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Tissue processing


Introduction

Proper handling of tissue specimens is critical to ensure that an accurate diagnosis is obtained from patient tissue samples. Whilst technological advances have streamlined processing, the principle steps remain the same: fixation, dehydration, clearing and infiltration. Regardless of the methodology, tissue samples requiring processing need to be placed in fixative as soon as possible after excision from the patient. This first step is essential to prevent autolysis, which could destroy diagnostic elements, and to prepare the tissue for the rigors of the reagents used in subsequent processing steps. This chapter will serve as a patient to pathologist guide for preparing tissue samples which ensures an accurate diagnosis with the best treatment outcomes for the patient.

Specimen tracking

To ensure accurate specimen tracking and maintain patient confidentiality, all tissue received in the laboratory must be given a unique identifier. Typically, an alphanumeric code is assigned to each sample. This code follows the specimen on its journey from the moment it is received in the laboratory to the final pathology report. Whilst chemical-resistant pens, pencils, slides and labels have been the standard, new technology utilizing barcodes, quick response (QR) codes and character recognition are readily available in most laboratories. Automated pre-labeling systems which permanently etch or emboss tissue cassettes help reduce human error. Many of these systems can track the particular run a block was processed on, offering greater information for auditing purposes. This type of data acquisition also assists in maintaining LEAN processes. Many histology laboratories in the USA are incorporating LEAN principles adapted from the Japanese manufacturing industry to streamline workflow, eliminate waste (either in materials or time), and foster a system of continuous improvement which will deliver a better “product”, i.e. quality tissue preparations which will allow faster diagnosis for improved patient outcomes. 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.

Factors influencing processing

When tissue is immersed in fluid, an interchange occurs between the tissue’s internal fluid and the surrounding solution. The rate of fluid exchange, or diffusion, is dependent upon the tissue size and density as well as the physical properties and concentration of the processing reagents. Several factors influence the rate at which diffusion occurs.

Viscosity

A solution with low viscosity has smaller sized molecules and a faster penetration rate. Conversely, solutions with larger molecules have high viscosity and the exchange rate is slower. Most of the solutions used in processing, dehydration and clearing have similar viscosities; embedding media however have varying viscosities. Melted paraffin wax, for example, has a low viscosity which enhances the impregnation rate.

Agitation

This increases the flow of solutions around the tissue. The mechanism for agitation in automated processors incorporates either vertical or rotary oscillation or pressurized removal and replacement of fluids at timed intervals.

Heat

An increase in temperature improves the fluid exchange and penetration rate, but this must be used sparingly to reduce the possibility of producing morphological heat artifact. Excessive exposure to heat can cause shrinkage and hardening of the tissue which negatively affects subsequent staining and immunohistochemistry.

Vacuum & pressure

These both increase fluid mobility, thus increasing the infiltration rate and decreasing the time necessary to complete each processing step. Vacuum aids the removal of air pockets in porous tissue, e.g. lung.

Processing solvent contamination

The number of blocks on each run, tissue type, size, frequency of the runs, use of sponges and cross-contamination of processor solvents will influence how often solutions should be rotated between stations or changed to maintain processing quality.

Tissue processing stages

  • Fixation – prevents autolysis and stabilizes tissue to maintain cellular structure.

  • Dehydration – removes water and unbound fixative from the tissue.

  • Clearing – displaces dehydrating solutions, making the tissue components receptive to the infiltrating medium.

  • Infiltration – permeates tissue with a support medium.

  • Embedding – orientation of the tissue sample in a support medium to create a tissue “block” suitable for sectioning.

Fixation

The first and most critical step in specimen handling is preservation or fixation of the tissue sample. Fixation denatures proteins rendering the cell and its components resistant to further autolysis. Complete fixation also allows the tissue to withstand the negative effects of subsequent processing reagents. Although samples are typically received in fixative, it is best to begin processing with a fixative station as the natural diffusion of water from the tissue will have diluted the fixative in the specimen container reducing the fixative’s efficacy.

Inadequately fixed tissue may exhibit zonal fixation due to the dehydrating solutions completing the fixation process towards the interior of the tissue. This may result in altered morphology and affect the staining characteristics of the tissue. The size and type of specimen in the tissue cassette determines the time needed for complete fixation and processing to occur. The tissue should be dissected to 2–4 mm in thickness and trimmed to a size that allows complete flow of reagents around the tissue in the cassette. Ideally tissue should be separated according to size and/or type, and processed using different schedules. The most commonly used reagent for the fixation of histological specimens is 10% neutral buffered formalin (NBF). Alcoholic formalin can be used to accelerate the penetration rate of the fixative and to shorten the time needed for complete fixation. Zinc formalin solutions are used for their ability to produce sharp nuclear detail and enhance staining capability. Non-formalin fixatives with glyoxal as the active ingredient have gained popularity for their added safety benefit (see Chapter 4 ).

Post-fixation treatment

Specimens fixed in alcoholic fixatives should be followed with alcohol to prevent re-introduction of water to the tissue specimen. The alcohol concentration will be dependent on the alcohol concentration of the fixative.

Picric acid fixatives, e.g. Bouin’s, will color the tissue bright yellow. A rinse of the tissue in 50-70% alcohol for 4-6 hours ( ) will remove excess fixative. Complete removal of the yellow color is essential to prevent the color from interfering with staining. This can also be accomplished by treating the cut tissue section on the glass slide with a dilute carbonate solution (see .)

A few drops of 1% eosin can be added to the specimen container 30 minutes prior to processing to assist in visualizing small tissue fragments during embedding. The pink color of the tissue remains during processing, but washes out during subsequent staining. Incomplete removal of the eosin can interfere with fluorescent procedures.

Dehydration

Dehydration displaces the residual fixative as well as cellular water. Water is found in the tissue in two forms, free and bound. The bound water molecule is an integral part of the macromolecules of the cell. Correct dehydration schedules during tissue processing should only remove the free water, leaving the bound water intact. Graded alcohols are used in dehydration to remove free water and keep the bound water in place. When tissues are exposed to heat or excessive time in the higher grade alcohols (95% or 100%), bound water is removed. The removal of the bound water will produce over-processing artifacts such as shrinkage, ‘parched earth’ effect and abnormal staining, as well as dry, brittle tissues during microtomy. For this reason, specimens are best processed through graded alcohols of increasing concentration. Incomplete dehydration will impair the penetration of the clearing reagents into the tissue, leaving the specimen soft and non-receptive to paraffin wax infiltration. Ethanol, reagent alcohol and isopropanol all have a strong history of working well in tissue processing. Glycol ether dehydrants are an effective alternative to alcohols in tissue processing. Unlike alcohol, glycol ethers are unable to act as a secondary fixative but their chemical properties prevent removal of bound water so over-processing is prevented. Due to their gentle nature they are used undiluted ( ).

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