Histopathology - Decalcification

Decalcification

the procedure whereby calcium or lime salts are removed from tissues following fixation

Acids and chelating agents

Decalcification is usually carried out by using (2):

Acids

is used to form a soluble calcium salt

Chelating agents

is used to bind to calcium ions

poor cutting

Inadequate decalcification may result in ______________

fine fret-saw

hand razor

Bones and calcified tissue is usually cut into small pieces using a ___________________, and trimmed with ________________

hand razor

permit complete penetration of the decalcifying solution with minimal surface damage

grating sensation

occurs when sectioned with a microtome knife

10% HCl, 1 hour

Remedy: the block can be placed face down on a pad of cotton gauze saturated with _______, for approximately ______________

Capable of removing calcium salts from tissues

A good decalcifying agent must be:

Acids

Chelating agents

Electrical ionization

Ion exchange resins

Calcium may be removed by the following agents (4):

Sulfurous acid

Hydrochloric acid

Trichloroacetic acid

Nitric acid

Formic acid

Citric acid

Chromic acid

Mineral acids that may be used for decalcifcation (7):

Nitric acid (5-10%)

Most common and very rapid decalcifying agent

Inhibit nuclear stains and destroys tissue

Formaldehyde and alcohol

To prevent progressive tissue damage and impaired staining nitric acid may be combined with ______________,_______________

Aqueous nitric acid

Formol nitric acid

Perenyi's fluid

Phloroglucin-nitirc acid

Types of nitiric acid (4):

Aqueous nitric acid

recommended for urgent biopsy, needle and small biopsy specimens to permit rapid diagnosis within 24 hours and less

12-24 hours

Decalcification time of aqueous nitric acid

Formol nitric acid

imparts a yellow color that will impair staining reaction of the cell

1-3 days

decalcification time of formol nitric acid

5% sodium sulfate, 0.1% Urea

Formol nitric acid is prevented by neutralizing the tissue with ___________ and addition of ________ to pure concentrated nitric acid

Perenyi's fluid

Relatively slow decalcifying agent for dense bones

2-7 days

Decalcification time of perenyi's fluid

ammonia

a precipitate is formed upon the addition of __________.

glacial acetic acid

Remedy for Perenyi's fluid

Ammonium oxalate

confirm the presence of calcium ---- signify that decalcification is still incomplete

Phloroglucin nitric acid

Most rapid decalcifying agent

12-24 hours

Decalcification time of phloroglucin nitric acid

trichloroacetic acid

does not require washing out

Very slow acting and a weak decalcifying agent

4-8 days

Decalcification time of trichloroacetic acid

90% alcohol

Trichloroacetic acid may be removed by several changes of ______________

Hydrochloric acid

recommended for surface decalcification of tissue blocks

Von Ebner's Fluid

Permits relatively good cytologic staing

recommended for teeth and small pieces of bone

Formic acid

Recommended for routine decalcification of post mortem research tissues

Citrate

addition of _______________ accelerates decalcification by cherlating the calcium liberated from the bone

Aqueous formic acid

Formic acid sodium citrate solution

Two types of formic acid (2):

Aqueous formic acid

suitable for most routine surgical specimen --- immunohistochemical staining

2-7 days

decalcification time of aqueous formic acid

Formic acid sodium citrate solution

Recommended for autopsy materials, bone marrow, cartilage and tissues studied for research purposes

Chromic acid (Flemming's Fluid)

an environmental toxin

highly corrosive to skin and mucous membranes

carcinogenic

6 days

decalcification time of citric acid

Chelating agents

are substances which combine with calcium ions and othe salts to form weakly dissociated complexes ------facilitate removal of calcium salts

EDTA (versene)

most common chelating agent

recommended for microscopic studies

1-3 weeks , 6-8 weeks

tissue is placed in EDTA from _______________ for small specimen, but may take ___________ or longer to totally decalcify

3 days

The solution should be changed every _______________

pH 7-7.4

EDTA is faster at what pH?

Magnesium chloride

EDTA inactivates alkaline phosphatase activity and can be restored by addition of ___________________

Ion exchange resin

hastens decalcification

X-ray method (physical)

The degree of decalcification may then be measured by

Electrophoresis

a process whereby positively charged calcium ions are attracted to a negative electrode

satisfactory for small bone fragments