Tissues from the body taken for diagnosis of disease processes must be processed in the histology laboratory to produce microscopic slides that are viewed under the microscope by pathologists. The techniques for processing the tissues, whether biopsies, larger specimens removed at surgery, or tissues from autopsy, are described below. The persons who do the tissue processing and make the glass microscopic slides are histotechnologists.
Tissues removed from the body for diagnosis arrive in the Pathology Department and are examined by a pathologist, pathology assistant, or pathology resident. Gross examination consists of describing the specimen and placing all or parts of it into a small plastic cassette which holds the tissue while it is being processed to a paraffin block. Initially, the cassettes are placed into a fixative.
Fixation - types of fixatives
The purpose of fixation is to preserve tissues permanently in as life-like a state as possible. Fixation should be carried out as soon as possible after removal of the tissues (in the case of surgical pathology) or soon after death (with autopsy) to prevent autolysis. There is no perfect fixative, though formaldehyde comes the closest. Therefore, a variety of fixatives are available for use, depending on the type of tissue present and features to be demonstrated.
There are five major groups of fixatives, classified according to mechanism of action:
- Oxidizing agents
Aldehydes include formaldehyde (formalin) and glutaraldehyde. Tissue is fixed by cross-linkages formed in the proteins, particularly between lysine residues. This cross-linkage does not harm the structure of proteins greatly, so that antigenicity is not lost. Therefore, formaldehyde is good for immunohistochemical techniques. Formalin penetrates tissue well, but is relatively slow. The standard solution is 10% neutral buffered formalin. A buffer prevents acidity that would promote autolysis and cause precipitation of formol-heme pigment in the tissues.
Glutaraldehyde causes deformation of alpha-helix structure in proteins so is not good for immunohistochemical staining. However, it fixes very quickly so is good for electron microscopy. It penetrates very poorly, but gives best overall cytoplasmic and nuclear detail. The standard solution is a 2% buffered glutaraldehyde
Mercurials fix tissue by an unknown mechanism. They contain mercuric chloride and include such well-known fixatives as B-5 and Zenker's. These fixatives penetrate relatively poorly and cause some tissue hardness, but are fast and give excellent nuclear detail. Their best application is for fixation of hematopoietic and reticuloendothelial tissues. Since they contain mercury, they must be disposed of carefully.
Alcohols, including methyl alcohol (methanol) and ethyl alcohol (ethanol), are protein denaturants and are not used routinely for tissues because they cause too much brittleness and hardness. However, they are very good for cytologic smears because they act quickly and give good nuclear detail. Spray cans of alcohol fixatives 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 specialized applications, but are used very infrequently.
Picrates include fixatives with picric acid. Foremost among these is Bouin's solution. It has an unknown mechanism of action. It does almost as well as mercurials with nuclear detail but does not cause as much hardness. Picric acid is an explosion hazard in dry form. As a solution, it stains everything it touches yellow, including skin.
Fixation - factors affecting fixation
There are a number of factors that will affect the fixation process:
- Time interval
Fixation is best carried out close to neutral pH, in the range of 6-8. Hypoxia of tissues lowers the pH, so there must be buffering capacity in the fixative to prevent excessive acidity. Acidity favors formation of formalin-heme pigment that appears as black, polarizable deposits in tissue. Common buffers include phosphate, bicarbonate, cacodylate, and veronal. Commercial formalin is buffered with phosphate at a pH of 7.
Penetration of tissues depends upon the diffusability of each individual fixative, which is a constant. Formalin and alcohol penetrate the best, and glutaraldehyde the worst. Mercurials and others are somewhere in between. One way to get around this problem is sectioning the tissues thinly (2 to 3 mm). Penetration into a thin section will occur more rapidly than for a thick section.
The volume of fixative is important. There should be a 10:1 ratio of fixative to tissue. Obviously, we often get away with less than this, but may not get ideal fixation. One way to partially solve the problem is to change the fixative at intervals to avoid exhaustion of the fixative. Agitation of the specimen in the fixative will also enhance fixation.
Increasing the temperature, as with all chemical reactions, will increase the speed of fixation, as long as you don't cook the tissue. Hot formalin will fix tissues faster, and this is often the first step on an automated tissue processor.
Concentration of fixative should be adjusted down to the lowest level possible, because you will expend less money for the fixative. Formalin is best at 10%; glutaraldehyde is generally made up at 0.25% to 4%. 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.
Fixatives - general usage
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. Ethanol (95%) is fast and cheap. 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 6-8 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. This is usually done with a series of alcohols, say 70% to 95% to 100%. 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. cold). 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. Newer processors have computers, not cam wheels, to control them and have sealed reagent wells to which a vacuum and/or heat can be applied.
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 former type allows custom sharpening to one's own satisfaction, but is expensive (more than $100 per blade). The latter cost about $1 per blade and are nearly as good. 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. Thin sections for electron microscopy (1/4 micron) are best done with a diamond knife which is very expensive ($2500).
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 more expensive the microtome ($15,000 to $30,000), the better and longer-lasting this mechanism will be.
Sectioning tissues is a real art and takes much skill and practice. Histotechnologists are the artists of the laboratory. It is important to have a properly fixed and embedded block or much artefact can be introduced in the sectioning. Common artefacts include tearing, ripping, "venetian blinds", holes, folding, etc. Once sections are cut, they are floated on a warm water bath that helps remove wrinkles. Then they are picked up on a glass microscopic slide.
The glass slides are then placed in a warm oven for about 15 minutes to help the section adhere to the slide. If this heat might harm such things as antigens for immunostaining, then this step can be bypassed and glue-coated slides used instead to pick up the sections.
At times during performance of surgical procedures, it is necessary to get a rapid diagnosis of a pathologic process. The surgeon may want to know if the margins of his resection for a malignant neoplasm are clear before closing, or an unexpected disease process may be found and require diagnosis to decide what to do next, or it may be necessary to determine if the appropriate tissue has been obtained for further workup of a disease process. This is accomplished through use of a frozen section. The piece(s) of tissue to be studied are snap frozen in a cold liquid or cold environment (-20 to -70 Celsius). Freezing makes the tissue solid enough to section with a microtome.
Frozen sections are performed with an instrument called a cryostat. The cryostat is just a refrigerated box containing a microtome. The temperature inside the cryostat is about -20 to -30 Celsius. The tissue sections are cut and picked up on a glass slide. The sections are then ready for staining.
The embedding process must be reversed in order to get the paraffin wax out of the tissue and allow water soluble dyes to penetrate the sections. Therefore, before any staining can be done, the slides are "deparaffinized" by running them through xylenes (or substitutes) to alcohols to water. There are no stains that can be done on tissues containing paraffin.
The staining process makes use of a variety of dyes that have been chosen for their ability to stain various cellular components of tissue. The routine stain is that of hematoxylin and eosion (H and E). Other stains are referred to as "special stains" because they are employed in specific situations according to the diagnostic need.
Frozen sections are stained by hand, because this is faster for one or a few individual sections. The stain is a "progressive" stain in which the section is left in contact with the stain until the desired tint is achieved. Staining a frozen section.
H and E staining
Hematoxylin is the oxidized product of the logwood tree known as hematein. Since this tree is very rare nowadays, most hematein is of the synthetic variety. In order to use it as a stain it must be "ripened" or oxidized. This can be done naturally by putting the hematein solution on the shelf and waiting several months, or by buying commercially ripened hematoxylin or by putting ripening agents in the hematein solution.
Hematoxylin will not directly stain tissues, but needs a "mordant" or link to the tissues. This is provided by a metal cation such as iron, aluminum, or tungsten. The variety of hematoxylins available for use is based partially on choice of metal ion used. They vary in intensity or hue. Hematoxylin, being a basic dye, has an affinity for the nucleic acids of the cell nucleus.
Hematoxylin stains are either "regressive" or "progressive". With a regressive stain, the slides are left in the solution for a set period of time and then taken back through a solution such as acid-alcohol that removes part of the stain. This method works best for large batches of slides to be stained and is more predictable on a day to day basis. With a progressive stain the slide is dipped in the hematoxylin until the desired intensity of staining is achieved, such as with a frozen section. This is simple for a single slide, but lends itself poorly to batch processing.
Eosin is an acidic dye with an affinity for cytoplasmic components of the cell. There are a variety of eosins that can be synthesized for use, varying in their hue, but they all work about the same. Eosin is much more forgiving than hematoxylin and is less of a problem in the lab. About the only problem you will see is overstaining, especially with decalcified tissues.