Histopathology (Decalcification)

DECALCIFICATION

The removal of calcium ions from the bone, teeth, calcified aorta, calcified tissues (tuberculous lungs, blood vessels, metastatic tissues) through histological process thereby making the bone flexible/soft/pliable and easy for pathological testing/investigation or routine processing.

Dystrophic calcification

- type of calcification on tissues that is associated with necrosis

Metastatic calcification

Calcification that occurs in the walls of blood vessels or in the kidney lung or elsewhere

Macerated and poor staining

- result if specimen is decalcified without fixation

Zinc Formalin mixtures, B5, formol acetic alcohol, Bouin's solution

Fixatives used for bone specimens if we want to preserve the bone marrow

20:1

Ratio of agent to tissue

18-30 deg C

optimum temperature range for decalcification

25 deg C

standard temperature for decalcification

forms soluble calcium salts with calcium moving it into the solution

Mechanism of action of acids in decalcification

ACID DECALCIFICATION

- most popular decalcifying agents belong to this type of decalcifying agent

Nitric Acid

- most common decalcifying agent

Nitric Acid

Fastest type of decalcifying agent but may impart a yellow color

Aqueous nitric acid solution

- nitric acid based decalcifying agent that is rapid and may be used in decalcifying urgent biopsies.

Formol Nitric Acid

- nitric acid based decalcifying agent that isalso rapid and may produce a relatively good nuclear staining and produces less tissue destruction compared to aqueous nitric acid

5% sodium sulfate

- solution that prevents the yellow discoloration causes by nitric acid decalcifying agents

Perenyi's fluid

- a nitric acid based decalcifying agent that is recommended for routine purposes and not urgent biopsies

- slow penetrating

- can avoid maceration

- made with nitric acid, chromic acid, and ethanol

Phloroglucin-nitric acid

- Most rapid nitric acid based decalcifying agent

Hydrochloic Acid

another type strong acid of decalcifying agent but is inferior to nitric acid because of its slower action but produces better nuclear staining

Von Ebner's Fluid

- Weakened hydrochloric acid decalcifying agent

Von Ebner's Fluid

- permits good cytologic staining and does not require washing out prior to dehydration

- recommended for small pieces of bone

- made of HCl, Distilled water, and Saturated Aqueous solution of NaCl

Weak acid decalcifying agents

- type of decalcifying agent that retains good nuclear staining

- is slower than strong acids but interfere less

- most common kind is formic acid based

Formic Acid

- moderate acting weak acid decalcifying agent

- can be used as both a fixative and decalcifyinga gent

- good for large bones as this is weaker and may accommodate the specimen longer

Evans and Krajian

- formic acid decalcifier that is buffered with sodium citrate

Kristensen

- formic acid decalcifier that is buffered with sodium formate

Gooding and Stewart

- a formic acid decalcifier that is buffered with 40% formalin

- can also fix and decalcify at the same time

Trichloroacetic Acid (TCA)

- weak acid decalcifying agent that is used for surface decalcification as it is very weak

- it is not used for urgent biopsies

Chromic Acid (Flemming Fluid)

- can be used as both a fixative and decalcifying agent

- used for small bone fragments

- inhibits nuclear staining with hematoxylin

- includes glacial acetic acid for nuclear staining

- carcinogenic

citric acid-citrate buffer solution

- permits excellent nuclear and cytoplasmic staining

- does not produce tissue cell distortion

- too slow for routine processes

CHELATING AGENTS

- a slow decalcifier

- used for research purposes

- used if DNA is needed to be preserved such as for Immunohistochemistry tests and PCR

14% EDTA

- concentration of EDTA needed for decalcifying agents to be tested through PCR

capturing calcium ions on the surface of apatite crystals

Mechanism of action of chelating agent decalcifying agents

Neutral EDTA

- most common chelating decalcifying agent

- marketed as Versene

pH 6.5-7.5 (7 is optimum)

Optimum pH range of EDTA for calcium to bind

Ion Exchange Resin

- kind of decalcifying agent that is trying to get positively charged calcium with a negative charged resin

- usually used with weak acids

Weak Acids (formic acid)

What type of acid decalcifying agent should be used with IER to accelerate reactions

Electrical Ionization

Decalcification method that makes use of electrophoresis

- used in research

-has grace effect on tissues

- also makes sue of the principle that calcium is positively charged and is attracted to the cathode