Tissue processing

6 Tissue processing



After the removal of a tissue sample from the patient, a series of physical and chemical processes must take place to ensure that the final microscopic slides produced are of a diagnostic quality. Tissues are exposed to a series of reagents that fix, dehydrate, clear, and infiltrate the tissue. The tissue is finally embedded in a medium that provides support for microtomy. The quality of the structural preservation of tissue components is determined by the choice of exposure times to the reagents during processing. Every step in tissue processing is important; from selection of the sample, determining the appropriate protocols and reagents to use, to staining and final diagnosis. Producing quality slides for diagnosis requires skills that are developed through continued practice and experience. As new technology and instrumentation develops, the role of the histology laboratory in patient care will continue to evolve, providing standardization of processes, increased productivity, and better utilization of the resources available. This chapter will provide an overview of the steps in the process and the reagents needed to prepare tissue for microscopic evaluation.

Labeling of tissues

A unique accession number or code should be assigned to every tissue sample as discussed in Chapter 5. This unique number should accompany the specimens throughout the entire laboratory process and may be electronically or manually generated. New technology has made bar code quick response (QR) and character recognition systems readily available in most laboratories. Automated pre-labeling systems that permanently etch or emboss tissue cassettes and slides, as well as chemically resistant pens, pencils, slides and labels, are routinely used in pathology laboratories. Regardless of whether an automated or manual labeling system is used, adequate policies and procedures must be in place to ensure positive identification of the tissue blocks and slides during processing, diagnosis, and filing.

Principles of tissue processing

Tissue processing is designed to remove all extractable water from the tissue, replacing it with a support medium that provides sufficient rigidity to enable sectioning of the tissue without parenchymal damage or distortion.

Factors influencing the rate of processing

When tissue is immersed in fluid, an interchange occurs between the fluid within the tissue and the surrounding fluid. The rate of fluid exchange is dependent upon the exposed surface of the tissue that is in contact with the processing reagents. Several factors influence the rate at which the interchange occurs: namely, agitation, heat, viscosity and vacuum.


The first stage of processing is the removal of ‘free’ unbound water and aqueous fixatives from the tissue components. Many dehydrating reagents are hydrophilic (‘water loving’), possessing strong polar groups that interact with the water molecules in the tissue by hydrogen bonding. Other reagents affect dehydration by repeated dilution of the aqueous tissue fluids. Dehydration should be accomplished slowly. If the concentration gradient is excessive, diffusion currents across the cell membranes may increase the possibility of cell distortion. For this reason, specimens are processed through a graded series of reagents of increasing concentration. Excessive dehydration may cause the tissue to become hard, brittle and shrunken. Incomplete dehydration will impair the penetration of the clearing reagents into the tissue, leaving the specimen soft and non-receptive to infiltration. There are numerous dehydrating agent; ethanol, ethanol acetone, methanol, isopropyl, glycol and denatured alcohols.

Dehydrating fluids


Clearing reagents act as an intermediary between the dehydration and infiltration solutions. They should be miscible with both solutions. Most clearants are hydrocarbons with refractive indices similar to protein. When the dehydrating agent has been entirely replaced by most of these solvents the tissue has a translucent appearance: hence the term ‘clearing agent’.

The criteria for choosing a suitable clearing agent are:

Most clearing agents are flammable liquids, which warrant caution in their use. The boiling point of the clearing agent gives an indication of its speed of replacement by melted paraffin wax. Fluids with a low boiling point are generally more readily replaced. Viscosity influences the speed of penetration of the clearing agent. Prolonged exposure to most clearing agents causes the tissue to become brittle. The time in the clearing agent should be closely monitored to ensure that dense tissue blocks are sufficiently cleared and smaller more fragile tissue blocks are not damaged. Cost should be considered, especially as it relates to disposal of the reagent. Since most clearing agents are aromatic hydrocarbons or short-chain aliphatic hydrocarbons, environmental issues must be addressed. Most institutions have a policy for the storage, disposal and safety requirements for all flammables used in the laboratory.

Clearing agents suitable for routine use

Infiltrating and embedding reagents

Paraffin wax

Paraffin wax continues to be the most popular infiltration and embedding medium in histopathology laboratories. Paraffin wax is a mixture of long-chained hydrocarbons produced in the cracking of mineral oil. Its properties are varied depending on the melting point used, ranging from 47 to 64°C. Paraffin wax permeates the tissue in liquid form and solidifies rapidly when cooled. The tissue is impregnated with the medium, forming a matrix and preventing distortion of the tissue structure during microtomy. It has a wide range of melting points, which is important for use in the different climatic regions of the world. To promote desirable ribboning during microtomy, paraffin wax of suitable hardness at room temperature should be chosen. Heating the paraffin wax to a high temperature alters the properties of the wax. Higher melting point paraffin wax provides better support for harder tissues, e.g. bone, can allow production of thinner sections, but may cause difficulty with ribboning. Lower melting point paraffin wax is softer and provides less support for harder tissues. It is more difficult to obtain thinner sections but ribboning is easier. Paraffin wax is inexpensive, provides quality sections and is easily adaptable to a variety of uses. Paraffin wax is compatible with most routine and special stains, as well as immunohistochemistry protocols.

Alternative embedding media

There are occasions when paraffin wax is an unsuitable medium for the type of tissue being processed including:


Resin is used exclusively as the embedding medium for electron microscopy (see Chapter 22), ultra-thin sectioning for high resolution and also for undecalcified bone (see Chapter 16).

Orientation of tissues

Specimen orientation during embedding is important for the demonstration of proper morphology. Incorrect orientation may result in diagnostic tissue elements being damaged during microscopy or not being evident for pathology review. Products are available that help ensure proper orientation: marking systems, tattoo dyes, biopsy bags, sponges, and papers. Orientation of the tissue should offer the least resistance of the tissue against the knife during sectioning. A margin of embedding medium around the tissue assures support of the tissue.

Tissues requiring special orientation include:

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Dec 13, 2017 | Posted by in HISTOLOGY | Comments Off on Tissue processing

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