Introduction to Pathology and Adaptive Changes PDF

1. Histopathological techniques in pathomorphological diagnostics Issues: Rules of conducting classes, credit receival (scoring), health and safety regulations and other regulations. Introduction into pathological techniques, types of histopathological pr...

1. Histopathological techniques in pathomorphological diagnostics Issues: Rules of conducting classes, credit receival (scoring), health and safety regulations and other regulations. Introduction into pathological techniques, types of histopathological procedures (intraoperative, postoperative, autopsy). Pre-laboratory and intra-laboratory preparation of a tissue material for histopathological examination. Fixing of tissue material (types, pros and cons of fixatives). Selection of suitable fixative for a specific tissue and type of examination. Methods for tissue processing. Preparation of special tissue material (bones/biopsy) for histopathological examination). Basic staining of histopathological slides. Exit test (max 3 points) Histopathological Techniques in Pathomorphological Diagnostics Histopathology is the study of diseased tissues at the microscopic level, which plays a crucial role in diagnosing diseases by examining tissue architecture and cellular details. Pathomorphological diagnostics utilises histopathological techniques to identify abnormalities in tissues and cells, often aiding in identifying cancers, infections, and inflammatory conditions. So basically, Histopathology involves studying diseased tissues at the microscopic level to observe changes in the structure and cellular details. Pathologists compare the histological features of healthy and diseased tissues to diagnose conditions, determine the extent of the disease, and identify abnormalities like infections, cancer, or inflammation. This 1. Introduction to Pathological Techniques Pathological techniques involve the preparation and examination of tissues to assess any pathological (disease-related) changes. They can be classified into several types based on when they are performed: Types of Histopathological Procedures: I. Intraoperative (Frozen Section): It is a fast technique used during surgery to give doctors immediate information about a tissue sample. It is performed during surgery to guide immediate clinical decisions. Tissue is quickly frozen, sectioned, stained, and analysed by the pathologist. What is it? During surgery, if a surgeon wants to know something specific about a piece of tissue—like whether it’s cancerous or if they’ve removed enough of a tumor—they take a small sample of the tissue and send it to the pathologist. The pathologist uses the frozen section technique to analyse it quickly, often within 15-20 minutes, while the patient is still in surgery. How does it work? 1. Freezing the Tissue: The tissue is rapidly frozen using a machine called a cryostat. Freezing makes the tissue hard enough to cut very thin slices. 2. Sectioning: Thin slices of the frozen tissue are cut using the cryostat. 3. Staining: The slices are then stained with dyes (like Hematoxylin and Eosin) to highlight the cells and tissue structure, making them visible under the microscope. 4. Microscopic Examination: The pathologist quickly looks at the tissue under a microscope to provide information to the surgeon. Why is it done?  Immediate Diagnosis: It helps surgeons make decisions right during the surgery. For example, if the tissue is cancerous, the surgeon might remove more tissue. If the tumor margin is clear (no more cancer cells at the edge of the tissue), the surgery might stop there. Saves Time: It avoids the need for another surgery by confirming diagnoses or treatment options right away. Limitations: Not as detailed: Frozen sections are not as perfect as regular (postoperative) tissue processing because freezing can sometimes distort the tissue a bit. But it’s good enough for urgent, on-the-spot decisions. Not for all tissues: Some types of tissues, like fatty tissues, may not freeze well and can be harder to evaluate. Summary: It’s like a quick test during surgery that allows the surgical team to make important decisions in real-time, without having to wait for the full, detailed tissue analysis that comes after the surgery. The intraoperative frozen section process is a method used by surgeons and pathologists to make quick, on-the-spot determinations concerning the pathology of a specific tissue. They use it to make on-the-spot diagnoses and decisions, such as whether a patient requires the removal of a portion of infected tissue or not. How it works: They take a piece of tissue and send it to pathology. Pathology then freezes the tissue (in a cryostat) so that it can be easily cut into tiny slices. They next stain the thinly cut sections with dyes (such as eosin and haematoxylin). They next compare the sample tissues with healthy tissues. The reason they do this is so that, for example, if the infected patient is in surgery, the surgeon can either continue the process (by cutting the rest of the diseased area during surgery) or leave it alone if it is not infected. As a result, its ability to perform in emergency situations is a plus, but one of its downsides is that it is not as detailed as post-operative care because it requires less time. Despite this, it remains helpful for making on-the-spot/urgent diagnoses.. Postoperative (Routine Histopathology): Tissue is processed and examined after surgery using standard methods. Purpose: Provides detailed and accurate diagnosis of removed tissues (e.g., tumor analysis). II. Postoperative (Routine Histopathology) Procedure: Postoperative histopathology, also known as routine histopathology, refers to the detailed examination of tissue that is removed during surgery after the procedure is completed. Here’s how the process works in simple terms: What is the Goal? The goal of postoperative histopathology is to thoroughly analyse the tissue under a microscope to figure out what disease is present, how advanced it might be, and whether or not the surgical procedure successfully removed the diseased tissue. The issue is processed and examined after surgery using standard methods For example, if a surgeon removes a tumor, the pathologist will look at the tumor closely to see: Is it cancerous or benign? How aggressive the cancer is, if it’s present. If the tumor was completely removed (are the "margins" of the tissue clear?). Steps Involved in Routine Histopathology: 1. Tissue Collection: After surgery, the removed tissue (for example, a lump or part of an organ) is sent to the pathology lab. This tissue could be anything from a small biopsy (tiny tissue sample) to a large specimen (like part of an organ).  2. Fixation: To prevent the tissue from rotting or breaking down, it is placed in a special chemical called a fixative (usually formalin). This helps preserve the structure of the tissue, keeping it as close to its natural state as possible. 3. Tissue Processing: After fixation, the tissue is prepared for microscopic examination. Here’s how: ○ Dehydration: The water is removed from the tissue using alcohol. ○ Clearing: The alcohol is replaced with a chemical (like xylene) that makes the tissue ready for the next step. ○ Embedding: The tissue is placed in paraffin wax, which hardens and provides support, making it easier to cut. 4. Sectioning: The tissue block is sliced into ultra-thin sections (like 3–5 microns thick). These slices are placed on glass slides. Kinda like slicing a loaf of bread super thin! 5. Staining: Since tissues are mostly transparent, they need to be stained with special dyes to see the different parts clearly under the microscope. The most common stain used is Hematoxylin and Eosin (H&E), which colors the nuclei of cells blue/purple and the rest of the tissue pink/red. 6. Microscopic Examination: A pathologist looks at the stained slides under a microscope. They will compare the tissue with normal (healthy) tissue to spot any abnormalities. They may look for: ○ Unusual cell shapes and sizes. ○ The way the cells are organised. ○ Whether there are signs of cancer, infection, or inflammation. 7. Report Generation: After carefully analysing the tissue, the pathologist writes a report. This report includes: ○ Diagnosis (e.g., cancer, benign growth, inflammation). ○ Description of how severe or advanced the disease is. ○ Comments on whether the tumor or diseased tissue was completely removed. 8. Communication with Surgeon: The pathologist sends the report to the surgeon or treating doctor, who then uses this information to plan the next steps in patient care, such as further treatment, additional surgery, or no further action if all is clear. Summary: The postoperative procedure, also known as routine histopathology, is a detailed process used to examine tissue samples collected during surgery. It helps pathologists determine whether the tissue is benign or cancerous, assess the severity of the disease (such as the stage of cancer), and check if the entire tumor has been removed. The procedure involves eight steps: First, the surgeon collects the tissue, then it is fixed in a solution like formalin to preserve its structure for laboratory analysis. In the lab, the tissue undergoes processing, which includes dehydration (removing water using alcohol), clearing (replacing the alcohol with a solution like xylene), and embedding (hardening the tissue in paraffin wax to make it easier to cut). Next, the tissue is sectioned into thin slices, which are then stained using dyes such as hematoxylin (to colour the nuclei blue/purple) and eosin (to stain the cytoplasm pink/red). This staining allows for easier examination under a microscope, as unstained tissues are nearly transparent. The pathologist then conducts a microscopic examination, identifying any abnormalities in the cells, such as changes in size, shape, or number. Finally, a report is written detailing the findings, and the pathologist consults with the surgeon to inform them of the results and guide any further treatment decisions III. Autopsy Procedure An autopsy is a medical procedure performed after death to determine the cause of death, investigate disease processes, or evaluate injuries. It involves a detailed examination of the body, both externally and internally, by a pathologist. Steps in an Autopsy: 1. External Examination: ○ The autopsy starts with a thorough examination of the outside of the body. ○ The pathologist looks at the skin, checks for any injuries, bruises, scars, surgical wounds, or abnormalities (e.g., rashes or discolorations). ○ They document the physical appearance of the body, including height, weight, and any visible marks. 2. Internal Examination: ○ After the external exam, the internal organs are examined. To do this, the pathologist makes a Y-shaped incision from the shoulders down to the chest and abdomen. The Y-shaped incision is used in autopsies  to provide a clear and effective way to access the internal organs while minimising damage to the surrounding tissues. The incision starts at the shoulders and extends down to the midline of the abdomen, forming a "Y" shape at the chest. This design allows the pathologist to open the chest and abdominal cavities simultaneously, facilitating a thorough examination of vital organs such as the heart, lungs, and liver, while also making it easier to close the incision afterward. This technique ensures that the internal examination is both comprehensive and efficient. ○ The chest and abdominal cavities are opened, and the rib cage is removed to access the organs inside (heart, lungs, liver, stomach, etc.). ○ Each organ is removed carefully, one by one, and examined. The organs are weighed, measured, and cut into sections to check for disease or abnormalities. 3. Brain Examination: ○ If necessary, the brain is also examined. To access it, the scalp is cut, and the skull is opened. ○ The brain is removed and checked for signs of trauma, bleeding, or disease (like stroke or tumors). 4. Microscopic Examination: ○ Small samples of tissues from different organs are taken during the autopsy. ○ These samples are processed in the histopathology lab, where they are stained and examined under a microscope to look for specific cellular changes (like infection, cancer, or other diseases). 5. Toxicology and Laboratory Testing: ○ Blood, urine, and other bodily fluids are collected during the autopsy. ○ These samples are tested for drugs, alcohol, or poisons (toxicology tests), which can sometimes help explain the cause of death. 6. Final Report: ○ After all the examinations and tests are completed, the pathologist compiles the findings in a report. ○ This report will detail the condition of the organs, any injuries or abnormalities, and give a conclusion on the cause of death (e.g., heart attack, infection, or trauma). ○ In some cases, it can also help answer questions about how the disease progressed before death. Types of Autopsies: Clinical Autopsy: Performed to understand the disease process and cause of death in hospital patients, often for medical research or quality control in healthcare. Forensic Autopsy: Performed in cases of suspicious or unexplained deaths (e.g., accidents, homicides). These autopsies help with legal investigations. Why Autopsies are Important: Clarifying the Cause of Death: It provides a clear and definitive understanding of why someone died, especially when it wasn’t obvious. Understanding Disease Progression: It helps doctors learn more about how diseases affect the body, which can improve medical treatments and knowledge. Legal and Public Health Reasons: In forensic cases, autopsies can determine if the death was due to unnatural causes (such as murder or accidents). They can also help detect outbreaks of infectious diseases or public health issues. Key Points: External and Internal Examination: Autopsies involve both looking at the outside of the body and examining internal organs. Organ and Tissue Study: All major organs are examined for disease or injury. Tissue samples are taken for microscopic study. Cause of Death: The primary goal is to determine the cause of death and understand any underlying medical conditions. Summary:- An autopsy is a medical procedure performed after death to determine the cause of death, evaluate injuries, or understand disease processes that may have affected the individual before they died. It involves a thorough examination of the body, both internally and externally. There are two main types of autopsies: forensic autopsies, which are conducted to determine the cause of death for legal purposes, and clinical autopsies, which are performed primarily for research and educational purposes within the medical field. There are six main steps in performing an autopsy: 1. External Examination: This involves inspecting the skin to check for bruises, discoloration, surgical scars, wounds, or other injuries. 2. Internal Examination: The pathologist makes a Y-shaped incision from shoulder to shoulder and down the abdomen. This allows for access to internal organs such as the liver, stomach, and heart. Each organ is examined for size, weight, and signs of disease or injury. 3. Brain Examination: An incision is made in the scalp, and a portion of the skull is removed to access the brain. This examination checks for any underlying issues, such as bleeding or trauma, that may have contributed to the individual's death. 4. Microscopic Examination: Tissue samples are taken from various organs and processed. This includes dehydrating the samples with alcohol, clearing them with chemicals, and embedding them in paraffin wax to facilitate cutting. The samples are then stained with dyes and examined under a microscope to look for cellular abnormalities. 5. Toxicological Examination: Samples of blood, urine, and other fluids are collected and analysed for substances that should not be present, such as drugs, alcohol, or poisons. 6. Report: Finally, the pathologist compiles a report detailing their findings, including the cause of death, any diseases or injuries found, and any other relevant information. 2. Pre-laboratory and Intra-laboratory Preparation of Tissue for Histopathological Examination Pre-laboratory (Specimen Collection and Handling): ○ Tissue samples are collected either surgically (biopsies, resections) or from autopsies. ○ 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): ○ Trimming: The tissue is trimmed to the appropriate size for processing. ○ Fixation: Stabilises the tissue to prevent autolysis (self-digestion) and putrefaction (decomposition by bacteria). ○ Processing: Tissue is dehydrated, cleared, and embedded for sectioning. ○ Embedding: Involves placing tissue in a solid medium (like paraffin wax) to facilitate slicing into thin sections. ○ Sectioning: Tissue is cut into ultra-thin sections (3–5 µm thick) for microscopic examination. ○ Staining: Different stains are used to highlight specific cell 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 cellular structure and proteins. Cons: Toxic, may cause shrinkage, and long-term exposure is hazardous. ○ Glutaraldehyde: Used mainly for electron microscopy. Pros: Excellent for ultrastructural preservation. Cons: Poor penetration, requires special handling. ○ Alcohol-based Fixatives (Methanol, Ethanol): Used for cytology and smears. Pros: Rapid action, excellent for fixing cytological specimens. Cons: Dehydrates tissue, leading to shrinkage and distortion.  ○ 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 be used for DNA analysis. Selection of Suitable Fixative for Tissue: ○ Formalin: Best for routine histology (large tissue specimens). ○ Glutaraldehyde: Preferred for electron microscopy. ○ Alcohol-based Fixatives: Used for cytological samples like pap smears and fine needle aspiration (FNA) samples. ○ Bouin's Solution: Used for delicate tissue like endocrine organs. 4. Methods for Tissue Processing Tissue processing is a series of steps to prepare tissue for microscopic examination, ensuring it is firm enough to be sectioned. Steps: ○ Dehydration: Removes water from the tissue using graded alcohol (e.g., 70%, 90%, 100% ethanol). ○ Clearing: Removes alcohol and replaces it with a substance (xylene 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 molten paraffin wax to harden it, providing support for sectioning. ○ Sectioning: Tissue is sliced into thin sections using a microtome. ○ Mounting: Thin sections are placed on glass slides. ○ Staining: The sections are stained for microscopic visualisation. 5. Preparation of Special Tissue Material (e.g., Bones/Biopsies) 1. Bone Preparation: Bone Tissue: Requires special preparation because bone is a hard tissue that must be decalcified (removal of calcium) before sectioning. ○ Decalcification: Bones are hard due to their mineral content (mainly calcium). Before they can be cut into thin slices, the calcium needs to be removed. This process is called decalcification. How It’s Done: The bone is soaked in a solution, usually an acid (like nitric acid) or a chelating agent (a substance that can bind to metal ions in a solution, forming a stable complex, like EDTA). This softens the bone by dissolving the calcium. Timing: Decalcification can take several hours to days, 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 to a suitable size for processing. This makes it easier to handle and embed. Embedding: After trimming, the bone is processed just like soft tissues. It is dehydrated, cleared, and embedded in paraffin wax, which allows for sectioning. Sectioning: The decalcified bone is then cut into very thin sections using a microtome, similar to other tissues. 2. Biopsy Preparation: Tissue Handling: Biopsies are small samples taken from a suspicious area of tissue (like skin, liver, or tumors). It’s essential to handle them carefully to preserve their structure. Fixation: Just like other tissues, biopsies must be fixed quickly after removal. This usually involves placing them in a fixative (like formalin) to prevent degradation. Processing: Biopsies undergo the same processing steps as other tissues: dehydration, clearing, and embedding in paraffin. Sectioning: The embedded biopsy can then be sliced into thin sections for microscopic examination. 3. Special Considerations: Orientation/ positioning: For both bones and biopsies, it’s essential to position the samples correctly before 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: Decalcification is crucial for bones to make them soft enough for sectioning. Biopsies must be fixed quickly to preserve cellular details. 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 tissue morphology. ○ 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. Specimen Collection: Proper labelling and handling of tissue. 2. Fixation: Stabilises and preserves tissue. 3. Processing: Dehydration, clearing, and embedding. 4. Sectioning: Thin slices of tissue for microscopic examination. 5. Staining: Application of dyes to highlight tissue structures. 6. Examination: Analysis under a microscope for diagnosis.