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Functional Considerations: Feulgen Microspectrophotometry

Feulgen microspectrophotometry is a technique that was developed to study DNA increases in developing cells and to analyze ploidy, i.e., the number of times the normal DNA content of a cell is multiplied (a normal, nondividing cell is said to be diploid; a sperm or egg cell is haploid). Recently, it has become a valuable tool for surgical pathologists in evaluating the metastatic potential of a malignant tumor and in making prognostic and treatment decisions. The technique of static cytometry of Feulgen-stained sections of tumors (contrasted with flow cytometry, which can only be used on isolated individual cells) uses microspectrophotometry coupled with a digitizing imaging system to measure the absorption of light at 560 nm by cells and cell clusters in Feulgen-stained sections. This technique allows the pathologist to describe ploidy patterns in specific adenocarcinomas (epithelial cancers) and has been particularly useful in studies of breast cancer, kidney cancer, colon and other gastrointestinal cancers, endometrial (uterine epithelium) cancer, and ovarian cancer. Adenocarcinomas that have a largely diploid pattern are said to be well differentiated and have a better prognosis than the same cancers with aneuploidy (nonintegral multiples of the haploid amount of DNA) and tetraploidy.

eral, a capture reagent, either a dye or a heavy metal, is used to trap or bind the reaction product of the enzyme by precipitation at the site of reaction. In a typical reaction to display a hydrolytic enzyme, the tissue section is placed in a solution containing a substrate (AB) and a trapping agent (T) that precipitates one of the products as follows:

where AT is the trapped end product and B is the hy-drolyzed substrate.

By using such methods, the lysosome (see page 32), first identified in differential centrifugation studies of cells, was equated with a vacuolar component seen in electron micrographs. In lightly fixed tissues, the acid hydrolases and esterases contained in lysosomes react with an appropriate substrate. The reaction mixture also contains lead ions to precipitate, e.g., lead phosphate derived from the action of acid phosphatase. The precipitated reaction product can then be observed with both light and electron microscopy.

Similar light and electron histochemical procedures have been developed to demonstrate alkaline phosphatase, adenosinetriphosphatases (ATPases) of many varieties (including the Na 7K1-ATPase that is the enzymatic basis of the sodium pump in cells and tissues), various esterases, and many respiratory enzymes (Fig. 1.3).

Enzyme Digestion

Enzyme digestion of a section adjacent to one stained for a specific component, such as glycogen, DNA, or RNA, can be used to confirm the identity of the stained material

Intracellular material that stains with the PAS reaction may be identified as glycogen by pretreatment of sections with diastase or amylase. Abolition of the staining after these treatments positively identifies the stained material as glycogen.

Similarly, pretreatment of tissue sections with de-oxyribonuclease (DNAse) will abolish the Feulgen staining in those sections, and treatment of sections of protein secretory epithelia with ribonuclease (RNAse) will abolish the staining of the ergastoplasm with basic dyes.

Enzyme Histochemistry

Histochemical methods are also used to identify and localize enzymes in cells and tissues

To localize enzymes in tissue sections, special care must be taken in fixation to preserve the enzyme activity. Usually, mild aldehyde fixation is the preferred method.

In these procedures, the reaction product of the enzyme activity, rather than the enzyme itself, is visualized. In gen-

Immunocytochemistry

A foreign protein or other antigen injected into an animal results in production of antibodies

An antibody is a protein produced by certain white blood cells that binds to the foreign substance that stimulated its production. In the laboratory, antibodies can be purified and conjugated (i.e., chemically bound) to a fluorescent dye such as fluorescein. This reagent can then be applied to sections of lightly fixed or frozen tissue on glass slides to localize the antigen in cells and tissues. The reaction can then be examined and photographed with a fluorescence microscope.

The specificity of the reaction between antigen and antibody is the underlying basis of immunocytochemistry

In a typical procedure, a specific protein, such as actin, is isolated from the muscle cells of one species, such as a rat, and injected into the circulation of another species, such as a rabbit. The actin stimulates the formation of antiactin antibodies that circulate in the bloodstream of the rabbit. The antibodies are then removed from the blood of the rabbit, conjugated with a fluorescent dye, and used to stain tissues or cells of the rat suspected of containing actin, such as fibroblasts in connective tissue. If actin is present, the antibodies bind to it, and the reaction is visualized by virtue of

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