Histology is the anatomical study of the microscopic structure of animal or plant tissues and cells. Histopathology is the study of diseased tissue. It is an important tool in the diagnosis of cancer and other diseases. Thin sections of tissue are processed and stained by a histotechnologist, and observed under a microscope by a pathologist. Many specific stains are available, and the ones that best highlight the features of interest are selected.
The examination of a tissue section involves several steps:
- Gross Examination
When the tissue sample (biopsy, or tissue removed during surgery, etc.) arrives in the laboratory, it is first examined and observed, before being placed in a cassette in order to be processed.
The purpose of fixation is to prevent tissue degradation and preserve tissues in a state as life-like as possible, in order to maintain clear and consistent morphological features of the cells and the sub-cellular components. It can be achieved by either physical or chemical methods, or combinations thereof. Aldehydes in phosphate buffered saline solution (4% to 10% formaldehyde for light microscopy; 0.5 to 4 % glutaraldehyde for electron microscopy) are often used. They preserve tissues or cells by cross-linking proteins. Dehydration, heat and salt formation may also be used for fixation. When electron microscopy is used, fixatives such as osmium tetroxide or uranyl acetate may be selected. Each fixative has benefits and drawbacks, such as shrinkage, swelling or hardening of the tissue, and must be carefully selected.
- Tissue Processing - Embedding
After tissue fixation, a series of processes must take place in order to ensure that quality slides are produced for diagnosis. Tissue processing is designed to provide sufficient rigidity to the tissue in order for sectioning to occur without tearing or distortion. The following steps are performed in tissue processing:
- Dehydration: removal of all unbound water and aqueous fixatives from the tissue. This process is usually performed in a stepwise manner, with successive dehydration baths of increasing concentration being used (for example, the first bath is 70% ethanol, followed by 90%, then 100%). Dehydrating agents include methanol, ethanol, isopropyl, glycol.
- Clearing: removal of the dehydrating chemical with a solvent miscible with paraffin (usually xylene, but others may be used, such as toluene, or derivatives of limonene)
- Embedding: Paraffin is used to infiltrate the tissue and form a block around the tissue. This process makes it easier to section tissues very thinly without tearing or damaging it.
Very thin sections (a few microns thick) of tissues are cut using a microtome. If frozen sections are used instead of a paraffin block, a cryostat is used. For light microscopy, sections are placed on a glass slide. For electron microscopy, sections are collected on 3mm diameter metal grids.
Biological tissue has little inherent contrast in either the light or electron microscope. Staining is employed to give both contrast to the tissue as well as highlighting particular features of interest. The staining process uses a variety of dyes selected for their ability to stain various cellular components of tissue. Hematoxylin and eosin (H&E) is the most commonly used stain in histology and histopathology. Hematoxylin stains nuclei blue; eosin stains the cytoplasm pink. Other stains are referred to as "special stains" because they are used for specific diagnostic requirements. Uranyl acetate and lead citrate are commonly used to impart contrast to tissue in the electron microscope.
Before the staining can be performed, the paraffin must be removed from slides in order to allow water soluble dyes to penetrate the sections. This is performed by running the slides through xylene to alcohols to water prior to staining.
- Coverslipping and Examination
The stained section on the glass slide must be covered with a coverslip in order to protect the tissue section from being damaged and to provide better optical quality for the examination. Examination of the stained slides is performed using a light microscope. Fluorescence microscopy, confocal microscopy or electron microscopy may also be used.
Immunohistochemistry (IHC) uses antibodies binding specifically to antigens in biological tissues to visualize proteins, carbohydrates, and lipids of interest. This technique will be discussed in a specific section
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