Tissue processingLeigh Winsor

INTRODUCTIONStabilised tissues must be adequately supported before they can be sectioned for microscopical examination. Whilst they may be sectioned following a range of preparatory freezing methods, tissues are more commonly taken through a series of reagents and finally infiltrated and embedded in a stable medium which when hard, provides the necessary support for microtomy. This treatment is termed tissue processing. Methods have evolved for a range of embedding media and applications (Table.1). Pre-eminent amongst these is the paraffin wax method, discussed here in detail, which is considered to be the most suitable for routine preparation, sectioning, staining and subsequent storage of large numbers of tissue samples.

The quality of structural preservation seen in the final stained and mounted section is largely determined by the choice of fixative and embedding medium. During tissue processing loss of cellular constituents and shrinkage or distortion should be minimal. After fixation, post-fixation and preparatory procedures, the four main stages in the paraffin method are dehydration, clearing, infiltration and embedding.

Tissue sampling and identificationTissue sampling generally follows standard protocols1,2 established by each laboratory for particular species and categories of specimens. Tissue blocks for processing should be as thin as is consistent with the purpose for which they are required, usually 1-2 mm thick for urgent specimens and rapid processing; 3-5 mm for routine material processed overnight. Specimens should not be tightly packed into processing cassettes or containers, but should have sufficient free space to facilitate fluid exchange. Small specimens and tissue fragments are processed in fine mesh containers, wrapped in lens tissue, sandwiched between sponge biopsy pads or more safely, double embedded in agar-paraffin wax.

Specimens are generally identified by a numbering system that is not bleached by subsequent fluid and solvent treatment. Examples include (a) a numbered card label generated by computer-printer, or handwritten in soft lead pencil or waterproof ink (b) colour coded plastic cassettes, machine or manually labelled.

Tissue marking and orientationMarking facilitates identification and correct orientation of particular tissue pieces or surfaces during embedding and subsequent microscopical examination. Tissue blocks are simply marked by cutting a notch on the reverse side of the block face to be sectioned, or by trimming the block to a particular shape. However dye marking is preferred for certain surgical specimens, small tissue pieces, and for serial sectioning orientation.

TISSUE MARKING SUBSTANCESCriteria3 for the selection of a suitable tissue marker are:

the marking substance must be relatively insoluble in fixative, processing reagents and embedding medium.

it must survive fixation and processing and not result in unacceptable contamination of the reagents and other tissues processed simultaneously.

it must remain on the surface of the specimen and not penetrate tissue. it should not react unfavourably with histological stains and must be clearly

identifiable both macroscopically and microscopically. for some purposes it may be important that the marker is either radiolucent or

radio-opaque.

Tissue markers are applied to the surface of the specimen using disposable swabs and allowed to dry.

India ink provides good black macro and microscopic marking, is resistant to processing, but takes 15-30 minutes to dry, and may spread beyond the marked area. Silver and gold inks are not recommended as they are solvent soluble4.

Silver nitrate (stick) provides a brown-black mark resistant to processing. Aqueous or alcoholic silver nitrate solutions behave like India ink and are not recommended.

Artists' grade pigments are radio-opaque, processing resistant and provide good macro and microscopic contrast. Prepare by finely grinding pigment (50% w/v) to a thin paste in acetone4,and store in tightly stoppered containers. These markers dry in 15-30 minutes.

Particulate pigments, 8% pigment w/v in 24% gelatine solution3, dry in less than 5 minutes, or in about 10 seconds on chilled specimens. Paprika, turmeric, henna, India ink, and Bismark brown are all inexpensive, strongly coloured processing-resistant pigments with distinctive microscopic particle morphology.

Alcian blue, 1% aqueous solution, is a rapid and reliable stain for marking resection margins of fixed breast5 and other biopsies. The specimen is dipped into the stain for a few seconds then blotted dry. Sufficient dye remains to mark resection margins.

Eosin, Erythrosin and Rose Bengal, 1-2% aqueous, are used to stain small translucent specimens. Tissues are stained for 5 minutes, rinsed in water then processed. Although some dye is lost in the dehydration alcohols, sufficient remains to render the tissues visible. Alternatively dye is incorporated in the 95% ethanol dehydrant, and tissues stained during the routine dehydration step.

Tissue marking dyes are available commercially and have been favourably evaluated6.

Completion of fixationTissues should be fixed before processing is initiated. Poorly fixed tissues are inadequately protected against the physical and chemical rigours of processing. Strategies commonly employed to ensure complete fixation of tissues include:

microwave irradiation of biopsy specimens in normal saline.7-9

continuing fixation on the tissue processor with one or more changes of the routine fixative, often at elevated temperatures of 40°C-60°C.

secondary fixation of tissues in formol sublimate on the tissue processor10, or in an alcoholic fixative which will complete fixation whilst initiating dehydration.

fixing in buffered phenol-formaldehyde pH 7.0 and pH 5.5 sequence at 40°C11.

Post fixation proceduresOn completion of fixation, tissues fixed in certain reagents must undergo special treatment.

Fixatives containing dichromate and chromium trioxideDichromates and chromium trioxide are reduced to insoluble green-brown chromic oxide in the higher alcohols and in dioxane. Tissues must be washed for 8-12 hours in running water before transferring to 60%-70% ethanol or dioxane.

Fixatives containing phosphatePhosphate salts precipitate in alcoholic solutions stronger than 70% ethanol, in dimethoxy propane, and in diethoxy propane. If they are deposited within tissues the precipitate can cause sectioning difficulties12. Tissues are rinsed free of fixative with water and processing initiated in 60%-70% ethanol.

Fixatives containing picric acidTissues fixed in non-alcoholic picric acid-based fixatives are washed in repeated 1-3 hourly changes of 50%-70% ethanol until the supernatant is faintly yellowish or clear. This may take 2-3 days. Specimens fixed in alcoholic picric acid fluids are washed in 80%-90% ethanol, as anhydrous conditions must be maintained. Picric acid retained in tissues can impede wax infiltration and exacerbate static electrification of ribbons during sectioning. It also has an adverse affect on stored wax embedded tissues13.

Fixatives containing ureaTissues fixed in urea containing fluids are washed overnight before transfer to 4% formaldehyde solution for storage. Urea complexes with formaldehyde to form insoluble urea-polymer pigments13.

Specific fixative requirementsCarnoy fixed tissues are near-anhydrous and are placed directly in absolute ethanol or in alcohol-transition solvent.

Heidenhain's SUSA fixed tissues are transferred directly to 95% ethanol, as trichloroacetic acid fixed collagen swells in aqueous solutions.

Tissues fixed in osmium tetroxide-based fixatives are washed for 5 hours in running water and dehydration initiated in 30% ethanol. Osmium tetroxide is reduced to black osmium in ethanol.

Principles of tissue processingTissue processing is concerned with the diffusion of various substances into and out of stabilised porous tissues. The diffusion process results from the thermodynamic tendency of processing reagents to equalise concentrations inside and outside blocks of tissue, thus generally conforming to Fick's Law: the rate of solution diffusion through tissues is proportional to the concentration gradient (the difference between the concentrations of the fluids inside and outside the tissue) as a multiple of temperature dependant constants for specific substances.

From this it can be seen that the significant variables in tissue processing are the operating conditions, particularly temperature, the characteristics and concentrations of the reagents and the properties of the tissue.

The tissueTissue porosity has a major impact on processing, subsequent microtomy and staining. Porosity at an ultrastructural level is determined by the nature and composition of the tissues, and the effects of fixatives, modifiers, and processing reagents to which the tissues are subjected. Tissue porosity involves natural and artefactual pores, and the swelling and shrinkage of the biopolymer matrix (Fig. 1)14-15. Even after fixation cell surfaces continue to act as osmotic membranes15. Irrespective of the fixative used, all tissues undergo limited shrinkage and hardening during dehydration, clearing and infiltration15-18 as well as staining and mounting15. Hardening generally results from tissue shrinkage, accompanied in most cases by decreased tissue porosity19. Fatty tissues usually require extended processing as lipids, such as myelin in brain tissues and general body fats, inhibit the diffusion of processing reagents.

FIXATIONIn general there is a tendency for tissues fixed in reagents that cause little initial shrinkage to undergo a greater degree of shrinkage during dehydration. Conversely tissues fixed in substances which cause considerable initial shrinkage, contract less during dehydration17,20,21.

Non-protein coagulant fixatives such as formaldehyde, result in little ultrastructural tissue damage14. However these agents tend to swell tissues15, and generally fail to give adequate protection from shrinkage and hardening during subsequent processing to paraffin wax (Fig. 2)15,17,20-23. Non-fixative salts, such as calcium chloride, incorporated in formaldehyde fixatives further stabilise tissues and as a consequence reduce processing-induced tissue shrinkage15,16.

Protein-coagulant fixatives such as ethanol, mercuric chloride or picric acid, shatter tissue ultrastructure14 but this may increase porosity. Tissues may shrink during fixation but are protected against further significant contraction and hardening during processing (Fig. 2)15,16,17,20-23. Secondary fixation of formaldehyde fixed tissues with coagulant fixative mixtures including formal sublimate, Helly's or Bouin's fluids, enhances the response of these tissues to processing, sectioning and staining10.

Duration of fixation determines the extent of tissue stabilisation and consequently porosity and influences reactivity in histological and immunohistochemical procedures. Underfixed tissues are inadequately protected against processing reagents and exhibit a range of artefacts13, including those associated with secondary fixation by the dehydrant. Prolonged exposure to primary and secondary fixatives during processing may impair tissue reactivity, particularly in immunohistochemical investigations.

SOLVENT EFFECTSLoss of certain substances from tissues during processing may also indirectly affect tissue porosity and result in shrinkage24. Even where the ultrastructure has not undergone disruption, tissue porosity can be increased for example, by the dissolution of lipid-rich structures such as membranes and fat droplets by processing solvents, which can then result in shrinkage25.

Some processing fluids such as glycerol for example, also affect tissues by increasing softness but decreasing porosity19 resulting in protracted clearing and infiltration times thus negating the original softening action. Other reagents including cedarwood oil19 maintain both softness and porosity and facilitate subsequent processing steps.

TISSUE MODIFIERSTissue modifiers such as phenol swell unfixed collagen and elastic fibres14, enhance protein polymer formation in formaldehyde fixed tissues11,26 coagulate proteins, and probably maintain or promote tissue porosity. These phenomena underlie the use of phenol and other surfactants to stabilise and soften hard tissues during fixation11,26-30 and processing11,26,29,31-32.

DENSITY AND THICKNESSVariable tissue density affects infiltration and subsequent microtomy33. Spongy, parenchymatous tissues are usually more rapidly infiltrated than hard and dense tissues. Block thickness also influences the rate of reagent diffusion and hence processing time. Tissue thickness should be optimised for particular processing schedules, or alternatively processing times are adjusted to accommodate thick, thin or large tissue blocks.

Processing reagentsThe chemical and physical properties of reagents which influence processing include polarity, concentration, miscibility with water, solvents and embedding media, evaporation rate, and viscosity (Tables 2, 3). Thermal conductivity, heat capacity, boiling point and the electromagnetic conductivity of reagents are particularly important in microwave-stimulated processing34.

POLARITYTo minimise tissue distortion there should be a gradual change in polarity of the processing fluid from highly polar aqueous fixatives and solutions to the embedding medium which is usually non-polar (hydrophobic)35. Tissues generally shrink when transferred to a fluid of relatively lower polarity35.

CONCENTRATIONIf the concentration gradient between fluid inside and outside the tissue is too high, rapid reagent diffusion and accompanying strong diffusion currents have the potential to shrink and disrupt tissues. For this reason specimens are almost always processed through a graded series of reagents of increasing concentration, the more delicate the tissues, the closer the gradations.

MISCIBILITYProcessing reagents which are miscible with water and with the embedding medium reduce the number of processing stages and are termed universal solvents. To avoid severe tissue shrinkage from concentration and polarity effects, they are often employed in a graded series.

Many transition solvents, for example xylene, are extremely water-intolerant, and are immiscible with hydrated alcohols. Couplers such as phenol mixed with a transition solvent permit clearing from 70%-95% alcohols36. Originally couplers were employed to overcome difficulties with hydrated higher alcohols. However they are now used in processing yolky or blood-filled tissues which harden excessively if fully dehydrated in absolute ethanol, and complement the use of tissue modifiers.

EVAPORATION RATEThe evaporation rate, rather than the vapour pressure or boiling point of a solvent, is the best predictor of the rate of elimination of a substance from molten infiltrating wax. Solvents with high evaporation rates are the most readily vaporised and are less likely to contaminate the infiltration medium.

VISCOSITYViscosity is the internal friction of a particular substance which affects rate of flow through tissues and is inversely proportional to temperature. It is particularly important in the clearing and infiltration stages of processing. Substances with high molecular weight, such as some transition solvents and waxes, have high viscosities and diffuse through tissues more slowly than, for example, the lower molecular weight, lower viscosity dehydrant alcohols.

If tissue shrinkage or swelling is to be avoided when the specimen moves from one processing step to the next, the fluid already in the tissues must diffuse outward through the tissue pores at the same rate as the fresh medium diffuses inwards. If the viscosity differential between fluid inside and outside the tissue is too great, shrinkage will result. Hence slow and gradual processing of tissues is necessary when viscous reagents are used (for example in nitrocellulose embedding methods).

EMBEDDING MEDIAInfiltrating and embedding media must fill all spaces within the tissue to support cellular components adequately during microtomy. Density of the hardened medium should approach that of the densest tissue component otherwise section deformation will result. The matrix must be elastic enough to recover sectioning deformation, and plastic enough

to facilitate thin sectioning30. Tissue-medium adhesion is enhanced if the embedding matrix has a fine uniform crystalline morphology which intimately contacts the tissue. Viscosity and melting point of the infiltration medium partly determine the duration and temperature of processing conditions.

Processing conditionsTemperature, pressure and agitation reduce the duration of tissue processing and improve the quality of infiltration.

TEMPERATUREAt low temperatures structural elements of tissues are stabilised against the destructive effects of solvent changes35. This is possibly because of the stiffening and strengthening effect of cold upon biopolymers resulting from diminution in thermal disruption of secondary bonds of the tissue constituents35. Unfortunately at low temperatures reagent viscosities increase and diffusion rates decrease, resulting in prolonged processing times.

Isothermally processed mammalian tissues show finer detail and less artefacts than those processed by the more practicable, common an-isothermic techniques19. Heat increases the kinetic energy of molecules and rate of diffusion, with a corresponding decrease in solution viscosity. The application of mild heat within the range 37°C to 45°C, during the dehydration and clearing steps considerably reduces processing times18,36, but may concomitantly increase shrinkage21. Tissue shrinkage during infiltration in paraffin wax results mainly from the effect of heat on collagen16.

High infiltration temperatures cause marked tissue shrinkage and hardening17,21 which can be avoided by maintaining embedding waxes 2-3°C above their melting points21. Prolonged immersion in paraffin wax at the correct temperature results in only slight tissue shrinkage16,37 though tissues such as blood, muscle and yolk may harden and become brittle. The extent to which tissues are affected during paraffin wax infiltration depends upon the combination of fixative, dehydrant and transition solvent used17,22,23,38 as well as the tissue type. Microwave stimulated processing involves complex molecular interactions, the key element of which is internal heating, with stimulation of diffusion34, and concomitant reduction in the duration of tissue processing.

PRESSURE AND VACUUMHigh pressure facilitates infiltration of dense specimens with viscous resinous embedding media at the block forming stage19, but is rarely employed for biological specimens. Positive pressures for fluid transfer that are encountered in closed system processors are probably too low to have a significant influence on tissue infiltration.

Vacuum applied during dehydration, clearing and infiltration stages improves the quality of processing. Tissues, particularly lung, are de-aerated, and the solvent boiling point is reduced, thus facilitating evaporation of the reagent from the molten infiltration medium. Duration of wax infiltration is dependent upon viscosity and is not reduced by the application of vacuum39.

AGITATIONFluid interchange between processing reagents and tissues is promoted by exposure of the maximum tissue surface area to reagents. If tissues are allowed to settle on the bottom of a container, remain static in the reagent, or are too tightly packed in the processor basket, tissue surface area available for fluid exchange will be restricted and the concentration gradient between the fluid inside and outside the tissues will be low. Reagent diffusion time is therefore increased and if the duration of processing is not correspondingly increased, inadequate processing will result.

During processing, tissues should be loosely packed, suspended and agitated within the medium to facilitate the exchange of dilute reagent from the tissues with the more concentrated reagent replacing it. Agitation of tissues and fluids in manual processing is achieved using rotors or magnetic stirrers. In automatic tissue processors, continual rotary or vertical motion of tissue containers, or tidal action and flow of processing fluids ensures adequate fluid exchange. Ideally tissue cassettes should be placed in processors so that the cassette perforations are perpendicular to the fluid flow. For efficient and effective processing there should be a specimen volume to processing fluid volume ratio of at least 1:50.

Alternate vacuum and positive pressure cycles during processing may provide some micro agitation within tissues, but this has yet to be substantiated. In ultrasonic stimulated processing40-41 tissues and fluids are subjected to high frequency agitation and associated phenomena, with simultaneous reduction in processing time.

DehydrationThe first step in processing is dehydration. Water is present in tissues in free and bound (molecular) forms. Tissues are processed to the embedding medium by removing some or all of the free water. During this procedure various cellular components are dissolved by dehydrating fluids. For example, certain lipids are extracted by anhydrous alcohols, and water soluble proteins are dissolved in the lower aqueous alcohols42.

Dehydration is effected as follows:

Dilution dehydration, the most commonly used method. Specimens are transferred through increasing concentrations of hydrophilic or water miscible fluids which dilute and eventually replace free water in the tissues.

Chemical dehydration, where the dehydrant, acidified dimethoxypropane or diethoxypropane, is hydrolysed by free water present in tissues to form acetone and methanol43-50 in an endothermic reaction.

Dehydration is necessary in all infiltration methods, except where tissues are simply externally supported by an aqueous embedding medium. Choice of a dehydrant is determined by the nature of the task, the embedding medium, processing method, and economic factors. Dehydrants differ in their capacity to cause tissue shrinkage (Fig. 3).

In the paraffin wax method, following any necessary post fixation treatment, dehydration from aqueous fixatives is usually initiated in 60%-70% ethanol, progressing through 90%-95% ethanol, then two or three changes of absolute ethanol before proceeding to the clearing stage.

Whilst well fixed tissues can be transferred directly to 95% ethanol,51 incompletely fixed tissues may exhibit artefacts if placed directly in higher alcohols. The dehydrant concentration at which processing is initiated depends largely upon the fixative employed. Following fixation in anhydrous fixatives such as Carnoy's fluid, for example dehydration is initiated in 100% ethanol. To minimise tissue distortion from diffusion currents, delicate specimens are dehydrated in a graded ethanol series from water through 10%-20%-50%-95%-100% ethanol.36

Duration of dehydration should be kept to the minimum consistent with the tissues being processed. Tissue blocks 1 mm thick should receive up to 30 minutes in each alcohol, blocks 5 mm thick require up to 90 minutes or longer in each change. Tissues may be held and stored indefinitely in 70% ethanol without harm.

Other dehydrants, including universal solvents, are used in a similar manner to that described for ethanol, though generally in different concentration increments.

Dehydrating agentsALCOHOLSThese are clear, colourless, flammable, hydrophilic liquids, miscible with water and, when anhydrous, with most organic solvents. In addition to their role as dehydrants, alcohols also act as secondary coagulant fixatives during tissue processing.

Ethanol is probably the most commonly used dehydrant in histology. It is supplied as 99.85% ethanol (absolute ethanol, 100 High Grade or Standard Grade) and as special Methylated Spirits (99.85% ethanol denatured with 2% methanol). Both are satisfactory for histological purposes. Ethyl alcohol formulations differ in standards and nomenclature worldwide and it may be necessary to consult various tables to ascertain the ethanol concentration.

Ethanol is a rapid, efficient and widely applicable dehydrant. It is normally a poor lipid solvent except under microwave processing conditions34. Ethanol dissolves nitrocellulose slowly unless combined in equal proportions (or better, 1:2)53 with diethyl ether. Processing times in absolute ethanol should be minimal. Progressive removal of bound water from carbohydrates and proteins during prolonged immersion in absolute ethanol causes tissues to harden excessively and become brittle19,22-23. Colloid, blood, collagen and yolky tissues are particularly affected19. The problem is exacerbated by heat during wax infiltration.

Anhydrous cupric sulphate added to the final absolute ethanol on a tissue processor scavenges any water present. The salt is self-indicating: white when anhydrous, blue when hydrated, and is only slightly soluble in ethanol. It is prepared by carefully heating

hydrated cupric sulphate until it turns white, on a hotplate at 250°C. Cool in a desiccator. Anhydrous calcium sulphate (Drierite) or molecular sieves act in a similar manner but are non-indicating. Solid drying agents are placed in a 1-2 cm thick layer in the reagent container, and covered with filter paper or fine sponge to avoid mixing with the specimens during tissue agitation.

Methanol is a good ethanol substitute54 but rarely used for routine processing because of its volatility, flammability and cost. It is a poor lipid solvent, and will not dissolve nitrocellulose unless mixed with acetone. In microwave processing it tends to harden tissues more than ethanol34.

Isopropanol was first suggested as an ethanol substitute during the prohibition era in the United States54. It is a universal solvent available as 99.8% (absolute) isopropanol, slightly slower in action and not as hygroscopic as ethanol, but a far superior lipid solvent. Isopropanol is completely miscible with water and most organic solvents, is fully miscible with melted paraffin wax55, and is readily expelled from tissues and wax baths. Isopropanol shrinks and hardens tissues less than ethanol54-57 and is used to dehydrate hard, dense tissues, which can remain in the solvent for extended periods without harm. To minimise shrinkage, fixed tissues are transferred via 60%-70% isopropanol or ethanol to absolute isopropanol.

Isopropyl alcohol has also been recommended as a xylene substitute58. In microwave stimulated processing, though unsatisfactory as a dehydrant, isopropanol is used as a transition solvent following ethanol dehydration34.

Isopropanol only dissolves nitrocellulose in the presence of esters59 such as methyl benzoate or methyl salicylate, and is used in methyl salicylate-based double-infiltration methods. It cannot be used as a dehydrant in alcohol-ether-celloidin techniques. Isopropanol is a solvent for some lipid-soluble dyes, but is not used in staining work stations as many other dyes are insoluble in this solvent.

Normal and tertiary butanols are universal solvents mainly used for small-scale manual processing of plant and animal tissues in teaching and research. Normal butanol is recommended for processing lightly chitinised arthropods38 and rodent tissues. It causes less hardening and shrinkage than ethanol,38,60 though this is offset by the prolonged processing schedules which may result in tissue shrinkage.18,22 N-butanol is poorly miscible with water and only slowly miscible with paraffin wax. It is flammable, with a penetrating camphor-like odour, and the vapours are eye irritants. Iso-butanol, with similar properties and processing characteristics22,23 is a less costly substitute for n-butanol. Tertiary-butanol is widely used in plant histology61 but rarely for animal tissues17,23. Below 26°C it is hygroscopic crystalline solid, a major disadvantage. In processing it is used in a similar manner to n-butanol.61

GLYCOL-ETHERSUnlike the alcohols, these reagents do not act as secondary fixatives, and apart from solvent effects do not appear to alter tissue reactivity.

2-Ethoxyethanol, ethylene glycol monoethyl ether, cellosolve or oxitol is used as a dehydrant preceding polyester wax embedding,19 for dehydration following dioxane-based fixation of hard animal tissues62, and in the agar-ester wax double embedding technique.

Ethoxyethanol is a colourless, nearly odourless flammable liquid, strongly hygroscopic, miscible with water and most organic solvents. Cellosolve dissolves nitrocellulose and tends to decompose on exposure to sunlight. It is rapid but non-hardening in action, and tissues can remain in it for years62. To avoid severe shrinkage, tissues are transferred from aqueous fixative or washing via 60%-70% ethanol into full strength cellosolve.

Dioxane, 1,4 diethylene dioxide causes less tissue shrinkage and hardening than ethanol17,22-23,62-63 and is excellent for tissues excessively hardened by ethanol-xylene processing. It has a rapid but gentle action, and is best used in a graded series. Tissues may remain in it for long periods without harm. It is a colourless, flammable universal solvent with an odour similar to butanol, freezes at 12°C, and is miscible with water, most organic solvents and paraffin wax. Dioxane dissolves mercuric chloride, but precipitates potassium dichromate and other salts. It is cumulatively toxic and a suspected carcinogen64.

Dioxane is expensive and is normally reclaimed by drying over a 10-20 mm layer of calcium oxide or anhydrous cupric sulphate. Calcium chloride should not be used as it reacts with dioxane and swells63. Dioxane is also recovered by freezing hydrated solvent in a spark-proofed refrigerator at 2-5°C. Water, which separates out, is decanted from the crystalline dioxane which is then thawed, finally dried over a solid dehydrant and reused65.

Explosive peroxides form in dioxane exposed to air. They accumulate in recycled solvent which should be periodically tested for the presence of peroxides.66

Polyethylene glycols (PEG) are water miscible polymers used to dehydrate and embed substances labile to the solvents and heat of the paraffin wax method. They are clear, viscous, slightly hydroscopic liquids or solids of low toxicity. Polyethylene glycols are miscible with most organic solvents and dissolve nitrocellulose. Dehydration is initiated in the low molecular weight liquid glycols. Tissues pass through glycols of increasing molecular weight and viscosity, and are finally embedded in a high molecular weight PEG which is solid at room temperature. Polyethylene glycol used for dehydration can be regenerated by heating at 104°C for 24 hours67.

OTHER DEHYDRANTSAcetone is a colourless flammable liquid with sharp characteristic ketonic odour, low toxicity and is freely miscible with water and organic solvents. It is a fast, effective dehydrant though it may cause tissue shrinkage; it may also act as a coagulant secondary fixative. Acetone is the best dehydrant for processing fatty specimens. Tissues are dehydrated through four changes of acetone, the last of which should always be fresh.

Tissues can be transferred directly from acetone to paraffin wax as the solvent boils off under vacuum68. However a transition solvent is normally interposed before the paraffin baths. Acetone is not recommended for microwave processing as it causes excessive nuclear shrinkage34.

Tetrahydrofuran is a colourless, highly volatile and flammable universal solvent with an offensive ethereal odour. It is completely miscible with water, most organic solvents, paraffin wax and mounting media. It dissolves mountants, but not most dyes. The solvent dehydrates rapidly causing little shrinkage or hardening, and is possibly the best of the universal solvents.69 It is less toxic than dioxane for which it can be substituted. Tissues are processed as in dioxane method. Tetrahydrofuran can form explosive peroxides which renders solvent recovery distillation dangerous66.

2,2 dimethoxypropane (DMP) and 2,2 diethoxypropane (DEP) are used for chemical dehydration of tissues.43-51 They are flammable and form peroxides.50 DMP and DEP are miscible with paraffin wax however methanol, one of the hydrolysis products, is not wax miscible and a post dehydration rinse in acetone,48 a transition solvent such as methyl salicylate49 or toluene should precede infiltration with wax. DMP shrinks tissues slightly less than DEP.51 Chemical dehydration is suitable for rapid manual processing or machine processing, and is comparable to conventional dehydration for tissue morphology and staining reactions.51 Acidified DMP/DEP can be reused several times, though dehydration times need to be extended. The reagent is stored at 4°C in a spark-proofed refrigerator.

Phenol, beechwood creosote and aniline facilitate dehydration when mixed with transition solvents, as in Weigert's carbol-xylol (xylene 75 ml and phenol 25 ml).36 The coupling action permits tissues and celloidin sections to be cleared from lower strength alcohols. Creosote and aniline are used less commonly though in a similar manner to phenol. Phenol consists of clear hygroscopic acicular crystals and is also available as 80% w/w liquefied phenol. It is soluble in water, alcohol and most organic solvents except petroleum ethers. Concentrated solutions coagulate nitrocellulose. On exposure to air and light, phenol and its solutions develop a pink to reddish discolouration. Containers must be protected from light and tightly sealed. Phenol crystals and 80% concentrate react violently with formaldehyde.

ClearingClearing is the transition step between dehydration and infiltration with the embedding medium. Many dehydrants are immiscible with paraffin wax, and a solvent (transition solvent, ante medium, or clearant) miscible with both the dehydrant and the embedding medium is used to facilitate the transition between dehydration and infiltration steps. Shrinkage occurs when tissues are transferred from the dehydrant to the transition solvent, and from transition solvent to wax17,18 (Fig 4). In the final stage shrinkage may result from the extraction of fat by the transition solvent.18

The term clearing arises because some solvents have high refractive indices (approaching that of dehydrated fixed tissue protein) and, on immersion, anhydrous tissues are rendered transparent or clear.70 This property is used to ascertain the endpoint and

duration of the clearing step. The presence of opaque areas indicates incomplete dehydration.

However, other solvents, notably chlorinated hydrocarbons, do not render tissues transparent and the clearing endpoint (generally when the specimen sinks in the solvent) must then be determined empirically. Transition solvents extract certain tissue substances such as lipids, but otherwise do not alter tissue reactivity nor behave as secondary fixatives during processing.

Choice of a clearing agent depends upon the following:

the type of tissues to be processed, and the type of processing to be undertaken the processor system to be used intended processing conditions such as temperature, vacuum and pressure safety factors cost and convenience.

Transition solventsHHYDROCARBONSThese are odourless flammable liquids with characteristic petroleum or aromatic odours, miscible with most organic solvents and with paraffin wax. They coagulate nitrocellulose.

Toluene and xylene clear rapidly and tissues are rendered transparent, facilitating clearing endpoint determination. Concerns over the exposure of personnel to xylene66 relate mainly to the use of the solvent in coverslipping rather than in processing and xylene substitutes can be used in these circumstances. Xylene hardens tissues fixed in non-protein coagulant fixatives and prolonged clearing in the solvent should be avoided. Tissues stabilised in protein coagulant fixatives (Bouin's or SUSA) are less affected.17,23 Benzene is more gentle and rapid than xylene and toluene and is probably the best transition solvent, though toxicity and possible carcinogenicity64 preclude its use in histology. Industrial grade xylene may contain nearly 25% of other solvents such as ethyl benzene, with traces of benzene, odorous mercaptans and hydrogen sulphide. Only the sulphur and benzene-free solvent-grade xylene should be used for histological purposes.

Petroleum solvents have a gentle, non-hardening action on tissues, clear more slowly than xylene and toluene, and do not render tissues transparent. Blends of aromatic, naphthenic and aliphatic solvents (each with varying toxicity, flammability and solvent action) can be used as xylene substitutes.66 Many of these solvents have a particularly strong petroleum odour which some people find objectionable. Toxic effects of petroleum solvents are broadly similar to those of pure hydrocarbons - skin degreasing, acute intoxication and narcosis in high concentrations. Blends with high aromatic and naphthene but reduced paraffin content such as Shell X3B71, are good, moderately toxic, high flash-point solvents. Those with high paraffin but little or no naphthene and aromatic content often have low flammability and toxicity, and a slow and gentle clearing

action. Kerosene, some xylene substitutes and Shellsol 1626 have properties intermediate between these two groups.

Chlorinated hydrocarbons are colourless solvents with sweet odours and are miscible with most organic solvents and with paraffin wax. They are good lipid solvents and do not dissolve nitrocellulose or render tissues transparent. Members of this group clear more slowly but harden far less than xylene. Although non-flammable, solvents in this group decompose in the presence of heat to form phosgene and hydrochloric acid. They are all narcotic and toxic to varying degrees. Chlorinated hydrocarbons are ozone destroying chemicals, and from January 1996 1,1,1 trichloroethane and carbon tetrachloride are banned from use under the Montreal Protocol.72

Chloroform is an expensive, heavy, highly volatile, slowly penetrating transition solvent. It causes less brittleness than xylene and is often used on dense tissues such as uterus and muscle32 which can be cleared overnight without undue hardening. Since chloroform attacks some plastics and sealants its use may be restricted in certain closed system processors.

Carbon tetrachloride has similar properties, but because of its high toxicity is now rarely used in histology.

Trichloroethane and other members of this group are commonly used as xylene73,74 and chloroform substitutes. They include 1,1,1 trichloroethane (1,1,1 TCE), present in Inhibisol; 1,1,1 TCE and perchloroethylene components of CNP30 and Histosol; and trichloroethylene. These solvents are stable to light but tend to slowly liberate hydrochloric acid on contact with water. They contain stabilisers to inhibit reactions with aluminium and its alloys.59 Although mildly toxic (except at high concentrations)64 the decision to substitute them for more toxic solvents must be soundly based (Table 7.3)75. Because of their high volatility, members of this group may achieve and exceed maximum allowable concentrations in poorly ventilated laboratories far more rapidly than xylene under the same conditions.75

ESTERSThese are colourless flammable solvents miscible with most organic solvents and with paraffin wax.

n-Butyl acetate is used as a xylene substitute76 and nitrocellulose solvent.

Amyl acetate, methyl benzoate and methyl salicylate are chiefly used as nitrocellulose solvents in double embedding techniques. They have low toxicity, but their strong penetrating odours necessitate good laboratory ventilation. They are ideal for manual processing as tissues may be left in them for extended periods without hardening. These esters are difficult to eliminate from paraffin wax and should be extracted from tissues with one or two brief changes of toluene or similar solvent before passing through two or three changes of wax. Methyl benzoate and methyl salicylate render tissues completely transparent and are used for clearing helminth parasites for examination and whole

mounting. Methyl salicylate clears tissues from 96% ethanol, hardens less and has a more pleasant odour than methyl benzoate. It causes minimal tissue shrinkage and hardening18 and tissues can remain in it indefinitely without harm. This ester is one of the best though expensive transition solvents.

TERPENESTerpenes are isoprene polymers found in essential oils originally derived from plants,77 though some are now synthesised. They are the earliest transition solvents to be used in histology78 and include turpentine and oils of bergamot, cedarwood, clove, lemon, origanum and sandalwood.61,79 In general the natural oils are not highly pure compounds but contain several substances.

Many terpenes clear tissues and celloidin sections from 80%-95% alcohol, render tissues transparent and have a slow gentle non-hardening action. Most are generally regarded as safe though some have particularly strong odours which can be overpowering, requiring good laboratory ventilation.

When used for processing hard, dense or chitinised non-mammalian tissues, terpenes may need to be diluted with the anhydrous dehydrant and with wax in a series, with terpene:dehydrant or wax ratios of 3:1, 1:1, 1:3 followed by three or four changes of pure wax. Tissue penetration is aided and shrinkage minimised by diluting viscous terpenes.

Terpenes have low evaporation rates and are difficult to eliminate from paraffin wax, necessitating one or two 30 minute changes of toluene or similar solvent to remove the terpene before infiltration with wax. Brief immersion in toluene does not negate the effectiveness of the terpene. Alternatively, tissues are given three, four or more changes of wax until the terpene has been eliminated. Although biodegradable, terpenes are not water miscible and should not be flushed away with water, but disposed of by recycling or incineration.

Cedarwood oil, largely composed of cedrene, rapidly clears tissues from 95% alcohol, hardens tissues the least of all the transition solvents, but is difficult to eliminate from tissues during wax infiltration. It is particularly useful for processing dense tissues such as uterus or scirrhous carcinomas, and has a role in forensic histopathology in processing the hardened skin margins of electrical burns and bullet wounds. Tissues can remain in cedarwood oil indefinitely without harm. Low viscosity refined oil should be used for clearing. Formation of crystalline cedrol in cedarwood oil can be overcome by the addition of 1 ml xylene or toluene to 80 ml cedarwood oil65. Cedarwood oil is expensive, but exhausted oil can be restored by filtering, then heating to 60°C under vacuum for 30-60 minutes80.

Limonene (d+ limonene) is derived from citrus fruit and is a component of various proprietary blends of transition solvents such as Clearene, Hemo-De and Histo-Clear marketed as xylene substitutes. It is less viscous than cedarwood oil and is similar to the esters in clearing action and in elimination from wax. Limonene may cause allergic skin reactions.81,82

Terpineol is a clear almost colourless mixture of isomers with a faint pleasant odour and very low evaporation rate. It clears tissues from 80%-90% alcohol with minimal hardening. Like the other terpenes it is difficult to eliminate from paraffin wax. It is a particularly useful substitute for cedarwood oil in manual processing and is also used in open-dish microscopic examination of cleared parasitic helminths. Tissues may remain in it indefinitely without harm.

Some of the specific safety requirements relating to processing solvents are summarised in Table 3.

Infiltration and embedding media and methodsIdeally an infiltrating and embedding medium should be:19

soluble in processing fluids suitable for sectioning and ribboning molten between 30°C and 60°C translucent or transparent; colourless stable homogeneous capable of flattening after ribboning non-toxic odourless easy to handle inexpensive

In addition the properties of the medium should approach those of the tissues to be sectioned with regard to density, elasticity, plasticity, viscosity and adhesion and should be harmless to the embedded material.

Various substances have been used to infiltrate and embed tissues for microtomy. None completely fulfil the foregoing criteria, and media are selected according to the nature of the task for which they are required.

Embedding is the process by which tissues are surrounded by a medium such as agar, gelatine, or wax which when solidified will provide sufficient external support during sectioning.

Infiltration is the saturation of tissue cavities and cells by a supporting substance which is generally, but not always, the medium in which they are finally embedded. Tissues are infiltrated by immersion in a substance such as a wax, which is fluid when hot and solid when cold. Alternatively, tissues can be infiltrated with a solution of a substance dissolved in a solvent, for example nitrocellulose in alcohol-ether, which solidifies on evaporation of the solvent to provide a firm mass suitable for sectioning.

Double embedding is the process by which tissues are first embedded or fully infiltrated with a supporting medium such as agar or nitrocellulose, then infiltrated a second time with wax in which they are also embedded.

Investment generally refers to the practice of embedding wax infiltrated tissues in another wax, such as Piccolyte-paraffin wax, modified to provide improved tissue support and sectioning qualities.

Vacuum infiltration is the impregnation of tissues by a molten medium under reduced pressure. The procedure assists the complete and rapid impregnation of tissues with wax, reduces the time tissues are subjected to high temperatures thus minimising heat-induced tissue hardening, facilitates complete removal of transition solvents, and prolongs the life of wax by reducing solvent contamination. Vacuum infiltration requires a vacuum infiltrator or embedding oven, consisting of wax baths, fluid trap and vacuum gauge, to which a vacuum of up to 760 mm Hg is applied using a water or mechanical pump. Modern tissue processors are equipped to deliver vacuum, or vacuum and pressure, to all or some reagent stations during the processing cycle.

Parffin waxPROPERTIESParaffin wax is a polycrystalline mixture of solid hydrocarbons produced during the refining of coal and mineral oils. It is about two thirds the density and slightly more elastic than dried protein.33

Wax hardness (viscosity) depends upon the molecular weight of the components and the ambient temperature. High molecular weight mixtures melt at higher temperatures than waxes comprised of lower molecular weight fractions. Paraffin wax is traditionally marketed by its melting points which range from 39°C to 68°C.

Tissue-wax adhesion depends upon crystal morphology of the embedding medium. Small, uniform sized crystals provide better physical support for specimens through close packing. Crystalline morphology of paraffin wax can be altered by incorporating additives which result in a less brittle, more homogeneous wax with good cutting characteristics. There is consequently less deformation during thin sectioning. Setting temperature does not appreciably affect crystal size.84-86

MODIFIED PARAFFIN WAXESThe properties of paraffin wax are improved for histological purposes by the inclusion of substances added alone or in combination to the wax:

improve ribboning: prolong heating of paraffin wax at high temperatures or use micro-crystalline wax

increase hardness: add stearic acid decrease melting point: add spermaceti or phenanthrene improve adhesion between specimen and wax (alter crystalline morphology): add

0.5% ceresin, 0.1-5% beeswax, rubber, asphalt, bayberry wax, or phenanthrene.87

Early histological wax formulations36,88-89 have largely been replaced by uniform, high quality proprietary blends of histological paraffin waxes. Additives recently incorporated in proprietary waxes include the following:Piccolyte 115, a thermoplastic terpene resin added at the rate of 5%-10% to the infiltrating wax,90 or to the final investing paraffin wax to improve tissue support for thin sectioning and facilitate flattening and expansion of sections on the waterbath.91-92 Piccolyte mixtures cannot be used in certain models of fluid-transfer type tissue processors.Plastic polymers such as polyethylene wax, added to improve adhesion, hardness and plasticity.93 Polymer waxes are incorporated in the majority of proprietary histological paraffin wax blends presently available.Dimethyl sulphoxide (DMSO) added to proprietary blends of plastic polymer paraffin waxes reduces infiltration times and facilitates thin sectioning.94 DMSO scavenges residual transition solvent and probably alters tissue permeability by substituting for or removing bound water thus improving infiltration. Some individuals who handle DMSO-paraffin wax may experience an unpleasant and annoying oyster or garlic taste probably caused by DMSO metabolites.95 Possible health risks associated with the use of DMSO-paraffin wax66 are minimal if correct laboratory hygiene is practised.

Embedding tissues in paraffin waxTissues are embedded by placing them in a mould filled with molten embedding medium which is then allowed to solidify. Embedding requirements and procedures are essentially the same for all waxes, and only the technique for paraffin wax is provided here in detail. At the completion of processing, tissues are held in clean paraffin wax which is free of solvent and particulate matter.

Requirements for embedding are as follows:

a supply of clean, filtered paraffin wax held at 2-4°C above its melting point. a cold plate to rapidly cool the wax. a supply of moulds in which to embed the tissues.

These elements are conveniently combined in commercially available embedding stations (Fig 5). Otherwise a wax dispenser, embedding oven and ice will suffice. There are four main mould systems and associated embedding protocols presently in use96 (Fig 6): traditional methods using paper boats; Leuckart or Dimmock embedding irons or metal containers; the Peel-a-way system using disposable plastic moulds and; systems using embedding rings or cassette-bases which become an integral part of the block and serve as the block holder in the microtome.

General Embedding ProcedureMETHOD1 Open the tissue cassette, check against worksheet entry to ensure the correct number of tissue pieces are present.2 Select the mould, there should be sufficient room for the tissue with allowance for at least a 2 mm surrounding margin of wax.

3 Fill the mould with paraffin wax.4 Using warm forceps select the tissue, taking care that it does not cool in the air; at the same time.5 Chill the mould on the cold plate, orienting the tissue and firming it into the wax with warmed forceps. This ensures that the correct orientation is maintained and the tissue surface to be sectioned is kept flat.6 Insert the identifying label or place the labelled embedding ring or cassette base onto the mould.7 Cool the block on the cold plate, or carefully submerge it under water when a thin skin has formed over the wax surface.8 Remove the block from the mould.9 Cross check block, label and worksheet.

ORIENTATION OF TISSUE IN THE BLOCKCorrect orientation of tissue in a mould is the most important step in embedding. Incorrect placement of tissues may result in diagnostically important tissue elements being missed or damaged during microtomy. In circumstances where precise orientation is essential tissue should be marked or agar double embedded. Usually tissues are embedded with the surface to be cut facing down in the mould. Some general considerations (Fig.7) are as follows:

elongate tissues are placed diagonally across the block tubular and walled specimens such as vas deferens, cysts and gastrointestinal

tissues are embedded so as to provide transverse sections showing all tissue layers tissues with an epithelial surface such as skin, are embedded to provide sections in

a plane at right angles to the surface (hairy or keratinised epithelia are oriented to face the knife diagonally)

multiple tissue pieces are aligned across the long axis of the mould, and not placed at random.

During cooling, paraffin wax shrinks up to 15%, causing compression in tissues.17 This compression is almost fully recovered when sections are floated on a warm waterbath6; compression resulting from microtomy is only partially recovered.

Processing methods and routine schedulesTissues are usually more rapidly processed by machine than by manual methods, although the latter can be accelerated by using microwave or ultrasonic stimulation. For routine purposes tissues are most conveniently processed through dehydration, clearing and infiltration stages automatically by machine. There are two broad types of automatic tissue processors - tissue-transfer and fluid-transfer types.

Automated tissue processingTISSUE-TRANSFER PROCESSORSThese processors are characterised by the transfer of tissues, contained within a basket, through a series of stationary reagents arranged in-line or in a circular carousel plan. The rotary or carousel is the most common model of automatic tissue processor, and was

invented by Arendt in 1909.79 It is provided with 9-10 reagent and 2-3 wax positions, with a capacity of 30-110 cassettes depending upon the model. Fluid agitation is achieved by vertical oscillation or rotary motion of the tissue basket. Processing schedules (Table 4) are card-notched, pin or touch pad programmed.

Tissue-transfer processors allow maximum flexibility in the choice of reagents and schedules that can be run on them, in particular, metal-corrosive fixatives, a wide range of solvents, and relatively viscous nitrocellulose solutions can all be accommodated. These machines have a rapid turn-around time for day/night processing. In more recent models (Fig.8) the tissue basket is enclosed within an integrated fume hood during agitation and transfer cycles thus overcoming the disadvantages of earlier styles.

FLUID-TRANSFER PROCESSORSIn fluid-transfer units (Fig.9) processing fluids are pumped to and from a retort in which the tissues remain stationary. There are 10-12 reagent stations with temperatures adjustable between 30-45°C, 3-4 paraffin wax stations with variable temperature settings between 48-68°C, and vacuum-pressure options for each station. Depending upon the model these machines can process 100-300 cassettes at any one time. Agitation is achieved by tidal action. Schedules are microprocessor programmed and controlled. Vacuum-pressure cycles coupled with heated reagents allow effective reductions in processing times and improved infiltration of dense tissues.

Fluid-transfer processors overcome the main drawbacks of the tissue-transfer machines. Tissues are unable to dry out within the sealed retort and reagent vapours are vented through filters or retained in a closed-loop system. Processors are provided with alert systems and diagnostic programs for troubleshooting and maintenance. Some models are unable to accept mercury or dichromate-based fixatives, certain solvents, for example chloroform, or wax additives such as Piccolyte. Representative schedules for rapid and overnight processing are provided in Table 5.

TISSUE RECOVERY PROCEDURESProcedures for recovery of tissues that have air dried because of mechanical or electrical failure of the processor are similar to those used for mummified specimens. Tissues accidentally returned into fixative or alcohol following wax infiltration are recovered by methods outlined in Table 6.

GENERAL CONSIDERATIONSBaskets and metal cassettes should be clean and wax-free.Tissues should not be packed too tightly in baskets so as to impede fluid exchange.Processors must be free of spilt fluids and wax accumulations to reduce hazards and to ensure mechanical reliability.Fluid levels must be higher than the specimen containers.Timing and delay mechanism must be correctly set and checked against the appropriate processing schedule.A processor log should be kept in which the number of specimens processed, processing reagent changes, temperature checks on the wax baths and the completion of the routine

maintenance schedule, is recorded as an integral part of the laboratory quality assurance program.

Manual tissue processingManual tissue processing is usually undertaken for the following reasons:

power failure or breakdown of a tissue processor a requirement for a non-standard processing schedule as for:

o rapid processing of an urgent specimen o delicate material o very large or thick tissue blocks o hard, dense tissues (nitrocellulose methods) o special diagnostic, teaching or research applications

small scale processing requirements resin embedding.

The main advantage of manual processing over automated methods lies in the flexibility of reagent selection, conditions and schedule design to provide optimum processing for small batches of tissues. Exposure of tissues to the deleterious effects of some reagents can be carefully monitored and regulated through observation and precise timing. There is usually considerable latitude in the processing times given in schedules although maximum rather than minimum times should be used, as it is better to extend processing rather than risk the problems of under processed tissues. Manual processing is accelerated using microwave ovens or ultrasonics.

Schedules for rapid manual processing using chemical dehydration (Table 7), one and two day routine processing (Table 8), and extended manual processing for large, thick, or hard tissues (Table 9) are provided.

Universal solvents with particularly favourable attributes, normally precluded from routine machine processing because of budgetary or safety constraints, can be successfully used in small volumes under controlled conditions for manual processing. Schedules are provided for manual processing using n-butanol (Table 10) and dioxane (Table 11).

Nonetheless manual processing can be time consuming and inconvenient. Care must be exercised so that tissues are left overnight in reagents that will cause minimal harm. A permanent series of solutions in wash bottles simplifies processing small single specimens. Tissues are processed in tubes and agitated on a rotor. Reagents are pipetted, or decanted through a fine sieve. Multiple specimens or large blocks are economically processed in large lidded jars of processing fluids. The specimen to reagent volume ratio should be at least 1:50. Agitation is provided by a magnetic-stirrer.

Dehydrated tissues float on the surface when transferred to higher density transition solvents such as chloroform or cedarwood oil. However, if placed in lower density mixtures of dehydrant-transition solvent before finally transferring to pure transition

solvent, tissues will remain submerged throughout the clearing stage. An alternative approach is to carefully layer the dehydrant onto the transition solvent and introduce the tissue into the upper layer. The tissue sinks as the dehydrant gradually replaces the transition solvent. Reagents are carefully decanted and the specimen placed in a fresh change of transition solvent.

Microwave-stimulated processingRapid manual microwave-stimulated paraffin wax processing of small batches of tissues gives excellent results which are comparable to tissues processed by longer automated non-microwave methods.34,97-101

Processing is undertaken in a dedicated microwave oven (Fig. 10) which is fitted with precise temperature control and timer, and an interlocked fume extraction system to preclude accidental solvent vapour ignition. Agitation is provided by an air-nitrogen system.

Domestic microwave ovens with a temperature probe and timer accurate to seconds are suitable for tissue processing. A turntable or in-built radiation disperser facilitates even reagent heating. Toxic and flammable solvent vapours generated during processing cannot always be adequately vented from these ovens and present an ignition hazard if the electrical system is unprotected. Ovens should therefore be used within a fume cupboard to minimise this problem. Calibration of domestic ovens is essential for optimum results and the accuracy of the temperature probe, duration of cycle time, and net power levels at various settings must be determined before the oven is used to process tissues.34

EQUIPMENTTissues are processed in conventional plastic cassettes, including those with (provided the metal lids lie below the fluid).9 Transparent glass or solvent-resistant plastic containers of about 200 ml capacity are ideal for processing batches of up to 14 cassettes per container.

FIXATIONFor rapid processing, tissues are fixed by microwave irradiation,9 or in 95% ethanol (600 ml)-polyethylene glycol PEG 400 (45 ml)102 from which specimens can be transferred directly to dehydrant. Formaldehyde-fixed tissues must be rinsed in running tap water for 5 minutes before microwave processing and an extra dehydration change incorporated in the schedule. Processing times for formaldehyde-fixed tissues need to be increased above those provided for coagulant-fixed tissues.34 Picric acid fixed tissues should not be microwave processed as there is an explosion risk even in well washed tissues.34 Processing schedules are provided in Table 12.

HINTS FOR MICROWAVE PROCESSING- Tissue blocks should be as thin as possible. Length and width are not as important.34

Process blocks of similar thickness together. Reagent volumes should be at least 50 times that of specimen volume.

The temperature probe should be placed centrally in processing baths. Use a dummy load to check heat generation should reagents boil on minimum

settings - an equal volume of reagent irradiated together with the primary load effectively halves the energy received by the primary load.

Pre-heat paraffin wax baths in a conventional oven. An increase in the number of cassettes or fluid volumes will require a

concomitant increase in power and or time to achieve the correct processing temperature.

Ultrasound-stimulated processingUltrasonics are used in histopathology to accelerate fixation, tissue processing for electron microscopy, the decalcification of bone, tissue softening in post-embedding adjuvants, improving the sensitivity of immunohistochemical reactions, conventional staining and for accelerated tissue processing. Unfixed tissue blocks 1-2 mm thick can be fixed and processed to paraffin wax using ultrasonic-stimulation in 1 hour 45 minutes.40-41

The most important effect of ultrasound at frequencies of 100 kHz-1 MHz is agitation. At lower frequencies cavitation phenomena and attendant heat, pressure and streaming effects may damage tissues and care must be exercised.

Processing is performed in reagent containers suspended in a detergent solution within the transducer tank of an ultrasonic cleaner operated at 50 watts. An immersion heater is used to elevate bath temperatures for paraffin wax infiltration. Tissues are placed in metal or plastic cassettes for processing.

Coagulant fixatives provide optimal stabilisation for ultrasonic-stimulated processing.40 Tissues are dehydrated in ethanol and cleared in toluene, or preferably methyl benzoate or methyl salicylate (Table 13). Cells and organelles such as cilia, microvilli and desmosomes are all well preserved. Old and friable specimens sometimes exhibit marginal distortion and erosion.

Alternative embedding media and processing methodsAlternative embedding media may provide optimum support for tissues in applications for which paraffin waxes are unsuited, for example when:

tissue components are heat or reagent labile hard or dense tissues are inadequately supported adhesion between specimen and wax is poor very thick or very thin sections are required sectioning whole organs such as lung or brain.

Resin embedding methods are now used for many of these applications. Non-resinous embedding media include those listed below.

AQUEOUS MEDIAAgar has a high melting point and low gelling temperature of agar make it ideal for

double embedding multiple small tissue fragments. Agar is generally unstained by overnight stains, but will stain with alcian blue.

Gelatine is used for simple embedding in a similar manner to agar. However the low melting point of gelatine (35-40°C) makes it unsuitable for double embedding. It is used in Gough and Wentworth's whole-organ sectioning method and its variants, or simply to support large tissue blocks for 1 mm thick sectioning and subsequent three-dimensional reconstruction.103 In phospholipid and enzyme studies tissues may be infiltrated and embedded in gelatine51,70 and the resulting blocks sectioned on a freezing microtome. This technique has now largely been superseded by other media used for cryotomy.

Sodium carboxy methyl cellulose (CMC) used as an embedding medium for whole body sectioning techniques, was first developed for autoradiograph studies104 and subsequently refined for histological and histochemical applications.105 Frozen tissues are transferred from coolant directly into 5% CMC, briefly placed under vacuum to remove trapped air, then frozen to a solid block for sectioning.

Polyvinyl alcohol (PVA) is a highly polar, water soluble medium suited to a variety of applications, in particular histochemical studies of lipids and enzymes.106-107 Tissues are infiltrated at elevated or room temperatures through an ascending series of aqueous PVA-glycerol solutions and embedded in 15% aqueous PVA which is slowly dried to produce a firm block. Sections are cut at 1-100 µm in the normal manner. Humidity control during microtomy is important. Processing schedules take 1-2 weeks or longer, restricting PVA to research applications. A low molecular weight, low viscosity PVA is necessary for this method.107 Renewed interest in PVA as an infiltrating and embedding medium for electron microscopy108 has resulted in refinements to the technique. Cross-linking PVA with glutaraldehyde provides a final hydrophobic block containing some 10% water, with improved sectioning characteristics and good preservation of lipids, proteins and carbohydrates. Tissues are infiltrated through aqueous PVA solutions at concentrations from 1%-25%. PVA of molecular weight 14kDa, 99% hydrolysed gives the most consistent results108, but must be fully hydrolysed with sodium hydroxide before use. The application of PVA to immunohistochemical studies is worthy of attention.

WATER-MISCIBLE MEDIAPolyethylene glycols (PEG) are water soluble media used for investigation of heat and solvent-labile lipids and proteins109-110, and to overcome tissue shrinkage, damage and distortion inherent in the paraffin wax technique. The polyethylene glycols, or Carbowaxes, are polymers of varying length (the numerical suffix denotes molecular weight). At room temperature PEG 200 and PEG 600 are syrupy liquids, PEG 1000 is soft and slippery, PEG 1500 is hard, and PEG 4000 is hard and brittle. In general they are less elastic, denser and somewhat harder than paraffin wax. Crystal slip is a bigger problem than in paraffin and sectioning deformation is mainly non-recoverable.33 Tissues are dehydrated by gradual infiltration through increasing concentrations of aqueous PEG solutions, to pure PEG in which they are finally embedded. Sections are cut in a low humidity environment, otherwise considerable difficulties arise. They are difficult to flatten without loss of tissue and adhere poorly to slides, leading to the development of

numerous flotation fluids.110 Low viscosity nitrocellulose (LVN)67 or water insoluble polyvinyl acetate resin110 incorporated into PEG dehydrating, infiltrating and embedding solutions allow water flotation of sections. The PEG dissolves, leaving the tissue in a thin film of LVN or PVA which is mounted on albumenised slides in the usual manner. These approaches (Table 14) surmount many of the previous problems inherent with PEG. The nitrocellulose is removed from sections by immersing in PEG 200 for 15 minutes.

Polyethylene Glycol-LVN Solutions67

REAGENTS REQUIRED1 Polyethylene glycol 95 g2 LVN HX 3-5 or 30-5 5-10 gThe LVN must be finely divided and dried to facilitate dissolution in the PEG media; 5% LVN is preferred as 10% LVN is near saturation in PEG.

METHODThe mixtures are heated to 60°C and stirred to aid solution.

Problems with this method include the high viscosity of infiltrating media necessitating slow agitation and uneven distribution of LVN in the final embedding mix which results in crazed blocks. These can be overcome mostly by thorough blending of the LVN and PEG. With current interest in immunohistochemistry, polyethylene glycols may warrant re-evaluation. However considerable time and patience are required when using these waxes.

Polyethylene glycol monostearate (Nonex 63B), a water soluble synthetic wax is used in a similar manner to polyethylene glycol and polyester waxes with application in histochemistry111 and botanical histology.112

WATER-TOLERANT MEDIADiethylene glycol distearate is a hard, brittle, water tolerant ester (m.p. 47-52°C). It has certain deficiencies when used for routine embedding,113 unless combined with other substances as in ester waxes. However it may be used unmodified for thin sectioning (0.5-2 µm) of freeze dried and osmium tetroxide fixed tissues for high resolution light microscopy.114-115 Tissues are dehydrated and cleared as in the paraffin wax method.

Ester waxes, developed by Steedman,116 and subsequently modified117-118 have low melting points, are hard at room temperature and have good adhesive properties. They are therefore ideal for supporting and serially sectioning refractory hard, chitinised material such as arthropods, and tissues which heat-harden excessively. They are also used for simple investment of paraffin blocks to be sectioned under hot conditions119 and in double embedding with agar. Ester waxes are no longer commercially available and must be prepared from the basic ingredients.

Ester Wax 1960118 (m.p. 48°C)REAGENTS REQUIRED1 Diethylene glycol distearate 60 g

2 Glyceryl monostearate 30 g3 300 polyethylene glycol distearate 10 g

METHOD1 Melt the diethylene glycol distearate and heat it until clear. Add the glyceryl monostearate and when dissolved add the polyethylene glycol distearate.2 Filter through coarse filter paper supported in a ring rather than a filter funnel.

In Tropical Ester wax 1960119 (m.p. 50°C) triethylene glycol monostearate 10 g is substituted in the forgoing formula for the 300 polyethylene glycol distearate. This modification permits sectioning and ribboning at room temperatures of 37.5°C. Tissues are infiltrated at 56-60°C.

Ester waxes are soluble in 95% ethanol, n-butanol, cellosolve and xylene. Tissues are usually dehydrated via 2-ethoxyethanol in which the waxes are soluble, obviating the need for a transition solvent. Ester waxes do not charge with static electricity, and have good ribboning, thin sectioning and glass adhesion properties.

Polyester wax, developed by Steedman120 is a ribboning, low melting point wax which reduces heat-induced artefacts. It is recommended for heat labile tissues,121 to minimise heat-induced hardening in difficult tissues and is an ideal medium for combined light and scanning electron microscopy of animal tissues.122 The properties of the wax facilitate immunohistochemical investigations as antigenic determinants are well preserved.123 The main advantages of this medium are low melting point and infiltration directly from 96% ethanol permitting a near isothermic processing schedule for mammalian tissues (Table 15). Polyester wax is no longer commercially available and must be prepared from the basic ingredients.

Polyester Wax 90/10 (m.p. 38°C)REAGENTS REQUIRED1 400 polyethylene glycol distearate 90 g2 1-hexadecanol (cetyl alcohol) 10 g

METHOD1 Melt the polyethylene glycol at 60 C, then mix in the cetyl alcohol.2 Cool to a solid from which working quantities are obtained.

The wax has good water tolerance and is soluble in most histological dehydrants and transition solvents. Because of its low melting point, non-volatile transition solvents such as cedarwood oil and other terpenes should not be used. Polyester wax adheres to metal embedding moulds and paper-boat or plastic peel-a-way moulds are recommended. Normally blocks are cut in cool room temperatures using a cooled knife. Polyester wax is more conveniently sectioned on a cryotome at -5°C to 22°C. Sections are affixed to gelatine subbed or aminoalkylsilane treated slides, or floated on amylopectin.120 Blocks and sections are stored at 4°C. Polyester wax is dissolved from sections with absolute ethanol. Steedman19 proposed simultaneous fixation and infiltration of tissues with picro-

formal-polyester wax and mercury-formal-polyester wax mixtures. These methods and principles present possibilities for immunohistochemistry, but appear to have received little attention.

HYDROPHOBIC MEDIANitrocelluloseCelloidin (C) and Low Viscosity Nitrocellulose (LVN), mixtures of di- and tri-nitrocellulose, are composed of yellowish-white matted filaments with the appearance of raw cotton. Nitrocellulose is insoluble in water, but soluble in absolute ethanol-diethyl ether, amyl acetate, methyl benzoate, methyl salicylate and 2-ethoxyethanol and is set by most hydrocarbon solvents. It is highly flammable, and must be kept alcohol-damped with n-butanol or as 8% solutions in ethanol-ether or 1% LVNC in methyl benzoate as it is explosive if detonated when dry. Celloidin solutions have a low tolerance of water and dehydration must be thorough. LVN tolerates up to 6% of water,124 has superior penetration and final block hardness and is supplied in various grades of viscosity and nitrogen content.53,67 Nitrocellulose tissue processing techniques are generally employed for sectioning hard tissues such as bone,125-126 for topographical studies of central nervous system tissues,127 or for delicate embryonic material. Tissues are processed at room temperature producing minimal and shrinkage and hardening. Immunohistochemical investigations such as immunophenotyping of lymphoid and non-lymphoid cells128 are possible on nitrocellulose processed tissues.

Double embedding and double infiltration methodsDouble embedding methods such as agar-paraffin embedding, are used when tissues require external support or particular pre-embedment orientation. Paraffin wax double infiltration methods provide hard tissues with additional support provided by substances such as agar or nitrocellulose, with the convenience and ease of wax microtomy.

AGAR-PARAFFIN WAX DOUBLE EMBEDDINGDouble embedding in agar-paraffin is a reliable and convenient method of handling minute and friable tissue fragments such as curettings and endoscopic biopsies, which can be lost during tissue processing.129-135 It also overcomes the difficulty of manipulating small tissue fragments during embedding and facilitates correct orientation and identification of tissues for histochemical and immunohistochemical control tissues.136

Agar Embedding MediumREAGENTS REQUIRED1 Agar (technical or microbiological grade) 1.5 - 3.0 g2 Distilled water 90 ml3 37% formaldehyde solution 10 ml

METHOD1 Dissolve the agar in the distilled water using a boiling water bath, autoclave or microwave oven.2 Add formaldehyde and mix well.3 Distribute 20 ml aliquot's into screw capped bottles. Store at 4°C.

4 For use melt the agar as previously indicated, cool to 50-60°C, and hold in a 60°C oven. Loosen container caps before remelting agar.5 Embed tissues by pipetting agar solution over tissue fragments correctly oriented on a clean flat surface129 or membrane filter.105

TECHNICAL NOTES1 Specimens can be embedded in silicon-rubber flat embedment moulds, moulds made from cut-down plastic syringes,130 or in metal Tissue-Tek type moulds.132,135

2 Unstabilized agar embedments sometimes disintegrate during processing and must be stabilised by the addition of 2.5%-4% formaldehyde to the medium, or by immersing blocks in 70% ethanol or fixative for 1-3 hours.

Agar-Paraffin Wax Double Embedding For Fragments, Biopsies And Friable Specimens135

REAGENT REQUIREDAgar (prepared as above)

METHOD1 Fill the appropriate sized Tissue-Tek base mould with agar medium cooled to approximately 40°C.2 Number embedding cassette, cross checked with request slip and specimen container.3 Place specimen in agar-filled mould and orientate.4 Allow agar to solidify for 5 minutes. Place embedding cassette on top of the mould to identify the tissue.5 When the agar block is solid, detach the specimen from the mould by sliding a scalpel down one side. Trim and notch the agar block as required, leaving a 3-5 mm width of agar surrounding the tissue. Process normally.

Agar-Paraffin Wax Double Embedding For Bone Marrow Aspirates And Cell Suspensions Using The Collodion Bag Technique137

REAGENT REQUIREDCollodion (add 4% nitrocellulose to 25 ml absolute ethanol : 75 ml diethyl ether)

METHOD1 Place 1-2 ml of collodion into a 10 ml glass centrifuge tube. Rotate the tube so that an even layer of collodion coats and sets on the inside surface; gentle blowing into the tube hastens the setting process.2 Fill the tube with 70% ethanol and stand for 5 minutes to harden the collodion. Drain the ethanol, rinse with water.3 Add fixed aspirate or suspension to tube.4 Centrifuge at 2000 rpm for 30 seconds.5 Decant supernatant fluid.6 Pipette 1-2 ml of agar, cooled to approximately 45°C, into the centrifuge tube.7 Rapidly resuspend the specimen and centrifuge at 2000 rpm for 1 minute.8 Allow the mass to cool for 5 minutes.9 Using fine forceps, carefully detach the collodion bag and contents from the tube.

10 Trim off unwanted collodion, and process as a normal specimen, taking care to orient and embed the specimen correctly.

AGAR - ESTER WAX DOUBLE INFILTRATIONDouble infiltration of tissues in agar and ester wax138-139 aids thin serial sectioning of chitinised tissues at 0.5-1.0 µm and is a possible alternative to pine resin-beeswax paraffin wax used to support plastic vascular prostheses for sectioning.140 The fine crystalline nature and hardness of ester wax improves tissue-wax adhesion and provides adequate support for thin serial sectioning.

Agar-Ester Wax Double Infiltration138

REAGENTS REQUIRED1 5% aqueous agarCellosolve (2-ethoxyethanol)

METHOD1 Infiltrate fixed tissues in 5% aqueous agar solution for 1 hour at 50-60°C.2 Orientate tissues in an agar filled mould as indicated previously. Allow the agar to set. Trim the block.3 Pass the block through the following series, 30 minutes in each solution: 30%, 50%, 70% ethanol; 90% ethanol plus cellosolve, 2:1; the same 1:2; pure cellosolve, 3 changes; cellosolve plus ester wax 1:1; pure ester wax, at least 2 changes, the last overnight.4 Embed as usual and cool rapidly.

TECHNICAL NOTEBuzzell141 dehydrates in dioxane, with amyl acetate as a transition solvent. Blocks are best cut using a retracting microtome.

NITROCELLULOSE - PARAFFIN WAX DOUBLE INFILTRATIONNitrocellulose-paraffin wax double embedding is mainly used for brain, friable tissues and decalcified bone and is particularly useful for whole body sections of small animals and chitinous tissues. It combines the plasticity and support provided by nitrocellulose with convenient handling and microtomy of the paraffin technique.

Tissues may be (a)infiltrated with thick nitrocellulose solutions and the resulting block trimmed, hardened in chloroform and infiltrated in paraffin wax, or (b)infiltrated with thin low viscosity nitrocellulose (LVN) solutions which are integrated into a normal processing schedule. Proprietary celloidin-ethanol-ether solutions provide a simple and convenient double-embedding method (Table 16). The principle drawbacks of this technique are prolonged exposure of tissues to absolute ethanol and the high flammability and volatility of diethyl ether precluding machine processing. Use of methyl benzoate or methyl salicylate as LVN solvents overcome these deficiencies.

In Peterfi's classic technique,142 tissues are dehydrated to absolute ethanol, infiltrated with 1% celloidin in methyl benzoate with 2-3 changes over 24-72 hours (until clear), hardened in three changes of toluene for 8 hours, then infiltrated as usual in paraffin wax.

Molnar's variation143 (Table 17) is shorter and more logical, and is suitable for manual or machine processing.

The n-butanol added to reduce the explosion hazard may contribute up to 30% of the weight of the nitrocellulose127 and must be taken into consideration when preparing solutions. Nitrocellulose dissolves slowly in methyl salicylate, and during preparation mixtures should be periodically shaken to assist dissolution. In recent times LVN has largely replaced celloidin.

Sections of double-embedded tissues may tend to wrinkle or curl on the waterbath. Floating on 95% ethanol or Ruyter's fluid139 softens the LVN and facilitates section flattening.

Methods for difficult tissuesHARD DENSE TISSUESTissues largely comprised of thickened keratin, dense collagen, closely packed smooth muscle fibres, colloid, areas of haemorrhage, thrombi or yolk, can be hardened excessively when processed on routine schedules and consequently, sections may crumble or shatter. Ideally the fixative, processing reagents, embedding medium and schedules should be selected to minimise hardening in these tissues. Despite careful processing hard tissues frequently require treatment with post-embedding adjuvants before microtomy.

Mammalian tissues such as uterus, scirrhous carcinoma, leiomyomas and keratinised tissues are softened by fixing in 4% phenol in a mixture of absolute ethanol (75 ml), water (10 ml) and chloroform (10 ml),29 or by treating fixed tissues using 4% aqueous phenol for 24-72 hours.29 Similar results are obtained by dehydrating tissues using phenol in the first bath of absolute ethanol, or in all dehydrant baths (Table 18).32 Transition solvents such as chlorinated hydrocarbons and terpenes are recommended as they do not exacerbate tissue hardness.

MUMMIFIED TISSUESMummified archaeological specimens for histology are first rehydrated then processed on a LVN-paraffin wax double-infiltration schedule using phenol-ethanol to soften the tissues and amyl acetate as the transition solvent.144-145

Methods For The Recovery Of Dried And Mummified TissuesREAGENTS REQUIREDSolution (after Sandison144)Absolute ethanol 30 mlFormaldehyde, 37% 0.5 mlSodium carbonate 0.2 gWater to 100 mlorVan Cleve & Ross' solution145

Trisodium phosphate 0.25 gWater 100 ml

METHOD1 Immerse tissues in either solution for 24-72 hours or more, depending upon the nature and thickness of the specimen (most tissues rehydrate and soften within 4-6 hours).2 Process re-hydrated tissues on a normal schedule beginning in 70% ethanol. Tissues dried and hardened over years, and mummified archaeological specimens should be rehydrated in Sandison's solution, then double infiltrated in phenol-amyl acetate-LVN-paraffin wax schedule.

YOLKY TISSUESYolk-rich gonads, and muscle of marine and freshwater fish and invertebrates are routinely fixed in FAACC fixative (formaldehyde 37%, 10 ml; glacial acetic acid, 5 ml; calcium chloride dihydrate, 1.3 g)146 and processed by the schedule given in Table 19. This fixative has similar fixation image, processing and sectioning characteristics to Bouin's fluid, but without the hazard, cost and inconvenience of picric acid used in this fixative. Buffered or saline formaldehyde fixatives cause excessive hardening of these tissues and are contra-indicated.

Steedman19 recommends polyester wax for minimising heat-induced hardening of difficult tissues.

FATTY TISSUESFatty tissues such as breast or lipoma may be inadequately processed in what is normally a successful schedule for other tissues. Ethanol is a poor fat solvent. To ensure complete dehydration, a superior fat solvent such as acetone or isopropanol should be inserted before the final absolute ethanol, and chloroform or 1,1,1,-trichloroethane used as the transition solvent.

AcknowledgmentsThe assistance of Laurie Reilly, Gary Doak and Savita Francis in the preparation of this chapter is gratefully acknowledged.

REFERENCESSAFETY DATA