Under Fixation - Histology

What is Fixation?

Fixation is a critical step in the preparation of biological tissues for microscopic examination. It involves treating the tissue with a chemical substance to preserve its structure and prevent degradation. The primary goal of fixation is to stabilize the tissue, prevent autolysis and putrefaction, and preserve the cellular and subcellular components as close to their living state as possible.

What is Under Fixation?

Under fixation refers to a situation where the tissue has not been adequately fixed. This can occur when the fixing agent does not penetrate the tissue sufficiently, or the tissue is not exposed to the fixative for an appropriate duration. As a result, the tissue may not be properly preserved, leading to various artifacts and inaccuracies in histological analysis.

Causes of Under Fixation

Several factors can lead to under fixation, including:

Insufficient Fixative Volume: The volume of fixative should be at least 10-20 times the volume of the tissue to ensure adequate penetration.
Inadequate Fixation Time: The tissue must be exposed to the fixative for an adequate period, typically 24-48 hours for formalin fixation.
Improper Tissue Size: Large or thick tissue sections can hinder the penetration of the fixative, leading to under fixation in the central areas.
Incorrect Fixative: Using an inappropriate fixative for the type of tissue or the specific analysis can result in poor fixation.

Consequences of Under Fixation

Under fixation can have several adverse effects on tissue samples, including:

Poor Morphology: The structural integrity of the tissue may be compromised, making it difficult to interpret histological features accurately.
Autolysis: Cellular enzymes can continue to degrade the tissue, leading to loss of cellular details.
Putrefaction: Bacterial action can cause tissue decay, further compromising the sample.
Staining Artifacts: Under fixed tissues often show uneven or poor staining, which can complicate the interpretation of histological sections.

How to Identify Under Fixation?

Under fixation can often be identified by observing the following signs:

Inconsistent Staining: Areas of the tissue may stain unevenly or exhibit weak staining.
Tissue Softness: Under fixed tissues are often softer and more difficult to section.
Presence of Autolytic Changes: Signs of cellular degradation, such as cytoplasmic granularity or nuclear breakdown, may be evident.

Preventing Under Fixation

To prevent under fixation, the following practices should be adopted:

Optimal Fixative Volume: Ensure the fixative volume is sufficient to cover and penetrate the tissue completely.
Adequate Fixation Time: Allow sufficient time for the fixative to act on the tissue, considering the type and size of the sample.
Proper Tissue Handling: Avoid thick sections and ensure tissues are adequately trimmed to facilitate fixative penetration.
Use of Correct Fixative: Select a fixative that is appropriate for the tissue type and the intended analysis.

Conclusion

Under fixation is a common issue in histological processing that can significantly impact the quality and reliability of tissue analysis. By understanding the causes, consequences, and preventive measures, histologists can ensure proper tissue fixation and obtain accurate and meaningful histological data.