This document discusses decalcification, which is the process of removing calcium from tissue using acids. It defines decalcification and lists criteria for complete decalcification. Various decalcification methods are described including strong acids, weak organic acids, ion exchange resins, chelating agents, electrophoretic techniques, and microwave techniques. Factors that influence decalcification time and methods for determining when decalcification is complete are provided. Post-decalcification treatment and surface decalcification are also summarized.

1. DECALCIFICATION
BY DR.AZEEM

2. DEFNITION
• THE PROCESS OF REMOVAL OF CALCIUM FROM TISSUE BY ACTION
ACID(i.e. demineralisation of hard tissue to soften them).
• Choice of demineralisation:
1. Urgency,
2. Degree of demineralisation,
3. Extent of investigation and
4. Staining technique.

3. CRITERIA FOR DECALCIFICATION
• COMPLETE CALCIUM REMOVAL FROM TISSUE.
• DOES NOT DAMAGE CELL.
• DOES NOT INTERFERE CELLS DURING STAINING.
• REASONABLE FAST DECALCIFICATION.

4. VARIOUS METHODS OF DECALCIFICATION
1.STRONG ACID:
• NITRIC ACID-AQUEOUS/FORMAL/PERENYI’S FLUID.
• HCL-AQUEOOUS.
2.WEAK/DILUTE ORGANIC ACIDS.
• FORMIC ACID-AQUEOUS/FORMALIN/SODIUM CITRATE.
• ACETIC ACID
• PICRIC ACID.
3.IRON EXCHANGE RESIN.
4.CHELATING AGENTS.
5.ELECTROPHORETIC TECHNIQUE.
6.MICROWAVE TECHNIQUE.

5. ACID DECALCIFIER
• ACID + CALCIUM=SOLUBLE CALCIUM SALTS.
• STAINING IS AFFECTED-INCREASE STAINING USE EHRLICH
HEMOTOXYLIN.
STRONG ACID WEAK ACID
1.10 ML ACID+ 90 ML DISTILLED WATER(HCL,NITRIC ACID)
2.5ML FORMALIN+15 ML NITRIC ACID+100 ML DW(FORMAL
NITRIC ACID)
3.10% NITRIC ACID 40ML+ABSOLUTE ALCOHOL
30ML+0.5%CHROMIC ACID 30 ML(PERENYI’S FLUID)
1.FORMIC ACID 10ML+ 90 ML DW(AQUEOUS FORMIC ACID)
2.5 ML FORMALIN+10 FORMIC ACID+100 ML DW(FORMAL
FORMIC ACID)
3.FORMIC ACID 35 ML+ 20% AQUEOUS TRISODIUM CITRATE
65 ML(FORMIC ACID SODIUM CITRATE)
4.BOUINS SOLUTION-SATURATED AQ. PICRIC ACID
500ML+FORMALIN 167ML + FORMIC ACID 33ML

6. STRONG ACID WEAK ACID
ADVANTAGE-
RAPID DECAL
DISADVANTAGE-
DAMAGE TISSUE,
INTERFERE STAINING(1DAY)
IHC INTERFERED
AND
LOSS OF ENZYMES.
NITRIC ACID-YELLOW, SO 0.1% UREA ADDED
OLD NITRIC ACID DAMAGE TISSUE-SO, FRESH ONE USED.
USES
NEEDLE AND SMALL BIOPSY RAPID DIAGNOSIS WITHIN 24 HRS
CORTICAL BONE SPECIMEN
AQUEOUS: FORMAL:
LITTLE TISSUE DAMAGE --------- INHIBIT MACERATION
STAINING --------- DISCOLORATION
TIME REQUIRED HUMAN RIB-24 HRS
ADVANTAGE-
FIXATION AND DECAL
2-4 DAYS
DISADVANTAGE-
FORMALIN INCREASED TO 25 ML SPEEDS DECAL
BUT TISSUE CELLULAR DETAILS INTERFERED.
TIME REQUIRED HUMAN RIB-3-4 DAYS

7. STRONG ACID WEAK ACID
AQUEOUS:
ADVANTAGE
LITTLE TISSUE DAMAGE
STAINING
TIME REQUIRED HUMAN RIB-24 HRS
FORMAL:
ADVANTAGE
INHIBIT MACERATION
DISCOLORATION
TIME REQUIRED HUMAN RIB-24 HRS
PERINYI’S FLUIDS:
ADVANTAGE
LITTLE HARDENING EFFECT-FOR SMALL DEPOSIT OF CALCIUM
CYTOLOGICAL PREPERATION.
SECTIONING EASIER
DISADVANTAGE
STOCK SOLUTION
VIOLET TINGE
SLOW DECAL ON LARGE BONE
CHEMICAL TEST FOR END POINT NOT CARRIED OUT –XRAY IS
USED
TIME REQUIRED HUMAN RIB-2-4 DAYS FOR FEMUR-14 DAYS
AQUEOUS:
ADVANTAGE
SMALL PIECE OF BONE-TEETH
GOOD NUCLEAR STAINING
IHC
TISSUE DAMAGE MINIMAL
DISADVANTAGE
SLOW
FORMIC ACID FORMALIN
ADVANTAGE
DECAL AND FIXATION
5ML FORMIC ACID TISSUE DAMAGE MINIMAL
DISADVANTAGE
LOSS OF CELLULAR DETAILS> 25ML.

8. ION EXCHANGE RESIN
• AMMONIUM FORM OF SULPHONATED POLYSTYRENE
• PROCEDURE:
• DECAL FLUID(20-30 TIMES THE SPECIMEN)
+
ION EXCHANGE RESIN LAYERED IN THE BOTTOM(NOT LESS THAN 10%)
• END POINT DECALCIFICATION BY X RAYS.
• ADVANTAGE:-
REDUCE TIME DECAL, REUSE TWICE WITH(N/10) HCL AND THRICE IN DW.
• DISADVANTAGE
LIMITED

9. CHELATING AGENTS EDTA-
SEQUESTRENE/VERSENE
• EDTA-BIND METALS(CALCIUM)
• PROCEDURE:
• 10% FORMALIN FIXED TISSUE
SALTS OF ETHYLENE DIAMINE TETRACETIC ACID FLUID (50 TIMES-5.5% EDTA BUFFERED WITH
PHOSPHATE TO PH7.4)-EDTA DISODIUM BIND TO CALCIUM IN TISSUES
+
CALCIUM AND MAGNESIUM IN TISSUE
EXCHANGE THRICE FLUIDS EVERY 4TH OR 5TH DAY.
END POINT DECALCIFICATION BY X RAYS AND CHEMICAL AGENTS.
• ADVANTAGE:-
ARTEFACT MINIMUM AND STAINING GOOD
• DISADVANTAGE
NOT USED IN LAB VERY SLOW.

10. ELECTROPHORETIC TECHNIQUE
• CATHODE ATTRACTING CALCIUM
• ELECTROLYTE-HCL8% AND FORMIC ACID 10%
• PROCEDURE:
• CATHODE-BRASS
+
ANODE-PLATINUM
+
ELECTROLYTE
• END POINT DECALCIFICATION BY X RAYS EVERY 2-3 HRS.
• ADVANTAGE:-
REDUCE TIME DECAL BY KEEPING PLATES TOGETHER
TIME REQUIRED FOR DECAL OF HUMAN RIBS-6-8 HRS.

11. MICROWAVE TECHNIQUE
• ADVANTAGE
• ACCLERATE DECAL.
• DISADVANTAGE:
MONITOR TIME AND TEMPERATURE-OVERCOME BY MICROTOME
TECHNOIQUES .

12. FACTORS INFLUENCING DECAL
• SUSPENSION OF CALCIFIED TISSUE
• AGE OF PATIENT, TYPE OF BONE, SIZE OF SPECIMEN AND SOLUTION.
• TEMPERATURE
• AGITATION
• VACUUM-ACCELERATED IN ION, ELECTROLYSIS, ULTRSONICS.
• CONCENTRATION AND VOLUME OF DECAL SOLUTION

13. DETERMINATION OF END POINTS.
• RADIOGRAPHY OF TISSUE-XRAY(EXCEPT MERCURIC CHLORIDE SPECIMEN).
• CHEMICAL TEST FOR CALCIUM(EXCEPT IN EDTA METHOD OR ACID DECAL IN HIGH CONC USED)-
CALCIUM OXALATE TEST
1. STEP 1-5ML SPECIMEN DECALCIFYING FLUID-LITMUS PAPER FOR PH
2. STEP2-NEUTRALIZE BY AMMONIUM HYDROXIDE UNTIL LITMUS TURNS NEUTRAL
3. STEP3-5ML SATURATED AMMONIUM OXALATE.
4. INTERPRET-
STEP 2 POSITIVE-WHITE PRECIPITATE-CHANGE OLD FLUID..TESTING TO BE STOPPED
STEP 2-IS NEGATIVE-THEN STEP3 –PRECIPITATE FORMS IT MEANS LESS CALCIUM-30 MINS…DECAL
COMPLETE..REPEAT AFTER 1 DAY.
• PHYSICAL TEST-(INACCURATE, UNRELIABLE AND DAMAGE TO TISSSUE)
• 1.INSERTING NEEDLE
• 2.BUBBLE TEST(CO2)=ACID+ CALCIUM CARBONATE =BUBBLE CO2.

14. TREATMENT FOLLOWING DECAL
• NEUTRALIZE ACID-WEAK ALKALI-
SATURATED LITHIUM CARBONATE
OR
5-10% AQUEOUS SODIUM CARBONATE
ORWASHING 2 CHANGES IN 70% ALCOHOL
• THOROUGH WASHING IN ALCOHOL 3-4 HRS
REMOVE ACID
LONGER WAX IMPREGNATION(58 DEGREE) FOR DECAL TISSUE
SPECIMEN

15. SURFACE DECALCIFICATION
• DEFINITION:
• REMOVAL OF SMALL FOCUS OF CALCIFICATION ON SURFACE OF
PARAFFFIN BLOCK
• PROCEDURE:
• BLOCK KEPT FACE DOWN IN 1-5% HCL, 10% FORMIC ACID FOR 15
MINS
• ADVANTAGE-PREVENT KNIFE DAMAGE AND TORN TISSUE