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Anatomic Pathology

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Biopsies and examination of tissues

Tissue Preparation | Techniques

Histopathology involves the examination of sampled tissues under the microscope. These may be small pieces of tissue obtained from a part of the body using a technique called biopsy or samples taken from whole organs or parts of organs removed during surgery.

Most biopsies are small samplings of the area of the body in which disease is suspected. These are called "incisional" biopsies and additional surgery or treatment may be recommended after the diagnosis is made.

Other biopsies may include the entire affected area, such as a skin mole. These are called "excisional" biopsies and examination of the adjacent, uninvolved margins helps to verify that the affected area has been completely removed.

The pathology laboratory also receives large whole organs, or parts of organs, removed during surgery, such as a uterus after a hysterectomy, the large bowel after a colectomy, or an amputation of an arm or leg. These specimens are examined as a whole (grossly) for size, shape, color, and/or presence of any external abnormalities. Then, smaller samples are taken for definitive microscopic evaluation.

 Tissue preparation

Formalin-fixed, paraffin-embedded (FFPE)
Biopsies and samples of tissue removed from organs are usually placed in formalin (diluted formaldehyde), which "fixes" the tissue by cross-linking proteins. This preserves the cellular architecture and also allows the tissue to survive the processing that comes next. Other types of fixatives may be used depending on the type of specimen or the cellular characteristics that need to be enhanced.

The tissue goes through various chemical steps (dehydration and dissolving of fat) in preparation for embedding into a paraffin (wax) block. The paraffin blocks are placed on a special machine that uses an extremely sharp knife (a microtome) to shave very thin pieces of tissue of about 5 µm (micrometers, or about 0.0002 inches) in thickness. The thin pieces are placed on a glass slide and stained with special reagents to highlight key aspects of the tissue.

Hematoxylin and eosin, also known as H&E, is the most widely used stain. It is a combination of a basic stain (hematoxylin) and an acidic stain (eosin). This reacts with acidic and basic cellular components on the slide to give, respectively, purple and pink colors to the tissues.

Frozen sections
When time is crucial (for instance, when a surgeon needs an answer while performing surgery), the pathologist will bypass the fixation, processing, and embedding in paraffin steps and perform a frozen section. The tissue is surrounded by a fluid containing polyethylene glycol and placed on a chilled metal block inside of a refrigerated device called a cryostat. Once the fluid has frozen, a laboratorian uses the microtome to thinly slice (section) the block. The thin slice is placed onto a glass slide, stained, and examined. The procedure usually takes 10-20 minutes. However, freezing of the tissue can result in some distortion of cells and some staining artifact. This is why frozen sections are often preliminary, with a final diagnosis based on the routine processing of tissue as noted above.

Touch preps
One additional method that can be done intra-operatively instead of freezing the tissue is making imprint smears (touch preps). The tissue is cut and pressed or smeared onto a glass slide, then stained and examined under the microscope. This process leaves individual cells from the tissue on the slide and avoids freezing artifacts. Imprint smears further reduce the time taken to examine tissue and are most often used to examine lymph nodes.

 Special techniques in histopathology 

Special staining
Pathologists use different special stains in addition to the routine hematoxylin and eosin (H&E) stain. These may highlight fat, different tissue fibers, mucus, microorganisms such as bacteria or fungi, proteins, or other biochemical substances that might be useful in identifying key elements that are characteristic of certain diseases.

Special stains can provide useful information but are somewhat limited in their ability to provide a definitive answer, whereas immunohistochemical stains are more specific in what they stain. This technique takes advantage of the unique properties of antibodies that have been developed to recognize specific components on or within cells. The antibodies are bound to certain stains or dyes that are readily visible when observed under a microscope. This allows a pathologist to select those antibodies designed to identify key cellular elements or tissue types that have been associated with certain diseases and assist in obtaining a final diagnosis.

Electron microscopy
Some medical situations require a higher level of microscopy than that provided by standard light microscopy. Examples include types of kidney disease (glomerulonephritis), pulmonary diseases (asbestosis), or aggressive cancers that lose their normal proteins. In these cases, a very powerful type of microscope called an electron microscope may be used. The electron microscope uses high voltage to create a wide beam of electrons that are directed at a specially stained sample. The electrons are either absorbed or scattered, creating a black and white image. It can magnify up to two million times, whereas the maximum power of a conventional light microscope is only one to two thousand times.

Genetic testing and other novel techniques
Recent advances have allowed pathologists to utilize formalin-fixed, paraffin-embedded (FFPE) tissue in a number of innovative ways. Specific areas of tissue can be removed (by micro-dissection) and the chemical constituents analyzed by mass spectrometry or other techniques.

Most often, genetic testing, which involves the study of chromosomes and their DNA, is done in the clinical pathology laboratory, but sometimes may be overseen by the anatomic pathology laboratory when tissue samples are employed. It can be a valuable tool as certain diseases may be caused by specific genetic abnormalities.

For example, sections of a chromosome may switch places (translocation) or may be missing (deletion). These chromosomal translocations and deletions can be detected using fluorescent in situ hybridization (FISH). DNA and RNA isolated from FFPE tissue can also be used to identify specific mutations or screened for abnormalities.

Because DNA and RNA from FFPE tissue are fragmented by tissue preparation (formalin fixation) and unprotected storage, mutation analysis is increasingly being performed using so-called "next generation" sequencing techniques in which millions of reactions are performed simultaneously on the short stretches of DNA, followed by the application of computer algorithms to determine the genome.

Most of these molecular techniques are currently utilized in order to identify mutations that help to guide therapy of malignant tumors, such as:

For a general discussion of these tests, see the article on Genetic Tests for Targeted Cancer Therapy.

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