Too high a concentration may adversely affect the tissues and produce artefact similar to excessive heat. Also very important is time interval from of removal of tissues to fixation.
The faster you can get the tissue and fix it, the better. Artefact will be introduced by drying, so if tissue is left out, please keep it moist with saline. The longer you wait, the more cellular organelles will be lost and the more nuclear shrinkage and artefactual clumping will occur. There are common usages for fixatives in the pathology laboratory based upon the nature of the fixatives, the type of tissue, and the histologic details to be demonstrated.
Formalin is used for all routine surgical pathology and autopsy tissues when an H and E slide is to be produced. Formalin is the most forgiving of all fixatives when conditions are not ideal, and there is no tissue that it will harm significantly.
Most clinicians and nurses can understand what formalin is and does and it smells bad enough that they are careful handling it. Zenker's fixatives are recommended for reticuloendothelial tissues including lymph nodes, spleen, thymus, and bone marrow.
Zenker's fixes nuclei very well and gives good detail. However, the mercury deposits must be removed dezenkerized before staining or black deposits will result in the sections. Bouin's solution is sometimes recommended for fixation of testis, GI tract, and endocrine tissue.
It does not do a bad job on hematopoietic tissues either, and doesn't require dezenkerizing before staining. Glutaraldehyde is recommended for fixation of tissues for electron microscopy. The glutaraldehyde must be cold and buffered and not more than 3 months old.
The tissue must be as fresh as possible and preferably sectioned within the glutaraldehyde at a thickness no more than 1 mm to enhance fixation. Alcohols, specifically ethanol, are used primarily for cytologic smears. Since smears are only a cell or so thick, there is no great problem from shrinkage, and since smears are not sectioned, there is no problem from induced brittleness.
For fixing frozen sections, you can use just about anything--though methanol and ethanol are the best. Once the tissue has been fixed, it must be processed into a form in which it can be made into thin microscopic sections.
The usual way this is done is with paraffin. Tissues embedded in paraffin, which is similar in density to tissue, can be sectioned at anywhere from 3 to 10 microns, usually routinely.
The technique of getting fixed tissue into paraffin is called tissue processing. The main steps in this process are dehydration and clearing. Wet fixed tissues in aqueous solutions cannot be directly infiltrated with paraffin. First, the water from the tissues must be removed by dehydration. Sometimes the first step is a mixture of formalin and alcohol.
Other dehydrants can be used, but have major disadvantages. Acetone is very fast, but a fire hazard, so is safe only for small, hand-processed sets of tissues. Dioxane can be used without clearing, but has toxic fumes. The next step is called "clearing" and consists of removal of the dehydrant with a substance that will be miscible with the embedding medium paraffin.
The commonest clearing agent is xylene. Toluene works well, and is more tolerant of small amounts of water left in the tissues, but is 3 times more expensive than xylene.
Chloroform used to be used, but is a health hazard, and is slow. Methyl salicylate is rarely used because it is expensive, but it smells nice it is oil of wintergreen.
There are newer clearing agents available for use. Many of them are based on limolene, a volatile oil found in citrus peels.
Another uses long chain aliphatic hydrocarbons Clearite. Although they represent less of a health hazard, they are less forgiving with poorly fixed, dehydrated, or sectioned tissues. Finally, the tissue is infiltrated with the embedding agent, almost always paraffin. Paraffins can be purchased that differ in melting point, for various hardnesses, depending upon the way the histotechnologist likes them and upon the climate warm vs.
A product called paraplast contains added plasticizers that make the paraffin blocks easier for some technicians to cut. A vacuum can be applied inside the tissue processor to assist penetration of the embedding agent. The above processes are almost always automated for the large volumes of routine tissues processed. Automation consists of an instrument that moves the tissues around through the various agents on a preset time scale.
The "technicon" tissue processor is one of the commonest and most reliable a mechanical processor with an electric motor that drives gears and cams , though no longer made. Tissues that come off the tissue processor are still in the cassettes and must be manually put into the blocks by a technician who must pick the tissues out of the cassette and pour molten paraffin over them.
This "embedding" process is very important, because the tissues must be aligned, or oriented, properly in the block of paraffin. Alternatives to paraffin embedding include various plastics that allow thinner sections. Such plastics include methyl methacrylate, glycol methacrylate, araldite, and epon.
Methyl methacrylate is very hard and therefore good for embedding undecalcified bone. Glycol methacrylate has the most widespread use since it is the easiest to work with. Araldite is about the same as methacrylate, but requires a more complex embedding process.
Epon is routinely used for electron microscopy where very thin sections are required. Plastics require special reagents for deydration and clearing that are expensive. For this reason, and because few tissues are plastic embedded, the processing is usually done by hand. A special microtome is required for sectioning these blocks.
Small blocks must be made, so the technique lends itself to small biopsies, such as bone marrow or liver. Once the tissues have been embedded, they must be cut into sections that can be placed on a slide. This is done with a microtome. The microtome is nothing more than a knife with a mechanism for advancing a paraffin block standard distances across it. There are three important necessities for proper sectioning: 1 a very sharp knife, 2 a very sharp knife, and 3 a very sharp knife. Knives are either of the standard thick metal variety or thin disposable variety like a disposable razor blade.
The advantage of the disposable blade becomes apparent when sectioning a block in which is hidden a metal wire or suture. Plastic blocks methacrylate, araldite, or epon are sectioned with glass or diamond knives. A glass knife can section down to about 1 micron.
Microtomes have a mechanism for advancing the block across the knife. Usually this distance can be set, for most paraffin embedded tissues at 6 to 8 microns. The reactions can be enhanced or selectively blocked by choosing a particular type of carbodiimide, pH, temperature, or catalyst.
Because peptide bonds may form as one result of the fixation reactions and subsequently these may be selectively broken using proteases, this group of compounds is thought to have some potential for use in immunohistochemistry as well as for routine histology.
Carbodiimides are already used for the preparation of immunogens. Diimidoesters are water-soluble compounds which cross-link amino groups of proteins and have been used for electron microscopy and immunohistochemistry. Chloro-s-triazides cyanuric chloride has been used for salivary gland mucins and immunofluorescence. Diisocyanates have been used to attach fluorescent tags to proteins, while Diethylpyrocarbonate DPC reacts with tryptophan residues and has been used as a vapour-phase fixative for freeze-dried tissue.
In an appropriate buffer solution it has been proposed as a fixative for small specimens. Maleimides appear to form some cross-links with proteins and Benzoquinone reacts with amines, amino acids and peptides and has been used to fix peptides in endocrine tissues for immunohistochemistr.
Mercuric chloride HgCl 2 was one of the first reagents used for tissue fixation. Although the mechanisms by which it fixes tissue are not fully understood it is known to react with amines, amides, amino acids and sulphydryl groups, the latter being prominent in its reaction with cysteine where it is thought to produce cross-links. It is a powerful protein coagulant which leaves tissue in a state which produces strong staining with acid dyes.
It reacts with phosphate residues of nucleic acids and effectively fixes nucleoproteins. There are several disadvantages to using fixatives containing mercuric chloride. Apart from the corrosive nature of mercuric chloride, mercury is highly toxic, can be absorbed through the skin and is a cumulative poison.
In most countries there are strict rules about disposal of mercury and compounds containing mercury. During fixation with fixatives containing mercuric chloride a crystalline or amorphous greenish-brown artefact pigment of mercury is randomly deposited in tissues. A subsequent treatment with sodium thiosulphate then removes residual iodine. Mercuric chloride-based fixatives tend to penetrate poorly and if fixation is prolonged tissues become very hard and are prone to shrinkage during processing.
In recent years a number of metal salts have been introduced as substitutes for mercuric chloride including salts of zinc and barium. Zinc chloride and zinc sulphate have been accepted fairly widely as being suitable and there are now many proprietary B-5 substitutes available. Zinc sulphate ZnSO 4 and zinc chloride ZnCl 2 are used as substitutes for mercuric chloride in a number of formulated and proprietary fixatives, the sulphate being more popular because it is potentially less corrosive than the chloride which has been reported as causing problems in tissue processors 6 see Part 4.
Zinc salts will react with a range of tissue end groups including amino, carboxyl and sulphydryl, forming reversible reaction products some of which can be removed with a citrate or EDTA wash.
Zinc is said to enhance fixation and staining, particularly of nuclei, in a similar way to mercuric chloride. It is claimed to have advantages in preserving immuno-reactivity when compared to formalin alone obviating the need for antigen retrieval for some epitopes. Zinc salts are far less toxic than mercury salts and disposal of zinc solutions should pose no problem 2, 6.
Picric acid or trinitro phenol C 6 H 2 NO 2 3 OH is a bright yellow crystalline substance that must be stored wet with water to avoid the risk of explosion by percussion or heating of the dry substance. It should be kept in a tightly sealed container and regularly checked to see that it is damp. Apart from being a component of fixatives picric acid is used as an acid dye in several stains eg.
If residual picric acid remains in tissue blocks after processing the staining characteristics of the tissue will be affected and will deteriorate in time.
Picric acid is a coagulant fixative which changes the charges on the ionisable side chains of proteins and disrupts electrostatic and hydrogen bonds. It forms salts picrates with basic groups of proteins causing coagulation. Mercuric chloride fixatives Non coagulant fixatives : They harden protein gels without separating the water from the protein in the gel.
Turbidity in the formalin is due to formation of paraformaldehyde which is formed due to polymerization of formaldehyde. This may be removed by filtration. Formic acid formed in Formaldehyde reduces the quality of routine staining, particularly nuclear, leaches out hemosiderin and promotes the formation of formalin pigment. This can be prevented by using formal saline to which handful of calcium carbonate , then shaken well and stored in jar containing a layer of marble chips.
Principle of formalin fixation Formalin acts by polymerizing action, i. Minimum time required for fixation 8 hours at room temperature. If excess of blood is present in tissues then formalin leads to formation of dark brown artifact pigment granules which are doubly retractile.
For fixation 2. Zenkers Best for fixation of blood forming or blood containing tissues i. This is really a compromise that appears to be widely accepted to produce good quality morphological preservation.
See Part 5 for further discussion. Time: The optimal time for fixation will vary between fixatives. For fixation to occur the fixative has to penetrate, by diffusion, to the centre of the specimen and then sufficient time has to be allowed for the reactions of fixation to occur. Both diffusion time and reaction time depend on the particular reagent used and the optimum time will vary from fixative to fixative. In busy diagnostic laboratories there is considerable pressure to reduce turnaround time and this can result in incompletely-fixed tissues being processed.
This can lead to poor quality sections showing tissue distortion and poor quality staining because poorly fixed tissue does not process well. Remember that if incompletely-fixed tissue is taken from formalin and placed in ethanol during processing, ethanol will continue to fix the tissue and the morphological picture at the centre of the specimen will be that of ethanol fixation. Penetration rate: The penetration rate of a fixing agent depends on its diffusion characteristics and varies from agent to agent.
This means that your formalin fixative should not be expected to penetrate more than say 1 mm in an hour and it will take approximately 25 hours to penetrate to the centre of a 10 mm thick specimen , i. Specimen dimensions: The preceding approximations emphasise the importance of specimen dimensions when fixing tissue.
A specimen should not be more than 4 mm thick. Ideally a 3 mm thick slice should provide excellent fixation and processing. It is useful to remember that the specimen cavity in a standard processing cassette is 5 mm deep.
Volume ratio: It is important to have an excess volume of fixative in relation to the total volume of tissue because with additive fixatives the effective concentration of reagent is depleted as fixation proceeds and in a small total volume this could have an effect on fixation quality. A fixative to tissue ratio of is considered the lowest acceptable ratio but I would advocate a target ratio of However pH can be important for other reasons as in the case of formaldehyde solutions, where breakdown of formaldehyde to form formic acid produces an acidic solution which in turn reacts with hemoglobin to produces an artefact pigment acid formaldehyde hematin.
The most popular formaldehyde solution in use today is therefore buffered to pH 6. For electron microscopy pH is more important and should match physiological pH.
Osmolality: The osmotic effects exerted by the fixative are again more important at the ultrastructural level than at the level of the light microscope because it is the phospholipid membranes that are easily damaged by excessively hypotonic or hypertonic solutions, but osmolality does have some relevance in routine histopathology.
Generally it is the osmolality of the vehicle buffer that is most important and in some formulations this is adjusted to resemble that of tissue fluid eg. Before fixation occurs cells can certainly be damaged by non-isotonic fluids such as water and if specimens cannot be immediately fixed they can be kept moist with gauze soaked in isotonic saline for a short time. It is not a good idea to hold tissue immersed in saline for extended periods.
There are a number of reagents that can be used to fix tissues. Formaldehyde, by far the most popular agent used for histopathology and glutaraldehyde, widely used for ultrastructural studies requiring electron microscopy, are described here. Other reagents are discussed in Part 3. For fixation, one part formalin is usually diluted with nine parts of water or buffer. In concentrated solutions formaldehyde exists as its monohydrate methylene glycol and as low molecular weight polymeric hydrates.
In its diluted form the monohydrate predominates.
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