HISTOPATH DECALCIFATION Flashcards
Removal of Calcium or lime salts from bones or calcified tissues following fixation
DECALCIFICATION
_____ solutions decalcify more rapidly but may destroy ____
more concentrated acid solutions ; tissue
decalcification ratio
20:1
____ hastens decalcification
heat and agitation
decalcification duration
1-2 days
tissue in decalcification
bones and teeth
criteria for decalcifying agent
Complete removal of ca salts
No damage to tissue
Lack of harmful effect on staining reactions
Speed for removal of Ca salts
types of decalcifying agents
Acid
Chelating agent
Ion exchange resins
Electrophoresis
Widely used for routine purpose
Acid
most common and a rapid decalcifying acid agent
5-10% Nitric Acid
5-10% Nitric Acid time
12 to 24 hours
Acid can be removed by
70% alcohol or formaldehyde
Imparts a yellow color due to formation of Nitrous Acid
nitric acid
5-10% nitric acid is used for
For urgent biopsies, for needle and small biopsies
For large and heavily mineralized cortical bone specimen
Yellow color may impair staining reaction
Produce less tissue distortion than 10% aqueous nitric acid
FORMOL-NITRIC ACID
FORMOL-NITRIC ACID is used for
For urgent biopsies
For nuclear staining
FORMOL-NITRIC ACID time
Time: 1-3 days
Decalcifies and soften tissues at the same time
Nuclear and cytoplasmic staining is good
Slow decalcifying agent for dense bones
PERENYI’S FLUID
Most rapid decalcifying agent
Nuclear staining is poor
PHLOROGLUCIN-NITRIC ACID
PHLOROGLUCIN-NITRIC ACID time
12-24 hours
Yellow color must be neutralized with
5% Sodium sulfate
in PHLOROGLUCIN-NITRIC ACID, When decalcification is complete..
the acid must be removed by 3 changes of 70% to 90% ethanol, when sections are cut the slides are brought to water and placed in 1% aqueous lithium carbonate for 1 hour and washed for 15 minutes then stained.
PERMITS good cytologic staining
Moderately rapid decalcifying agent
Does not require washing out
HYDROCHLORIC ACID
HYDROCHLORIC ACID is used for
For teeth and small pieces of bone
The extent of decalcification cant be measured by a
chemical test
Fixative and decalcifying agent
Excellent nuclear and cytoplasmic staining
Formic Acid
Formic Acid is used for
For small pieces of bones and teeth
For immunohistochemical staining
Formic acid Requires..
neutralization with 5% Na sulfate and washing out to remove the acid from the tissue
Good nuclear staining
Does not require washing
Weak agent, not used for dense tissues and suitable for small spicules of bone
Not recommended for urgent biopsies
TRICLOROACETIC ACID
Weak decalcifying agent
Suitable for minute bone spicules
SULFUROUS ACID
Used ad fixative and decalcifying agent
For minute bone spicules
Nuclear staining with hematoxylin is inhibited
Chromic aid is environmental toxin
Highly corrosive to skin, mucous membrane
carcinogenic
CHROMIC ACID (Flemming’s Fluid)
COMBINE WITH Calcium salts and other salts to form complexes and to facilitate removal of calcium
Excellent for immunohistochemical or enzyme staining and for electron microscopy
Chelating Agents
common chelating agent
EDTA
Chelating Agents duration
1-3 weeks for small specimens
6-8 weeks or longer for dense bones
in Chelating Agents, the pH is adjusted to.
7 to 7.4
Ammonium form of polysterene resin hastens decalcification by removing calcium ions from formic acid-containing decalcifying solutions
Not recommended for fluids containing mineral acids such as Nitric acid or Hydrochloric acid
Cellular detail is well-preserved
Extent of decalcification can be measured by routine chemical test
ION EXCHANGE RESIN
Is a process whereby positively charged calcium ions are attracted to negative electrode and subsequently removed from the decalcifying solution.
ELECTROPHORESIS
in ELECTROPHORESIS, the time required for decalcification is thereby..
shortened due to the heat and electrolytic reaction produced in the process.
Acid can be removed from tissues or neutralized chemically by immersing the decalcified bone in either saturated lithium carbonate solution or 5-10% aqueous Na bicarbonate solution for several hours
POST-DECALCIFICATION
in post-decalcification ___ is used for rinsing
running tap water
how long is running tap water
30 min for small; 1-4 hrs for larger specimen
Measuring Extent of Decalcification
Physical or Mechanical Test
X-Ray or Radiological Method
Chemical Method/Calcium Oxalate Test
Done by touching or bending the tissue
Pricking the tissue with fine needle or probe
Physical or Mechanical Test
Ideal, most sensitive and expensive
Detects smallest focus of Calcium
X-ray or Radiological Method
Detects Calcium in acid solution by precipitation of insoluble Calcium hydroxide or Calcium oxalate
Cloudiness/ppts indicates Ca is present
Chemical Method/ Calcium Oxalate Test
