DECALCIFICATION OF BONY AND HARD TISSUE FOR HISTOPATHOLOGY
PROCESSING
PREPARED BY MR. ABHIJIT DAS
INTRODUCTION:
Decalcification of bony and hard tissue is an
essential step in histopathology processing, especially when examining tissue
samples like bone or teeth.
These tissues contain a significant
amount of calcium, which makes them too hard to be sectioned with standard microtomes.
Decalcification removes the calcium,
softening the tissue for proper cutting and examination under a microscope.
FACTORS CONTROLLING THE RATE OF
DECALCIFICATION:
Ø Type
of Decalcifying Agent: Strong acids like nitric acid act
faster than milder acids like formic acid or chelating agents like EDTA.
Ø Concentration
of Decalcifying Agent: Higher concentrations speed up
decalcification but can damage tissue if too harsh.
Ø Temperature:
Higher temperatures increase the rate but may also cause tissue damage. Room
temperature is typically used to balance speed and safety.
Ø Size
and Thickness of Tissue: Thinner and smaller tissue samples
decalcify faster than larger ones.
Ø Agitation:
Stirring or gently agitating the decalcifying solution can accelerate the
process.
Ø Type
of Tissue: Dense bones take longer to decalcify than softer
bones or cartilage.
METHODS OF DECALCIFICATION:
There are two primary
methods of decalcification:
1.
Acid Decalcification:
Ø Uses
strong (e.g., nitric acid) or weak acids (e.g., formic acid) to dissolve
calcium from tissues.
Ø Fast
but may damage tissue if not carefully monitored.
2.
Chelating Agent Decalcification:
Ø Uses
chelating agents like EDTA that bind and remove calcium ions.
Ø Slower
but preserves tissue structure better, making it ideal for delicate samples.
SURFACE DECALCIFICATION:
Surface decalcification targets only the tissue
surface. The tissue block is quickly treated with a decalcifying agent on the outer layer, then rinsed and sectioned. This
preserves the inner tissue while softening the outer calcified part.
END POINT DETERMINATION OF DECALCIFICATION:
End-point determination of decalcification ensures that all calcium has been removed without
over-processing the tissue. Common methods include:
1.
Physical Testing: Bending or probing the
tissue with a needle to check softness.
2.
Chemical Testing: Testing the decalcifying solution for calcium presence.
3.
X-ray Method: X-raying
the sample to check for remaining calcium.