Histopathology involves study of diseased tissues thereby aiding diagnosis of tumors. Histopathological examination of tissues involves three stages - surgery, biopsy or autopsy.
After collecting the diseased tissues, they are first stabilized by placing in a fixative, to prevent decay. Formalin (10% formaldehyde in water) is the fixative used normally.
- The stabilized sample tissues are first transferred to a container where permitted reagents are allowed to act on the tissues.
- Concentrated ethanol helps to dehydrate the tissues. Toluene or Xylene is used after the above procedure to immerse the tissues followed by hot liquid paraffin wax. This process takes 12 to 16 hours, during which paraffin replaces the water in the tissue turning it soft and moist. Now the sample is ready for embedding.
- The tissue is transferred and set in a mold. During this process of embedding, additional paraffin is added to prepare a hard paraffin block.
- Microtome is used to section the embedded tissues into very thin sections of about 3-7 micrometer in thickness. These thin layers or sections are now ready for microscopic study.
- One or more pigments are used to stain the sections to view the details clearly. A combination of hematoxylin and eosin is used to stain the tissues. The cytoplasm pink is got by using eosin and nuclei blue by hematoxylin. Saffron, silver salts and some artificial dyes are the other compounds used to color tissue sections. For staining specific proteins, carbohydrates and liquids, antibodies are also used. The technique known as immunohistochemistry has helped scientists in the microscopic studies to identify different categories of cells specifically.
- A medically qualified expert or pathologist examines the histological slides microscopically.