6

Tissue Processing
Lena T. Spencer and John D. Bancroft

INTRODUCTION
After the removal of a tissue sample from the patient, a series of processes must take place to ensure the nal microscope slides are of a diagnostic quality. Tissues are exposed to a series of reagents that x, dehydrate, clear, and inltrate, with nal embedding in a medium that provides support for the tissue. The quality of the structural preservation of tissue components is determined by the choice of reagent and exposure times to the reagents during processing. Each step in the tissue processing is important from procurement of the specimen and the selection of the sample, determining the appropriate protocols and reagents to use, to staining and nal diagnosis. Producing quality slides for diagnosis is not an accident; it 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.

system is used, adequate policies and procedures have to be in place to ensure positive identication of the tissue blocks and slides during processing, diagnosis, and ling.

Completion of xation before processing

Fixation is the most important step in the processing of the tissue sample. If xation is not complete prior to processing, stations should be designated on the processor for this purpose. If tissue is inadequately xed, the subsequent dehydration solutions may complete the process, possibly altering the staining characteristics of the tissue. The size and type of specimen in the tissue cassette determines the time needed for complete xation and processing. The tissue should be dissected to 34 mm in thickness; a rule of thumb for most specimens is the size of a small coin. Care must be taken not to overll the cassette during gross dissection, impeding the ow of reagents around the tissue. If possible, larger or smaller pieces of tissue should be separated and processed using different schedules.

Labeling of tissues
A unique identication number or code is assigned to the tissue sample accessioned in the laboratory. This number may be electronically or manually generated and should accompany the specimens throughout the entire laboratory process, including documentation in the pathology report. Recent technology has made bar code and character recognition systems readily available to 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

Post-xation treatment
Special xation techniques may require additional steps before processing is initiated. Picric acid xatives form water-soluble picrates making it necessary to place the tissue cassettes directly into 70% alcohol for processing. Alcoholic xatives, such as Carnoys uid, should be placed directly into 100% alcohol. To help in the visualization of small fragments of tissue during embedding, a few drops of 1% eosin can be added to the specimen container 30 minutes prior to processing. The pink

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Tissue processing

coloration of the tissue remains during processing, but washes out during subsequent staining.

Viscosity
Viscosity is the property of resistance to the ow of a uid. The smaller the size of the molecules in the solution, the faster the rate of uid penetration (low viscosity). Conversely, if the molecular size is larger, the rate of exchange is slower (high viscosity). Most of the solutions used in processing, dehydrates and clearants, have similar viscosities, with the exception of cedar wood oil. Embedding mediums have varying viscosities. Parafn has a lower viscosity in the uid (melted) state, enhancing the rapidity of the impregnation.

PRINCIPLES OF TISSUE PROCESSING

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

dehydration: removal of water and xative from the tissue clearing: removal of dehydrating solutions, making the tissue components receptive to the inltrating medium inltrating: permeating the tissue with a support medium embedding: orienting the tissue sample in a support medium and allowing it to solidify.

Vacuum
Using reduced pressure to increase the rate of inltration decreases the time necessary to complete each step in the processing of tissue samples. Vacuum will remove reagents from the tissue only if they are more volatile than the reagent it is replacing. Vacuum used on the automated processor should not exceed 15 inches of Hg (mercury) to prevent damage and deterioration to the tissue. Vacuum can aid in the removal of trapped air in porous tissue. Impregnation time of dense, fatty tissue can be greatly reduced with the addition of vacuum during processing.

Factors inuencing the rate of processing

When tissue is immersed in uid, interchange occurs between the uid within the tissue and the surrounding uid. Several factors, discussed below, inuence the rate at which interchange occurs.

Fixation
Preserving cells and tissue components with minimal distortion is the most important step in the processing of tissue samples and is discussed in detail in Chapter 4. Fixation stabilizes proteins, rendering the cell and its components resistant to further autolysis by inactivating lysosomal enzymes, and changes the tissues receptiveness to further processing. Fixation must be complete before subsequent steps in the processing schedule are initiated.

Agitation
The rate of uid exchange is dependent upon the exposed surface of the tissue that is in contact with the processing reagent. Agitation increases the ow of fresh solutions around the tissue. Automated processors incorporate vertical or rotary oscillation or pressurized removal and replacement of uids at timed intervals as the mechanism for agitation. Efcient agitation can reduce the overall processing time by 30%.

DEHYDRATION
The rst stage of processing is the removal of unbound water and aqueous xatives from the tissue components. Many dehydrating reagents are hydrophilic (water loving), possessing strong polar groups that interact with the water molecules in the tissue. Other reagents affect dehydration by repeated dilution of the aqueous tissue uids. Dehydration should be accomplished slowly. If the concentration gradient between the uid

Heat
Heat increases the rate of penetration and uid exchange. It must be used sparingly to reduce the possibility of shrinkage, hardening, and brittleness of the tissue. Temperatures limited to 45C can be used effectively; higher temperatures may be deleterious to subsequent immunohistochemical staining.

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inside and outside the tissue is excessive, diffusion currents cross the cell membranes during uid exchange, increasing the possibility of cell distortion. For this reason specimens are always 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 prohibit the penetration of the clearing reagents into the tissue, leaving the specimen soft and non-receptive to inltration. There are numerous dehydrating agents: ethanol, ethanol acetone, methanol, isopropyl, glycol, and denatured alcohols. If the dehydrant of choice is ethanol, the tissue is rst immersed in 70% ethanol in water, followed by 95% and 100% solutions. For delicate tissue it is recommended that the processing start in 30% ethanol.

Butyl alcohol (butanol)

This is used primarily for plant and animal histology; it is a slow dehydrant with less shrinkage and hardening of the tissue.

Acetone CH3COCH3
Acetone is a clear, colorless, ammable uid, miscible with water, ethanol, and most organic solvents. It is rapid in action, with poor penetration, and causes brittleness in tissues if use is prolonged. Acetone removes lipids from tissue during processing.

Additives to dehydrating agents

When added to dehydrating agents, phenol acts as a softening agent for hard tissues such as tendon, nail, dense brous tissue, and keratin masses. A 4% solution is added to each of the 95% ethanol stations. Alternatively, hard tissue can be immersed in a glycerolalcohol mixture.

Dehydrating uids
Ethanol C2H5OH
This is a clear, colorless, ammable liquid. It is hydrophilic, miscible with water and other organic solvents, fast acting, and reliable. Ethanol is taxable, controlled by the federal government, and requires careful record keeping. Graded concentrations of ethanol are used for dehydration. Ethanol ensures total dehydration, making it the reagent of choice for the processing of electron microscopy specimens.

Universal solvents
Universal solvents dehydrate and clear during tissue processing. Dioxane, tertiary butanol, and tetrahydrofuran are considered to be universal solvents; they are not recommended for processing delicate tissues due to their hardening properties (Carson 1977; Sheehan & Hrapchak 1980). For safety issues see Chapter 2.

Industrial methylated spirit (denatured alcohol)

This has the same physical properties as ethanol. Denatured alcohol consists of ethanol, with the addition of methanol (about 1%), isopropyl alcohol, or a combination of alcohols. For purposes of tissue processing it is used in the same manner as ethanol.

CLEARING
A clearing reagent acts as an intermediary between the dehydration and inltration solutions. It 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:

Methanol
This is a clear, colorless and ammable uid which is highly toxic; it is miscible with water, ethanol, and most organic solvents. It can be substituted for ethanol.

Propan-2-ol, isopropyl alcohol CH3CHOHCH3

Isopropyl alcohol is miscible with water, ethanol, and most organic solvents. It is often used in microwave processing schedules. Isopropyl alcohol does not cause over-hardening or shrinkage of the tissue.

rapid removal of dehydrating agent ease of removal by melted parafn minimal tissue damage ammability toxicity cost.

Most clearing agents are ammable liquids, which warrants caution in their use. The boiling point of the

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clearing agent gives an indication of its speed of replacement by melted parafn. Fluids with a low boiling point are generally more readily replaced. Viscosity inuences 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 sufciently cleared and smaller, more fragile, tissue blocks are not damaged (Carson 1977; Sheehan & Hrapchak 1980; Luna 1992). 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 have to be addressed. Most institutions have a policy for the storage, disposal, and safety requirements for all ammables used in the laboratory.

posal is dependent upon the water treatment centers standards at the site of the laboratory. Their main disadvantages are a strong pungent odor and small tissue deposits of minerals such as copper or calcium, which may dissolve.

Safety
Safe handling of common histological chemicals is discussed in Chapter 2. Every histology laboratory should have a chemical hygiene plan that incorporates specic work practices to protect workers from potentially hazardous chemicals. Information sheets should be available for all the chemicals used in the laboratory. The basic information includes exposure limits, target organs, storage, disposal, and how to handle spills. These sheets are placed in an easily accessible place for quick reference.

Clearing agents suitable for routine use

Xylene
A ammable, colorless, liquid with a characteristic petroleum or aromatic odor, miscible with most organic solvents and parafn. Suitable for blocks that are less than 5 mm in thickness. Over-exposure during processing will cause over-hardening. Xylene is commonly used in routine histology laboratories and is recyclable.

RECYCLING REAGENTS
Distillation equipment is used in many laboratories to recycle alcohol and xylene using fractional distillation heat to separate different waste products in the solvents by boiling points; the component with the highest boiling point is puried. Advantages include:

Toluene
Similar properties to xylene, although it is less damaging with prolonged immersion of tissue. It is ammable and more volatile than xylene.

Chloroform
Chloroform is slower in action than xylene but causes less brittleness. Thicker tissue blocks can be processed, greater than 1 mm in thickness. Tissues placed in chloroform do not become translucent. It is non-ammable but highly toxic, and phosgene gas is given off when chloroform is heated. Often used when processing specimens of the central nervous system.

reduced cost rapid efcient eliminates the need to have chemicals removed by a waste disposal company, and environmentally unsafe chemicals are not being sent to land-lls for disposal.

The recycled reagents must be tested for quality (Dapson & Dapson 2005).

PARAFFIN WAX
Parafn continues to be the most popular inltration and embedding medium in the histology laboratory. The tissue is impregnated with wax, which forms a matrix preventing tissue structure distortion during microtomy. Parafn wax has a wide range of melting points, which is important for use in the different climatic regions of the world. It is inexpensive, provides quality sections,

Methyl benzoate and methyl salicylate

These are slow-acting clearing agents and can be used when double embedding techniques are required.

Citrus fruit oilslimonene reagents

Limonene reagents are extracts from orange and lemon rinds; they are non-toxic and miscible with water. Dis-

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and is easily adaptable to a variety of uses. It can be used for most routine and special stains.

The advantages of using an embedding system are:

Parafn wax properties

Parafn wax is a mixture of long-chain hydrocarbons produced in the cracking of mineral oil. Its properties are varied depending on the melting point; low melting point parafn is usually softer, higher melting point parafn is usually harder, which can effect microtomy. Melting points range from 40 to 70C. Heating the parafn to a high temperature alters the properties of the wax. To promote good ribboning during microtomy, parafn wax of suitable hardness at room temperature should be chosen.

ease of use speed tissue and holder are rmly attached, creating a single unit blocks led immediately after sectioning permanent identication.

Cassettes and molds that accommodate larger or smaller specimens may be purchased from scientic supply companies.

Quality control
Temperature of all parafn dispensers, otation water baths, and automated processors are carefully monitored and documented. The histology laboratory should have a policy and procedure manual that addresses quality issues and corrective actions.

Parafn wax additives

Parafn waxes that contain plasticizers or other resin additives are commercially available, providing a selection that is appropriate for most laboratories. These mixtures create parafn with the desired hardness for the tissue to be embedded. Substances that were added to parafn in the past included beeswax, rubber, ceresin, plastic polymers, and diethylene glycol distearate. Many of these additives have a higher melting point than parafn wax, consequently making the tissue more brittle.

Orientation of tissues
Specimen orientation during embedding is important for the demonstration of proper morphology. Improper orientation may result in diagnostic tissue elements being damaged during microscopy (see Chapter 5). 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. Most tissue are embedded at; the margin of embedding medium around the tissue will assure support of the tissue. Tissues requiring special orientation include:

Embedding tissue in parafn wax

Embedding involves the enclosing of properly processed, correctly oriented specimens in a support medium that provides external support during microscopy. The embedding medium must ll all the spaces within the tissue, supporting cellular components. It should provide elasticity, resisting section distortion while facilitating sectioning. Most laboratories use embedding centers, consisting of three modules: a parafn dispenser, a cold plate, and a heated storage area for molds and tissue cassettes. Parafn is dispensed automatically from a nozzle into a suitably sized mold. The tissue is oriented in the mold; a cassette is attached, producing a at block face with parallel sides. The mold is placed on a small cooling area to allow the parafn to solidify. The quick cooling of the wax ensures a small crystalline structure, producing fewer artifacts when sectioning the tissue.

Tubular structures: arteries, veins, fallopian tubes, and vas deferenscut in cross-section of the lumen. Skin, intestine, gallbladder, and other epithelial biopsiescut in a plane at right angles to the surface, and oriented so the epithelial surface is cut last, minimizing compression and distortion of the epithelial layer. Muscle biopsiessections containing both transverse and longitudinal planes. Multiple pieces of a tissueoriented side by side with the epithelial surface facing in the same direction.

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AUTOMATED TISSUE PROCESSING

The basic principle for tissue processing requires the exchange of uids using a series of solutions for a predetermined length of time in a controlled environment. For decades instrumentation used in tissue processing was relatively unchanged. Recent advances now include specialty microwave ovens and the emergence of constant through-put processors.

Tissue processors
The carousel-type processor (tissue transfer) and the selfcontained uid exchange systems were the rst automated tissue processors used in the histology laboratory. This type of processor transported tissue blocks contained in baskets through a series of reagents housed in stationary containers. The length of time the specimens were submerged in each reagent container was electronically programmed. Earlier models accomplished this step by notching the face of a clock disc. Vertical oscillation or the mechanical raising and lowering of the tissue into the reagent containers provided the agitation needed for the processing of the tissue. The enclosed, self-contained vacuum tissue processor later became the mainstay in most laboratories. A microprocessor is used to program the instrument. Tissues are loaded into a retort chamber where they remain throughout the process. Reagents and melted parafn are moved sequentially into and out of the retort chamber using vacuum and pressure. Each step is customized by adding time, temperature, or vacuum/pressure. The advantages of this system are that vacuum and heat can be applied at any stage, customized schedules for tissue processing produced, uid spillage contained, and fumes eliminated. These processors employ alarm systems and diagnostic programs for trouble-shooting instrumentation malfunction. Specially designed microwave ovens for tissue processing are now common. The microwave oven shortens the processing time from hours to minutes. Microwave exposure stimulates the diffusion of the solutions into the tissue by increasing the internal heat of the specimen, accelerating the reaction time. Tissues are manually transferred from container to container of reagent. Most laboratory microwave ovens contain precise temperature controls, timers, and fume extraction systems. The

time for processing is dependent on the thickness and density of the specimen. Reagents used for microwave processing include ethanol, isopropanol or proprietary mixtures of alcohol, and parafn. Graded concentration of solutions is not required. Clearing agents are not necessary because the temperature of the nal parafn step facilitates evaporation of the alcohols from the tissue. Xylene and formalin are not used in this process, which eliminates toxic fumes and carcinogens. Properly controlled processing provides uncompromised morphology and antigenicity of the specimens. Increased efciency through improved turnaround times, environmentally friendly reagents, and greater protability due to reduction in number and volume of reagents, are advantages of this system. Disadvantages of the system are: the process is labor intensive because the solutions are manually manipulated, the cost of laboratory-grade microwaves may be prohibitive, and proper use of the microwave oven requires calibration and monitoring (Kok & Boon 1992; Willis & Minshew 2005). Recent advances in technology have led to the development of an enclosed processor, called a continuous input, rapid tissue processor, that uses microwave technology, vacuum inltration, and proprietary reagents described as being molecular-friendly. The tissue cassettes are moved through four stations that contain acetone, isopropanol, polyethylene glycol, mineral oil, and parafn. Microwaves and agitation are used to accelerate the diffusion of solvents in the tissue. A patented microwave technology is utilized operating at a continuous low power instead of pulsing high levels of microwave energy. The advantage of this system is the acceptance of tissues into the system at timed intervals, improving turnaround time. The reagents used are environmentally safe, eliminating toxic vapors in the laboratory. The morphology and quality of the specimens is consistent with that of traditional tissue processing. Disadvantages include the cost of the processor and that grossing of the tissue sample requires standardized specimen dissection (Morales et al 2004).

Processor maintenance
Every institution should have a policy outlining the rotation and changing of solutions for each tissue processor. The numbers, sizes, types of tissue processed

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and the reagents used will play a role in the determination of this policy. Solutions should be carefully monitored to ensure quality. Every manufacturer has a handbook outlining a preventive maintenance schedule.

Automated processing schedules

Overnight schedules for tissue processing are still popular in laboratories, but schedules have changed to reect the emphasis on reducing turnaround time for the specimen. Rapid processing for small biopsies or stat specimens is easily accommodated.

Important maintenance tips

Any spillage or overow should be wiped away immediately Accumulation of wax on any surface should be removed Temperature of the parafn bath should be set to 3C above the melting point of the parafn Timing should be checked when placing tissue cassettes in the processor, especially when delayed schedules are selected.

Overnight processing
For many laboratories, this is considered the routine processing schedule. Tissues continue xation by being submerged in 10% formalin, buffered or unbuffered. The process may include alcoholic formalin, varying concentrations of alcohol, xylene, or a xylene substitute, followed by inltration in parafn. Schedules are customized for the tissues being processed; factors inuencing the processing schedule include the end-time required, reagents used, the inclusion of heat and vacuum, the size and number of tissues. The schedule in Table 6.1 can be modied, adjusting times from the various stations, keeping in mind the endtime needed for completion and prior xation.

Advantages of the newer technology in processing:

Custom programs specic to tissues being processed; addition of vacuum, agitation, or heat at any stage Rapid schedules Fluid and fume containment Environmentally friendly reagents Delay schedules.

Specialized tissues
Tissues such as brain, eyes, and bone require specialized processing (see Chapter 19 for brain, Chapters 18 and 29 for bone). A schedule for eyes is shown in Table 6.2.

Table 6.1 Station 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Overnight processing Reagents 10% Formalin 10% Formalin 50% Alcohol/formalin 70% Alcohol 95% Alcohol 95% Alcohol 100% Alcohol 100% Alcohol Xylene Xylene Parafn Parafn Parafn Parafn Time 1h 1h 1h 30 min 30 min 40 min 40 min 40 min 40 min 40 min 30 min 20 min 20 min 40 min Pressure/Vacuum On On On On On On On On On On On On On On Temp 38 C 38C 38C 38C 38C 38C 38C 38C 38C 38C 60C 60C 60C 60C

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Schedule for processing eyes

Eyes require special processing; this is dictated by the delicate nature of some parts of the structures and toughness of others. Ideally, a separate processor should be dedicated for tissues that require special handling because of the reagents used. The eye must be thoroughly xed, prior to dissection and subsequent processing. Phenol is added to the lower percentage alcohols to soften the sclera and lens. Reagents are selected that provide the best dehydration and clearing of the tissue (chloroform has been used as the clearing agent because it is less harsh than xylene and causes minimal shrinkage), keeping the retina attached. Large tissue cassettes and molds are specically made for use in processing eyes.

lines of any residual parafn. The clean cycle will ush the lines using xylene, 100% alcohol, and water.

MANUAL TISSUE PROCESSING

Manual tissue processing is rarely used today. There can be circumstances requiring the tissue sample to be manually processed, including:

Power failure or equipment malfunction Large tissue samples requiring more time than can be allocated on an automated processor Small biopsies, such as transplant specimens, needing a rapid diagnosis.

Manual processing schedules Rapid processing schedules for small biopsies

Recently excised endoscopic biopsies and needle biopsies can be adequately processed in 25 hours using heat (3745C) and vacuum. Tissues requiring dissection should be trimmed to 2 mm in thickness. Most small specimens will x prior to processing. If xation is not complete, processing should begin in a station containing 10% formalin. Table 6.3 shows an example of a shortened process for an enclosed processor. The enclosed processor drain time is approximately 35 minutes at each station. The program can be amended, changing times at various stations; drain times should be taken into consideration when determining the end time. After each run the instrument must be cleaned to purge the The schedule in Table 6.4 is adaptable for large, dense tissue blocks. Times should be extended in each container, or more containers may be added to the schedule.

Table 6.3

Short processing schedule for biopsies Time 10 10 10 10 10 10 10 10 10 10 10 min min min min min min min min min min min Pressure/ Vacuum Temp On On On On On On On On On On On 38C 38C 38C 38C 38C 38C 38C 38C 38C 38C 58C

Station Reagents 1 2 3 4 5 6 7 8 9 10 11 10% Formalin 10% Formalin 70% Alcohol 95% Alcohol 95% Alcohol 100% Alcohol 100% Alcohol Xylene Xylene Parafn Parafn

Table 6.2 Station 1 2 3 4 5 6 7 8 9 10 11 12

Processing eyes Reagents 10% Formalin 4% Phenol/70% Alcohol 4% Phenol/70% Alcohol 95% Alcohol 95% Alcohol 100% Alcohol 100% Alcohol 100% Alcohol/Chloroform Chloroform Chloroform Parafn Parafn Time 0h 1h 1h 1h 1h 1.5 h 1.5 h 2h 2h 2h 2h 3h

Table 6.4 biopsies Step

Manual tissue processing for small Time 10 10 10 10 10 min min min min min

10% Formalin 95% Alcohol 100% Alcohol Xylene Parafn

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1. Place tissue in cassette. Drop cassette in 10% formalin. Formalin container placed under warm tap water. 2. Remove tissue cassette from formalin and place in container with 95% alcohol, on a stir plate with a stir bar. 3. Continue through 100% alcohol and xylene using stir plate and stir bar. 4. Place tissue cassette in melted parafn. 5. Embed as usual.

needed to house the processing reagents and the limited use these types of section have in neuropathology. It is included here for historical purposes only.

Restoration of tissue dried in processing

Despite precautions taken during processing, technical or mechanical malfunctions may occur, resulting in tissue drying out prior to parafn impregnation. The tissue will never be regarded as normal, but the following treatment may help provide slides of diagnostic quality.

ALTERNATIVE EMBEDDING MEDIA

There are occasions when parafn is an unsuitable medium for the type of section required, including:

Tissue restoration
70% ethanol Glycerol Dithionite 70 ml 30 ml 1g

Processing reagents remove or destroy tissue components, the object of investigation Sections are required to be thinner The use of heat may adversely affect tissue The inltrating medium is not sufciently hard to support the tissue.

Resin
Resin is used exclusively as the embedding medium for electron microscopy (see Chapter 30), ultra-thin sectioning for high resolution and for undecalcied bone (see Chapters 18 and 29).

Tissues remain in the solution for several hours or overnight. Processing begins with the dehydrating solutions and continues to completion. Tissue may be difcult to section; coated or plus slides should be used.

Summary
Technological advances have been made in the instrumentation of tissue processors, in part due to increased workload, the demand for faster turnaround time for diagnostic samples, and shortages in the workforce. The addition of microprocessors, microwaves, and environmentally friendly chemicals are only a few of the improvements that will eventually revolutionize tissue processing.

Agar
Agar gel alone does not provide sufcient support for sectioning of tissues. Its main use is as a cohesive agent for small friable pieces of tissue after xation. Fragments of tissue are embedded in melted agar, allowed to solidify, and trimmed for routine processing. One method providing superior results is as follows: lter the xative with the tissue fragments through a Millipore lter using suction, carefully pour melted agar into the tube, allow solidication of the agar, and follow with routine processing and embedding in parafn.

Acknowledgments
This chapter is a development of the chapter that appeared in the rst ve editions. In those editions, Tissue processing was written by Keith Gordon and Paul Bradbury, with successful merging for later editions by Graeme Anderson and John Bancroft. Our acknowledgments go to the previous contributors.

Gelatin
Gelatin is used primarily in the production of sections of whole organs in the GoughWentworth technique and in frozen sectioning.

Celloidin
The use of celloidin or LVN (low viscosity nitrocellulose) is discouraged because of the special requirements

REFERENCES
Carson F.L. (1977) Histotechnology, a self-instructional text, 2nd edn. Chicago: ASCP Press, pp. 2642.

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Dapson J.C., Dapson R.W. (2005) Hazardous materials in the histopathology laboratory, regulations, risks, handling and disposal, 4th edn. Battle Creek, MI: Anatech, pp. 157164. Kok L.P., Boon M.E. (1992) Microwave cookbook of microscopists, 3rd edn. Leiden: Coulomb Press. Luna L.G. (1992) Histopathologic methods and color atlas of special stains and tissue artifacts. Downers Grove: Johnson Printers, pp. 166. Morales A.R., Nassiri M., Kanhoush R. et al. (2004) Experience with an automated microwave-assisted rapid tissue processing method: validation of histologic quality and

impact on the timeliness of diagnostic surgical pathology. American Journal of Clinical Pathology 121:528536. Sheehan D.C., Hrapchak B. (1980) Theory and practice of histotechnology, 2nd edn. St Louis: C.V. Mosby, pp. 5985. Vernon S.E. (2005) Continuous throughput rapid tissue processing revolutionizes histopathology workow. Laboratory Medicine 36:300302. Willis D., Minshew J. (2005) The whole enchilada with the rice and beans. Ft. Lauderdale: National Society for Histotechnology.