Why is Proper Tissue Handling Important?
Proper tissue handling is crucial in histology to ensure the integrity and quality of the tissue samples. Mishandling can lead to artifact formation, degradation, and loss of cellular detail, which can significantly impact the accuracy of diagnostic and research outcomes. Correct handling techniques preserve the morphology and cellular architecture, allowing for reliable analysis.
Collection
Tissue collection should be done using sterile instruments to prevent contamination. It's essential to minimize the time between collection and fixation to prevent autolysis and degradation. The tissue should be placed immediately into an appropriate fixative.
Fixation
Fixation is a vital step that preserves tissue morphology by stabilizing proteins and preventing decomposition. Common fixatives include formalin and paraformaldehyde. The choice of fixative depends on the type of analysis intended. Proper fixation requires adequate volume (at least 10 times the volume of the tissue) and sufficient time.
Trimming
After fixation, tissues are trimmed to appropriate sizes to fit into cassettes. Trimming should be done carefully to avoid damaging the tissue. It's crucial to consider the orientation of the tissue to ensure relevant areas are exposed for sectioning.
Processing
Tissue processing involves dehydration, clearing, and infiltration with paraffin. Dehydration is typically done using graded alcohols, followed by clearing agents like xylene. Finally, tissues are infiltrated with paraffin wax to provide support for sectioning.
Embedding
Embedding involves placing the tissue in a mold with molten paraffin. Proper orientation is critical to ensure the tissue sections will be representative of the desired area. The paraffin block is then cooled and solidified.
Sectioning
Sectioning is the process of cutting thin slices of the embedded tissue using a microtome. Sections are usually cut at 3-5 micrometers for routine histology. Sharp blades and correct technique are essential to obtain high-quality sections without artifacts.
Staining
Staining enhances the contrast of tissue structures, making them visible under the microscope. Hematoxylin and eosin (H&E) staining is the most commonly used routine stain. Special stains and immunohistochemistry may be used for specific cellular components or pathogens.
How Should Tissues be Stored?
Proper storage of tissue samples is essential to maintain their integrity for future analysis. Fresh tissues can be stored short-term in a refrigerator at 4°C. For long-term storage, fixed tissues can be kept in fixative at room temperature, or as paraffin blocks. Frozen tissues should be stored at -80°C or in liquid nitrogen for preservation of nucleic acids and proteins.
Autolysis: Delay in fixation can lead to enzymatic degradation of tissues.
Over-fixation: Prolonged fixation can cause hardening and shrinkage of tissues, affecting morphology.
Improper Orientation: Incorrect orientation during embedding can result in non-representative sections.
Inadequate Dehydration: Insufficient dehydration can lead to poor infiltration and section quality.
Contamination: Use of non-sterile instruments or improper handling can introduce contaminants.
Are There Specific Guidelines for Handling Different Tissue Types?
Yes, different tissue types may require specific handling protocols. For example, fatty tissues require longer processing times due to their lipid content, while delicate tissues like brain require milder fixation and processing to preserve fine structures. It's essential to follow standardized protocols for each tissue type to ensure optimal results.
Conclusion
Proper tissue handling in histology is a multifaceted process that requires attention to detail at each step. From collection to storage, each phase must be carried out meticulously to preserve tissue integrity and ensure accurate diagnostic and research outcomes. By adhering to best practices and understanding the specific needs of different tissues, histologists can achieve high-quality results and contribute valuable insights to the field of biomedical sciences.
