You are on page 1of 10


Tissue Processing
Lena T. Spencer and John D. Bancroft

After the removal of a tissue sample from the patient, a series of processes must take place to ensure the final microscope slides are of a diagnostic quality. Tissues are exposed to a series of reagents that fix, dehydrate, clear, and infiltrate, with final 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 final 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 identification of the tissue blocks and slides during processing, diagnosis, and filing.

Completion of fixation before processing
Fixation is the most important step in the processing of the tissue sample. If fixation is not complete prior to processing, stations should be designated on the processor for this purpose. If tissue is inadequately fixed, 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 fixation 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 overfill the cassette during gross dissection, impeding the flow 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 identification 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-fixation treatment
Special fixation techniques may require additional steps before processing is initiated. Picric acid fixatives form water-soluble picrates making it necessary to place the tissue cassettes directly into 70% alcohol for processing. Alcoholic fixatives, such as Carnoy’s fluid, 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


making the tissue components receptive to the infiltrating medium infiltrating: permeating the tissue with a support medium embedding: orienting the tissue sample in a support medium and allowing it to solidify. hardening. higher temperatures may be deleterious to subsequent immunohistochemical staining. Embedding mediums have varying viscosities. Vacuum used on the automated processor should not exceed 15 inches of Hg (mercury) to prevent damage and deterioration to the tissue. Factors influencing the rate of processing When tissue is immersed in fluid. influence the rate at which interchange occurs. replacing it with a support medium that provides sufficient rigidity to enable sectioning of the tissue without damage or distortion. Fixation stabilizes proteins. Conversely. enhancing the rapidity of the impregnation. Agitation increases the flow of fresh solutions around the tissue. Agitation The rate of fluid exchange is dependent upon the exposed surface of the tissue that is in contact with the processing reagent. dehydrates and clearants. and changes the tissues receptiveness to further processing. if the molecular size is larger. The smaller the size of the molecules in the solution. . Paraffin has a lower viscosity in the fluid (melted) state. Vacuum will remove reagents from the tissue only if they are more volatile than the reagent it is replacing. the faster the rate of fluid penetration (low viscosity). rendering the cell and its components resistant to further autolysis by inactivating lysosomal enzymes. It must be used sparingly to reduce the possibility of shrinkage. Vacuum can aid in the removal of trapped air in porous tissue. Temperatures limited to 45°C can be used effectively. fatty tissue can be greatly reduced with the addition of vacuum during processing. have similar viscosities. Impregnation time of dense. Many dehydrating reagents are hydrophilic (‘water loving’). Viscosity Viscosity is the property of resistance to the flow of a fluid. Automated processors incorporate vertical or rotary oscillation or pressurized removal and replacement of fluids at timed intervals as the mechanism for agitation. Efficient agitation can reduce the overall processing time by 30%. with the exception of cedar wood oil. Other reagents affect dehydration by repeated dilution of the aqueous tissue fluids. discussed below. Dehydration should be accomplished slowly. DEHYDRATION The first stage of processing is the removal of unbound water and aqueous fixatives from the tissue components. Vacuum Using reduced pressure to increase the rate of infiltration decreases the time necessary to complete each step in the processing of tissue samples. the rate of exchange is slower (high viscosity). Fixation must be complete before subsequent steps in the processing schedule are initiated. Most of the solutions used in processing. If the concentration gradient between the fluid Heat Heat increases the rate of penetration and fluid exchange. 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. but washes out during subsequent staining. and brittleness of the tissue.84 Tissue processing coloration of the tissue remains during processing. Stages of tissue processing are: • • • • dehydration: removal of water and fixative from the tissue clearing: removal of dehydrating solutions. PRINCIPLES OF TISSUE PROCESSING Tissue processing is designed to remove all extractable water from the tissue. interchange occurs between the fluid within the tissue and the surrounding fluid. possessing strong polar groups that interact with the water molecules in the tissue. Several factors.

Propan-2-ol. There are numerous dehydrating agents: ethanol. Most clearing agents are flammable liquids. The criteria for choosing a suitable clearing agent are: Methanol This is a clear. It is hydrophilic. making it the reagent of choice for the processing of electron microscopy specimens. flammable fluid. it is miscible with water. isopropyl alcohol CH3CHOHCH3 Isopropyl alcohol is miscible with water. diffusion currents cross the cell membranes during fluid exchange. ethanol acetone. leaving the specimen soft and non-receptive to infiltration. Isopropyl alcohol does not cause over-hardening or shrinkage of the tissue. colorless. it is a slow dehydrant with less shrinkage and hardening of the tissue. controlled by the federal government. increasing the possibility of cell distortion. Most clearants are hydrocarbons with refractive indices similar to protein. Universal solvents Universal solvents dehydrate and clear during tissue processing. which warrants caution in their use. dense fibrous tissue. For safety issues see Chapter 2. Denatured alcohol consists of ethanol. It can be substituted for ethanol. hard tissue can be immersed in a glycerol–alcohol mixture. Ethanol ensures total dehydration. colorless. hence the term ‘clearing agent’. and most organic solvents. It should be miscible with both solutions. For purposes of tissue processing it is used in the same manner as ethanol. Graded concentrations of ethanol are used for dehydration. phenol acts as a softening agent for hard tissues such as tendon. and denatured alcohols. Sheehan & Hrapchak 1980). Excessive dehydration may cause the tissue to become hard. For this reason specimens are always processed through a graded series of reagents of increasing concentration. When the dehydrating agent has been entirely replaced by most of these solvents the tissue has a translucent appearance. ethanol. colorless and flammable fluid which is highly toxic. and most organic solvents. flammable liquid. Dioxane. and requires careful record keeping. If the dehydrant of choice is ethanol. For delicate tissue it is recommended that the processing start in 30% ethanol. or a combination of alcohols. ethanol. Acetone removes lipids from tissue during processing. and most organic solvents.Clearing 85 inside and outside the tissue is excessive. and tetrahydrofuran are considered to be universal solvents. Dehydrating fluids Ethanol C2H5OH This is a clear. ethanol. Alternatively. brittle. CLEARING A clearing reagent acts as an intermediary between the dehydration and infiltration solutions. miscible with water. It is rapid in action. The boiling point of the . followed by 95% and 100% solutions. with poor penetration. It is often used in microwave processing schedules. Industrial methylated spirit (denatured alcohol) This has the same physical properties as ethanol. they are not recommended for processing delicate tissues due to their hardening properties (Carson 1977. the tissue is first immersed in 70% ethanol in water. • • • • • • rapid removal of dehydrating agent ease of removal by melted paraffin minimal tissue damage flammability toxicity cost. A 4% solution is added to each of the 95% ethanol stations. miscible with water and other organic solvents. fast acting. methanol. with the addition of methanol (about 1%). and shrunken. Acetone CH3COCH3 Acetone is a clear. isopropyl. Incomplete dehydration will prohibit the penetration of the clearing reagents into the tissue. Additives to dehydrating agents When added to dehydrating agents. and causes brittleness in tissues if use is prolonged. nail. and reliable. Ethanol is taxable. and keratin masses. tertiary butanol. isopropyl alcohol. glycol. Butyl alcohol (butanol) This is used primarily for plant and animal histology.

Prolonged exposure to most clearing agents causes the tissue to become brittle. tissue blocks are not damaged (Carson 1977. disposal. Luna 1992). Citrus fruit oils—limonene reagents Limonene reagents are extracts from orange and lemon rinds. colorless. which forms a matrix preventing tissue structure distortion during microtomy. The recycled reagents must be tested for quality (Dapson & Dapson 2005).86 Tissue processing clearing agent gives an indication of its speed of replacement by melted paraffin. These sheets are placed in an easily accessible place for quick reference. and phosgene gas is given off when chloroform is heated. Paraffin wax has a wide range of melting points. PARAFFIN WAX Paraffin continues to be the most popular infiltration and embedding medium in the histology laboratory. provides quality sections. they are non-toxic and miscible with water. greater than 1 mm in thickness. Most institutions have a policy for the storage. Safety Safe handling of common histological chemicals is discussed in Chapter 2. It is inexpensive. which is important for use in the different climatic regions of the world. and how to handle spills. 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. Clearing agents suitable for routine use Xylene A flammable. Thicker tissue blocks can be processed. although it is less damaging with prolonged immersion of tissue. Over-exposure during processing will cause over-hardening. and safety requirements for all flammables used in the laboratory. Since most clearing agents are aromatic hydrocarbons or short-chain aliphatic hydrocarbons. Advantages include: Toluene Similar properties to xylene. The basic information includes exposure limits. the component with the highest boiling point is purified. Tissues placed in chloroform do not become translucent. Their main disadvantages are a strong pungent odor and small tissue deposits of minerals such as copper or calcium. Viscosity influences the speed of penetration of the clearing agent. which may dissolve. disposal. Suitable for blocks that are less than 5 mm in thickness. The tissue is impregnated with wax. Xylene is commonly used in routine histology laboratories and is recyclable. The time in the clearing agent should be closely monitored to ensure that dense tissue blocks are sufficiently cleared and smaller. • • • • • reduced cost rapid efficient eliminates the need to have chemicals removed by a waste disposal company. Methyl benzoate and methyl salicylate These are slow-acting clearing agents and can be used when double embedding techniques are required. Cost should be considered. It is flammable and more volatile than xylene. It is non-flammable but highly toxic. more fragile. Chloroform Chloroform is slower in action than xylene but causes less brittleness. Sheehan & Hrapchak 1980. environmental issues have to be addressed. Dis- . and environmentally unsafe chemicals are not being sent to land-fills for disposal. liquid with a characteristic petroleum or aromatic odor. especially as it relates to disposal of the reagent. Information sheets should be available for all the chemicals used in the laboratory. Fluids with a low boiling point are generally more readily replaced. storage. target organs. Often used when processing specimens of the central nervous system. posal is dependent upon the water treatment center’s standards at the site of the laboratory. miscible with most organic solvents and paraffin. Every histology laboratory should have a chemical hygiene plan that incorporates specific work practices to protect workers from potentially hazardous chemicals.

supporting cellular components. Multiple pieces of a tissue—oriented side by side with the epithelial surface facing in the same direction. and diethylene glycol distearate. ceresin. Many of these additives have a higher melting point than paraffin wax. and automated processors are carefully monitored and documented. biopsy bags. and vas deferens—cut in cross-section of the lumen. • • • • Tubular structures: arteries. consequently making the tissue more brittle. which can effect microtomy. plastic polymers. and other epithelial biopsies—cut in a plane at right angles to the surface. Muscle biopsies—sections containing both transverse and longitudinal planes. producing fewer artifacts when sectioning the tissue. Paraffin wax additives Paraffin waxes that contain plasticizers or other resin additives are commercially available. the margin of embedding medium around the tissue will assure support of the tissue. and papers. Melting points range from 40 to 70°C. gallbladder. Heating the paraffin to a high temperature alters the properties of the wax. Its properties are varied depending on the melting point. Quality control Temperature of all paraffin dispensers. Paraffin is dispensed automatically from a nozzle into a suitably sized mold. The mold is placed on a small cooling area to allow the paraffin to solidify. sponges. paraffin wax of suitable hardness at room temperature should be chosen. fallopian tubes. Improper orientation may result in diagnostic tissue elements being damaged during microscopy (see Chapter 5). Most tissue are embedded flat. Products are available that help ensure proper orientation: marking systems. Orientation of the tissue should offer the least resistance of the tissue against the knife during sectioning. To promote good ribboning during microtomy. Substances that were added to paraffin in the past included beeswax. It should provide elasticity. higher melting point paraffin is usually harder. consisting of three modules: a paraffin dispenser. It can be used for most routine and special stains. The histology laboratory should have a policy and procedure manual that addresses quality issues and corrective actions. low melting point paraffin is usually softer. Cassettes and molds that accommodate larger or smaller specimens may be purchased from scientific supply companies. providing a selection that is appropriate for most laboratories. Tissues requiring special orientation include: Embedding tissue in paraffin wax Embedding involves the enclosing of properly processed. producing a flat block face with parallel sides. Most laboratories use embedding centers. The tissue is oriented in the mold. tattoo dyes. flotation water baths. and a heated storage area for molds and tissue cassettes. • • • • • ease of use speed tissue and holder are firmly attached. The advantages of using an embedding system are: Paraffin wax properties Paraffin wax is a mixture of long-chain hydrocarbons produced in the cracking of mineral oil. creating a single unit blocks filed immediately after sectioning permanent identification. Orientation of tissues Specimen orientation during embedding is important for the demonstration of proper morphology. The quick cooling of the wax ensures a small crystalline structure. veins. resisting section distortion while facilitating sectioning.Paraffin wax 87 and is easily adaptable to a variety of uses. intestine. The embedding medium must fill all the spaces within the tissue. correctly oriented specimens in a support medium that provides external support during microscopy. and oriented so the epithelial surface is cut last. These mixtures create paraffin with the desired hardness for the tissue to be embedded. Skin. rubber. . a cold plate. a cassette is attached. minimizing compression and distortion of the epithelial layer.

The tissue cassettes are moved through four stations that contain acetone. Willis & Minshew 2005). and paraffin. Disadvantages of the system are: the process is labor intensive because the solutions are manually manipulated. A patented microwave technology is utilized operating at a continuous low power instead of pulsing high levels of microwave energy. The length of time the specimens were submerged in each reagent container was electronically programmed. The time for processing is dependent on the thickness and density of the specimen. Microwaves and agitation are used to accelerate the diffusion of solvents in the tissue. and proper use of the microwave oven requires calibration and monitoring (Kok & Boon 1992. Recent advances in technology have led to the development of an enclosed processor. This type of processor transported tissue blocks contained in baskets through a series of reagents housed in stationary containers. The microwave oven shortens the processing time from hours to minutes. Xylene and formalin are not used in this process. types of tissue processed . Earlier models accomplished this step by notching the face of a clock disc. isopropanol or proprietary mixtures of alcohol. Disadvantages include the cost of the processor and that grossing of the tissue sample requires standardized specimen dissection (Morales et al 2004). and greater profitability due to reduction in number and volume of reagents. Reagents used for microwave processing include ethanol. isopropanol. For decades instrumentation used in tissue processing was relatively unchanged. that uses microwave technology. accelerating the reaction time. Recent advances now include specialty microwave ovens and the emergence of constant through-put processors. and paraffin. and fume extraction systems. 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. rapid tissue processor.88 Tissue processing AUTOMATED TISSUE PROCESSING The basic principle for tissue processing requires the exchange of fluids using a series of solutions for a predetermined length of time in a controlled environment. timers. Microwave exposure stimulates the diffusion of the solutions into the tissue by increasing the internal heat of the specimen. sizes. vacuum infiltration. are advantages of this system. The numbers. environmentally friendly reagents. The morphology and quality of the specimens is consistent with that of traditional tissue processing. Graded concentration of solutions is not required. These processors employ alarm systems and diagnostic programs for trouble-shooting instrumentation malfunction. The enclosed. Each step is customized by adding time. Specially designed microwave ovens for tissue processing are now common. polyethylene glycol. Increased efficiency through improved turnaround times. A microprocessor is used to program the instrument. called a continuous input. The advantages of this system are that vacuum and heat can be applied at any stage. Tissues are manually transferred from container to container of reagent. and proprietary reagents described as being ‘molecular-friendly’. eliminating toxic vapors in the laboratory. or vacuum/pressure. and fumes eliminated. self-contained vacuum tissue processor later became the mainstay in most laboratories. customized schedules for tissue processing produced. The reagents used are environmentally safe. mineral oil. The advantage of this system is the acceptance of tissues into the system at timed intervals. which eliminates toxic fumes and carcinogens. Reagents and melted paraffin are moved sequentially into and out of the retort chamber using vacuum and pressure. temperature. Tissues are loaded into a retort chamber where they remain throughout the process. fluid spillage contained. Tissue processors The carousel-type processor (tissue transfer) and the selfcontained fluid exchange systems were the first automated tissue processors used in the histology laboratory. the cost of laboratory-grade microwaves may be prohibitive. Clearing agents are not necessary because the temperature of the final paraffin step facilitates evaporation of the alcohols from the tissue. Most laboratory microwave ovens contain precise temperature controls. Properly controlled processing provides uncompromised morphology and antigenicity of the specimens. improving turnaround time. Processor maintenance Every institution should have a policy outlining the rotation and changing of solutions for each tissue processor.

Solutions should be carefully monitored to ensure quality.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 Paraffin Paraffin Paraffin Paraffin 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 38°C 38°C 38°C 38°C 38°C 38°C 38°C 38°C 38°C 60°C 60°C 60°C 60°C . followed by infiltration in paraffin. or a xylene substitute. addition of vacuum. Table 6. Advantages of the newer technology in processing: • • • • • Custom programs specific to tissues being processed. A schedule for eyes is shown in Table 6. the inclusion of heat and vacuum. Tissues continue fixation by being submerged in 10% formalin. Important maintenance tips • • • • Any spillage or overflow should be wiped away immediately Accumulation of wax on any surface should be removed Temperature of the paraffin bath should be set to 3°C above the melting point of the paraffin Timing should be checked when placing tissue cassettes in the processor. Rapid processing for small biopsies or stat specimens is easily accommodated. agitation. adjusting times from the various stations. Schedules are customized for the tissues being processed. reagents used.Automated tissue processing 89 and the reagents used will play a role in the determination of this policy. xylene. Automated processing schedules Overnight schedules for tissue processing are still popular in laboratories. this is considered the routine processing schedule. factors influencing the processing schedule include the end-time required. The process may include alcoholic formalin. buffered or unbuffered. the size and number of tissues. Overnight processing For many laboratories. especially when delayed schedules are selected. Specialized tissues Tissues such as brain. Every manufacturer has a handbook outlining a preventive maintenance schedule. but schedules have changed to reflect the emphasis on reducing turnaround time for the specimen. keeping in mind the endtime needed for completion and prior fixation. The schedule in Table 6. Chapters 18 and 29 for bone). varying concentrations of alcohol. eyes.1 can be modified.2. or heat at any stage Rapid schedules Fluid and fume containment Environmentally friendly reagents Delay schedules. and bone require specialized processing (see Chapter 19 for brain.

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.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 38°C 38°C 38°C 38°C 38°C 38°C 38°C 38°C 38°C 38°C 58°C 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 Paraffin Paraffin Table 6. or more containers may be added to the schedule. If fixation is not complete.3 shows an example of a shortened process for an enclosed processor. changing times at various stations. The program can be amended.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 Paraffin Paraffin Time 0h 1h 1h 1h 1h 1. 100% alcohol.5 h 2h 2h 2h 2h 3h Table 6. After each run the instrument must be cleaned to purge the The schedule in Table 6.4 is adaptable for large. Manual processing schedules Rapid processing schedules for small biopsies Recently excised endoscopic biopsies and needle biopsies can be adequately processed in 2–5 hours using heat (37–45°C) and vacuum.5 h 1. this is dictated by the delicate nature of some parts of the structures and toughness of others. Most small specimens will fix prior to processing. Ideally. Large tissue cassettes and molds are specifically made for use in processing eyes.90 Tissue processing Schedule for processing eyes Eyes require special processing. a separate processor should be dedicated for tissues that require special handling because of the reagents used. 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). prior to dissection and subsequent processing. The enclosed processor drain time is approximately 3–5 minutes at each station. drain times should be taken into consideration when determining the end time. Tissues requiring dissection should be trimmed to 2 mm in thickness. Phenol is added to the lower percentage alcohols to soften the sclera and lens. lines of any residual paraffin. MANUAL TISSUE PROCESSING Manual tissue processing is rarely used today. processing should begin in a station containing 10% formalin.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 Paraffin . Table 6. keeping the retina attached. and water. dense tissue blocks. Table 6. The clean cycle will flush the lines using xylene. needing a rapid diagnosis. Times should be extended in each container. The eye must be thoroughly fixed.

in part due to increased workload. Remove tissue cassette from formalin and place in container with 95% alcohol. It is included here for historical purposes only. 2. Acknowledgments This chapter is a development of the chapter that appeared in the first five editions. One method providing superior results is as follows: filter the fixative with the tissue fragments through a Millipore filter using suction. and shortages in the workforce. 5. 4. allowed to solidify. the demand for faster turnaround time for diagnostic samples. resulting in tissue drying out prior to paraffin impregnation. Fragments of tissue are embedded in melted agar. and environmentally friendly chemicals are only a few of the improvements that will eventually revolutionize tissue processing. with successful merging for later editions by Graeme Anderson and John Bancroft. The addition of microprocessors. Chicago: ASCP Press. Resin Resin is used exclusively as the embedding medium for electron microscopy (see Chapter 30). 26–42. Gelatin Gelatin is used primarily in the production of sections of whole organs in the Gough–Wentworth technique and in frozen sectioning. Tissue may be difficult to section. and follow with routine processing and embedding in paraffin.L. Drop cassette in 10% formalin. In those editions. Summary Technological advances have been made in the instrumentation of tissue processors. the object of investigation Sections are required to be thinner The use of heat may adversely affect tissue The infiltrating medium is not sufficiently hard to support the tissue. Continue through 100% alcohol and xylene using stir plate and stir bar. Place tissue in cassette. microwaves. ultra-thin sectioning for high resolution and for undecalcified bone (see Chapters 18 and 29). Processing begins with the dehydrating solutions and continues to completion. but the following treatment may help provide slides of diagnostic quality. Agar Agar gel alone does not provide sufficient support for sectioning of tissues. ALTERNATIVE EMBEDDING MEDIA There are occasions when paraffin is an unsuitable medium for the type of section required. 2nd edn. Celloidin The use of celloidin or LVN (low viscosity nitrocellulose) is discouraged because of the special requirements REFERENCES Carson F. carefully pour melted agar into the tube. Embed as usual. (1977) Histotechnology. Formalin container placed under warm tap water. pp. and trimmed for routine processing. Place tissue cassette in melted paraffin. allow solidification of the agar. including: Tissue restoration 70% ethanol Glycerol Dithionite 70 ml 30 ml 1g • • • • Processing reagents remove or destroy tissue components. Tissues remain in the solution for several hours or overnight. a self-instructional text.References 91 1. on a stir plate with a stir bar. . Our acknowledgments go to the previous contributors. Restoration of tissue dried in processing Despite precautions taken during processing. The tissue will never be regarded as normal. 3. Its main use is as a cohesive agent for small friable pieces of tissue after fixation. Tissue processing was written by Keith Gordon and Paul Bradbury. technical or mechanical malfunctions may occur. coated or plus slides should be used. needed to house the processing reagents and the limited use these types of section have in neuropathology.

handling and disposal.G. Sheehan D. Luna L.C. Nassiri M. Morales A. American Journal of Clinical Pathology 121:528–536. pp. (1992) Microwave cookbook of microscopists. (2005) Continuous throughput rapid tissue processing revolutionizes histopathology workflow. Laboratory Medicine 36:300–302. risks.E. 3rd edn. .P..W. Dapson R. et al. (2005) The whole enchilada with the rice and beans. pp.. 2nd edn.. regulations. Willis D.C. (2005) Hazardous materials in the histopathology laboratory. (2004) Experience with an automated microwave-assisted rapid tissue processing method: validation of histologic quality and impact on the timeliness of diagnostic surgical pathology. 59–85. (1980) Theory and practice of histotechnology.V. 4th edn.. Kanhoush R. 1–66. Battle Creek. 157–164.E. pp. MI: Anatech.. Vernon S. Kok L. Mosby. Lauderdale: National Society for Histotechnology.R. Leiden: Coulomb Press. Hrapchak B. (1992) Histopathologic methods and color atlas of special stains and tissue artifacts. Downers Grove: Johnson Printers.92 Tissue processing Dapson J. St Louis: C.. Ft. Minshew J. Boon M.