What are Fixation and Processing Artifacts?
In histology, artifacts refer to structures or features that are seen in tissue sections but are not present in the living tissue. These artifacts can arise during tissue processing, which includes
fixation, dehydration, clearing, embedding, cutting, and staining. Artifacts can obscure or distort the true structure of the tissue and may lead to misinterpretation.
Common Fixation Artifacts
Fixation is the first and one of the most crucial steps in tissue processing. It involves stabilizing the tissue to prevent degradation. However, improper fixation can introduce artifacts such as:- Shrinkage: Tissue shrinkage can occur if the fixative is too strong or if the fixation time is too long. This can result in gaps between cells and can distort the tissue architecture.
- Swelling: Some fixatives can cause tissue swelling, altering the cellular morphology and making it difficult to interpret the tissue structure.
- Autolysis: Inadequate fixation can lead to autolysis, where cells start to digest themselves, resulting in a loss of cellular detail.
- Precipitation: Chemical fixatives can cause precipitates to form within the tissue, which can appear as granular artifacts under the microscope.
Processing Artifacts
Processing artifacts occur during the steps following fixation, including dehydration, clearing, and embedding. Common processing artifacts include:- Tissue Distortion: During dehydration and clearing, rapid changes in solvents can cause tissue distortion. Gradual changes in solvent concentrations can help minimize this.
- Cracking: Improper embedding, especially with paraffin, can lead to cracking of the tissue. This is often due to rapid cooling or uneven embedding.
- Sectioning Artifacts: During microtomy, the process of cutting thin tissue sections, artifacts such as chatter (alternating thick and thin sections), folds, and knife marks can be introduced.
- Staining Artifacts: Uneven or incomplete staining can occur due to improper washing or reagent contamination, leading to misleading tissue appearance.
- Fixation: Use the appropriate fixative and fixation time for the tissue type. Ensure that the fixative penetrates the tissue evenly and that the tissue is not too thick.
- Dehydration and Clearing: Gradually increase the concentration of alcohol during dehydration and use fresh clearing agents to avoid tissue distortion.
- Embedding: Ensure that the embedding medium (e.g., paraffin) is pure and that the tissue is properly oriented and fully infiltrated.
- Sectioning: Use a sharp microtome blade and ensure the tissue block is properly chilled to reduce sectioning artifacts.
- Staining: Use fresh reagents, follow standardized protocols, and ensure thorough washing between staining steps to achieve uniform staining.
Conclusion
Understanding and minimizing fixation and processing artifacts is essential in histology to ensure accurate interpretation of tissue sections. By following best practices and being aware of potential sources of artifacts, histologists can produce high-quality tissue sections that accurately reflect the true structure and function of the tissue.
