Histopathological Techniques in Pathomorphological Diagnostics PDF
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Nicolaus Copernicus University in Toruń
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Summary
This document provides a detailed overview of histopathological techniques used in diagnostic pathology. It covers topic such as tissue preparation for histology, various types of fixatives and their usage, tissue processing methods and special tissue preparations like bone and biopsies.
Full Transcript
. Pre-laboratory and Intra-laboratory Preparation of Tissue for Histopathological 2 Examination Pre-laboratory (Specimen Collection and Handling): ○ issue samples are collected either surgically (biopsies, resections) or from autopsies....
. Pre-laboratory and Intra-laboratory Preparation of Tissue for Histopathological 2 Examination Pre-laboratory (Specimen Collection and Handling): ○ issue samples are collected either surgically (biopsies, resections) or from autopsies. T ○ Proper identification, labelling, and documentation of the specimen are critical. ○ Tissue should be placed in an appropriate medium or fixative to prevent degradation. Intra-laboratory (Tissue Handling and Preparation): ○ rimming:The tissue is trimmed to the appropriatesize for processing. T ○ Fixation:Stabilises the tissue to prevent autolysis(self-digestion) and putrefaction (decomposition by bacteria). ○ Processing:Tissue isdehydrated,cleared, andembeddedfor sectioning. ○ Embedding:Involvesplacing tissue in a solid medium(like paraffin wax) to facilitate slicing into thin sections. ○ Sectioning:Tissue is cut intoultra-thin sections(3–5 µm thick) for microscopic examination. ○ Staining:Different stains are used to highlight specificcell structures or tissue components. 3. Fixation of Tissue Material Fixation is a critical step in preserving tissue morphology and preventing degradation. Types of Fixatives: ○ Formaldehyde (Formalin):The most commonly used fixative(10% neutral-buffered formalin). Pros:Good tissue preservation, maintains cellularstructure and proteins. Cons:Toxic, may cause shrinkage, and long-term exposureis hazardous. ○ Glutaraldehyde:Used mainly for electron microscopy. Pros:Excellent for ultrastructural preservation. Cons:Poor penetration, requires special handling. ○ Alcohol-based Fixatives (Methanol, Ethanol):Usedfor cytology and smears. Pros:Rapid action, excellent for fixing cytologicalspecimens. Cons:Dehydrates tissue, leading to shrinkage anddistortion. ○ Bouin’s Solution:Fixative used for delicate tissues(e.g., biopsies). Pros:Preserves soft tissues well, good for histochemistry. Cons:May cause yellowing of the tissue, cannot beused for DNA analysis. Selection of Suitable Fixative for Tissue: ○ Formalin:Best for routine histology (large tissuespecimens). ○ Glutaraldehyde:Preferred forelectron microscopy. ○ Alcohol-based Fixatives:Used forcytological sampleslike pap smears andfine needle aspiration (FNA) samples. ○ Bouin's Solution:Used for delicate tissue likeendocrineorgans. 4. Methods for Tissue Processing issue processing is a series of steps to prepare tissue for microscopic examination, ensuring it is firm enough to be T sectioned. Steps: ○ ehydration:Removes water from the tissue usinggradedalcohol (e.g.,70%, 90%, 100% ethanol). D ○ Clearing:Removesalcoholand replaces it with a substance(x ylene or toluene) that is miscible with paraffin. We remove the alcohol simply because if it stays on the tissue, paraffin wax won't be able to properly embed in the tissue and get to all areas needed in the tissue. And also to prevent shrinkage of the tissue from alcohol, we introduce clearing mediums. ○ Embedding:Tissue is infiltrated with moltenparaffinwaxtohardenit, providing support for sectioning. ○ Sectioning:Tissue is sliced into thin sections usingamicrotome. ○ ounting:Thin sections are placed on glass slides. M ○ Staining:The sections are stained for microscopicvisualisation. 5. Preparation of Special Tissue Material (e.g., Bones/Biopsies) 1. Bone Preparation: Bone Tissue:Requires special preparation becausebone is a hard tissue that must bedecalcified(removalof calcium) beforesectioning. ○ ecalcification:Bones are hard due to their mineralcontent (mainly calcium). Before they can be cut D into thin slices, the calcium needs to be removed. This process is calleddecalcification. How It’s Done:The bone issoaked in a solution, usuallyanacid(like nitric acid) or achelating agent(a substance that can bind to metalions in a solution, forming a stable complex, like EDTA). This softens the bone by dissolving the calcium. Timing:Decalcification can take several hours todays, depending on the size of the bone and the solution used. It’s crucial to monitor this step closely to avoid over-decalcifying, which can damage the tissue. Trimming:Once decalcified, the bone is trimmed toa suitable size for processing. This makes it easier to handle and embed. Embedding:After trimming, the bone is processed justlike soft tissues. It is dehydrated, cleared, and embedded in paraffin wax, which allows for sectioning. Sectioning:The decalcified bone is then cut intovery thin sections using a microtome, similar to other tissues. 2. Biopsy Preparation: issue Handling:Biopsies are small samples takenfrom a suspicious area of tissue (like skin, liver, or tumors). T It’s essential to handle them carefully to preserve their structure. Fixation:Just like other tissues, biopsies must befixed quickly after removal. This usually involves placing them in a fixative (like formalin) to prevent degradation. Processing:Biopsies undergo the same processing stepsas other tissues: dehydration, clearing, and embedding in paraffin. Sectioning:The embedded biopsy can then be slicedinto thin sections for microscopic examination. 3. Special Considerations: rientation/ positioning:For both bones and biopsies,it’s essential to position the samples correctly before O embedding. This ensures that the most informative areas of the tissue are available for examination. Staining:Once sectioned, the samples are stained(often with H&E) to highlight cellular structures, making it easier to identify any abnormalities. Key Points: ecalcification is crucial for bonesto make themsoft enough for sectioning. D Biopsies must be fixed quicklyto preserve cellulardetails. Both types of tissues are processed similarly, but require specific handling techniques to ensure accuracy in diagnosis. 6. Basic Staining of Histopathological Slides Staining is critical for visualising cellular and tissue structures under the microscope. Common Stains: ○ Hematoxylin and Eosin (H&E Staining): Hematoxylin:Stains nuclei blue/purple. Eosin:Stains cytoplasm and extracellular matrix pink/red. Purpose:Standard stain for visualising general tissuemorphology. ○ Periodic Acid-Schiff (PAS Staining): Stains carbohydrates (e.g., glycogen, mucins) in tissues. Purpose:Highlights basement membranes, fungal organisms,and glycogen storage diseases. ○ Masson's Trichrome: Differentiates between muscle fibres (red), collagen (blue/green), and nuclei (black). Purpose:Used in liver biopsies to assess fibrosis. ○ Immunohistochemistry (IHC): Uses antibodies to target specific antigens in the tissue. Purpose:Identifies specific cell types, proteins,and receptors (e.g., for cancer diagnosis and subtyping). Summary of Key Histopathological Steps . 1 pecimen Collection:Proper labelling and handlingof tissue. S 2. Fixation:Stabilises and preserves tissue. 3. Processing:Dehydration, clearing, and embedding. 4. Sectioning:Thin slices of tissue for microscopicexamination. 5. Staining:Application of dyes to highlight tissuestructures. 6. Examination:Analysisunder a microscope for diagnosis.
