Histopathology refers to the microscopic examination of tissue in order to study the manifestations of disease. Specifically, in clinical medicine, histopathology refers to the examination of a biopsy or surgical specimen by a pathologist, after the specimen has been processed and histological sections have been placed onto glass slides.
Collection of tissues
Histopathological examination of tissues starts with surgery, biopsy, or autopsy. The tissue is removed from the body or plant, and then placed in a fixative which stabilizes the tissues to prevent decay. The most common fixative is formalin (10% formaldehyde in water).
Preparation for histology
The tissue is then prepared using histology procedures for viewing under a microscope using one of two method of fixation - chemical fixation or frozen section.
Chemical Fixation Tissue Processing
The samples are transferred to a cassette, a container designed to allow reagents to freely act on the tissue inside. This cassette is immersed in multiple baths of progressively more concentrated ethanol, to dehydrate the tissue, followed by toluene or xylene, and finally extremely hot liquid (usually paraffin). During this 12 to 16 hour process, paraffin will replace the water in the tissue, turning soft, moist tissues into a sample miscible with paraffin, a type of wax. This process is known as tissue processing.
The processed tissue is then taken out of the cassette and set in a mold. Through this process of embedding, additional paraffin is added to create a paraffin block which is attached to the outside of the cassette.
The process of embedding then allows the sectioning of tissues into very thin (2 - 7 micrometer) sections using a microtome. The microtome slices the tissue ready for microscopic examination. The slices are thinner than the average cell, and are layered on a glass slide for staining.
Frozen Section Tissue Processing
The second method of histology processing is called frozen section histology. In this method, the tissue is frozen and sliced thinly using a microtome mounted in a refrigeration device called the cryostat. The thin frozen sections are mounted on a glass slide, dried, and stained using the same staining techniques as traditional wax embedded sections. The advantages of this method is rapid processing time, less equipment requirement, and less need for ventilation in the laboratory. The disadvantage is the poor quality of the final slide. It is used less in diagnostic pathology, but more in determining margin of a tumor during surgery.
Staining of the Processed Histology Slides
This can be done to slides processed by the chemical fixation or frozen section slides. To see the tissue under a microscope, the sections are stained with one or more pigments. The aim of staining is to reveal cellular components; counterstains are used to provide contrast.
The most commonly used stain in histopathology is a combination of hematoxylin and eosin. Hematoxylin is used to stain nuclei blue, while eosin stains cytoplasm and the extracellular connective tissue matrix pink. There are hundreds of various other techniques which have been used to selectively stain cells. Other compounds used to color tissue sections include safranin, Oil Red O, congo red, silver salts and artificial dyes. Histochemistry refers to the science of using chemical reactions between laboratory chemicals and components within tissue. A commonly performed histochemical technique is the Perls Prussian Blue reaction, used to demonstrate iron deposits in diseases like Hemochromatosis.
Recently, antibodies have been used to stain particular proteins, lipids and carbohydrates. Called immunohistochemistry, this technique has greatly increased the ability to specifically identify categories of cells under a microscope. Other advanced techniques include in situ hybridization to identify specific DNA or RNA molecules. These antibody staining methods often require the use of frozen section histology. Digital cameras are increasingly used to capture histopathological images.
The histological slides are examined under a microscope by a pathologist, a medically qualified specialist. This medical diagnosis is formulated as a pathology report describing the histological findings and the opinion of the pathologist. In the case of cancer, this represents the tissue diagnosis required for most treatment protocols.
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