Rationale: Processing is a secondary step in the histology process. Tissue after fixation will go through several steps that will allow some structural format to the tissue that will allow the tissue to be sectioned and stain for diagnostic purposes.

Once completed this lecture, the student should be able to: a)Describe the different dehydrants, clearing and infiltrating agents b)Learn the difference between processing options c)Describe the proper and effective way of embedding tissues d)Learn the terms open processor and closed processor.

INTRODUCTION (Carson Book Pages 26 – 35) All tissues must be adequately supported before they can be sectioned for microscopical examination. The exceptions to this are frozen sections which are sectioned following a range of preparatory freezing methods. Permanent 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. The most widely used method for routine preparation; sectioning, staining and subsequent storage of large numbers of tissue samples is the paraffin wax method, which we will discuss in the embedding lecture. Principles of tissue processing Tissue processing is concerned with the diffusion of various substances into and out of stabilize porous tissues. The diffusion process results from the thermodynamic tendency of processing reagents to equalize 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. Dehydration (Removal of Water) The 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 alcohols. Dehydration is effected as follows: 1

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 hydrolyzed by free water present in tissues to form acetone and methanol 43-50 in an endothermic reaction.Dehydration is necessary in all infiltration methods, except where tissues are simply externallysupported by an aqueous embedding medium. Choice of a dehydrant is determined by the natureof the task, the embedding medium, processing method, and economic factors. Dehydrants differ in their capacity to cause tissue shrinkage. 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. While well fixed tissues can be transferred directly to 95% ethanol, incompletely fixed tissues may exhibit artifacts 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 minimize tissue distortion from diffusion currents, delicate specimens are dehydrated in a graded ethanol series from water through 10%-20%-50%-95%-100% ethanol. 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 agents ALCOHOLS These are clear, colorless, 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 conditions. Ethanol dissolves nitrocellulose slowly unless combined in equal proportions (or better, 1:2) 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 brittle. Colloid, blood, collagen and yolky tissues are particularly affected. The problem is exacerbated by heat during wax infiltration. Methanol is a good ethanol substitute but rarely used for routine processing because of its volatility, flammability and cost. It is a poor lipid solvent, and will not dissolve nitrocellulose 2

unless mixed with acetone. In microwave processing it tends to harden tissues more than ethanol. Isopropanol was first suggested as an ethanol substitute during the prohibition era in the United States. It is a universal solvent available as 99.8% (absolute) isopropanol, slightly slower in action and not as hydroscopic as ethanol, but a far superior lipid solvent. Isopropanol is completely miscible with water and most organic solvents, is fully miscible with melted paraffin wax, and is readily expelled from tissues and wax baths. Isopropanol shrinks and hardens tissues less than ethanol and is used to dehydrate hard, dense tissues, which can remain in the solvent for extended periods without harm. To minimize shrinkage, fixed tissues are transferred via 60%70% isopropanol or ethanol to absolute isopropanol. Isopropyl alcohol has also been recommended as a xylene substitute. In microwave stimulated processing, though unsatisfactory as a dehydrant, isopropanol is used as a transition solvent following ethanol dehydration. Isopropanol only dissolves nitrocellulose in the presence of esters 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 lipidsoluble 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 chitinized arthropods and rodent tissues. It causes less hardening and shrinkage than ethanol, though this is offset by the prolonged processing schedules which may result in tissue shrinkage. N-butanol is poorly miscible with water and only slowly miscible with paraffin wax. It is flammable, with a penetrating camphor-like odor, and the vapors are eye irritants. Iso-butanol, with similar properties and processing characteristics is a less costly substitute for n-butanol. Tertiary-butanol is widely used in plant histology but rarely for animal tissues. Below 26°C it is hygroscopic crystalline solid, a major disadvantage. In processing it is used in a similar manner to n-butanol. GLYCOL-ETHERS Unlike 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, for dehydration following dioxane-based fixation of hard animal tissues, and in the agar-ester wax double embedding technique. Ethoxyethanol is a colorless, nearly odorless 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 ethanol 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 colorless, flammable universal solvent with an odor 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 carcinogen 3


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 swells. 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 reused Explosive peroxides form in dioxane exposed to air. They accumulate in recycled solvent which should be periodically tested for the presence of peroxides. 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 hours. OTHER DEHYDRANTS Acetone is a colorless flammable liquid with sharp characteristic ketonic odor, 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 vacuum. However a transition solvent is normally interposed before the paraffin baths. Acetone is not recommended for microwave processing as it causes excessive nuclear shrinkage. Tetrahydrofuran is a colorless, highly volatile and flammable universal solvent with an offensive ethereal odor. 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. 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 dangerous

2,2 dimethoxypropane (DMP) and 2,2 diethoxypropane (DEP) are used for chemical dehydration of tissues. They are flammable and form peroxides. 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, a transition solvent such as methyl salicylate or toluene should precede infiltration with wax. DMP shrinks tissues slightly less than DEP. Chemical dehydration is suitable for rapid manual processing or machine processing, and is comparable to conventional dehydration for tissue morphology and staining reactions. 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 4

solvents, as in Weigert's carbol-xylol (xylene 75 ml and phenol 25 ml). 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 discoloration. Containers must be protected from light and tightly sealed. Phenol crystals and 80% concentrate react violently with formaldehyde. Clearing (Removal of Alcohol) Clearing 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 wax. In the final stage shrinkage may result from the extraction of fat by the transition solvent. 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. 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 solvents HYDROCARBONS These are odorless flammable liquids with characteristic petroleum or aromatic odors, 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 xylenerelate 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 stabilized in protein coagulant fixatives (Bouin's or SUSA) are less affected. Benzene is more gentle and rapid than xylene and toluene and is probably the best transition solvent, though toxicity and possible carcinogenicity 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 5

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. Many of these solvents have a particularly strong petroleum odor 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 colorless solvents with sweet odors 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. 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 muscle 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 xylene 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 stabilizers to inhibit reactions with aluminum and its alloys. Although mildly toxic (except at high concentrations) the decision to substitute them for more toxic solvents must be soundly based. 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. ESTERS These are colorless flammable solvents miscible with most organic solvents and with paraffin wax. n-Butyl acetate is used as a xylene substitute 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 odors 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 6

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

TERPENES Terpenes are isoprene polymers found in essential oils originally derived from plants, though some are now synthesized. They are the earliest transition solvents to be used in histology and include turpentine and oils of bergamot, cedarwood, clove, lemon, origanum and sandalwood. 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 odors which can be overpowering, requiring good laboratory ventilation. When used for processing hard, dense or chitinized 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 minimized 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 oil. Cedarwood oil is expensive, but exhausted oil can be restored by filtering, then heating to 60°C under vacuum for 30-60 minutes. 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. Terpineol is a clear almost colorless mixture of isomers with a faint pleasant odor and very low evaporation rate. It clears tissues from 80%-90% alcohol with minimal hardening. Like the other 7

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 helminthes. Tissues may remain in it indefinitely without harm. Infiltration and embedding media and methods Ideally an infiltrating and embedding medium should be: • soluble in processing fluids • suitable for sectioning and ribboning • molten between 30°C and 60°C • translucent or transparent; colorless • stable • homogeneous • capable of flattening after ribboning • non-toxic • odorless • 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 fulfill 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, gelatin, or wax which when solidified will provide sufficient external support during sectioning. Infiltration (Interpenetration) 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 minimizing heat-induced tissue hardening, facilitates complete removal of transition solvents, and prolongs the life of wax by reducing 8

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. Paraffin wax PROPERTIES Paraffin 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. 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. MODIFIED PARAFFIN WAXES The 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 microcrystalline 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. Early histological wax formulationshave 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, or to the final investing paraffin wax to improve tissue support for thin sectioning and facilitate flattening and expansion of sections on the waterbath. 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. 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. DMSO scavenges residual transition solvent and probably alters tissue permeability by substituting for or removing bound water thus 9

improving infiltration. Some individuals who handle DMSO-paraffin wax may experience an unpleasant and annoying oyster or garlic taste probably caused by DMSO metabolites. Possible health risks associated with the use of DMSO-paraffin wax are minimal if correct laboratory hygiene is practiced. Processing conditions Temperature, pressure and agitation reduce the duration of tissue processing and improve the quality of infiltration. TEMPERATURE At low temperatures structural elements of tissues are stabilized against the destructive effects of solvent changes. 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 constituents. 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 artifacts than those processed by the more practicable, common an-isothermic techniques. 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 times, but may concomitantly increase shrinkage. Tissue shrinkage during infiltration in paraffin wax results mainly from the effect of heat on collagen. High infiltration temperatures cause marked tissue shrinkage and hardeningwhich can be avoided by maintaining embedding waxes 2-3°C above their melting points. Prolonged immersion in paraffin wax at the correct temperature results in only slight tissue shrinkage 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 usedas well as the tissue type. Microwave stimulated processing involves complex molecular interactions, the key element of which is internal heating, with stimulation of diffusion, and concomitant reduction in the duration of tissue processing. PRESSURE AND VACUUM High pressure facilitates infiltration of dense specimens with viscous resinous embedding media at the block forming stage, 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 vacuum. AGITATION Fluid 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 10

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 processing tissues and fluids are subjected to high frequency agitation and associated phenomena, with simultaneous reduction in processing time.

Processing methods and routine schedules Tissues 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 - tissuetransfer and fluid-transfer types. Automated tissue processing TISSUE-TRANSFER PROCESSORS These processors are characterized 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. 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 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 the tissue basket is enclosed within an integrated fume hood during agitation and transfer cycles thus overcoming the disadvantages of earlier styles. FLUID-TRANSFER PROCESSORS In fluid-transfer units, 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 3045°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 11

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 vapors 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. TISSUE RECOVERY PROCEDURES Procedures 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

GENERAL CONSIDERATIONS • Baskets 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 processing Manual 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: • rapid processing of an urgent specimen • delicate material • very large or thick tissue blocks • hard, dense tissues (nitrocellulose methods) • 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 12

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. Universal solvents with particularly favorable 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. 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 dehydranttransition 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 processing Rapid 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. Processing is undertaken in a dedicated microwave oven which is fitted with precise temperature control and timer, and an interlocked fume extraction system to preclude accidental solvent vapor 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 vapors 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 minimize 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. HINTS FOR MICROWAVE PROCESSING • Tissue blocks should be as thin as possible. Length and width are not as important. • 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. 13

• 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. WATER-MISCIBLE MEDIA Polyethylene glycols (PEG) are water soluble media used for investigation of heat and solventlabile lipids and proteins, 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 nonrecoverable. 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. Low viscosity nitrocellulose (LVN) or water insoluble polyvinyl acetate resin 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 albumenized slides in the usual manner. These approaches surmount many of the previous problems inherent with PEG. The nitrocellulose is removed from sections by immersing in PEG 200 for 15 minutes. 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 histochemistry and botanical histology. WATER-TOLERANT MEDIA Diethylene glycol distearate is a hard, brittle, water tolerant ester (m.p. 47-52°C). It has certain deficiencies when used for routine embedding, 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. Tissues are dehydrated and cleared as in the paraffin wax method. Ester waxes, developed by Steedman, and subsequently modifiedhave low melting points, are hard at room temperature and have good adhesive properties. They are therefore ideal for supporting and serially sectioning refractory hard, criticized 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 conditions and in double embedding with agar. Ester waxes are 14

no longer commercially available and must be prepared from the basic ingredients. Ester Wax 1960 (m.p. 48°C) Polyester wax, developed by Steedman is a ribboning, low melting point wax which reduces heat-induced artifacts. It is recommended for heat labile tissues, to minimize heat-induced hardening in difficult tissues and is an ideal medium for combined light and scanning electron microscopy of animal tissues. The properties of the wax facilitate immunohistochemical investigations as antigenic determinants are well preserved. 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. Polyester wax is no longer commercially available and must be prepared from the basic ingredients. HYDROPHOBIC MEDIA Nitrocellulose Celloidin (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, has superior penetration and final block hardness and is supplied in various grades of viscosity and nitrogen content. Nitrocellulose tissue processing techniques are generally employed for sectioning hard tissues such as bone, for topographical studies of central nervous system tissues, 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 cells are possible on nitrocellulose processed tissues. Methods for difficult tissues HARD DENSE TISSUES Tissues largely comprised of thickened keratin, dense collagen, closely packed smooth muscle fibers, colloid, areas of hemorrhage, 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 minimize hardening in these tissues. Despite careful processing hard tissues frequently require treatment with post-embedding adjuvant before microtomy. Mammalian tissues such as uterus, scirrhous carcinoma, leiomyomas and keratinized tissues are softened by fixing in 4% phenol in a mixture of absolute ethanol (75 ml), water (10 ml) and chloroform (10 ml), or by treating fixed tissues using 4% aqueous phenol for 24-72 hours. Similar results are obtained by dehydrating tissues using phenol in the first bath of absolute ethanol, or in all dehydrant baths. Transition solvents such as chlorinated hydrocarbons and terpenes are recommended as they do not exacerbate tissue hardness.

UNDERPROCESSED TISSUE Carson Book, Page 26, Fig.2-1 15

• Bone not dehydrated or cleared sufficiently • Poor Infiltration • Center of block is soft (White area), indication of • Area not well infiltrated with paraffin • Block cannot sectioned.


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