Decalcification Flashcards
The procedure whereby calcium or lime salts are removed from tissues following fixation
decalcification
Purpose of Decalcification
To ensure and facilitate the
normal cutting of sections
To prevent obscuring the
microanatomic detail of sections
Types of D-Agents
- Based on strong mineral oils
- Based on weaker organic acids
- Composed of chelating agents
Most common
and fastest
decalcifying agent
10% Aqueous Nitric
Acid Solution
Most Rapid
Decalcifying
Agent so far
Phloroglucin
Moderately rapid
decalcifying agent
Von Ebner’s Fluid
Moderate acting decalcifying agent
Formic Acid
Recommended for
urgent biopsies
Formol
Recommended for
urgent works
Phloroglucin
easily removed by
70% alcohol
10% Aqueous Nitric
Acid Solution
Imparts yellow color which will impair
staining reaction
10% Aqueous Nitric
Acid Solution
The solution should be used inside the fumehood
Formol
Maceration is avoided due to the presence of chromic acid and
alcohol
Perenyi’s Fluid
Safer to handle than nitric acid or hydrochloric acid
Formic Acid
Recommended for autopsy materials, bone marrow, cartilage and
tissues studied for research purposes
Formic Acid – Sodium
Citrate Solution
Relatively slow
decalcifying agent
for dense bones
Perenyi’s Fluid
Recommended for routine decalcification of postmortem research tissues
Formic Acid
Suitable only for
small spicules of
bone
Trichloroacetic Acid
Decalcifies and
softens tissue at
the same time
Perenyi’s Fluid
Weak decalcifying agent; not used for dense tissues
Trichloroacetic Acid
Suitable only for
minute pieces of
bone
Sulfurous Acid
Recommended for
routine purposes
Perenyi’s Fluid
Greater distortion
of Tissues
Hydrochloric Acid
recommend for surface decalcification of the tissue block
1% solution in 70% alcohol
Used to decalcify
minute bone
spicules
Chromic Acid
(Flemming’s Fluid)
Does not produce
cell or tissue
distortion
Citric Acid – Citrate
Buffer Solution
Forms precipitates at the bottom, which requires frequent changes of solution
Chromic Acid
(Flemming’s Fluid)
Suitable for routine surgical specimens, when
immunohistochemical staining is needed
Formic Acid
Nuclear staining
with hematoxylin
is inhibited
Chromic Acid
(Flemming’s Fluid)
Good nuclear staining
- 10% aqueous nitric acid solution
- formol - nitric acid
- hydrochloric acid
- trichloroacetic acid
- formic acid
Good (P) / Excellent (CF) nuclear and cytoplasmic staining
- Perenyi
- Citric acid - Citrate buffer solution
- Formic acid
Good cytologic staining
- von ebner’s fluid
Poor nuclear staining
Phloroglucin
Recommended for routine purposes
Perenyi’s fluid
Not recommended for routine purposes
- formic acid - sodium citrate soln
- citric acid
Not recommended for urgent work
- formic acid
- trichloroacetic acid
Does not require washing
- Trichloroacetic acid
- Von ebner’s fluid
Recommended for teeth and small pieces of bone
- Formic acid
- Von ebner’s fluid
Fixative and Decalcifying agent
- Formic acid
- Chromic acid
EDTA commercial name
Versene
Recommended for
detailed microscopic
studies
EDTA
DT for small specimen
1-3 weeks
DT for dense cortical bone
6-8 weeks to decalcify
EDTA inactivates alkaline phosphatase activity, which can be restored by
adding magnesium chloride
In IER, the degree of decalcification may
be measured by
physical or X-ray
method.
A process whereby positively charged calcium ions are attracted to a
negative electrode and subsequently removed from the decalcifying
solution
Electrophoresis
satisfactory for small bone fragments, processing only a
limited number of specimens at a time
Electrophoresis
temperature of impaired nuclear staining of Van Gieson’s stain for collagen fibers
37C
temperature of tissue that will undergo complete digestion within 24-48 hours
55ºC
ideal time for
decalcification
24-48 hours
DT for dense bones tissue
14 days longer
Done by touching with fingers to determine the
consistency of tissue
Physical or Mechanical Test
causes damage and distortion of tissue.
pricking
Best method for determining
complete decalcification
X-Ray or Radiological Method
Not recommended on Tissue fixed
in mercuric chloride
X-Ray or Radiological Method
Simple, reliable and convenient method for routine purpose
Chemical Method
(Calcium Oxalate Test)
Detect Calcium in the decalcifying solution
by
precipitation of insoluble calcium
hydroxide or calcium oxalates
may act both as a decalcifying agent and tissue softener
Perenyi’s Fluid
Tissue Softeners
4% Aqueous
Phenol Solution
Molliflex
2% Hydrochloric Acid
1% Hydrocloric Acid
in 70% Alcohol
The removal of acid from tissue or neutralized chemically by
immersing the specimen either saturated lithium carbonate solution
or 5-10% aqueous sodium bicarbonate soln for several hours
Post-Decalcification
