Decalcification

Decalcification

• is the removal of calcium ions from a bone or calcified tissue through a histological process that makes them flexible and easier to cut.

• adjusts the hard substance of bones to the softness of paraffin embedding medium.

• enables the histotechnologist to cut soft sections of the bone using the microtome, so that they can be processed like any other soft tissue of the body

Bones

are the main object of decalcification in a surgical pathology laboratory

True

True or False: Decalcification is a lengthy procedure

2–3 mm

A low speed saw may be sufficient to routinely and rapidly reduce undecalcified surgical specimens of hard tissue, to a thickness of?

Strong mineral acids, such as 10% hydrogen chloride (HCl), or weak organic acids, such as 5-10% formic acid (HCOOH), form soluble calcium salts in an ion exchange that moves calcium into the decalcifying solution

Principle of decalcification

True

True or False: Decalcification should be done after fixation and before impregnation, to ensure and facilitate the normal cutting of sections and to prevent obscuring the microanatomic detail of such sections by bone dust and other cellular debris.

True

True or False: Poorly-fixed specimens become macerated during decalcification and stain poorly afterwards. This is very noticeable in areas containing bone marrow. It is therefore common practice for laboratories to extend fixation times for bone specimens before commencing decalcification

Acid decalcifying agents

are the most widely used agents for routine decalcification of large amounts of bony tissues because they are stable, readily available, and relatively inexpensive as compared to other decalcifying agents.

Strong acids

are the most rapid in action but if used longer than necessary will rapidly cause a loss of nuclear staining and can macerate tissues

True

True or False: Rapid decalcifying agents are more likely to adversely affect any subsequent staining

Nitric acid

• is the most common and the fastest decalcifying agent used so far, utilized both as a simple solution or combined with other reagents.

• This may be used as simple aqueous solutions with recommended concentrations of 5 10%.

• Disadvantage: inhibiting nuclear stains and destroying tissues

Hydrochloric acid (HCI)

is inferior compared to nitric acid in its role as a decalcifying agent because of its slower action and greater distortion of tissue produced on the decalcified section. However, it produces good nuclear staining and if used in 1% solution with 70% alcohol, may be recommended for surface decalcification of the tissue blocks

Weak acids

are popular and are widely used for decalcification

Formic acid

• is a moderate-acting decalcifying agent which produces better nuclear staining with less tissue distortion, and is safer to handle than nitric acid or hydrochloric acid

• recommended for routine decalcification of postmortem research tissues, although not suitable for urgent examinations

Chelating agents

are substances which combine with calcium ions and other salts (e.g. iron and magnesium deposits) to form weakly dissociated complexes and facilitate removal of calcium salt.

ethylene diamine tetra acetic acid (EDTA)

• The most common chelating agent in the market

• recommended only for detailed microscopic studies

• it does not act like inorganic or organic acids but it binds metallic ions, notably calcium and magnesium

Sonication with EDTA

• has been successfully used to accelerate decalcification of trephine specimens for subsequent molecular analysis.

• During the process, the temperature must be carefully controlled.

Ion exchange resin

• hastens decalcification by removing calcium ions from formic acid-containing decalcifying solutions, thereby increasing solubility from the tissue.

• It is not recommended for fluids containing mineral acids such as nitric acid or hydrochloric acid.

Electrophoresis

is a process whereby positively charged calcium ions are attracted to a negative electrode and subsequently removed from the decalcifying solution

microwave oven

has been used quite often for tissue processing, but there are very few studies describing its use in decalcification of bone or teeth

Microwave oven decalcification

• is faster than routine decalcification irrespective of the decalcifying agents used

• is a novel technique compared to the manual method. In this method, hard tissues are placed in the decalcifying agent in a microwave oven for intermittent periods with regular changes of the solution till the end point is reached

• Concentration

• Fluid access

• Size and consistency

• Agitation

• Temperature

Factors influencing the rate of decalcification

True

True or False: more concentrated acid solutions decalcify bone more rapidly, but are more harmful to the tissue

20:1

The recommended ratio of fluid to tissue volume for decalcification is?

False

True or False: Decrease in size and consistency of tissues will require longer periods for complete decalcification.

Gentle fluid agitation

is achieved by low-speed rotation, rocking, stirring or bubbling air into the solution

18°C -30°C

The optimum temperature so far recommended for decalcification is the room temperature range of?

True

True or False: Prolonged decalcification of tissue is liable to prevent hydrolysis and lead to maceration and destruction of tissue components which are poorly stained

1. Physical tests

2. chemical test

3. X-ray

There are several methods to check if the end point of decalcification has been reached:

Physical tests

• require manipulation, bending, probing or trimming of the specimen to “feel” for remaining calcified areas

• it is generally considered to be unreliable

chemical test

• can be applied when some acid decalcifiers are used (particularly formic acid).

• This test is cumbersome and useless in the every- day practice of surgical pathology when many samples are placed in the decalcification solution simultaneously

X-ray

• This is a very expensive although the most ideal, most sensitive and most reliable method of determining extent of decalcification due to its ability to detect even the smallest focus of calcium which appears opaque in an X-ray plate

• not recommended for mercuric chloride-fixed tissues due to the latter's characteristic radio-opacity which will interfere with the correct interpretation of the plate

Surface decalcification

This is a method of dealing with small unexpected deposits of calcium that may be encountered in paraffin blocks

Perenyi's fluid

may act both as a decalcifying agent and tissue softener

12-24 hours

To soften unduly hard tissues, selected portions are left in the fluid for?

4% aqueous phenol solution

Washing out and immersion of fixed tissues in —- for 1-3 days may also cause considerable tissue softening and easier sectioning of blocks without producing marked deleterious effects and tissue distortion

1. Molliflex

2. 2% hydrochloric acid

3. 1% hydrochloric acid in 70% alcohol

Other substances which may be used as tissue softeners are?

Molliflex

Tissues immersed in —- may appear swollen and soapy

Van Gieson's stain

At 37°C, there will be impaired nuclear staining of —- for collagen fibers

24-48 hours

At 55°C, the tissue will undergo complete digestion within?

Microwave, sonication and electrolytic methods

produce heat, and must be carefully monitored to prevent excessive temperatures that damage tissue

3-5 mm

Uniform —- sections are optimal for decalcification with or without microwave- accelerated processing

Microwave irradiation

has been shown to speed up the process of decalcification significantly–from days to hours

• Formic acid 88% 100 ml

• Concentrated hydrochloric acid 80 ml

• Distilled water 1000 ml

Solution Used for Electrolytic Decalcification

Electrolytic Decalcification

This method is satisfactory for small bone fragments, processing only a limited number of specimens at a time

Neutral EDTA

acts slowly but causes little tissue damage. Conventional stains are largely unaffected

• immunohistochemistry (IHC)

• Fluorescent In Situ Hybridization (FISH)

• Polymerase Chain Reaction (PCR)

It is more appropriate for research applications where very high quality morphology is required or particular molecular elements must be preserved for techniques such as?

commercial name of EDTA