Tissue Processing

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Tissue Processing
  HISTOLOGY LECTURE #14 INTRODUCTION TO TISSUE PROCESSING Rationale : Processing is a secondary step in the histology process. Tissue after fixation willgo through several steps that will allow some structural format to the tissue that will allow thetissue to be sectioned and stain for diagnostic purposes. Objective: Once completed this lecture, the student should be able to:a) Describe the different dehydrants, clearing and infiltrating agentsb) Learn the difference between processing optionsc) Describe the proper and effective way of embedding tissuesd) Learn the terms open processor and closed processor.  TISSUE PROCESSING   INTRODUCTION(Carson Book Pages 26 – 35) All tissues must be adequately supported before they can be sectioned for microscopicalexamination. 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 thenecessary support for microtomy. This treatment is termed tissue processing. Methods haveevolved for a range of embedding media and applications. The most widely used method forroutine preparation; sectioning, staining and subsequent storage of large numbers of tissuesamples is the paraffin wax method, which we will discuss in the embedding lecture.1  2 Principles of tissue processing  Tissue processing is concerned with the diffusion of various substances into and out of stabilizeporous tissues. The diffusion process results from the thermodynamic tendency of processingreagents to equalize concentrations inside and outside blocks of tissue, thus generallyconforming to Fick's Law: the rate of solution diffusion through tissues is proportional to theconcentration gradient (the difference between the concentrations of the fluids inside and outsidethe 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 operatingconditions, particularly temperature, the characteristics and concentrations of the reagents andthe 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 dehydratingfluids. For example, certain lipids are extracted by anhydrous alcohols, and water solubleproteins are dissolved in the lower aqueous alcohols.   Dehydration is effected as follows:Dilution dehydration, the most commonly used method. Specimens are transferred throughincreasing concentrations of hydrophilic or water miscible fluids which dilute and eventuallyreplace free water in the tissues.Chemical dehydration, where the dehydrant, acidified dimethoxypropane or diethoxypropane, ishydrolyzed by free water present in tissues to form acetone and methanol 43-50 in anendothermic 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 differin their capacity to cause tissue shrinkage. In the paraffin wax method, following any necessarypost 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 ethanolbefore proceeding to the clearing stage. While well fixed tissues can be transferred directly to95% ethanol, incompletely fixed tissues may exhibit artifacts if placed directly in higheralcohols. The dehydrant concentration at which processing is initiated depends largely upon thefixative employed. Following fixation in anhydrous fixatives such as Carnoy's fluid, for exampledehydration is initiated in 100% ethanol. To minimize tissue distortion from diffusion currents, delicate specimens are dehydrated in agraded ethanol series from water through 10%-20%-50%-95%-100% ethanol.Duration of dehydration should be kept to the minimum consistent with the tissues beingprocessed. Tissue blocks 1 mm thick should receive up to 30 minutes in each alcohol, blocks 5mm thick require up to 90 minutes or longer in each change. Tissues may be held and storedindefinitely in 70% ethanol without harm. Other dehydrants, including universal solvents, areused in a similar manner to that described for ethanol, though generally in different concentrationincrements.  3 Dehydrating agents ALCOHOLS  These are clear, colorless, flammable, hydrophilic liquids, miscible with water and, whenanhydrous, with most organic solvents. In addition to their role as dehydrants, alcohols also actas 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 benecessary to consult various tables to ascertain the ethanol concentration.Ethanol is a rapid, efficient and widely applicable dehydrant. It is normally a poor lipid solventexcept under microwave processing conditions. Ethanol dissolves nitrocellulose slowly unlesscombined in equal proportions (or better, 1:2) with diethyl ether. Processing times in absoluteethanol should be minimal. Progressive removal of bound water from carbohydrates and proteinsduring prolonged immersion in absolute ethanol causes tissues to harden excessively and becomebrittle. Colloid, blood, collagen and yolky tissues are particularly affected. The problem isexacerbated by heat during wax infiltration. Methanol is a good ethanol substitute but rarely used for routine processing because of itsvolatility, flammability and cost. It is a poor lipid solvent, and will not dissolve nitrocelluloseunless 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 UnitedStates. It is a universal solvent available as 99.8% (absolute) isopropanol, slightly slower inaction and not as hydroscopic as ethanol, but a far superior lipid solvent. Isopropanol iscompletely miscible with water and most organic solvents, is fully miscible with melted paraffinwax, and is readily expelled from tissues and wax baths. Isopropanol shrinks and hardens tissuesless than ethanol and is used to dehydrate hard, dense tissues, which can remain in the solvent forextended periods without harm. To minimize shrinkage, fixed tissues are transferred via 60%-70% isopropanol or ethanol to absolute isopropanol. Isopropyl alcohol has also beenrecommended as a xylene substitute. In microwave stimulated processing, though unsatisfactoryas 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 ormethyl salicylate, and is used in methyl salicylate-based double-infiltration methods. It cannot beused 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 thissolvent. Normal and tertiary butanols are universal solvents mainly used for small-scale manualprocessing of plant and animal tissues in teaching and research. Normal butanol is recommendedfor processing lightly chitinized arthropods and rodent tissues. It causes less hardening andshrinkage than ethanol, though this is offset by the prolonged processing schedules which mayresult in tissue shrinkage. N-butanol is poorly miscible with water and only slowly miscible withparaffin wax. It is flammable, with a penetrating camphor-like odor, and the vapors are eyeirritants. Iso-butanol, with similar properties and processing characteristics is a less costlysubstitute for n-butanol. Tertiary-butanol is widely used in plant histology but rarely for animaltissues. Below 26°C it is hygroscopic crystalline solid, a major disadvantage. In processing it isused in a similar manner to n-butanol.  4 GLYCOL-ETHERS Unlike the alcohols, these reagents do not act as secondary fixatives, and apart from solventeffects do not appear to alter tissue reactivity. 2-Ethoxyethanol , ethylene glycol monoethyl ether, cellosolve or oxitol is used as a dehydrantpreceding polyester wax embedding, for dehydration following dioxane-based fixation of hardanimal tissues, and in the agar-ester wax double embedding technique.Ethoxyethanol is a colorless, nearly odorless flammable liquid, strongly hygroscopic, misciblewith water and most organic solvents. Cellosolve dissolves nitrocellulose and tends todecompose on exposure to sunlight. It is rapid but non-hardening in action, and tissues canremain in it for years62. To avoid severe shrinkage, tissues are transferred from aqueous fixativeor washing via 60%-70% ethanol into full strength cellosolve. Dioxane , 1,4 diethylene dioxide causes less tissue shrinkage and hardening than ethanol and isexcellent for tissues excessively hardened by ethanol-xylene processing. It has a rapid but gentleaction, and is best used in a graded series. Tissues may remain in it for long periods withoutharm. It is a colorless, flammable universal solvent with an odor similar to butanol, freezes at12°C, and is miscible with water, most organic solvents and paraffin wax. Dioxane dissolvesmercuric chloride, but precipitates potassium dichromate and other salts. It is cumulatively toxicand a suspected carcinogen . Dioxane is expensive and is normally reclaimed by drying over a10-20 mm layer of calcium oxide or anhydrous cupric sulphate. Calcium chloride should not beused as it reacts with dioxane and swells. Dioxane is also recovered by freezing hydrated solventin a spark-proofed refrigerator at 2-5°C. Water, which separates out, is decanted from thecrystalline dioxane which is then thawed, finally dried over a solid dehydrant and reusedExplosive peroxides form in dioxane exposed to air. They accumulate in recycled solvent whichshould be periodically tested for the presence of peroxides. Polyethylene glycols (PEG) are water miscible polymers used to dehydrate and embedsubstances 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 withmost organic solvents and dissolve nitrocellulose. Dehydration is initiated in the low molecularweight 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 DEHYDRANTSAcetone is a colorless flammable liquid with sharp characteristic ketonic odor, low toxicity andis freely miscible with water and organic solvents. It is a fast, effective dehydrant though it maycause tissue shrinkage; it may also act as a coagulant secondary fixative. Acetone is the bestdehydrant 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 undervacuum. However a transition solvent is normally interposed before the paraffin baths. Acetoneis not recommended for microwave processing as it causes excessive nuclear shrinkage.
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