SAS 4: Decalcification Flashcards
is the removal of calcium ions from a bone or calcified tissue through a histological process that makes them flexible and easier to cut
Decalcification
The principle of decalcification is
Strong mineral acids - 10% hydrogen chloride
Weak organic acids - 5-10% formic acid
the recommended ratio of fluid to tissue volume for decalcification is
20:1
Temperature of impaired nuclear staining of Van Gienson’s stain for collagen fibers
37 degree celsius
Temperature of tissue will undergo complete digestion within 24-48 hours
55 degree celsius
Calcium may be removed by the ff:
Acids
Chelating agents
Ion exchange resins
electrical ionization
What are the acid decalcifying agents
- Nitric acid
- Hydrochloric acid
- Formic acid
- Trichloroacetic acid
- Sulfurous acid
- Chromic acid
- Citric acid
most common and fastest decalcifying agent used
Nitric acid
Disadvantage of Nitric acid
progressive tissue damage
acts as both tissue softener and decalcifying agent
Perenyi’s fluid
recommended for routine purposes
Perenyi’s fluid
decalcifying time for Phloroglucin-Nitric acid
12-24 hours
most rapid decalcifying agent
Phloroglucin-Nitric acid
it is rapid in action, produces minimum distortion of tissues and produces good nuclear staining
Aqueous nitric add solution 10%
it is composed of concentrated nitric acid, formaldehyde, and distilled water
Formol-Nitrci acid
Decalcifying time of Formic-Nitric acid
1-3 days
it is recommended for routine decalcification of postmortem research tissues
Formic Acid
Decalcifying time of Formic acid
2-7 days
Both fixative and decalcifying agent
Formic acid
it is inferior compared to nitric acidin its role as a decalcifying agent because of its slower action and greater distortion of tissues produced
Hydrochloric acid
Weak decalcifying agent, not used for dense bone tissues
Trichloroacetic acid
Decalcifying time of Trichloroacetic acid
4-8 days
Very weak decalcifying agent, suitable for minute pieces of bone
Sulfurous acid
Both fixative and decalcifying agent and may be used for decalcifying minute bone spicules
Chromic acid (Flemming’s fluid)
It permits excellent nuclear and cytoplasmic staining and doesn’t produce well distortion
Citric acid
Decalcifying time of Citric acid
6 days
substances which combine with calcium ions and other salts to form weakly dissociated complexes and facilitate removal or calcium salt
Chelating agents
hastens decalcification by removing calcium ions from formic-acid containing decalcifying solutions
Ion exchange resin
It is a process whereby positively charged calcium ions attracted to the negative electrode and subsequently removed from the decalcifying solution
electrophoresis
This method is satisfactory for small bone fragments, processing only a limited number of specimens at a time
Electrophoresis
It has been used quite often for tissue processing, but there are very few studies describing its use in decalcification of bone or teeth
Microwave oven decalcification
Factors influencing decalcification
- concentration
- fluid access
- size and consistency
- agitation
- temperature
The 3 ways to measure extent of decalcification
- Physical/Mechanical test
- X-ray or Radiological method
- Chemical method
it requires manipulation, bending, probing and trimming of the specimen to feel for remaining calcified areas
Physical/Mechanical test
very expensive, “Most ideal, Most sensitive, most reliable” and it’s not recommended for mercuric chloride fixed tissue
X-ray or Radiological method
The decalcifying agent is usually changed every 24-48 hours and the chemical test is done on the discarded fluid
Chemical method
it is simple, reliable, and recommended for routine purposes
Chemical method
What are the tissue softeners:
- 4% aq. phenol
- molliflex
- 2% HCl
- 1% HCl in 70% alcohol
- Perenyi’s fluid - decalcifying agent and tissue softener at the sane time
