The Histology Laboratory—What the Pathologist Needs to Know—From Tissue Cassettes to Glass Slides

Tissue Processing

After tissues are sufficiently fixed in formalin, the cassettes are loaded into a tissue processor. Automated processing consists of three steps and usually requires several hours to complete. Rapid methods have also been developed that are particularly suited to small specimens and specialty practices.

Dehydration: The water in the tissue is replaced by alcohol. Nonaqueous embedding media (such as paraffin) cannot penetrate tissues containing water.

Clearing: The alcohol is replaced by a clearing agent that makes the tissue receptive to infiltration by the embedding medium. The clearing agent must be miscible with both alcohol and the embedding medium. Because xylene (a common clearing agent) has a high refractive index, the tissue will also become transparent (“cleared”).

Infiltration: The xylene is replaced by paraffin or another embedding medium. The paraffin stiffens the tissue, which allows very thin sections (only a few microns in thickness) to be cut with a microtome.

Factors That May Interfere With Optimal Tissue Processing

Fatty tissue. Fixatives, and especially dehydrants, penetrate fatty tissues slowly. Fatty tissue must be sliced very thin to fix and dehydrate well. Typical examples are breast tissue and adipocytic tumors.

Tissue too thick or large for the cassette. It is often tempting for prosectors to stuff cassettes with tissue because it is faster than thinly sectioning tissue and fewer cassettes are required. However, fixatives and processing solutions cannot gain access to the tissue. The tissue will not process well and may remain soft. This is referred to as a “wet” section. It is often impossible to section such tissue or to obtain a thin even section. This outcome can have a significant adverse effect on patient care if the tissue can never be examined (e.g., lymph nodes on a tumor resection). Tissue sections should be no thicker than 0.2–0.3 cm when placed into a cassette. Although it is possible to remove tissue from paraffin and fix for a longer period of time, this process is labor intensive and may not improve the quality of tissue preservation.

Cross sections of tissues (e.g., colon, skin). Specimens that require specific orientation (e.g., a complete cross section perpendicular to the surface of the tissue) should be oriented appropriately in the cassette. Sponges placed in a cassette are sometimes helpful in holding tissue flat.

Multiple small tissue fragments. Fragments of tissue small enough to be lost through the holes in the cassette (0.1–0.2 cm) must be placed in a specimen bag or wrapped in lens paper. This also aids in identifying all tissue fragments for embedding.

Type of fixative. The type of fixative should be specified if not the usual fixative used by the laboratory. Some fixatives (e.g., those containing mercury) require special techniques for processing to remove precipitates and pigments. Anaqueous processing (e.g., to demonstrate uric acid crystals) is often accomplished by hand and not in tissue processors.