DECALCIFICATION
➢ Removal of calcium ions or lime salts from the organic extracellular matrix, calcified collagen and surrounding
tissues of bones (these ions & salts are difficult to catch)
➢ NOT A MANDATORY STEP in tissue processing
➢ Spx are immersed in a decalcifying agent
➢ We never use CONCENTRATED solns cos it’s damaging to tissues
➢ Both decalcification and processing depend on bone thickness.
➢ Ideal thickness is 1-3mm
➢ Must be done following fixation
➢ (Volume) Ratio of fluid to tissue 20:1 (10-20x the volume of the spx)
➢ Heat and constant agitation speeds up decalcification (makes decalcification time shorter)
➢ More concentrated acid solutions decalcify more rapidly but may destroy tissue
➢ Optimal temperature is at ROOM TEMPERATURE
➢ Autotechnicon cannot do decalcification so this tissue process is done MANUALLY.
4 METHODS OF DECALCIFICATION
Electrolytic Method / o Most rapid method because by electricity, calcium is removed
Electrophoresis / Electric o Placing the spx in acid and apply electric current
Ionization o This method is dependent upon the supply of electric current
o The positively charged calcium ions are attracted to negative electrodes from the
decalcifying solution (the +charge calcium ions are attracted to the -charge
electrons, thus facilitating calcium removal)
o The process is longer compared to electrolytic method
o Duration: 1-14 days
Ion Exchange resin o Spx is placed in a container, at the bottom of the container, place an ammonium
form of polystyrene resin which will increase tissue solubility to facilitate removal
of calcium.
Not carried out
o So ibig sabihin, ibababad mo parin yung spx pero at the bottom of the container ay
in the lab
may resin.
because they
are not suited
o Calcium is rapidly removed by the decalcifying solution containing formic acid,
for biopsies so thereby increasingly solubility from tissues
the process is • EDTA:
VERY LONG - Is an example of chelating agent
- A very good anticoagulant/Potent anticoagulant
Use of Chelating agent: - However, it is a weak decalcifying agent
- Small specimens: 1-3wks
EDTA
- Dense tissues: 6-8wks
• Not a good decalcifying agent but it is considered as the best decalcifier in electron
microscopy (EM) and immuno-histochemistry
• Disadvantage: Inactivates alkaline phosphatase
Use of Acid • Most common method
• Done MANUALLY (get a container, place a decalcifying acid, and immerse the spx to
be decalcified)
1. Nitric acid
- 5-10% Nitric acid is the routine clearing agent when acid is used
- Most commonly used acid
- Concentrated Nitric acid MUST be diluted to produce 5-10% soln
- To dilute, use DISTILLED WATER to produce ________ (sometimes formalin
kaya magiging formol nitric)
➢ Removal of calcium ions or lime salts from the organic extracellular matrix, calcified collagen and surrounding
tissues of bones (these ions & salts are difficult to catch)
➢ NOT A MANDATORY STEP in tissue processing
➢ Spx are immersed in a decalcifying agent
➢ We never use CONCENTRATED solns cos it’s damaging to tissues
➢ Both decalcification and processing depend on bone thickness.
➢ Ideal thickness is 1-3mm
➢ Must be done following fixation
➢ (Volume) Ratio of fluid to tissue 20:1 (10-20x the volume of the spx)
➢ Heat and constant agitation speeds up decalcification (makes decalcification time shorter)
➢ More concentrated acid solutions decalcify more rapidly but may destroy tissue
➢ Optimal temperature is at ROOM TEMPERATURE
➢ Autotechnicon cannot do decalcification so this tissue process is done MANUALLY.
4 METHODS OF DECALCIFICATION
Electrolytic Method / o Most rapid method because by electricity, calcium is removed
Electrophoresis / Electric o Placing the spx in acid and apply electric current
Ionization o This method is dependent upon the supply of electric current
o The positively charged calcium ions are attracted to negative electrodes from the
decalcifying solution (the +charge calcium ions are attracted to the -charge
electrons, thus facilitating calcium removal)
o The process is longer compared to electrolytic method
o Duration: 1-14 days
Ion Exchange resin o Spx is placed in a container, at the bottom of the container, place an ammonium
form of polystyrene resin which will increase tissue solubility to facilitate removal
of calcium.
Not carried out
o So ibig sabihin, ibababad mo parin yung spx pero at the bottom of the container ay
in the lab
may resin.
because they
are not suited
o Calcium is rapidly removed by the decalcifying solution containing formic acid,
for biopsies so thereby increasingly solubility from tissues
the process is • EDTA:
VERY LONG - Is an example of chelating agent
- A very good anticoagulant/Potent anticoagulant
Use of Chelating agent: - However, it is a weak decalcifying agent
- Small specimens: 1-3wks
EDTA
- Dense tissues: 6-8wks
• Not a good decalcifying agent but it is considered as the best decalcifier in electron
microscopy (EM) and immuno-histochemistry
• Disadvantage: Inactivates alkaline phosphatase
Use of Acid • Most common method
• Done MANUALLY (get a container, place a decalcifying acid, and immerse the spx to
be decalcified)
1. Nitric acid
- 5-10% Nitric acid is the routine clearing agent when acid is used
- Most commonly used acid
- Concentrated Nitric acid MUST be diluted to produce 5-10% soln
- To dilute, use DISTILLED WATER to produce ________ (sometimes formalin
kaya magiging formol nitric)
