The principle of tissue processing is to prepare fixed tissues for microscopic examination by embedding them in a solid medium, typically paraffin wax, through a series of steps that progressively remove water and replace it with a substance miscible with both water and the embedding medium. This involves three key sequential stages: dehydration, clearing, and infiltration.
Detailed Explanation of Tissue Processing Steps
Tissue processing aims to create a specimen that can be thinly sectioned for microscopic analysis while preserving cellular structures. This is accomplished through the following stages:
-
Dehydration: This process removes water from the tissue. Water is incompatible with paraffin wax, the most common embedding medium. Dehydration is typically achieved by immersing the tissue in a series of increasing concentrations of alcohol (e.g., ethanol), gradually replacing the water within the tissue. For example, the tissue might be moved sequentially through 70%, 80%, 90%, 95%, and finally, 100% ethanol solutions.
-
Clearing: The clearing stage replaces the dehydrating agent (alcohol) with a solvent that is miscible with both alcohol and the embedding medium (paraffin). This is crucial because the alcohol remaining in the tissue would prevent proper infiltration of the paraffin wax. Common clearing agents include xylene, toluene, and limonene. Clearing also often makes the tissue translucent.
-
Infiltration: This step replaces the clearing agent with the embedding medium, typically molten paraffin wax. The tissue is immersed in several changes of paraffin wax at a controlled temperature (usually around 58-62°C) to ensure complete penetration of the wax into the tissue. This infiltration provides support for the tissue during sectioning, allowing for thin, uniform slices to be cut. Vacuum infiltration is often used to improve penetration.
Importance of Each Step
| Step | Purpose | Consequences of Inadequate Processing |
|---|---|---|
| Dehydration | Removal of water to allow for subsequent infiltration by organic solvents and embedding media. | Incomplete infiltration, poor sectioning, tissue damage during sectioning, and poor staining. |
| Clearing | Removal of the dehydrating agent and replacement with a solvent miscible with both alcohol and paraffin. | Incomplete infiltration, soft or mushy tissue, difficulty in sectioning, and potential for tissue distortion. |
| Infiltration | Replacement of the clearing agent with the embedding medium to provide support during sectioning. | Inadequate support for sectioning, brittle tissue, crumbling during sectioning, and poor quality sections unsuitable for diagnosis. |
Alternative Embedding Media
While paraffin wax is the most common embedding medium, other options exist, including:
- Resins: Used for electron microscopy and for harder tissues that are difficult to section in paraffin.
- Cryomedia: Water-based, used for frozen sections when rapid diagnosis or preservation of certain antigens or enzymes is required.
Conclusion
In summary, tissue processing relies on the sequential steps of dehydration, clearing, and infiltration to transform fixed tissue into a state suitable for embedding and subsequent thin sectioning, enabling detailed microscopic analysis. Proper adherence to the principles of each step is essential for producing high-quality histological slides for accurate diagnosis and research.
