CHAPTER 3
DECALCIFICATION
Learning Objectives:
At the end of this chapter, students should know the decalcification of hard bony tissues.
Specifically, to;
1.
Define decalcification.
2.
Discuss the importance of decalcification.
3.
Determine and discuss the steps before decalcification.
4.
Know the types of decalcification process.
5.
Know the characteristics of an ideal decalcifying agent.
6. Discuss the application, advantages and disadvantages of each decalcifying process and
the agents used;
A. Mineral Acids
B. Ion Exchange Resins
C. Chelation
D. Electrolytic
7.
8.
Know the block surface decalcification.
Know and discuss the decalcification end point determination methods.
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DECALCIFICATION
• Decalcification is a process of complete
removal of calcium salt from the tissues like
bone and teeth and other calcified tissues
(E.G. tuberculous lungs, arterioschlerotic
vessels) after fixation.
• Decalcification is done to assure that the
specimen is soft enough to allow cutting with
the microtome knife.
• Unless the tissues is completely decalcified,
sections will be torn and ragged and may
damage the cutting edge of microtome knife,.
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STEPS BEFORE DECALCIFICATION
1. Slice tissue samples at about 4-5 mm
thick. Calcified tissue needs 2-3 hours
only, for complete decalcification.
Slice
Tissue
2. Fixative of choice for bone or bone
marrow is Zenker formal or Bouin's
fluid. Unfixed tissue tends to be
damaged 4 times greater during
decalcification than a properly fixed
tissue.
Fix
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DECALCIFICATION PROCESS TYPES
1. Dissolution of calcium by a
diluted mineral ACID.
2. Removal of calcium by diluted
mineral with ION EXCHANGE
RESIN to keep the decalcifying
fluid calcium free.
3. Using Chelating agents (EDTA)
MINERAL
ACID
ION
EXCHANGE
RESIN
DECAL
CIFICA
TION
CHELATING
AGENT
ELECTRICAL
CURRENT
4. Electrolytic removal of calcium
ions from tissue by use of
ELECTRIC CURRENT.
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GOOD DECALCIFYING AGENT CRITERIA
1. Completely removal of calcium.
2. Absence of damage to tissue cells.
3. Subsequent staining not altered.
4. Shorter time.
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MINERAL ACIDS
MINERAL ACIDS:
A. STRONG ACID (Nitric Acid , Hydrochloric Acid)
B. WEAK ACID (Formic Acid, Acetic Acid, Picric Acid)
Nitric
Hydro
chloric
CALCIUM
Formic
dissolution
Picric
Acetic
• NITRIC ACID:
Very rapid, but if used for longer than 24-48 hours cause deterioration of stainability
specially of the nucleus. Choice for temporal bones.
Example:
1. PERENYI’S FLUID (nitric acid, absolute alcohol, chromic acid)
Slow for decalcifying hard bone but excellent fluid for small deposits of calcium eg.
calcified arteries, coin lesions and calcified glands.
2. Formalin Nitric acid
Nitric acid causes serious deterioration of nuclear stainability which partially inhibited
by formaldehyde.
MINERAL ACIDS
• HYDROCHLORIC ACID:
Slower than nitric acid but still rapid. Fairly good nuclear staining.
• FORMIC ACID:
Extensively used as acid decalcifier. Nuclear staining in better, but requires
neutralization and thorough washing prior to dehydration.
Example:
1.
Gooding and Stelwart's fluid. (Formic acid, Formalin)
2.
Evans Krajian fluid (Trisodium citrate, formic acid). At pH 2-3.
3.
Formic Acid Sodium Citrate - Gives better staining result than nitric
acid method, since its less harsh on the cellular properties. Therefore even
with over exposure of tissue, it causes little loss of staining qualities.
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SURFACE DECALCIFICATION
 The surface of the block
to be decalcified is
trimmed with scalpel.
 The block is then placed
in acid solution at 1%
hydrochloric acid face
downwards.
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ION EXCHANGE RESINS
• ION EXCHANGE RESINS are used to remove
calcium ion FROM FLUID.
• The resins is an ammoniated salt of
sulfonated resin along with various
concentrations of formic acid are used.
• After use, the resin may be regenerated by
with dilute N/10 HCL followed by distilled
water.
• ADVANTAGES:
1. faster decalcification
2. tissue preservation
3. cellular details better preserved
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CHELATING AGENTS
 CHELATING AGENTS are organic
compounds which binds certain
metals. Ethylene-diamene-tetraacetic acid, disodium salt called
VERSENATE has the power of
capturing calcium.
 This is a slow process but has
little or no effect on other tissue
elements. Some enzymes are
still
active
after
EDTA
decalcification.
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ELECTROLYTIC AGENT
• Electrolytic method the electrolyte
APPARATUS attracts calcium ions to a
negative electrode with added
decalcifying solution (HCL, Formic
acid, Distilled water).
FOLLOW-UP STEPS:
1. Neutralization: tissues are de-acidified
or neutralized by treatment with alkali
(lithium or sodium sulphate).
2. Washing: removes acid or alkali which
would otherwise interfere with staining.
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DECALCIFICATION END POINT
DETERMINATION METHODS
1. FLEXIBILITY
Bending, needling or by use of scalpel if it bends easily
that means decalcification is complete. This is
unreliable, and may cause damage and distortion.
2. X-RAY
Best method but very costly. Tissue fixed in mercuric
chloride containing fixatives cannot be tested as they
will be radio opaque.
3. CHEMICAL
It is done to detect calcium FROM the decalcifying fluid.
If calcium is tested negative, decalcification in
considered complete.
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