METHODS OF DETECTING ENDPOINT OF DECALCIFICATION

Mr. Muguongo • X-RAY

• PHYSICAL METHODS

• CHEMICAL METHODS.

X-RAY METHOD

• This is the method of choice but it cannot be used on tissues that have been fixed with fixatives containing mercuric chloride.

• This is because it causes radio opaqueness.

• It also allows of even metal implants not suspected to be present in the tissue.

PHYSICAL METHOD

• These include:

probing with a fine needle or pin.

Palpating & binding the tissue consistently.

• This is the most inaccurate method of detection of endpoint of decalcification.

• This is because it depends on the hands of the technologist.

• It also causes tissue damage.

CHEMICAL METHOD

• Relies on the detection of calcium in the decalcifying fluid. Once no further calcium is detected decalcification is complete.

• The best time to check endpoint of decalcification is usually 24hrs for the initial stages.

• The fluid to be used is that which just about to be discarded.

TECHNIQUE

1. Put 5mls of used decalcifying fluid in a chemically clean test. 2. Add a piece of blue litmus paper which turns red due to the

acidity of the decalcifying fluid. 3. Add conc. ammonium soln drop by drop until the litmus paper

turns blue indicating alkalinity. if soln turns cloudy due to formation of calcium hydroxide decalcification is not complete and it is not necessary to go on further with the test.

4. If the solution remains clear add 0.5 mls of 5% ammonium oxalate allow to stand for 30 minutes.

5. If turbidity forms due to formation of calcium oxalate, decalcification is incomplete. The tissue therefore requires further decalcification

6. If soln remains clear ,decalcification is complete.

• Note ; when using chemical method to

determine endpoint of decalcification, it is important that the decalcifying fluid be prepared in distilled water .

• Failure to observe this would lead to false positive results because of the presence of calcium in natural water.

TISSUE PROCESSING

• Before a tissue block is sliced in to thin sections , it must have undergone preparatory processes including:

1.Dehydartion.

2. Clearing/ dealcoholization.

3.Infliltration.

4. Embedding.

DEHYDRATION

• This is the complete removal of all traces of water from a tissue block using alcohol.

• The water maybe natural or may have been introduced during fixation or when washing the tissue.

• Common dehydrants include:

Acetone

Butyl alcohol( butanol)

Ethyl alcohol(ethanol)

Dioxane

Isopropyl alcohol (propanol)

Cotd’

• Dehydration process is accomplished by passing fixed tissue blocks through a series of ascending grades of alcohol.

• The first batch usually varies between 30% to 70% depending on the consistency of the tissue.

• Delicate tissues such as brain are started at 30% while hard tissues such as the brain are started at 70% alcohol.

• Tissues from alcoholic fixatives are started at high concentrations.

• Absolute alcohol shld never be used when starting cause this could cause distortion ,shrinkage,hardening and rapture of the cell membrane .

Contd’

• The tissue shld stay in each batch long enough to allow complete dehydration.

• The incomplete dehydration results into tissues that are not infiltrated hence poor section.

• The common range of alcohol is usually; 70%,90%,95% and 100%.

DEHYDARNTS.

1. Acetone; It is a quick dehydrant but not normally used in routine work coz it causes shrinkage and hardening.

2. Butanol; slow dehydrant which is miscible with alcohol

3. Dioxane; it has an advantage of being miscible with both water and molten wax.

It causes little shrinkage however not routinely used coz its fumes are highly toxic.

Cotd’

• Propanol ; used as a substitute for ethyl alcohol although it is never used with celloidine.

Its cheaper than ethanol.

CLEARING(DEALCOHOLIZATION)

• This is the complete removal alcohol from tissue blocks using an antimedium.

• Alcohol is removed from tissues because it is immiscible with molten paraffin wax .

• This also makes the tissues transparent and also raises the R.I of tissue .

Main clearing agents;

This include; 1.Xylene 2.Toulene 3. Benzene 4.Cedar wood oil. 5. Chloroform. 6.Carbon tetrachloride. 7. Aniline oil. 8.Carbon disulphide.

Cotd’

1. Xylene

Common clearing agent in the histolab.

Adva; cheap to buy.

Rapid in action

Makes tissues transparent.

Disdva;

makes tissues brittle if left for too long.

Cond’

2.Toluene Adva; does not make tissues brittle. Disadva; produces toxic flames. 3.Benzene Clears tissues of up to 2 to 5 mm thick within 3hrs. Adva; fairly rapid in action does not make tissues brittle. causes little shrinkage easily eliminated from paraffin wax. Disadva; Highly flammable.

Contd’

4.Cedar wood oil;

Clears tissues upto 5mm thick within 3hrs.

Is very tolerant and does not cause shrinkage even when left indefinitely.

It is however very slow for routine work.

Contd’

5.chlorofrom

Tolerant and it does not harden the tissue.

recommended for tough tissues e.g. skin and calcified tissue.

also useful for large specimen.

Disadva; does not make tissue transparent.

INFILTRATION

This process entails completely saturating the tissue in the medium to be used for embedding.

This helps provide internal support for the tissue.

Wax is the commonest impregnating medium for routine work.

It gives sufficient support for easy sectioning of the tissue.

Contd’

Clearing agent is eliminated from the tissue by diffusion into the melted paraffin wax(in the embedding medium)

The wax in turn diffuses into the tissue to replace the clearing agent.

Number of changes depend on;

1. Density of the work.

2. Size of the block.

Contd’

Wax used shld be of suitable melting point.

The melting point of the wax varies with nature of the tissue.

Hard tissues require wax of higher melting point than soft tissue .

Mostly this ranges between 50 to 60 degrees Celsius.

Contd’

Complete wax impregnation is necessary for the production of good sections , however;

Prolonged and very high temperatures may lead to overhardening of tissue ;this is detrimental to tissue sectioning.

Inadequate impregnation leads to ultimate poor penetration of the medium of embedded tissue block which being inadequately supported by the wax cracks or crumbles during section cutting.

Contd’

The wax impregnation oven is usually electrically heated .

A reliable thermostat should maintain the temp at 2 to 3 degrees above the melting point of the wax.

The interior of the oven should be large enough.

Contd’

Tissue density; dense tissues require longer immersion in molten paraffin to ensure complete impregnation e.g. bone and fibrinomas.

Size of tissue block; time required for thorough impregnation depends on thickness of the tissue , apiece of 5mm thickness for example takes an average of three hrs while that of 5mm takes 10 hrs.

EMBEDDING

Involves immersing the tissue in block of molten paraffin wax in a mould and allowing the wax to solidify in the mould so that a block of wax containing the tissue is produced.

The aim of blocking is to provide external support to the tissue for the purpose of sectioning with a microtome.

EXAMPLES OF EMBEDDING MEDIA

1.Paraffin wax.

Forms of paraffin wax;

paraplast

Paraplast plus

Bio loid.

Tisssue mat

TECHNIQUE FOR EMBEDDING(manual)

1. Fill the mold with molten paraffin wax.

2. Warm a pair of blunt – nosed forceps and use them to transfer the tissue from the paraffin bath to the mould.

3. Warm the forceps again and orientate the tissue until it is lying on the desired plane.

4. Remove the corresponding label from the paraffin bath and place it against the side of the mould adjacent to the tissue.

Contd’

5. Blow on the surface until a thin film of wax has solidified.

6. Transfer the mould into a container of cold water and immerse it gently.

The mould shld remain immersed until the wax hardens.

This may take 10 to 30 mins but may be hastened by using running tap water.

Contd’

2.Celloidin.

3.Esther wax

4.LVN (low viscosity nitrocellulose)

5.Water soluble wax

6.Synthtic resins.

7.Gelatin

1.PARAFFIN WAX

Available in a wide range of melting points ranging from 45-60.

Waxes with low melting points are softer than those with higher melting points.

There are various methods of impregnating tissue using paraffin wax;

1.Manual method

2.Automatic tissue processing

3.Vacuum impregnation.

Contd’ During manual processing, at least 4 changes of

wax are required at intervals of 15 minutes to

Make sure that the there is complete removal of the clearing agent.

During automatic tissue processing by use of a tissue processing machine only two changes are required because the tissues are agitated in the wax which accelerates the removal of the clearing agents.

Contd’

In both methods tissues should receive adequate time in molten paraffin wax but the period should not be prolonged as such treatment makes the tissue suffer from effects of excessive shrinkage and hardening which causes difficulty during sectioning.

Incomplete infiltration leads to;

1. Incomplete removal of clearing agent from the tissue.

2. Tissues become soft and crumble.

3. Wax fails to penetrate tissue thoroughly.

Note; the ultimate result of poor or incomplete infiltration is poor sectioning.

Vacuum infiltration.

Tissue like the skin ,lungs and bones and tough tissues like the tendons and ligaments are impregnated in paraffin wax under negative pressure in a vacuum embedding oven.

The vacuum embedding oven consists of a heavy brass chamber with a thick plate glass lid.

The lid rests on a rubber washer to effect air tight seal when in use.

Contd’

It has screw valves in the upper part of the chamber to allow exhaustion and readmission of air.

The greater part of the chamber is enclosed in a water jacket.

The temperature is thermostatically controlled at about 2-4 c above the melting point of the paraffin wax used.

Generally ; vacuum infiltration is the infiltrative act of cleared tissue blocks in molten paraffin wax under negative pressure.

Contd’

It is used in cases the following purposes are intended;

Quicken removal /displacement of clearing agent from the tissue.

Quicken penetration of molten paraffin wax into the tissues.

ADVANTAGES OF VAUUM INFILTRATION.

1. It is good for urgent biopsies for quick reporting.

2. Good for spongy tissues e.g. the lungs

3. Good for adipose tissue which becomes spongy after fats have been dissolved in alcohol.

4. Its good for dense tissues e.g. liver, spleen and the brain.

5. Good for hard tissues like the brain.

6. Good for tough tissues like the uterus and fibroids.

Contd’

7. There is little shrinkage of tissue.

8. There is much less distortion of cell consistency.

Disava; it is quite expensive.

TECHNIQUE.

1. Fix and dehydrate the tissue.

2. Clear in two changes of xylene 1hr each.

3. Place tissues in molten paraffin wax in the vacuum chamber and make the chamber airtight.

4. Exhaust the air slowly by means of a vacuum pump or a venturi sunction pump until there is negative pressure of between 400-500mmHG.

5. Leave for 15 mins then slowly re-admit air until normal atmospheric pressure is attained.

Contd’ 6. Place the tissue in a fresh paraffin wax.

7. Repeat steps 4 & 5.

8. Place the tissue in fresh paraffin wax and leave for 30-45 minutes.

9. Bring to normal atmospheric pressure then embedd the tissue.

Common paraffin wax additives.

1. Bee wax

2. Resins

3. Rubber

4. Plastic wax

5. Bay berry wax

6. Stearic acid

note; these additives aid in making the paraffin wax get more elasticity and also be able to support the tissue better hence enhancing better tissue sectioning.

Other forms of paraffin wax

1. Paraplast .

It is a mixture of highly purified paraffin wax and several plastic polymers . This gives it the advantage of being more elastic.

2.Paraplast plus;

Composed of purified paraffin and plastic polymers .

It also contains dimethyl sulphoxide which gives it a more penetrating power than the paraplast .

It is supplied in form of pellets.

Cntd’

3.biloid; it is similar to paraffin wax but contains plastic polymers.

It is recommended for thin walled tissues.

4.Tissue mat; product of paraffin wax which contains rubber.

It is used in same way as paraffin wax and paraplast.

It is supplied inform of sheets and mats.

Advantages of paraffin wax

1. Good cutting quality.

2. Blocks are durable.

3. Easy storage of tissue blocks.

4. sections ribbon well.

5. Has a reasonable processing time.

6. Sections can cut as thin as 3um.

Disadvantages of paraffin wax

1. Tissues harden and shrink if the required time is prolonged.

2. Thick sections of large specimens are difficult to cut.

3. Soft tissues e.g. liver and spleen tend to crack during sectioning.

2. Esther wax.

It is good for impregnation of bone tissues and insects.

It is an embedding media that sets to become harder than paraffin wax with a lower melting point of between 46-48c.

It is not water soluble but is soluble in 95% ethanol and usual clearing agents.

Because of its solubility with 95% alcohol it is possible to impregnate