6Tissue 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 fi nal microscope slides are of a diagnostic quality. Tissues are exposed to a series of reagents that fi x, dehydrate, clear, and infi ltrate, with fi nal embedding in a medium that provides support for the tissue. The quality of the structural preservation of tissue components is deter-mined 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 speci-men and the selection of the sample, determining the appropriate protocols and reagents to use, to staining and fi nal diagnosis. Producing quality slides for diagno-sis 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.

Labeling of tissues

A unique identifi cation 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 labora-tory 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

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

Completion of fi xation before processing

Fixation is the most important step in the processing of the tissue sample. If fi xation is not complete prior to pro-cessing, stations should be designated on the processor for this purpose. If tissue is inadequately fi xed, the sub-sequent 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 fi xation and processing. The tissue should be dissected to 3–4 mm in thickness; a rule of thumb for most specimens is the size of a small ‘coin’. Care must be taken not to overfi ll the cassette during gross dissection, impeding the fl ow of reagents around the tissue. If possible, larger or smaller pieces of tissue should be separated and processed using different schedules.

Post-fi xation treatment

Special fi xation techniques may require additional steps before processing is initiated. Picric acid fi xatives form water-soluble picrates making it necessary to place the tissue cassettes directly into 70% alcohol for processing. Alcoholic fi xatives, such as Carnoy’s fl uid, should be placed directly into 100% alcohol. To help in the visual-ization 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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84 Tissue processing

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

PRINCIPLES OF TISSUE PROCESSING

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

Stages of tissue processing are:

• dehydration: removal of water and fi xative from the tissue

• clearing: removal of dehydrating solutions, making the tissue components receptive to the infi ltrating medium

• infi ltrating: permeating the tissue with a support medium

• embedding: orienting the tissue sample in a support medium and allowing it to solidify.

Factors infl uencing the rate of processing

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

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

HeatHeat increases the rate of penetration and fl uid exchange. It must be used sparingly to reduce the possibility of shrinkage, hardening, and brittleness of the tissue. Tem-peratures limited to 45°C can be used effectively; higher temperatures may be deleterious to subsequent immuno-histochemical staining.

ViscosityViscosity is the property of resistance to the fl ow of a fl uid. The smaller the size of the molecules in the solu-tion, the faster the rate of fl uid penetration (low viscos-ity). Conversely, if the molecular size is larger, the rate of exchange is slower (high viscosity). Most of the solu-tions used in processing, dehydrates and clearants, have similar viscosities, with the exception of cedar wood oil. Embedding mediums have varying viscosities. Paraffi n has a lower viscosity in the fl uid (melted) state, enhanc-ing the rapidity of the impregnation.

VacuumUsing reduced pressure to increase the rate of infi ltration 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.

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 receptive-ness to further processing. Fixation must be complete before subsequent steps in the processing schedule are initiated.

DEHYDRATION

The fi rst stage of processing is the removal of unbound water and aqueous fi 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 fl uids. Dehydration should be accomplished slowly. If the concentration gradient between the fl uid

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inside and outside the tissue is excessive, diffusion currents cross the cell membranes during fl 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 infi ltration. There are numerous dehydrating agents: ethanol, ethanol acetone, methanol, isopropyl, glycol, and denatured alcohols. If the dehydrant of choice is ethanol, the tissue is fi 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.

Dehydrating fl uids

Ethanol C2H5OHThis is a clear, colorless, fl ammable liquid. It is hydro-philic, 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.

Industrial methylated spirit (denatured alcohol)This has the same physical properties as ethanol. Dena-tured alcohol consists of ethanol, with the addition of methanol (about 1%), isopropyl alcohol, or a combina-tion of alcohols. For purposes of tissue processing it is used in the same manner as ethanol.

MethanolThis is a clear, colorless and fl ammable fl 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.

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, fl ammable fl uid, miscible with water, ethanol, and most organic solvents. It is rapid in action, with poor penetration, and causes brit-tleness in tissues if use is prolonged. Acetone removes lipids from tissue during processing.

Additives to dehydrating agentsWhen added to dehydrating agents, phenol acts as a soft-ening agent for hard tissues such as tendon, nail, dense fi 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 glycerol–alcohol mixture.

Universal solventsUniversal solvents dehydrate and clear during tissue processing. Dioxane, tertiary butanol, and tetrahydro-furan 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.

CLEARING

A clearing reagent acts as an intermediary between the dehydration and infi ltration solutions. It should be mis-cible with both solutions. Most clearants are hydrocar-bons 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:

• rapid removal of dehydrating agent

• ease of removal by melted paraffi n

• minimal tissue damage

• fl ammability

• toxicity

• cost.

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

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86 Tissue processing

clearing agent gives an indication of its speed of replace-ment by melted paraffi n. Fluids with a low boiling point are generally more readily replaced. Viscosity infl uences 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 suffi ciently 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 fl ammables used in the laboratory.

Clearing agents suitable for routine use

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

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

ChloroformChloroform 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 chloro-form do not become translucent. It is non-fl ammable but highly toxic, and phosgene gas is given off when chloroform is heated. Often used when processing speci-mens of the central nervous system.

Methyl benzoate and methyl salicylateThese are slow-acting clearing agents and can be used when double embedding techniques are required.

Citrus fruit oils—limonene reagentsLimonene reagents are extracts from orange and lemon rinds; they are non-toxic and miscible with water. Dis-

posal is dependent upon the water treatment center’s standards at the site of the laboratory. Their main disad-vantages 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 dis-cussed in Chapter 2. Every histology laboratory should have a chemical hygiene plan that incorporates specifi c work practices to protect workers from potentially haz-ardous chemicals. Information sheets should be avail-able 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.

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 purifi ed.Advantages include:

• reduced cost

• rapid

• effi cient

• eliminates the need to have chemicals removed by a waste disposal company, and

• environmentally unsafe chemicals are not being sent to land-fi lls for disposal.

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

PARAFFIN WAX

Paraffi n continues to be the most popular infi ltration and embedding medium in the histology laboratory. The tissue is impregnated with wax, which forms a matrix preventing tissue structure distortion during microtomy. Paraffi n 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,

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

Paraffi n wax properties

Paraffi n 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 paraffi n is usually softer, higher melting point paraffi n is usually harder, which can effect microtomy. Melting points range from 40 to 70°C. Heating the paraffi n to a high temperature alters the properties of the wax. To promote good ribboning during microtomy, paraffi n wax of suitable hardness at room temperature should be chosen.

Paraffi n wax additives

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

Embedding tissue in paraffi n 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 fi 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 paraffi n dispenser, a cold plate, and a heated storage area for molds and tissue cassettes. Paraffi n is dispensed automatically from a nozzle into a suitably sized mold. The tissue is oriented in the mold; a cassette is attached, producing a fl at block face with parallel sides. The mold is placed on a small cooling area to allow the paraffi n to solidify. The quick cooling of the wax ensures a small crystalline structure, producing fewer artifacts when sectioning the tissue.

The advantages of using an embedding system are:

• ease of use

• speed

• tissue and holder are fi rmly attached, creating a single unit

• blocks fi led immediately after sectioning

• permanent identifi cation.

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

Quality control

Temperature of all paraffi n dispensers, fl otation water baths, and automated processors are carefully moni-tored and documented. The histology laboratory should have a policy and procedure manual that addresses quality issues and corrective actions.

Orientation of tissues

Specimen orientation during embedding is important for the demonstration of proper morphology. Improper ori-entation 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 resis-tance of the tissue against the knife during sectioning. Most tissue are embedded fl at; the margin of embedding medium around the tissue will assure support of the tissue.

Tissues requiring special orientation include:

• Tubular structures: arteries, veins, fallopian tubes, and vas deferens—cut in cross-section of the lumen.

• Skin, intestine, gallbladder, and other epithelial biopsies—cut 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 biopsies—sections containing both trans-verse and longitudinal planes.

• Multiple pieces of a tissue—oriented side by side with the epithelial surface facing in the same direction.

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88 Tissue processing

AUTOMATED TISSUE PROCESSING

The basic principle for tissue processing requires the exchange of fl uids using a series of solutions for a pre-determined 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 con-stant through-put processors.

Tissue processors

The carousel-type processor (tissue transfer) and the self-contained fl uid exchange systems were the fi rst auto-mated tissue processors used in the histology laboratory. This type of processor transported tissue blocks con-tained in baskets through a series of reagents housed in stationary containers. The length of time the specimens were submerged in each reagent container was electron-ically programmed. Earlier models accomplished this step by notching the face of a clock disc. Vertical oscilla-tion 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 micro-processor is used to program the instrument. Tissues are loaded into a retort chamber where they remain through-out the process. Reagents and melted paraffi n 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 process-ing produced, fl uid spillage contained, and fumes eliminated. These processors employ alarm systems and diagnostic programs for trouble-shooting instrumenta-tion malfunction.

Specially designed microwave ovens for tissue pro-cessing 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 tempera-ture 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 paraffi n. Graded concentration of solutions is not required. Clearing agents are not nec-essary because the temperature of the fi nal paraffi n 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 con-trolled processing provides uncompromised morphology and antigenicity of the specimens. Increased effi ciency through improved turnaround times, environmentally friendly reagents, and greater profi tability due to reduc-tion 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 man-ually manipulated, the cost of laboratory-grade micro-waves 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 devel-opment of an enclosed processor, called a continuous input, rapid tissue processor, that uses microwave tech-nology, vacuum infi ltration, and proprietary reagents described as being ‘molecular-friendly’. The tissue cas-settes are moved through four stations that contain acetone, isopropanol, polyethylene glycol, mineral oil, and paraffi n. Microwaves and agitation are used to accelerate the diffusion of solvents in the tissue. A pat-ented 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 determina-tion of this policy. Solutions should be carefully monitored to ensure quality. Every manufacturer has a handbook outlining a preventive maintenance schedule.

Important maintenance tips

• Any spillage or overfl ow should be wiped away immediately

• Accumulation of wax on any surface should be removed

• Temperature of the paraffi n bath should be set to 3°C above the melting point of the paraffi n

• Timing should be checked when placing tissue cassettes in the processor, especially when delayed schedules are selected.

Advantages of the newer technology in processing:

• Custom programs specifi c to tissues being pro-cessed; addition of vacuum, agitation, or heat at any stage

• Rapid schedules

• Fluid and fume containment

• Environmentally friendly reagents

• Delay schedules.

Automated processing schedules

Overnight schedules for tissue processing are still popular in laboratories, but schedules have changed to refl ect the emphasis on reducing turnaround time for the speci-men. Rapid processing for small biopsies or stat speci-mens is easily accommodated.

Overnight processingFor many laboratories, this is considered the routine processing schedule. Tissues continue fi 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 infi ltration in paraffi n.

Schedules are customized for the tissues being pro-cessed; factors infl uencing the processing schedule include the end-time required, reagents used, the inclu-sion of heat and vacuum, the size and number of tissues. The schedule in Table 6.1 can be modifi ed, adjusting times from the various stations, keeping in mind the end-time needed for completion and prior fi xation.

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 Overnight processing

Station Reagents Time Pressure/Vacuum Temp

1 10% Formalin 1 h On 38° C 2 10% Formalin 1 h On 38°C 3 50% Alcohol/formalin 1 h On 38°C 4 70% Alcohol 30 min On 38°C 5 95% Alcohol 30 min On 38°C 6 95% Alcohol 40 min On 38°C 7 100% Alcohol 40 min On 38°C 8 100% Alcohol 40 min On 38°C 9 Xylene 40 min On 38°C10 Xylene 40 min On 38°C11 Paraffi n 30 min On 60°C12 Paraffi n 20 min On 60°C13 Paraffi n 20 min On 60°C14 Paraffi n 40 min On 60°C

Automated tissue processing 89

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90 Tissue processing

Schedule for processing eyesEyes require special processing; this is dictated by the delicate nature of some parts of the structures and tough-ness of others. Ideally, a separate processor should be dedicated for tissues that require special handling because of the reagents used. The eye must be thor-oughly fi xed, prior to dissection and subsequent pro-cessing. 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 shrink-age), keeping the retina attached. Large tissue cassettes and molds are specifi cally made for use in processing eyes.

Rapid processing schedules for small biopsiesRecently excised endoscopic biopsies and needle biopsies can be adequately processed in 2–5 hours using heat (37–45°C) and vacuum. Tissues requiring dissection should be trimmed to 2 mm in thickness. Most small specimens will fi x prior to processing. If fi xation is not complete, processing should begin in a station contain-ing 10% formalin. Table 6.3 shows an example of a shortened process for an enclosed processor. The enclosed processor drain time is approximately 3–5 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

lines of any residual paraffi n. The clean cycle will fl 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 man-ually 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 schedulesThe schedule in Table 6.4 is adaptable for large, dense tissue blocks. Times should be extended in each con-tainer, or more containers may be added to the schedule.

Table 6.2 Processing eyes

Station Reagents Time

1 10% Formalin 0 h 2 4% Phenol/70% Alcohol 1 h 3 4% Phenol/70% Alcohol 1 h 4 95% Alcohol 1 h 5 95% Alcohol 1 h 6 100% Alcohol 1.5 h 7 100% Alcohol 1.5 h 8 100% Alcohol/Chloroform 2 h 9 Chloroform 2 h10 Chloroform 2 h11 Paraffi n 2 h12 Paraffi n 3 h

Table 6.3 Short processing schedule for biopsies

Station Reagents Time

Pressure/

Vacuum Temp

1 10% Formalin 10 min On 38°C 2 10% Formalin 10 min On 38°C 3 70% Alcohol 10 min On 38°C 4 95% Alcohol 10 min On 38°C 5 95% Alcohol 10 min On 38°C 6 100% Alcohol 10 min On 38°C 7 100% Alcohol 10 min On 38°C 8 Xylene 10 min On 38°C 9 Xylene 10 min On 38°C10 Paraffi n 10 min On 38°C11 Paraffi n 10 min On 58°C

Table 6.4 Manual tissue processing for small

biopsies

Step Time

10% Formalin 10 min95% Alcohol 10 min100% Alcohol 10 minXylene 10 minParaffi n 10 min

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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 paraffi n.5. Embed as usual.

ALTERNATIVE EMBEDDING MEDIA

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

• Processing reagents remove or destroy tissue com-ponents, the object of investigation

• Sections are required to be thinner

• The use of heat may adversely affect tissue

• The infi ltrating medium is not suffi ciently hard to support the tissue.

ResinResin is used exclusively as the embedding medium for electron microscopy (see Chapter 30), ultra-thin section-ing for high resolution and for undecalcifi ed bone (see Chapters 18 and 29).

AgarAgar gel alone does not provide suffi cient support for sectioning of tissues. Its main use is as a cohesive agent for small friable pieces of tissue after fi xation. Frag-ments of tissue are embedded in melted agar, allowed to solidify, and trimmed for routine processing. One method providing superior results is as follows: fi lter the fi xative with the tissue fragments through a Millipore fi lter using suction, carefully pour melted agar into the tube, allow solidifi cation of the agar, and follow with routine processing and embedding in paraffi n.

GelatinGelatin is used primarily in the production of sections of whole organs in the Gough–Wentworth technique and in frozen sectioning.

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

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 paraffi n impregnation. The tissue will never be regarded as normal, but the follow-ing treatment may help provide slides of diagnostic quality.

Tissue restoration

70% ethanol 70 mlGlycerol 30 mlDithionite 1 g

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

Summary

Technological advances have been made in the instru-mentation 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 environ-mentally friendly chemicals are only a few of the improvements that will eventually revolutionize tissue processing.

Acknowledgments

This chapter is a development of the chapter that appeared in the fi rst fi 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 acknowledg-ments go to the previous contributors.

REFERENCESCarson F.L. (1977) Histotechnology, a self-instructional

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Dapson J.C., Dapson R.W. (2005) Hazardous materials in the histopathology laboratory, regulations, risks, han-dling and disposal, 4th edn. Battle Creek, MI: Anatech, pp. 157–164.

Kok L.P., Boon M.E. (1992) Microwave cookbook of micros-copists, 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. 1–66.

Morales A.R., Nassiri M., Kanhoush R. et al. (2004) Experi-ence with an automated microwave-assisted rapid tissue processing method: validation of histologic quality and

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Sheehan D.C., Hrapchak B. (1980) Theory and practice of histotechnology, 2nd edn. St Louis: C.V. Mosby, pp. 59–85.

Vernon S.E. (2005) Continuous throughput rapid tissue processing revolutionizes histopathology workfl ow. Laboratory Medicine 36:300–302.

Willis D., Minshew J. (2005) The whole enchilada with the rice and beans. Ft. Lauderdale: National Society for Histotechnology.

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