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Tissue Processing Protocols

To ensure the quality of microscopic slides for patient diagnosis, many variables & parameters must be explored

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Vol. 22 • Issue 9 • Page 20

Cover Story

Processing of tissue specimens for patient diagnosis has been somewhat overlooked in many ways. What actually goes on inside the tissue processor? What does each of the processing reagents do to the tissue? Which instrument platform is recommended? What processing cycle should be used? What tissue processing work flow is needed and how should it be implemented?

Principle

Tissue processing is the next step after fixation. The tissue sections will go through the process of dehydration, clearing and infiltration, exposing tissues to various chemicals for certain processes. During tissue processing, one step will build upon another. Although many references include the embedding process in tissue processing, embedding is really a separate process after paraffin infiltration.

Dehydration

After proper fixation, the tissue sections begin the process of dehydration, or the gradual removal of water. At the molecular level, water is present in free and bound tissues. The free water is removed; dehydration is accomplished by using graded alcohols.

During tissue processing, many cellular entities or structures are also removed such as fats, lipids (by the higher grade alcohols) and proteins (by the lower alcohols). Particular dehydrating reagents can be used to help with tissue shrinkage. Most dehydration processes usually begin in 70% alcohol, then at least two stations of 95% and at least two stations of 100% alcohol. Thorough and complete fixation must occur before the tissues are submitted to processing such as dehydration. If tissues are poorly fixed, the alcohol will continue the fixation process only to create possible artifacts and alter morphology.

As well, the time that tissues should remain in the dehydrating reagents should be tested and validated. Biopsies should be validated on a short processing cycle, whereas the larger tissues should be on at least a 10-12 hour processing cycle or a rapid processing cycle. Thin tissue sections (1-2 mm in thickness) usually require about 30 minutes per station on a conventional processor. Thicker tissue sections (3-4 mm in thickness) usually require about 60 minutes or more per station on a conventional processor.

For most histology laboratories, either ethyl (ethanol), isopropyl (isopropanol) or a "reagent grade" alcohol is preferred. Ethyl alcohol can cause hardening and brittle effects on collagen, connective tissues, colloid tissues and even bloody tissues. Isopropyl alcohol is a great substitute for ethyl alcohol in tissue processing and actually hardens tissue less than ethyl alcohol. Isopropyl can be used as a clearant and tissues can go directly to paraffin.

Acetone is a rapid dehydrate and can cause some shrinkage if the time is not controlled. Tissues that contain a lot of fats and lipids can be dehydrated with acetone. Acetone can also be used as a clearant.

Clearing

Clearing is the removal of the alcohol after dehydration and serves as a transition step between dehydration and paraffin infiltration. The term clearing is derived from the fact that some solvents have a high index of refraction and actually render tissues "clear" or transparent, which is the clearing endpoint. Xylene is the most commonly used hydrocarbon for the clearing station in conventional tissue processing. Some xylene grades contain benzene; xylene for routine histology should be benzene and sulphur free. The time that tissue remains in xylene should be controlled to ­eliminate over-hardening of the tissues.

Xylene substitutes work well, although they do not tolerate water. As a rule, the xylene substitutes must be used in a series of three stations on the tissue processor. Xylene substitutes can be slow but offer good results as a clearant.

Infiltration

Infiltration, sometimes referred to as "interpenetration," is the final stage of tissue processing prior to the embedding step. Infiltration is the submerging of tissue into a medium that will internally support tissue spaces and cell walls for internal support during microtomy. For most routine histology tissue processing, a fine grade of paraffin is used. Paraffin is a petroleum-based substance that is crystalline in structure. The melting points of paraffin can range from 38-68°C depending on its use. Additives such as rubber, bayberry, beeswax, stearic acid, resins (piccolyte 115), and plastic polymers (dimethyl sulphoxide - DSMO) assist with the sectioning process in microtomy. For histology purposes, the paraffin is usually melted 2-3°C above the melting point.

Parameters

• Agitation: During the tissue processing cycle, there must be some type of fluid agitation to move reagents through and around the tissue sections. Methods range from the up and down motion, magnetic stirrers in the processing retort, to fluid exchange. In recent years, the pressure and vacuum technology provided unique agitation of reagents for improved tissue processing. Ideally, tissue cassettes should be arranged fairly loose in the processing rack to allow thorough reagent flow in, around and through the tissue cassette.

• Heat: The application of heat during the dehydration and clearing steps of tissue processing will considerably reduce processing times. Care must be taken to maintain the heat temperatures no higher than 37°C, as high temperatures may affect immunohistochemistry. The temperature for the paraffin infiltration step should be no more than about 2-3°C above the melting point of the paraffin. If the paraffin temperature exceeds the recommended temperature settings, shrinkage, hardness and brittleness will also occur in the tissue sections. Paraffin temperatures of the tissue processor paraffin tanks must be taken and recorded at least daily.

• Vacuum and Pressure: Vacuum used during the dehydration and clearing stages of tissue processing is an advantage especially to porous tissues. Pressure can also add some advantage to the tissues in these stages. Together, vacuum and pressure can pull reagents through and around the tissues to insure more rapid and complete tissue processing.

Tissue Processor Platforms

Two tissue processing platforms are used: conventional and rapid (includes microwave processing). Conventional tissue processors are classified as the usual overnight processors, allowing 8-10 tissue processing programs, 12 or more reagent stations plus paraffin stations, choice of heat, vacuum and/or pressure and other options. Usually these were the carousel rotating processors later advancing to the enclosed processors. The fixation step can be delayed from a few hours to a few days.

Newer processors have two retorts available to operate two processing cycles at the same time for better utilization of the instrument, thus providing a Lean and Six Sigma workflow process.

Rapid (microwave) tissue processors have been available to assist with better turnaround time (TAT); many have been ­incorporated in the Lean and Six Sigma workflow processes. These require the change in daily application of tissue processing and many of these processors implement the "continuous flow" concept to handle the tissue specimens in a one-piece flow methodology. In most cases the tissues can be processed and slides prepared in the same day.

A few of these rapid processors incorporate at least two processing cycles to accommodate either biopsies or thin sections up to fatty or larger tissues. A reduction of reagents can be used and graded alcohols and xylene are eliminated. These instruments are truly tissue processors -- no online fixation or delay times. Special rapid processing reagents are used, even those to allow the fixation and processing of tissues for molecular assays. Controlled microwave technology is used in many of these rapid processors.

Quality Control

The tissue processing cycles and reagents require specific quality control (QC) measures. The proper changing or rotation of the processing reagents should be on a set schedule in most cases. Documentation of these changes or rotations must be recorded.

As well, a hydrometer must be used to check the strength of the diluted alcohols and recorded. Care must be taken to ensure that the correct reagent is poured into the correct reagent container to avoid processing errors. Temperatures of the paraffins should be checked and recorded daily. Preventative maintenance schedules should be established at least once or twice annually. The quality and even grade of the processing reagents should be adhered to for the proper processing of the tissues.

Validation

New tissue processor instruments are often installed, a processing program loaded and tissue specimens processed. However, there must be a validation process before a tissue processor is put into use to identify and record, for example, the tissue processor platform, what tissue types will be processed, which processing protocol or cycle will be implemented, technical training for each operator, the workflow process, reagents, processing cycles, etc.

One method to validate the tissue types is to use a multi-tissue block and variety of tissues in one block, all the same size. This is not perfect but can save time. If two processing cycles are available, both will need to be validated. The thickness also needs to be validated. Standardization will play a role in the validation process. Upon completion, the slides need to be reviewed by both technical staff and pathologists, then documented.

M. Lamar Jones is with Carolinas College of Health Sciences, Charlotte, NC; and Davidson County Community College, Lexington, NC.




     

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