Histopathology involves a multi-step process primarily centered around examining tissue samples under a microscope to diagnose diseases. Here's a breakdown of the process:
1. Tissue Acquisition (Specimen Collection):
- The process begins with obtaining a tissue sample from the patient. This can be achieved through various methods, including:
- Biopsy: Removing a small piece of tissue for examination. Biopsies can be incisional (removing a portion of a lesion), excisional (removing the entire lesion), or core needle biopsies (using a needle to extract a cylindrical tissue sample).
- Surgical Resection: Removing a larger tissue mass during surgery.
2. Fixation:
- Immediately after removal, the tissue is placed in a fixative, most commonly formalin (10% neutral buffered formalin).
- Purpose of Fixation:
- Preserves Tissue: Prevents autolysis (self-destruction) and putrefaction (decay).
- Hardens Tissue: Makes it easier to handle and cut.
- Maintains Structure: Preserves the cellular architecture and prevents degradation.
3. Processing:
- Dehydration: The fixed tissue is dehydrated by immersing it in a series of increasing concentrations of alcohol (e.g., 70%, 90%, 100%). This removes water from the tissue.
- Clearing: Alcohol is then replaced with a clearing agent, such as xylene, which is miscible with both alcohol and the embedding medium. This makes the tissue translucent.
- Infiltration/Embedding: The tissue is infiltrated with a supporting medium, typically molten paraffin wax. The wax impregnates the tissue, providing support for thin sectioning. The tissue is then embedded in a block of paraffin wax.
4. Sectioning:
- The paraffin block is trimmed and mounted on a microtome.
- Microtome: A specialized instrument that uses a sharp blade to cut the tissue into extremely thin sections, typically 3-5 micrometers thick.
- Floating: Sections are floated on a warm water bath to remove wrinkles.
5. Staining:
- The most common staining method is Hematoxylin and Eosin (H&E) staining.
- Hematoxylin: Stains acidic structures (e.g., nuclei) blue or purple.
- Eosin: Stains basic structures (e.g., cytoplasm) pink or red.
- Other Stains: Special stains may be used to highlight specific tissue components or microorganisms (e.g., Periodic acid-Schiff (PAS) stain for carbohydrates, trichrome stain for collagen).
6. Mounting:
- The stained tissue sections are mounted onto glass slides using a mounting medium and coverslip. This protects the tissue and provides a clear view under the microscope.
7. Microscopic Examination and Diagnosis:
- A histopathologist examines the stained slides under a microscope.
- The pathologist analyzes the cellular and tissue architecture, looking for abnormalities that indicate disease.
- Based on the microscopic findings, the pathologist writes a pathology report that includes a diagnosis and other relevant information. This report is then sent to the clinician who ordered the test, to help guide patient care.
Summary Table:
| Step | Description | Purpose |
|---|---|---|
| Tissue Acquisition | Obtaining a tissue sample (biopsy or surgical resection). | Obtaining material for analysis. |
| Fixation | Immersing the tissue in formalin. | Preserving tissue structure and preventing degradation. |
| Processing | Dehydration, clearing, and embedding in paraffin wax. | Preparing the tissue for thin sectioning. |
| Sectioning | Cutting the tissue into thin slices using a microtome. | Creating sections thin enough for microscopic examination. |
| Staining | Applying stains (e.g., H&E) to highlight cellular components. | Enhancing visibility and differentiation of tissue structures. |
| Mounting | Attaching the stained section to a glass slide. | Protecting the tissue and providing a clear view under the microscope. |
| Microscopic Exam | Pathologist examines the slide and makes a diagnosis. | Identifying abnormalities and determining the presence of disease. |
