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Clinical Laboratory Science: Histology and Cytology Histology Specimen Accessioning Gross Examination Fixation - Types of Fixatives The objective of fixation is to preserve cells and tissue constituents in as close a life-like state as possible and to allow them to undergo further preparative procedures without change. Fixation arrests autolysis and bacterial decomposition and stabilizes the cellular and tissue constituents so that they withstand the subsequent stages of tissue processing. Asid
  Clinical Laboratory Science: Histology and Cytology   Histology   Specimen AccessioningGross ExaminationFixation - Types of FixativesThe objective of fixation is to preserve cells and tissue constituents in as close alife-like state as possible and to allow them to undergo further preparativeprocedures without change. Fixation arrests autolysis and bacterialdecomposition and stabilizes the cellular and tissue constituents so that theywithstand the subsequent stages of tissue processing. Aside from theserequirements for the production of tissue sections, increasing interest in cellconstituents and the extensive use of immunohistochemistry to augmenthistological diagnosis has imposed additional requirements. Fixation should alsoprovide for the preservation of tissue substances and proteins. Fixation is,therefore, the first step and the foundation in a sequence of events thatculminates in the final examination of a tissue section.It is relevant to point out that fixation in itself constitutes a major artefact. Theliving cell is fluid or in a semi-fluid state, whereas fixation produces coagulationof tissue proteins and constituents, a necessary event to prevent their loss ordiffusion during tissue processing; the passage through hypertonic and hypotonicsolutions during tissue processing would otherwise disrupt the cells. Forexample, if fresh unfixed tissues were washed for prolonged periods in runningwater, severe and irreparable damage and cell lysis would result. In contrast, if the tissues were first fixed in formalin, subsequent immersion in water isgenerally harmless.A large variety of fixatives is now available, but no single substance or knowncombination of substances has the ability to preserve and allow thedemonstration of every tissue component. It is for this reason that some fixativeshave only special and limited applications, and in other instances, a mixture of two or more reagents is necessary to employ the special properties of each. Theselection of an appropriate fixative is based on considerations such as thestructures and entities to be demonstrated and the effects of short-term and long-  term storage. Each fixative has advantages and disadvantages, some arerestrictive while others are multipurpose. The requirements of a large through-put diagnostic laboratory are also quite different from those of a researchlaboratory with small numbers of specimens for specialised structural analysisand less requirement for urgency.Over the years, various classifications of fixatives have been proposed, withmajor divisions according to function as coagulants and non-coagulants, oraccording to their chemical nature into three general categories which includealcoholic, aldehydic and heavy metal fixatives. A modification of Hopwood'sclassification is shown below: 1. Aldehydes, such as formaldehyde, glutaraldehyde.2. Oxidizing agents: metallic ions and complexes,such as osmium tetroxide, chromic acid.3. Protein-denaturing agents, such as acetic acid, methyl alcohol (methanol), ethyl alcohol(ethanol).4. Unknown mechanism, such as mercuric chloride, picric acid.5. Combined reagents.6. Microwaves.7. Miscellaneous: excluded volume fixation, vapour fixation. Aldehydes include formaldehyde (formalin) and glutaraldehyde. Tissue is fixedby cross-linkages formed in the proteins, particularly between lysine residues.This cross-linkage does not harm the structure of proteins greatly, so thatantigenicity is not lost. Therefore, formaldehyde is good for immunoperoxidasetechniques. Formalin penetrates tissue well, but is relatively slow. The standardsolution is 10% neutral buffered formalin (4% formaldehyde). A buffer preventsacidity that would promote autolysis and cause precipitation of formol-hemepigment in the tissues.Glutaraldehyde causes deformation of alpha-helix structure in proteins so is notgood for immunoperoxidase staining. However, it fixes very quickly so is goodfor electron microscopy. It penetrates very poorly, but gives best overallcytoplasmic and nuclear detail. The standard solution is a 2% bufferedglutaraldehyde.  M ercurials fix tissue by an unknown mechanism. They contain mercuric chlorideand include such well-known fixatives as B-5 and Zenker's. These fixativespenetrate relatively poorly and cause some tissue hardness, but are fast and giveexcellent nuclear detail. Their best application is for fixation of hematopoieticand reticuloendothelial tissues. Since they contain mercury, they must bedisposed of carefully.Alcohols, including methyl alcohol (methanol) and ethyl alcohol (ethanol), areprotein denaturants and are not used routinely for tissues because they cause toomuch brittleness and hardness. However, they are very good for cytologic smearsbecause they act quickly and give good nuclear detail. Spray cans of alcoholfixatives are marketed to physicians doing PAP smears, but cheap hairsprays do just as well.Oxidizing agents include permanganate fixatives (potassium permanganate),dichromate fixatives (potassium dichromate), and osmium tetroxide. They cross-link proteins, but cause extensive denaturation. Some of them have specializedapplications, but are used very infrequently.Picrates include fixatives with picric acid. Foremost among these is Bouin'ssolution. It has an unknown mechanism of action. It does almost as well asmercurials with nuclear detail but does not cause as much hardness. Picric acidis an explosion hazard in dry form. As a solution, it stains everything it touchesyellow, including skin.Tissue ProcessingStabilized tissues must be adequately supported before they can be sectioned formicroscopic examination. While they may be sectioned following a range of preparatory freezing methods, tissues are more commonly taken through a seriesof reagents and finally infiltrated and embedded in a stable medium which whenhard, provides the necessary support for sectioning by microtomy. Thistreatment is termed tissue processing. M ethods have evolved for a range of embedding media and applications. Pre-eminent amongst these is the paraffinwax method, 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 mountedsection is largely determined by the choice of fixative and embedding medium.During tissue processing, loss of cellular constituents and shrinkage or distortionshould be minimal. After fixation, post-fixation and preparatory procedures, thefour main stages in the paraffin method are dehydration, clearing, infiltrationand embedding.  The first step in processing is dehydration. Water is present in tissues in free andbound (molecular) forms. Tissues are processed to the embedding medium byremoving some or all of the free water. During this procedure various cellularcomponents are dissolved by dehydrating fluids. For example, certain lipids areextracted by anhydrous alcohols, and water soluble proteins are dissolved in thelower aqueous alcohols.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 absoluteethanol before proceeding to the clearing stage. Duration of dehydration shouldbe kept to the minimum consistent with the tissues being processed. Tissue blocks1 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 andstored indefinitely in 70% ethanol without harm.Clearing is the transition step between dehydration and infiltration with theembedding medium. M any dehydrants are immiscible with paraffin wax, and asolvent (transition solvent, ante medium, or clearant) miscible with both thedehydrant and the embedding medium is used to facilitate the transition betweendehydration and infiltration steps. Shrinkage occurs when tissues are transferredfrom 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 thetransition solvent. The term clearing arises because some solvents have highrefractive indices (approaching that of dehydrated fixed tissue protein) and, onimmersion, anhydrous tissues are rendered transparent or clear. This property isused to ascertain the endpoint and duration of the clearing step. The presence of opaque areas indicates incomplete dehydration.Xylene clears rapidly and tissues are rendered transparent, facilitating clearingendpoint 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.Infiltration is the saturation of tissue cavities and cells by a supporting substancewhich is generally, but not always, the medium in which they are finallyembedded. Tissues are infiltrated by immersion in a substance such as a wax,which is fluid when hot and solid when cold. Alternatively, tissues can beinfiltrated with a solution of a substance dissolved in a solvent, for examplenitrocellulose in alcohol-ether, which solidifies on evaporation of the solvent toprovide a firm mass suitable for sectioning.
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