DECALCIFICATION Flashcards
The procedure whereby __ are removed from tissues following fixation.
It should be done after fixation and before impregnation to ensure and facilitate the normal cutting of sections.
Decalcification
- calcium or lime salts
Decalcification
Acids
Chelating Agents
Ion Exchange
Electrophoresis
Purpose of decalcification
To ensure and facilitate the normal cutting of sections
To prevent obscuring the microanatomic detail of sections
Most widely used agents for routine decalcification. Stable, easily available and relatively inexpensive
Acid decalcifying agent
Acid decalcifying agent
Chromic Acid
Nitric Acid
Hydrochloric acid
Formic acid
Trichloroacetic acid
Sulfurous acid
Citric acid
ü Prolonged decalcification may lead to tissue distortion
ü Produces __ and __
ü Seriously damage tissue stainability
ü Imparts __ which will impair
staining reaction
10% Aquaeous Nitric acid Solution
- minimum distortion of tissues and good nuclear staining
- yellow color
Decalcification Time __
Most common and fastest decalcifying agent
Rapid in action
easily removed by _
10% Aquaeous Nitric acid Solution
12-24 hours
70% alcohol
- Produces less tissue destruction than 10% aqueous nitric acid
- The solution should be used inside the fumehood
Formol- Nitric Acid
Decalcification time: __
reco for urgent biopsies
__ is relatively good
Formol- Nitric acid
1-3 days
nuclear staining
- _ and __ is good
ü Maceration is avoided due to the presence of __ and __
ü Can not be determined by chemical test
Perenyi’s Fluid
- Nuclear and cytoplasmic staining
- chromic acid and alcohol
Decalcification Time __
Relatively slow decalcifying agent for dense bones
Recommended for routine purposes
Decalcifies and softens tissue at the same time
Perenyi’s Fluid
2-7 days
Decalcification Time __
Most Rapid decalcifying agent so far
Poor Nuclear staining
Recommended for urgent works
Phloroglucin- Nitric acid
12-24 hrs
Slower action
Greater distortion of Tissues
ü Inferior to __ in its role as a decalcifying agent
ü Produces ___
ü ___ - recommend for surface decalcification of the tissue block
Hydrochloric acid
- nitric acid
- good nuclear staining
- 1% solution in 70% alcohol
Permits relatively_
Does not require washing out before dehydration
Moderately rapid decalcifying agent
Recommended for __ and _
Von Ebner’s Fluid
- good cytologic staining
- teeth and small pieces of bone
- Moderate acting decalcifying agent
ü Safer to handle than __ or __
ü Recommended for routine decalcification of __
ü Suitable for routine surgical specimens, when __staining is needed
Formic Acid
- nitric acid or hydrochloric acid
– post mortem research studies
- immuno histochemical
Decalcification time __
May be used as a fixative and decalcifying agent
Relatively slow not for urgent works
Recommended for __ and __
It permits _ and _
Requires neutralization with __ and _
Formic Acid
- 2-7 days
- teeth and small pieces of bone
excellent nuclear and cytoplasmic staining
5% sodium sulfate and washing out
Decalcification time __
Relatively slow; not recommended for routine purposes
Requires neutralization with __
Formic Acid- Sodium Citrate Solution
- 3-14 days
- 5% sodium sulfate
ü Permits better nuclear staining than nitric acid method
ü Recommended for __,__,__ and __ studied for research purposes
Formic Acid- Sodium Citrate Solution
- autopsy materials, bone marrow, cartilage and tissues
Decalcification Time __
Very slow-acting, not recommended for urgent works
Permits good nuclear staining
Suitable only for small spicules of bone
Trichloroacetic acid
4-8 days
Does not require washing out
Weak decalcifying agent; not used for dense tissues
Trichloroacetic acid
Very Weak Decalcifying Agent
Suitable only for minute pieces of bone
Sulfurous acid
May be used as a fixative and a decalcifying agent
Used to decalcify minute bone spicules
Nuclear staining with __ is inhibited
Chromic acid ( Flemmings Fluid)
- hematoxylin
Decalcification Time___
Too slow action for routine purposes
Permits excellent nuclear and cytoplasmic staining
Does not produce cell or tissue distortion
Citric acid citrate buffer solution
6 days
Chelating agent
Edta
Cellular detail is well-preserved Daily washing of solution is eliminated Permits excellent statining results
The tissue may stay for __
The degree of decalcification may be measured by __ or _
Ion exchange resin
- 1-14 days
physical or X-ray method
ü Ammonium form of polystyrene resin that hastens decalcification by removing calcium ions from formic acid-containing decalcifying solutions.
ü A layer (1/2 thick) is spread over the bottom of the container and the specimen is placed on top of it. Then the decalcifying agent is added, usually 20-30X the volume of the tissue.
Ion exchange resin
Commercial name: _
Recommended for detailed microscopic studies
Very slow Decalcifying Agent
Permits excellent staining results
EDTA
- Versene
Combines with calcium ions and other salts to form weakly dissociated complexes and facilitate removal of calcium salts
ü For small specimen, __
ü For dense cortical bone, it will take __to decalcify
- is an excellent bone decalcifier for __ , __ and __
ü inactivates alkaline phosphatase activity, which can be restored by adding __
EDTA
1-3 weeks
6-8 weeks
- immunohistochemical, enzyme staining and electron
microscopy
magnesium chloride
A process whereby positively charged calcium ions are attracted to a negative electrode and subsequently removed from the decalcifying solution
ü D.T. short due to the _ and_
ü Same principles with Chelating agent:
ü this process utilizes electricity and is dependent upon a supply of direct current to remove the calcium deposits.
ü This method is satisfactory for ___, processing only a limited number of specimens at a time
ü Good cytologic and histologic details are not always preserved.
Electrophoresis
- heat and electrolytic reaction
- small bone fragments
factors influencing rate of decalcification
concentration
volume
time
temperature
__ and __ will increase the speed of the process
High concentration and greater amount of fluid
- _ impaired nuclear staining of_ for collagen fibers
ü __ tissue will undergo complete digestion within 24-48 hours
ü RT range of __
Temperature
37oC
Van Gieson’s stain
55oC
18-30 C
Increase in size and consistency of tissue require longer period.
Ratio 20:1
Volume
ü __ ideal timefor decalcification
ü Dense bone tissue–__
TIME
24-48 hours
14dayslonger
Measuring the extent of decalcification
physical or mechanical test
xray or radiological method
chemical method ( calcium oxalate test)
- Done by touching with fingers to determine the consistency of tissue.
ü Bending, needling or by use of scalpel if it bends easily that means decalcification is complete.
ü __, causes damage and distortion of tissue.
physical or mechanical test
Pricking
Best method for determining complete decalcification. Not recommended on Tissue fixed in __ . (radio opacity)
xray or radiological method
- mercuric chloride
Simple, reliable and convenient method for routine purposes.
Detect Calcium in the decalcifying solution by precipitation of insoluble __ or __
chemical method ( calcium oxalate test)
- calcium hydroxide or calcium oxalates
The removal of acid from tissue or neutralized chemical by immersing the specimen either __ or__ for several hours.
ü Simply rinse the decalcified specimen with running tap water.
post decalcification
saturated lithium carbonate solution or 5-10% aqueous sodium bicarbonate soln
How to soften unduly Hard Tissues?
Selected portions are left in the fluid for 12-24 hours and dehydrated in the same or manner
Submerge the cut surface of the block in the fluid for 1-2 hours before sectioning, to facilitate easier cutting of tissues
may act both as a decalcifying agent and tissue softener
perenyis fluid
Washing out and immersion of fixed tissues + 4% Aqueous Phenol Solution for 1-3 days =
- Considerable Tissue Softening
- Easier Block Sectioning without producing marked delerious effects & tissue distortion
Other substances which may be used as Tissue Softeners:
Molliflex
2% Hydrochloric Acid
1% Hydrocloric Acid in 70% Alcohol
good nuclear staining
10% Aquaeous Nitric acid Solution
formol-nitric acid
hydrochloric acid
formic acid- scs
trichloroacetic acid
good nuclear and cytoplasmic staining
citric acid- cbs
formic acid
perenyis fluid
good cytologic staining
von ebner
poor nuclear staining
phloroglucin nitric acid
reco for routine purposes
perenyis fluid
citirc acid cbs (too slow)
cannot be determined by chemical test
perenyis fluid
phloroglucin
chromc acid- flemming fluid
recommended for
teeth and small
pieces of bone
von ebner
formic acid
May be used as a
fixative and
decalcifying agen
formic acid
chromic acid flemmings fluid
