Histology

Histology is the study of microscopic anatomy using slides of tissue sections. It is a crucial aspect of determining diagnosis and prognosis for many diseases, as well as an educational tool for understanding the structure of the human body and the mechanisms of disease. When histology is used to study diseased tissue, it is called histopathology.

There are many techniques for getting the tissue specimen from the body to the microscope slide. The first step is fixation, whereby the tissue is soaked in a chemical that stops all metabolic processes. The most [Page 855]commonly used fixative is formalin, which contains formaldehyde and phosphate-buffered saline. The sample is then washed, put in multiple solutions of progressively more concentrated alcohol to remove the water, and washed with an organic solvent to remove the alcohol. The second step is embedding, whereby the specimen is impregnated with melted paraffin wax and set to cool. The washing and embedding process takes at least 12 hours when using standard procedures. The tissue-containing paraffin block is then cut into very thin slices of two to eight micrometers and mounted on slides. In the final step, the slide is stained with a dye so the tissue can be visualized.

There are several dyes that are used to highlight different features of the cells. The most commonly used one is hematoxylin and eosin (H&E). Hematoxylin stains the nuclei of cells blue while eosin stains the cytoplasm pink. Other dyes include crystal violet, which stains gram-positive bacteria dark blue, and periodic acid Schiff (PAS), which stains carbohydrates pink. Masson's trichrome stains keratin red, collagen and bone blue-green, cytoplasm pink, and nuclei black. Silver staining is used to highlight DNA and proteins like type III collagen. To highlight fat, Sudan stain is used. To localize specific proteins or antigens, the tissue is exposed to antigen-specific antibodies conjugated to a fluorescent dye.

Alternative techniques of fixation and embedding are sometimes when they are needed. For example, to retain membrane structures for electron microscopy, histologists use fixatives containing heavy metals that bind to phospholipids. Tissue can also be frozen and cut using a cryostat. This technique is called cryosection, and is used when the analysis of a specimen needs to be performed quickly. Cryosection is used during intraoperative consultation, which is when during a surgery, histology is used to look at tissue that has been removed to determine how to proceed. For example, if a cancer is suspected to have spread, a sample of the suspected metastasis (spreading cancer) is given to a pathologist to determine if it is cancer. If so, the surgry is usually not curative, and the surgeon will choose a less aggressive surgery.

Another use of histology is to determine the nature of infectious agents, for example whether a bacterial infection is gram-positive or gram-negative will allow the more proper choice of antibiotic. Histological mechanisms are also used to screen for diseases, such as the analysis of cervical scrapings collected during a Pap smear to determine if the endothelium is progressing toward cervical cancer.

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