Tissue processing is the technique by which fixed tissues are made suitable for embedding within a supportive medium such as molten paraffin wax. It is the next step after fixation and a very important step because poor tissue processing can significantly affect tissue sectioning and staining.
Aims of tissue processing
To provide sufficient rigidity to the tissue so that it can easily be cut into thin section.
Principle of processing
To enable the penetration of molting paraffin wax into the tissue for rigidity and adequate support for easy cutting. This is done by removing the water within the tissue and replacing it with paraffin wax.
What are the steps of tissue processing
Tissue processing consists of three sequential steps: Dehydration, clearing, and infiltration.
- Dehydration
Water is immiscible with wax. Therefore, therefore, to infiltrate the tissue with wax, water is removed from the tissue.
- Clearing:
This step is needed to clear the dehydrating agent and to facilitate the transition of dehydration and impregnation stage. The clear substance is usually miscible to both the impregnating medium and dehydrating agent.
- Infiltration and impregnation
The tissue is infiltrated with a supporting medium. This medium provides adequate rigidity of the tissue to make thin section.
Factors that Influence Tissue Processing
- Size of the tissue sample
The smaller the size of the tissue, better is the infiltration of the embedding medium. Optimum thickness of the tissue should be kept as 3–4 mm only.
- Agitation:
Agitation causes a continuous removal of fluid from the surface by fresh medium. The tissue gets a better contact with the surrounding medium if it is completely immersed and agitated. This has a better effect of action of fluid on the tissue. Too much agitation may damage the soft and delicate tissue.
- Heat
Heat increases the rate of penetration of the fluid within the tissue. Note that, Low heat or temperature impedes the whole process while overheating can produce hard and brittle tissue.
- Viscosity
Viscosity also affect tissue processing especially the viscosity of the embedding media. Higher viscosity of the medium low down the penetration rate in the tissue and heat reduces the viscosity of the medium and helps in better penetration.
- Vacuum
Application of negative pressure facilitates tissue processing. Vacuum helps to remove the entrapped air from the tissue and thereby enhances the penetration of fluid within the tissue. Negative pressure also increases the volatility of the clearing agent and therefore helps to remove the fluid from the tissue.
Tissue Processing Methods
There are two method involved: Simple manual and automated processor.
Manual tissue processing is mostly done in small laboratory handling a handful number of tissue while automated tissue processing is common in equipped and large laboratories.
Automated tissue processor
Automated tissue processor: The basic principle of tissue processor is to transfer the tissue in different fluid for a specified time in a desired environment. So far, there are two types of tissue processor.
1. Tissue transfer processor
- Here, the bucket of tissue is transferred from a carousel to other after a specified time. There are also several containers of reagents.
- The tissue remains in a basket which contains 30-100 cassettes.
- The basket containing the tissue is then submerged in the specific containers for a particular time which is automatically transferred to the next container.
- A gentle agitation is created by rotatory movement or vertical oscillation of the tissue in the basket which causes a continuous removal of fluid from the surface by fresh medium.
- A microprocessor determines the time schedule and transfer of tissue in each container.
- A gentle agitation is created by vertical oscillation or by rotatory movement of the tissue basket.
2. Fluid transfer processor
This is a closed system processor where tissues are kept in the container. The container is periodically filled with particular fluid. The fluid is pumped out from the container containing the tissue after a certain period. The container is again filled with the fluid required for the next step. The fluid transfer processor has some advantages such as the vacuum pressure that makes the system faster and efficient and there is no chance for tissue drying.
Manual Tissue Processor
As earlier mentioned, manual tissue processors are rarely used in routine laboratory but has the following advantages.
- Small number of samples can be processed
- Monitoring of each steps in possible
- It can be useful when automated tissue processor is not working that is, serves as back up or emergency tissue processor.
- It is possible to select the reagents of choice with flexibility in time duration.
The only disadvantages of manual tissue processing is the fact that the procedure is time consuming therefore inconvenient.
Microwave Processing
This method is ideal for small number of delicate tissues as it reduces the time of processing significantly and may be used for all the steps of tissue processing.
The microwave oven usually has the following compartment.
- A proper exhaust to remove the toxic gas.
- A system to control the temperature
- A system to control the time duration of particular temperature.
Precautions of Tissue Processing
- The amount of fluid should be adequate and the fluid level should be always higher than the tissue level.
- The bulk of the tissue should be optimum for adequate penetration of fluid.
- The tissue basket and cassettes should be clean and any spillage of wax must be cleaned.
- The temperature of the infiltrating medium should always be optimum and it is preferable to keep the temperature 3–4 °C above the melting point.
- There should be a proper record of the change of fluid, number of tissues processed, etc.
Overnight processing time schedule
- 50% ethanol: 1 h
- 70% ethanol: 1 h
- 90% ethanol: 1 h
- Absolute alcohol: 1 h
- Absolute alcohol: 1 h
- Absolute alcohol: 1 h
- Xylene/toluene: 1 h
- Xylene/toluene: 1 h
- Xylene/toluene: 1 h
- Paraffin wax: 1 h
- Paraffin wax: 1 h
- Paraffin wax: 1 h
Reference:
- Aziz SJ, Zeman-Pocrnich CE. Tissue Processing. Methods Mol Biol. 2022;2422:47-63. doi: 10.1007/978-1-0716-1948-3_4. PMID: 34859398.
- P. Dey, Basic and Advanced Laboratory Techniques in Histopathology and Cytology,https://doi.org/10.1007/978-981-10-8252-8_2
