Tissue Processing Steps An Overview

Tissue Processing Steps

The word tissue handling alludes to the treatment of the tissue. This is not the only technique employed for tissue sections can also be produced by means of cryostat or freezing microtome on frozen tissues. The marking must be a full-confirmation framework.

To ensure these most laboratories have a numbering system for each specimen. as soon as the specimen is received it is given a specific individual number, which is also recorded in the register with the details like patient name, name of the doctor referring it, nature of the tissue noted.

Principle of Tissue Processing

Embedded in a solid medium with the help of first remove the tissue water which is then replaced by any medium such as paraffin wax so that the tissue is soft to enable the microtome knife to cut the section. 

Most of the tissue fixation is aqueous fixatives so before the tissue can be embedded in paraffin wax it is necessary that the water and some of the lipid tissue fluids be removed completely by a process called dehydration.

Following Steps of Tissue Processing

1.  Fixation
2.  Dehydration
3.  Clearing
4.  Infiltration and impregnation

1. Fixation

Usually, the tissue that is received at the laboratory is already fixed but before processing further check it the fixation is complete.

2. Dehydration

The other routine tissue specimen may use 70% alcohol. A higher concentration of alcohol initially is inadvisable because this may cause a very rapid removal of water that may produce cell shrinkage. For routine biopsy and autopsy tissue of 4-7nm thickness 70%, 80%, 90%, 95%, and absolute alcohol are sufficient to give reasonably satisfactory results.

Use Of Solid Dehydrates

Anhydrous copper sulfate is used in a higher grade of dehydrating alcohols. Anhydrous copper sulfate is white, it removes water from the alcohol which in turn has been diluted upon absorption of water from the tissues.

The change of color of copper sulfate from white to blue indicates that both alcohol and water should be changed. The utilization of copper sulfate improves the procedure of lack of hydration and furthermore delays the life of liquor.

Other Dehydrating Agents

1. Acetone 
2. Dioxane
3. Isopropyl alcohol

3. Clearing

Most of these issues have a similar refractive index to that of protein therefore the tissue is left translucent. The clearing agent is required when the dehydrating agent is not miscible with the impregnating medium

1. Xylene: It has rapid action. Biopsy specimens of 3-4 mm. Immersion time must not be prolonged otherwise the tissue becomes brilliant.

2. Toluene and Benzene: Toluene and benzene are similar in properties to xylene but are less damaging to the tissue on prolonged exposure.

3. Chloroform:

• It does not affect the refractive index of the tissue that is not rendered translucent.
• It is expensive.
• It is inflammable.

4. Carbon Tetrachloride: It has comparable properties to chloroform yet is less expensive. 

5. Cedarwood oil: It is useful for the treatment of fragile tissues as it has the least solidifying impact.

• It is very slow in action.
• It is very expensive.

Techniques of Clearing

If the tissue is cleared in chloroform a carbon tetrachloride it may be left overnight. 

4. Infiltration And Impregnation

It is the process of the complete elimination of clearing agents through surrounding melted paraffin wax. Afterword wax is diffuse inside the tissue is called impregnation.

Impregnation with Wax

Impregnation with paraffin wax happens in a wax shower warmed to 50-60 degrees Celcius relying on the dissolving purpose of the wax being used. Frequent check of the temperature of paraffin baths is required since temperature 5-degree Celcius above the melting point of the paraffin will cause tissue shrinkage and hardening.

Properties of Paraffin Wax

1. Easy to prepare a large number of tissue blocks in a comparatively short time.
2. Minimum supervision is required.
3. It is cheaper than other impregnating media.

Points to be remembered during the use of Paraffin Wax

1. It ought not to contain water, which makes it take shape and turn it white.
2. The wax has to be filtered before using bye use of ordinary filter paper.
3. Higher softening point waxes are difficult to lace.

For impregnation the wax bath has to be kept at a high temperature, making the tissue hard, too low melting point wax may not be hard enough to support the tissue during cutting.

Technique of Impregnation

The amount of wax should be 25-50 times the volume of the tissue. The tissue must be submitted to three changes in wax. The temperature of the wax bath should be 2-3 degrees Celcius above the melting point of the wax.

Time of Impregnation

Depends on the following three factors: and

1. The size and type of tissue.
2. The clearing agent employed.
3. The use of a vacuum embedding oven.

1. Size and type of tissue: 

If even small amounts of clearing agent so more change of wax this will cause crystallization and produce crumbing of the sections during cutting.

CNS needs twice as long as soft tissue like the liver. Tissue like muscle and stringy tissue tend ko to over solidify and get fragile in wax to a base. The reduction of time can be achieved by using a vacuum embedding medium.

2. Use of vacuum embedding oven:

With the use of a normal paraffin bath, 2 changes of paraffin wax for a period of 2 hours are needed but by using a vacuum embedding oven this time may be halved.