Decalcification- MIDTERMS L7

DECALCIFICATION

• The procedure whereby calcium or lime salts are removed from tissues following fixation.

• It should be done after fixation and before impregnation to ensure and facilitate the normal cutting of sections

• Decalcification 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.

• A lengthy procedure, as bone pieces have to be left in the decalcifying agent for several days or even weeks, depending on the size of the tissue.

• Make sure that the tissue has been adequately fixed and rinsed well to prevent any undesired reaction with the decalcifying agent.

BONES

Are the main object of decalcification in a surgical pathology laboratory

STRONG MINERAL ACID

10% HCl

WEAK ORGANIC ACID

5-10% Formic acid

BUFFERED FORMALIN

Is a satisfactory fixative for bone but where the preservation of bone marrow is important, some laboratories use alternatives.

ALTERNATIVES FOR BUFFERED FORMALIN

1. Zinc formalin mixture

2. Formol Acetic Acid (Davidson’s fixative)

3. Bouin’s solution

THOROUGH FIXING PRIOR TO DECALCIFICATION

In order to protect the cellular and fibrous elements of bone from damage caused by the acids used as decalcifying agents.

INADEQUATE DECALCIFICATION

May result in poor cutting of hard tissues and damage to the knife edge during sectioning.

POORLY- FIXED SPECIMENS

Become macerated during decalcification and stain poorly afterwards. This is very noticeable in areas containing bone marrow.

HIGH- QUALITY FINE- TOOTH SAWS

Should be used to prepare bone slices

COARSE SAWS

Can cause considerable mechanical damage and force bone fragments into the soft tissues present in the specimen.

PURPOSE OF DECALCIFICATION

• To ensure and facilitate the normal cutting of sections

• To prevent obscuring the microanatomic detail of sections

CHARACTERISTICS OF A GOOD DECALCIFYING AGENT

• Must be capable of removing calcium salts from tissue completely.

• Must not cause any destruction to cells and tissue component.

• Has a good staining capacity

CALCIUM MAY BE REMOVED BY ANY OF THE FOLLOWING AGENTS

• Acids

• Chelating agents

• Ion exchange resins

• Electrophoresis

ACID DECALCIFYING AGENTS

Most widely used agents for routine decalcification. Stable, easily available and relatively inexpensive

EXAMPLES OF DECALCIFYING AGENTS

• 10% Aqueous Nitric Acid Solution

• Formol – Nitric Acid

• Perenyi’s Fluid

• Phloroglucin– Nitric Acid

• Hydrochloric Acid

• Von Ebner’s Fluid

• Formic Acid

• Formic Acid – Sodium Citrate Solution

• Trichloroacetic Acid

• Sulfurous Acid

• Chromic Acid (Flemming’s Fluid)

10% AQUEOUS NITRIC ACID SOLUTION

• Recommended concentrations would be 5% - 10%

• Rapid in action

• Produces minimum distortion of tissues and good nuclear staining

DECALCIFICATION TIME OF 10% AQUEOUS NITRIC ACID SOLUTION

12-24 hrs

COMPOSITION OF 10% AQUEOUS NITRIC ACID SOLUTION

Concentrated Nitric Acid + Distilled Water

FORMOL NITRIC ACID

• Rapid acting; recommended for urgent biopsies

• Nuclear staining is relatively good

• Produces less tissue destruction than 10% aqueous nitric acid

DECALCIFICATION TIME OF FORMOL NITRIC ACID

1-3 days

COMPOSITION OF FORMOL NITRIC ACID

Concentrated nitric acid + 40% formaldehyde + distilled water

PARENYI’S FLUID

• Recommended for routine purposes

• Decalcifies and softens tissue at the same time

• Nuclear and cytoplasmic staining is good

• Maceration is avoided due to the presence of chromic acid and alcohol

DECALCIFICATION TIME OF PARENYI’S FLUID

2-7 days

COMPOSITION OF PARENYI’S FLUID

10% Nitric acid + 0.5% Chromic acid + Absolute ethyl alcohol

PHLOROGLUCIN- NITRIC ACID

• Most rapid decalcifying agent so far

• Recommended for urgent works

• Nuclear staining is poor

• Prolonged decalcification produces extreme tissue distortion

• Complete decalcification cannot be determined by chemical means

DECALCIFICATION TIME OF PHLOROGLUCIN- NITRIC ACID

12-24 hrs.

COMPOSITION OF PHLOROGLUCIN- NITRIC ACID

Concentrated nitric acid + 10% Nitric acid + Phloroglucin

HYDROCHLORIC ACID

• Inferior to nitric acid in its role as a decalcifying agent because of its slower action and greater distortion of tissues

• Produces good nuclear staining

• Recommended for surface decalcification of tissue blocks

VON EBNER’S FLUID

• Permits relatively good cytologic staining

• Moderately rapid decalcifying agent

• Does not require washing out before dehydration

• Recommended for teeth and small pieces of bone

COMPOSITION OF VON EBNER’S FLUID

Distilled water + HCl + Sodium Chloride

FORMIC ACID

• Moderate acting decalcifying agent which produces better nuclear staining with less tissue distortion. May be used as a fixative and decalcifying agent

• Safer to handle than nitric acid or hydrochloric acid

• Recommended for routine decalcification of postmortem research tissues

• Recommended for small pieces of bones and teeth

• Suitable for routine surgical specimens, when immunohistochemical staining is needed

• Relatively slow, not suitable for urgent works

• Requires neutralization with 5% sodium sulfate and washing out

DECALCIFICATION TIME OF FORMIC ACID

2-7 days

FORMIC ACID- SODIUM CITRATE SOLUTION

• Permits better nuclear staining than nitric acid method

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

DECALCIFICATION TIME OF FORMIC ACID- SODIUM CITRATE SOLUTION

3-14 days

COMPOSITION OF FORMIC ACID- SODIUM CITRATE SOLUTION

Aqueous Sodium Citrate + Formic saline

TRICHLOROACETIC ACID

• Permits good nuclear staining

• Does not require washing out

• Suitable only for small spicules of bone

• Weak decalcifying agent

DECALCIFICATION TIME OF TRICHLOROACETIC ACID

4-8 days

COMPOSITION OF TRICHLOROACETIC ACID

TCA acid + 10% formol saline

SULFUROUS ACID

Very weak decalcifying agent suitable only for minute pieces of bone

CHROMIC ACID (FLEMMING’S FLUID)

• May be used as a fixative and a decalcifying agent

• Used to decalcify minute bone spicules

• Nuclear staining with hematoxylin is inhibited

• Forms precipitates at the bottom, which requires frequent changes of solution

• Degree of decalcification cannot be measure by routine chemical test

• Carcinogenic and environmental toxin and corrosive to skin and mucous membranes

Ethylene Diamine Tetraacetic Acid (EDTA)- CHELATING AGENTS

• Combines with calcium ions and other salts to form weakly dissociated complexes and facilitate removal of calcium salts

• Commercial name: Versene

• Recommended for detailed microscopic studies It is a very slow decalcifying agents

• EDTA is used for immunohistochemical, enzyme staining and electron microscopy

• EDTA inactivates alkaline phosphatase activity

EDTA FOR SMALL SPECIMEN

1-3 weeks

EDTA FOR DENSE CORTICAL BONE

It will take 6-8 weeks to decalcify

pH of EDTA

6.5 – 7.4

ION EXCHANGE RESIN

• Ammonium form of polystyrene resin that hastens decalcification by removing calcium ions from formic acid containing decalcifying solutions.

• Not recommended for fluids containing mineral acids such as nitric acid or hydrochloric acid

• A layer of ion exchange resin (1/2 thick) is spread over the bottom of the container and the specimen is placed on top of it. Then the decalcifying agent is added.

• The tissue may stay for 1 – 14 days.The degree of decalcification may be measured by physical or X-ray method

ELECTROPHORESIS/ ELECTRICAL IONIZATION

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

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

• Good cytologic and histologic details are not always preserved.

ELECTROPHORESIS USES

88% formic acid + concentrate HCl + Distilled water

FACTORS INFLUENCING RATE OF DECALCIFICATION

• Structure

• Temperature

• Volume

• Time

STRUCTURE

High conc and greater amount of fluid will increase the speed of the process

TEMPERATURE

• 37*C

• 55*C

• Room Temperature

37*C

Impaired nuclear staining of Van Gieson’s stain for collagen fibers

55*C

Tissue will undergo complete digestion within 24-48 hours

ROOM TEMPERATURE

Range of 18-30ºC

VOLUME

Ratio 20:1

TIME

• 24-48 hours ideal time for decalcification

• Dense bone tissue – 14 days longer

MEASURING THE EXTENT OF DECALCIFICATION

• Physical or Mechanical Test

• X- Ray or Radiologic Method

• Chemical Method (Calcium Oxalate Test)

PHYSICAL OR MECHANICAL TEST

• Done by touching w/ the fingers to determine the consistency of tissue.

• Bending, needling or by use of scalpel if it bends easily that means decalcification is complete.

• Pricking, causes damage and distortion of tissue.

X- RAY OR RADIOLOGIC METHOD

• Best method for determining complete decalcification.

• Not recommended on Tissue fixed in mercuric chloride. (radio opacity)

CHEMICAL METHOD (CALCIUM OXALATE TEST)

• Simple, reliable and convenient method for routine purposes

• Detect Ca in the decalcifying solution by precipitation of insoluble calcium hydroxide or calcium oxalates

POST DECALCIFICATION

• The removal of acid from tissue or neutralized chemically by immersing the specimen either saturated lithium carbonate solution or 5 10% aqueous sodium bicarbonate solution for several hours.

• Simply rinse the decalcified specimens with running tap water.

TISSUE SOFTENERS

Unduly hard tissues which are liable to damage the microtome knives may require tissue softeners, aside from decalcification.

KINDS OF TISSUE SOFTENERS

• Perenyi’s fluid

• Moliflex

• 2% HCl

• 1% HCl in 70% Alcohol