Histopathology Tissue Processing Techniques and Principles

Posted on Feb 15, 2026 in Medicine & Health

Aims of Tissue Processing

The basic aim of tissue processing is to provide sufficient rigidity to the tissue to be cut into thin sections for microscopic examination.

Principle of Processing

In tissue processing, the water within the tissue is removed, and another medium (usually paraffin wax) is impregnated into the tissue to provide adequate support.

Essential Steps in Tissue Processing

Dehydration: In this step, water is removed from the tissue. Water is immiscible with wax; therefore, to infiltrate the tissue with wax, it is necessary to remove water.
Clearing: This is needed to clear the dehydrating agent and facilitate the transition between the dehydration and impregnation stages. The clearing substance is usually miscible with both the dehydrating agent and the impregnating medium.
Infiltration and Impregnation: In this stage, the tissue is infiltrated with a supporting medium suitable for providing adequate rigidity to the tissue to make a thin section.

Dehydration Procedures

Every tissue contains some amount of free or unbound water molecules. As the commonly used supporting medium (paraffin) is not miscible with water, it is necessary to remove the free water molecules from the tissue for the successful impregnation of the supporting medium.

Dehydration should be done gradually from low to high concentrations of dehydration fluid. The tissue should be kept in the dehydration fluid for an optimal time because too much time in the dehydrating fluid may cause the tissue to become hard and brittle.

Ethanol as a Dehydrating Agent

Ethanol (ethyl alcohol) is the most popular and commonly used dehydrating agent. It is a clear, colorless, and flammable liquid.
It is a relatively rapid and efficient dehydrating agent.
As a dehydrating agent, ethyl alcohol is used in 50%, 70%, 90%, and 100% concentrations.
For delicate tissue, dehydration may be started with a 30% concentration of ethyl alcohol.
In a routine laboratory, 70%, 90%, and 100% alcohol for 2 hours each are sufficient for tissue dehydration.

Clearing and Transition Stages

After removing the free water molecules from the tissue, the next step of processing is to remove the dehydrating agent itself because many dehydrating agents are not miscible with the impregnating material (paraffin wax). The clearing agent should be miscible with both the dehydrating agent and the embedding medium.

Aims of Clearing

Removal of the dehydrating agent (e.g., alcohol) to facilitate the impregnation of paraffin wax.
To make the tissue clear and improve microscopic examination.

Characteristics of an Ideal Clearing Agent

Low viscosity and high penetration rate.
Low melting point.
Miscible with both alcohol and molten wax.
No tissue damage and less toxic.
Less flammable and inexpensive.

Clearing Agent Specifications

Volume: 40 times the volume of the specimen.
Total Duration: Smaller biopsy (1 hour); Larger tissue (Three changes in xylene or toluene, 60 minutes each).
End Point Detection: Tissue becomes transparent.
Prolonged Exposure: May result in brittle and more friable tissue.
Common Agents: Xylene, toluene, chloroform, amyl nitrate, cedarwood oil, and limonene.

Infiltration and Impregnation

Impregnation is the saturation of tissue cavities and cells by a supporting substance.

Ideal Impregnating Medium

Miscible with the clearing agent.
Liquid at high temperatures and solid at room temperature.
Non-toxic, cheap, homogenous, and stable.

Paraffin Wax Medium

This is the most popular, universally accepted embedding medium for tissue processing. It is a non-toxic and inexpensive medium. The melting point of paraffin wax varies from 39°C to 70°C.

Advantages: Tissue blocks can be stored for a long duration; it is non-toxic, cheap, and safe.
Disadvantages: Tissue shrinkage and hardening occur in cases of prolonged impregnation; it takes a long duration for the impregnation of bone and eye tissues.

Vacuum Impregnation Method

Vacuum impregnation helps to impregnate the molten medium into the tissue under decreased pressure. This is a rapid method to impregnate wax within the tissue. The vacuum impregnation method is specifically applied in the processing of the lung and spleen.

Tissue Processing Methods

Automated Tissue Processors

The basic principle of a tissue processor is to transfer the tissue through different fluids for a specified time in the desired environment. There are two types:

Tissue Transfer Process: Here, tissue is transferred from one carousel to another after a specified time. The basket containing the tissue is submerged in a specific container for a particular time and then transferred to the next container automatically.
Fluid Transfer Process: This is a completely closed processor. Tissue is kept in a container, and the container is periodically filled with a particular fluid. After a certain period, the fluid is pumped out.

Advantages of Automated Systems: Vacuum pressure makes the system faster and more efficient. As this is a closed system, there is no chance of tissue drying.

Manual Tissue Processing

This is rarely used in routine laboratories but offers specific benefits:

A small number of samples can be processed in a small laboratory.
Careful monitoring in each step is possible.
Useful in emergencies when the automated processor is not working.
Allows for the selection of reagents with flexibility in time duration.
Disadvantages: Inconvenience and the time-consuming nature of the procedure.

Rapid Tissue Processing

In the rapid tissue processing system, the entire process takes only 2-3 hours instead of the standard 8-hour duration.

Advantages: Rapid results, equally good quality tissue sections, and no night shift required for technologists.
Disadvantages: A large quantity of cases is needed for effective use; fatty tissue can be processed rapidly.

Overall Precautions and Schedules

General Precautions

The bulk of the tissue should be optimum for adequate penetration of fluid.
The amount of fluid should be adequate, and the fluid level should always be higher than the tissue level.
The tissue basket and cassettes should be clean; any spillage of wax should be removed.
The temperature of the infiltrating medium should be optimum (preferably 3 to 4°C above the melting point).
There should be a proper record of fluid changes and the number of tissues processed.

Time Schedule for Overnight Processing

First Formalin (1 jar): 1 minute
70% Alcohol: 2 hours
80% Alcohol: 2 hours
90% Alcohol: 2 hours
Absolute Alcohol: 2 hours
Xylene 1: 1.5 hours
Xylene 2: 1.5 hours
Wax 1: 2 hours
Wax 2: 2 hours