Decalcification Flashcards
What is decalcification
The process of removing minerals from bone or other calcified tissues such as teeth. This is carried out without distorting cells and connective tissue, and without leaving any harmful effects on staining reactions
Importance of decalcification
Softening of the tissue so that it can be sectioned in the gross room and using a microtome. It ensures good quality paraffin sections while preserving all the essential microscopic elements.
Results of over decalcification and under decalcification
under - difficulty sectioninng the tissue
over - lack of nuclear staining, stains very poorly
what is calcified tissue made of
Mad eup of the strong and hard mineral hydroxyapatite. This is an inorganic material
What should be carried out before decalcification
- full fixation
- skin and surrounding tissue removed and separately fixed
- section cut prior using fine thoothed hacksaw
When are undecalcified bone sections examined?
for the diagnosis of metabolic bone disease
What 4 factors influence which decalcifier is used
- urgency of the case
- degree of mineralization
- extent of investigation required
- staining reactions to be carried out
What 2 methods can be used for decalcification
Acid solutions ; nitric acid, HCl or formic acid
or chelating agents such as ethylenediaminetetraacetic acid
How do acids decalcify tissue
they lower the pH causing excess hydrogen ions to combine with hydroxyl ions to give water and calcium ions, which will form salts
Talk about acid decalcification method and how different acids work
They are commonly used at solutions of 5-10%, the stronger the acidity the more maceration of the tissue (swelling, loss of staining ability and poorer morphology)
- HCl and HNO3 - act rapidly. Nitric acid can lead to serious deterioration of tissue. HCl can form a carcinogen (bis chloromethyl ether) if previous formaldehyde is not well washed out.
- formic acid is slower acting.
What 2 methods can be implemented with acids to make decalcification more efficient?
Ion exchange resin - makes use of formic acid over a layer of an ammoniated salt of a sulphonated resin. The calcium is rapdily removed from the solution into the resin.
Electrolytic method - mixture of formic acid and HCl are placed in a glass jar. The specimen is suspended by a platinum wire anode in the jar with temp kept between 30-45 degrees
advantages of the ion exchange resin
well preserved cell detail, fast process, elimination of daily solution change and resin can be reused
How are chelating agents used
they have the property of binding to certain metals such as EDTA which binds to calcium ions for removal. pH is kept between 5 and 7.2
Advantages and disadvantages of chelating agents
adv - can take place at neutral pH, no damage to the tissue and preservation is excellent, allows for staining with most staining techniques.
dis - very slow acting
How can the end point of decalcification be determined
Mechanical, chemical or radiographic methods
mechanical method of finding end point of decalcification
probe the specimen with a needle and scrape the section surface. This creates histological artefacts and overall inaccurate
chemical method of end point determination
Depends on the precipitation of calcium oxalate
- 5mls of the used decalcifying fluid is made neutral to litmus paper using concentrated ammonium hydroxide.
- the resulting solution is mixed well and allowed to stand for 30 minutes
Results
- turbidity from calcium oxalate shows presence of calcium
- no turbidity shows that decalcification is complete
imp that decalcifying fluid is changed after each chemical check
radiographic check of end point
considered as being the most accurate. you x ray the specimen. Cannot be carried out on metallic fixed tissue as specimen will be rendered radiopaque.
What should be done to specimen after decalcification and how is surface decalcification carried out
- washed well with running water to remove excess acid
- surface decalcficiation with 1-5% HCl
Explain how the process of decalcification is carried out in practice in all
- properly fixed specimen
- fixative is properly washed out
- volume of solution used must be 20:1 with the specimen
- Acid mixture is cahnges every 24-48 hours and EDTA solutions every 2-5 days to ensure no depletion of solution
- once ready, the speciment is thoroughly rised with tap water to neutralise the acid decalcifying solution
what factors infleunce decalcification
- agitation
- tissue size (ideally 3mm)
- solution (aqeuous used, alcoholic would aid in preventing undue swelling byt they render the decalcifying agent almost inefficnet and suppress ionisation)
- concentration of solution (reminder that more conc more damaging)
- volume of solution (20:1)
- reaction temp (at 60 degrees bone will be completely macerated in acid, not EDTA tho)
