Lec 6: Decalcification Flashcards
Enumerate the steps for Conventional Tissue Processing
- Fixation
- Decalcification
- Dehydration
- Clearing
- Infiltration
- Embedding
- Trimming/Cleaning
- Sectioning
- Staining
- Mounting
- Labeling
The removal of calcium ions from a bone or calcified tissue through a histological process.
Decalcification
The process which makes the bone or calcified tissue flexible and easy to cut.
Decalcification
Tissues that include Decalcification
- bones
- teeth
- calcified tumor
- calcified heart valve
The substance that makes the bone hard
Calcium
- It forms soluble calcium salts in anion exchange which moves calcium from the bone into the decalcifying solution.
- It sequesters metallic ions including calcium in aqueous solutions.
- Strong Mineral Acid or Weak Organic Acid
- Chelating agent
Criteria of a good Decalcifying Agent
- Complete removal of calcium
- absence of damage to tissue cells or fibers
- Non impairment of subsequent staining technique
- Reasonable speed of the calcification
What are the factors affecting the rate of decalcification (5)
- concentration
- fluid access
- size & consistency
- agitation
- temperature
Factors affecting Decalcification Rate:
[TRUE or FALSE]
1. Increased temperature = slows down Decalcification = increases tissue damage
2. The bigger the tissue, the longer the decalcifying period.
3. The denser the tissue, the faster the decalcifying period.
4. Higher concentration = slower Decalcification
- FALSE
- TRUE
- FALSE
- FALSE
Factors affecting Decalcification Rate:
- Higher concentration does what to the tissue?
- The optimum temperature recommended.
- Recommended Fluid to Tissue ratio
- How does gentle agitation & low-speed stirring/rocking affect Decalcification Rate?
- How long do dense bone tissues usually take to complete Decalcification?
- Decalcifies rapidly, but more harm to tissue
- RT (18–30°C)
- 20:1
- Increase Decalcification Rate
- 14 days +
Procedure of Decalcification
- Selection of tissue
- Fixation
- Decalcification
- Acid Neutralization
- Thorough washing
In tissue selection, what is the ideal thickness for:
a.) dense bone
b.) soft bone
a.) 2 to 5 mm
b.) 4 to 6 mm
When cutting large specimen:
1.) What saw is used?
2.) What saw is NOT advisable to use?
- Large fine-toothed bone saw
- Coarse Saw
When cutting a small specimen, what is used?
Geological cutting machine with diamond impregnated cutting disc
It protects the cellular elements of the bone from damage caused by acids in decalcifying agents.
Fixation
FIXATION:
- Fixative for EM
- Fixatives for good preservation of Bone Marrow
- Fixatives for Tooth specimens
- Fixatives for Bone
- Glutaraldehyde
-
a.) Zinc formalin mixture
b.) B-5, formol-acetic alcohol (Davidson’s solution)
c.) Bouin’s solution - 15% formic acid
- Buffered formalin
The three main types of decalcifying agents
- strong mineral acids
- weak organic acids
- chelating agent
The 2 methods of Decalcification
- Acid method
» strong mineral acids
» weak organic acids - Chelation
» chelating agent
Strong Mineral Acid:
- most common
- fastest
- recommended for routine purposes
- at higher concentration, inhibits nuclear stain & damages tissue
Nitric Acid
NITRIC ACID:
- Decalcification time: 12 to 24 hours
- easily remove by 70% alcohol
- recommended for urgent biopsy
- produces good nuclear staining
- can be used for large or heavily mineralized cortical bone specimen
Aqueous Nitric Acid Solution 10%
NITRIC ACID:
- Decalcification time: 12 to 24 hours
- imparts a yellow color (impairs staining reaction)
- damages tissue & tissue antigens
Aqueous Nitric Acid Solution 10%
NITRIC ACID:
- Decalcification time: 1 to 3 days
- less tissue damage than 10% Aqueous Nitric Acid
- recommended for urgent biopsy
- good nuclear staining
- imparts a yellow color
Formol-Nitric Acid
NITRIC ACID:
- Decalcification time: 2 to 7 days
- decalcifies and softens tissue
- maceration is avoided (presence of chromic acid & alcohol)
- recommended for routine purposes
- good Nuclear & Cytoplasmic staining
- slow for dense bones
Perenyi’s fluid
NITRIC ACID:
- Decalcification time: 12 to 24 hours
- most rapid
- poor Nuclear staining
- imparts yellow color
Phoroglucin-Nitric Acid
- does NOT require washing out before dehydration
- recommended for teeth and small pieces of bone
Von Ebner’s Fluid
FORMIC ACID:
- Decalcification time: 2 to 7 days
- fixative & decalcifier
- excellent Nuclear & Cytoplasmic staining
- recommended for teeth & small pieces of bone
- for most routine surgical specimens
- slow
- requires Neutralization (5% Sodium suilfate & wash)
10% Formic Acid
FORMIC ACID:
- Decalcification time: 3 to 14 days
- slow
- better nuclear staining than Nitric Acid
- recommended for autopsy materials, bone marrow, cartilage and tissue studies for research purposes
Formic acid - sodium citrate solution
- Decalcification time: 4 to 8 days
- good nuclear staining
- does NOT require washing out
- NOT used for dense tissues
- only for small spicules of bone
Trichloroacetic acid
- a fixative & decalcifying agent
- for minute bone spicules
- inhibits nuclear staining
- forms precipitate
- forms insoluble pigments (dehydration)
- environmental toxin & carcinogenic
Chromic acid (Flemming’s Fluid)
Chromic acid
- pH
- Decalcification time
- 4.5
- 6 days
Most common chelating agent in the market
EDTA
EDTA:
- Action of EDTA
- Commercial name
- Duration of tissue in EDTA
a. Small specimens
b. Dense cortical bone - Solution should be changed every ___ days.
- Captures and combines with calcium to form an insoluble non-ionized complex.
- Versene
-
a. 1 to 3 weeks
b. 6 to 8 weeks or longer - Every 3 days
The effects of the following to the tissue:
1. Over Decalcification
2. Under Decalcification
- Over Decalcification
• spoils the staining of basophilic elements
• causes maceration of the softer tissue elements - Under Decalcification
• interferes with proper sectioning & staining
It is the process where the chemicals used in decalcification will be neutralized in order to prevent a reaction of the chemicals to the succeeding pathologic processes
Acid Neutralization
The chemical solutions used to accomplish Chemical neutralization
- saturated lithium carbonate solution
- 5 to 10% aqueous sodium bicarbonate solution
How long are the following rinsed in tap water?
a.) small sample
b.) larger specimens
a.) 30 minutes
b.) 1 to 4 hours
