellow colour
Causes very little hydrolysis
Needle and Small Biopsy's to allow for rapid
diagnosis
Its popular especially for small specimens that are
not densely decalcifed

22. Perenyi's Fluid (1882)
• 10% Nitric Acid 40ml
• Absolute Ethanol 30ml
• 0.5% Chromic Acid 30ml
1. Mix shortly before use
2. Chromic acid must be collected for proper
disposal.
3. Its popular especially for small specimens that are
not densely decalcifed

23. To Note about Strong Acids
•Strong acids are more damaging to:
1.Tissue antigens for immunohistochemical
staining
2.Enzymes may be completely lost.
3.Strong acids are used for needle & small
biopsy specimens to permit rapid diagnosis
within 24 hours.

24. Weak Acid Decalcifiers
Decalcifier Formula
Formic Acid 5% in dH₂0
Evans & Krajian Formic Acid 25 ml
Sodium Citrate 10g
dH₂0 75ml
Kristensen Formic Acid 18ml
Sodium Formate 3.5g
dH₂0 82ml
Gooding & Stewart Formic Acid 2-25ml
40% Formaldehyde 5ml
dH₂0 75ml
Acetic Acid Used as components of decalcifiers
Picric Acid Used as components of decalcifiers

25. Weak Decalcifiers II
Weak, organic acids e.g. formic, acetic, picric.
Acetic & picric acid cause tissue swelling & are not used
alone as primary decalcifiers
but are found as components of Carnoy’s & Bouin’s
fixatives

26. Formic Acid
Formic acid is the only weak acid used extensively as a decalcifier
•aqueous (5-10%)
•buffered or combined with formalin.
Formic acid solutions are either:
The formalin/10% formic acid mixture simultaneously fixes & decalcifies.
•Very small bone pieces
•Jamshidi needle biopsies.
•Formic acid gentle & slower than Hcl or nitric acid
•suitable for most routine surgical specimens, particularly for immuno histochemistry.
•Decalcification usually complete within 2-7days.
Recommended for –

27. Aqueous Formic Acid
1. Well fixed 2-5mm thick blocks are placed in – concentrated formic acid 5-
25ml – Distilled water 100ml – 40% formaldehyde (optional) 5 ml
2. Change daily until decalcification is complete ( 1-7 days for an average
blocks depending on concentration of acid).
3. Replace fluid with 5% sodium sulfate overnight
4. Wash 12 -24 hrs in running tap water.
5. Dehydrate in graded alcohols ,clear in chloroform or toluene and embed in
wax

28. Formic Acid Sodium Citrate
Sodium citrate solution
•100g Sodium citrate in Distilled water 500ml
Stock formic acid
•250 ml of Conc. formic acid in 250ml of distilled
water

29. Decalcification
Procedure
• Quantity of decal soln
>20 vol. of specimen.
• Wash the decalcified
specimen for 24-48 hrs
–to remove the decal
soln.

30. Other Decalcifying Fluids
Jenkins fluid Absolute alcohol
• 73ml Distilled Water,
• 10ml Chloroform,
• 10ml Glacial Acetic Acid
• 3ml Hydrochloric Acid 4ml
Trichloroacitic Acid
• Formal saline (10%) –
• 95 ml Tricloroacitic acid - 5 gm
This is used for small biopsies. The process of decalcification is slow hence cannot be used for
dense bone or big bony pieces

31. Von –Ebners Fluid
Time Taken 3-5
Days
Formula:
Saturated Aq.
Sodium Chloride
50ml
Distilled Water
50ml
Hydrochloric
Acid 8ml

32. Ion Exchange
Resins
• remove the calcium ions from the fluid
• ensures a more rapid rate of solubility of
the calcium from the tissue
• reduction in the time of decalcification.
Used to:
• Well preserved cellular detail
• Faster decalcification
• Elimination of the daily solution change
• Resin can be reused by removing excess
acid.
Advantages :

33. Ion Exchange Resin II
Tissue is placed in a bottle in a mixture of 10% or
20% resin and formic acid.
Resin used is ammonium form of sulphonated
polystyrene resin.
The volume of fluid is 20 – 30 times that of the
specimen.
After use, resin may be regenerated by washing
twice with dilute N/10 HCl , followed by 3 washes in
distilled water.

34. Electrophoretic Decalcification
• First described in 1947.
• Attraction of the calcium ions to a negative electrode in addition to the
solution of the calcium in the electrolyte.
Advantage
I. Shortened time for complete decalcification.
II. Better preservation of soft tissue details.
Disadvantage
I. Limited no. of specimen processed at a time.

35. Electrophoretic
Decalcification
II

36. Electrophoretic
Decalcification
III
Temperature of the reaction- 30" to 45" C.
Solutions changed after 8 hours of use to
ensure maximum speed of decalcification.
Tissues are rinsed well in alkaline water &
immersed in lithium carbonate before staining.
Lithium carbonate treatment of a cut section
will neutralize any remaining acid in the tissue

37. Decalcification Histological
Techniques
Advantages
• It preserves the enzyme activity
• It also preserves the nucleic acids and
polysaccharides. It can be done by:
1. Buffer mixture
2. Chelating agents

38. Buffer Mixtures
1. Citric acid – citrate buffer (pH 4.5)
2. Molar hydrochloric acid – citrate buffer (pH 4.5) Lorch’s citrate hydrochloric acid buffer
(pH 4.5)
3. Acetate buffer (pH 4.5)
• Calcium salts may be removed from bone when placed into a buffered solution of citrate, pH
4.5.
• Daily changes of the buffer are necessary and the decalcification progress checked by chemical
oxalate test.

39. Chelating agents
• Chelating agents are the organic
compounds that have the power of
binding with certain metals.
Advantages
1. It shows a minimum of artefact
2. Section stained by most techniques
with first class results.
Disadvantages-
1. Slow process as calcium is removed
layer by layer from the hydroxyapatite
lattice.

40. EDTA Chelating Agent
• First described by HILLMAN & LEE (1953)
• Commonly used agent is EDTA.
• Binds to metallic ions like Calcium & Magnesium
• Ionized calcium on the outside of the apatite crystal , the crystal becomes
progressively smaller during decalcification.
• Slow process that does not damage tissues or their stainability, also pH sensitive.
• Excellent bone decalcifier for immunohistochemical or enzyme staining & electron
microscopy.

41. Surface Decalcification
• Needed when partially decalcified bone/unsuspected mineral deposits in soft
tissue are found during paraffin sectioning.
• After finding a calcification, the exposed surface in a paraffin block is placed
face side down in 5% HCL for 1hour or 10% formic acid for 15 to 60 minutes.
• Rinsed to remove the corrosive acids & re sectioned

42. End Point Decalcification
Probing the tissue
with the needle
Chemical tests Bubble test Radiography

43. End Point
Decalcification II
Physical tests require manipulation, bending probing
or trimming of the specimen to “feel” for remaining
calcified areas.
Chemical test- (calcium oxalate test) 5 ml of
decalcified fluid are neutralized with 0.5N sodium
hydroxide, 1 ml of 5 g/dl ammonium oxalate is added.
Appearance of turbidity indicates presence of calcium.
* Not done for EDTA Decalcification

44. End Point Decalcification III
Bubble test Acids - reacts with
calcium carbonate in bone to
produce carbon dioxide , seen as
a layer of bubbles on the bone
surface.
Bubble test is subjective & unreliable, tiny
bubbles indicate less calcium present.
Radiography
Faxitron machine with exposure setting of 10-
110 kv, 3ma tube current And kodak x-omat x ray
film is used.
Vinten Instruments Ltd,Jessamy Rd ,Weybridge
England

45. Neutralisation of Acids
• Saturated Lithium Carbonate Solution or
• 5-10% Aqueous Sodium Bicarbonate Solution for several
hours.
Chemical neutralization is
accomplished by immersing
decalcified bone into either
Many laboratories recommend
rinsing the specimens in tap
water for a few hours.
Culling(1974) recommended
washing in two changes of 70%
alcohol for 12- 18 hour before
continuing with dehydration

46. Processing
Decalcified
Bone
Oversized, thick bone slabs require an extended processing
schedule to obtain adequate de-hydration, clearing and paraffin
infiltration. I.e.., 3 changes of wax under vaccum of 2 hours
Small bone and needle biopsies containing little cortical bone
can be processed with soft tissues.
Decalcified bone sectioning -made easier after infiltration and
embedding in harder paraffin to give firmer support.

47. Processing Decalcified Bone II
• If a bone sample still appears chalky, mushy and crumbles out of block during sectioning,
then:
I. Dehydration, clearing or paraffin infiltration may be incomplete.
II. Blocks can be melted down, and re- infiltrated with paraffin for up to eight hours
to see if this improves sectioning.
III. Reversing processing by melting paraffin from bone and going back through 2
changes of xylene, 2 changes of 100% alcohol to remove residual water and then
reprocessing back in to paraffin.
IV. Double embedding procedure can produce better results than paraffin wax alone.

48. Microtomy of Decalcified Bones
• Base sledge microtome & wedge
shaped steel or tungsten carbide edged
knife
• An optimal section thickness for bone is
same as soft tissues, 4-5µm or up to 6-
7µm is accepted.
• Bone marrow biopsies should be cut at
2-3µm for marrow cell identification

49. Microtomy of Decalcified Bones II
• The floating water bath may need to be
hotter than for soft tissues as bone has
the tendency to crinkle when cut.
• Lifted onto the chrome-gelatin coated
slides .

50. Artefcats
• Artefacts Under decalcification
• Inability to section
• Incomplete infiltration of paraffin
• Staining characteristics
• Bone dust
• Remedy- surface decal, redecal

51. Artefacts II
• Over decalcification
• Nuclear detail lost or severely compromised
• Disruption of cell membrane and cytologic properties
• Loss of glycogen
• Swelling of tissue, especially collagen
• Staining characteristics
• Recalcification

53. Recent Advances
• Introduction of ultrasonic energization in decalcification
• Decalcification of bone specimens of 2-5 mm thickness can
be achieved in 5 hours or less when the decalcifying fluids
are agitated by ultrasonic energization.
• Acid or chelating decalcifiers may be used and the
application of combined fixation-chelation permits
routinely many histochemical procedures previously
requiring special handling.

54. Recent Advances II
• 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 will accelerate the
process.
• The solution should be changed after each cycle. The temperature
restriction between 42-45°C for best results