Histology & Cytology Assignment Notes

Tissue specimens start their journey at the receiving area of the anatomical pathology laboratory. They are sent in containers immersed in fixative solution and accompanied by a request form that lists the patient information and history, along with a description of the type of tissue and the location it was obtained from. The specimens are logged in the Laboratory-Information System (LIS), and are given a unique patient accession number which identifies each specimen for each patient. This unique accession number for a histology specimen generally starts with a letter that identifies the process by which the tissue was obtained such as; surgical, autopsy, biopsy etc., followed by two numbers identifying the year of collection and then the patient number. This accession number is printed on the best buffered cassettes which are given next to the grossing department, where they are used to house the selected tissue for “tissue processing”. Tissues contained in these cassettes should not exceed a thickness of 5mm. Once Tissue is received, logged and accessioned, it is examined macroscopically by a pathology assistant. Here, tissue is described, measured, and weighed: its consistency is determined and a decision is made on whether the optional step of decalcification is required to remove calcium. When a malignancy is suspected during the grossing process, the specimen is often painted with ink in order to mark the margins of the specimen. Different type inks can be used to identify different areas if needed. When sections are made and processed, the ink will mark the actual margin on the slide. Once pieces of the tissue are selected for examination, they are placed in numbered tissue cassettes, and sent for paraffin processing. 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 to prevent autolysis & putrefaction. There is no perfect fixative, however formaldehyde comes the closest. Therefore, the use of the other available fixatives, depends on the tissue type and features to be demonstrated. Fixation is best carried out at a pH close to neutral, in the range of 6 to 8. Hypoxia of tissues lowers the pH, therefore fixative pH must be neutral to prevent excessive acidity. Acidity in unbuffered formalin will cause pigment that appears as colored dark-brown deposits in tissue. Concentrated formalin is usually diluted by the addition of distilled or RO water and then buffered with phosphate at a pH of 7-8.Speed of fixation of tissues depends upon two factors; the penetration rate of the individual fixative, and the size of the tissue. For example, formalin penetrates the tissue at a rate of 1mm/hr. Formalin and alcohol are known to penetrate the best, and glutaraldehyde the worst. Mercurials and others are somewhere in between. Two factors that can speed-up the penetration process are; heat & agitation. The volume of the fixative is also important, it should be 15 to 20 times the volume of tissue. The concentration of the fixative is best at its minimum level possible, for two reasons; first, it is cheaper and second; high concentrations may damage the tissues. Neutral Buffered Formalin is best used at a concentration of 10%. Glutaraldehyde is generally made up at 0.25% to 4%. In order for a tissue to be examined microscopically, it must be placed on a slide in extremely thin sections. This cannot be achieved without molding the tissue in a form to make such sectioning possible. The tissue is impregnated and infiltrated with paraffin wax in order to be sectioned in sections ranging from 4-6 microns. The technique of getting fixed tissue into paraffin is called fluid exchange. The main steps in this process are; dehydration then clearing before infiltration with wax. In tissue processing, the first step after fixation is “dehydration”. Tissues, which are naturally aqueous, cannot be directly infiltrated with paraffin as paraffin & water are immiscible and would produce undesirable ice crystals. Therefore, the water from the tissues must be removed by dehydration. This is usually done with a series of ascending alcohol concentrations, say 70% to 95% to 100%. The next step is called "clearing", which is the controlled removal of the dehydrating chemical, and replacing it with a substance that is miscible with paraffin. The most common clearing agent is xylene. In the final step, the tissue is infiltrated with paraffin. Various paraffins with different melting points are available for purchase, for varying degrees of hardness, depending on histotechnologist preference and on the climate of the surrounding environment (warm vs. cold). A popular product called Paraplast contains additive plasticizers that make the paraffin blocks easier to cut. A vacuum, which applies negative pressure, is used inside the tissue processor to speed-up the penetration of wax into the tissue. The above tissue processing is done using automated tissue processors, with those that run on the “fluid exchange” principle being preferred. These processors are computerised and can be programed for various tissues and applications. The compartment that houses the tissue cassettes is sealed, in order to minimize the exposure of lab professionals to harmful chemical fumes. After the paraffin processing is complete, the tissues need to be manually embedded into metal molds. This "embedding" process is very important, because the various types of tissues must be aligned and oriented in the mold according to the anatomy of the tissue (muscle, skin, tubal tissue). This will allow the unique structures of the various tissues to be examined under the microscope by the pathologist. Once the tissues have been embedded into the molds, the result is a tissue block. This block will make sectioning of the tissue easier. A microtome is used to cut tissue into sections of about 4 to 6 micrometers. These sections are placed on a slide before it is stained. Prior to mounting the tissue block for the process of sectioning, the block is placed on ice. This allows the paraffin in the block and the tissue to harden, which produces better sections than when the paraffin is soft. The microtome is a relatively simple instrument made of a knife with a mechanism for advancing the paraffin block according to a set distance determined by the operator to produce the desired section thickness. 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, otherwise, artefact can be introduced in the sectioning. Common artefacts include tearing, ripping, holes, folding, etc. Once sections are cut into a ribbon, they are floated on a warm waterbath which is kept at 5℃ below the melting point of the paraffin wax used. The floatation on the surface of the water, helps remove wrinkles which may have formed in the ribbon. A section of the ribbon is then picked up on an albumin treated glass slide. Albumin helps to better adhere the tissue on the slide. The glass slides are then placed in a special oven maintained at a temperature of 58℃ to 62℃ for about 15 minutes. This removes the water and also helps the section adhere to the slide. Now, before we are able to stain the tissue, the paraffin process, which we did earlier, must be reversed. This means, we need to remove the wax out of the tissue and add water back into the tissue. Without doing so, the water-based dyes, such as hematoxylin, would be unable to stain the tissue. In other words, before any staining can be done, the slides are "deparaffinised" by subjecting them to xylene (or a substitute). Then they are “rehydrated” by dipping them in descending concentrations of alcohol. 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 commonly used in histology is the Hematoxylin and Eosin (H & E). Other stains are referred to as "special stains" because they are employed in specific situations according to diagnostic need. Hematoxylin is the oxidized product of the logwood tree. This product is 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 adding ripening (oxidizing) agents, such as sodium iodate, to the hematein solution. In order for hematoxylin to bind to the tissue, it requires a chemical referred to as a"mordant". This is provided by a metal cation such as aluminum. Hematoxylin, being a basic dye, has an affinity for the nucleic acids of the cell nucleus. On the other hand, eosin is an acidic dye with an affinity for the cytoplasmic components of the cell. Hematoxylin stains are either "regressive" or "progressive". With a regressive stain, the slides are overstained with hematoxylin and then decolourized by a solution such as acid-alcohol that removes excess stain, and then counterstained, with eosin. This method works best for large batches of slides. With a progressive stain the slide is dipped in the hematoxylin until the desired intensity of staining is achieved, rinsed to remove excess stain (no decolourizing agent), then counterstained. Progressive stains are best used for single slide staining such as with a frozen section. In histology, the stained slide must be cover-slipped. Traditionally this has been done by a cover-glass. A new method uses a plastic tape. Cover-slipping protects the tissue from being scratched, and provides better optical quality for viewing under the microscope. It preserves the tissue section for years to come. Again, the stained slide must go through a process to reverse what occurred when the paraffin sections were brought to water. The stained slide is taken through ascending series of alcohol solutions to remove the water, then through the clearing agent xylene, before applying mounting medium under the glass coverslip, or a plastic tape. Some tissues are calcified and will not section properly with paraffin embedding. Examples of naturally calcified tissues in the body are; bone & teeth, but other tissues such as, breast & lungs may also contain calcified areas in certain diseases. This calcium must be removed prior to processing the tissue. A variety of agents or techniques have been used to decalcify tissue and none of them work perfectly. This includes; mineral acids, ion exchange, and electrolysis. Strong mineral acids such as nitric acid are commonly used with dense bone because it will remove large quantities of calcium at a rapid rate. Unfortunately, these strong acids also damage cellular morphology, so are not recommended for delicate tissues such as bone marrow. Another chemical known for its use as an anticoagulant in lavender tubes called EDTA, is also used as a decalcifier. Organic acids such as acetic and formic acid are better suited to bone marrow, since they are not as harsh. However, they act more slowly on dense cortical bone. Formic acid in a 10% concentration is the best all-around decalcifier. Some commercial solutions are available that combine formic acid with formalin to fix and decalcify tissues at the same time. Electrolysis has been tried in experimental situations where calcium had to be removed with the least tissue damage. It is slow and not suited for routine daily use. There are two areas of cytology; gynecological and non-gynecological, in which specimens are prepared for examination for the presence of cancerous and precancerous cells. The name Papanicolaou is closely associated with human cytology, cervical screening, and the staining of specimen material. In 1943 George N. Papanicolaou published the results of his research into the diagnosis of cervical cancer, which involved fixing cervical cells while still moist, and staining them in the following three competing dyes, Hematoxylin, OG6 & eosin. This staining technique, known also as the Pap stain, is also used for non-gynecological cytology material. For instance, specimens of sputum or urine, containing squamous epithelial or similar cells, demonstrate excellent results when stained with the Papanicolaou technique. In gynecological cytology, cells are collected from the outer and inner surfaces of the cervical canal. In the traditional pap testing, cells were smeared on the slide directly and manually, by the physician. The smear must be fixed immediately while it is still wet. This is done with Cytospray which is made largely of alcohol-based. This direct technique yields more endocervical cells, but blood and other inflammatory cell may be present in the smears, and this may lead to difficulties in the diagnostic microscopic evaluation. In the new, recently developed methods, referred to as; Liquid Based Cytology (LBC), two brand are available for use; Thinprep and Surepath. In LBC, the specimen material is collected with a brush or a broom. Whichever device is used, it is placed in the transport container, which is filled with alcohol-based preserving medium. This medium clears mucus, blood and inflammatory cells, providing a very clean microscopic image. Once received in the lab, the vial containing the cells undergoes an automated process to create a smear with a monolayer of evenly distributed cells that is representative of all the variety of cells collected. The slides are usually fixed by exposing them to 95% ethanol for a minimum period of 15 minutes. Another advantage of these methods is, that the area to be evaluated is smaller and exhibits a tidy distribution of cells. The two methods are of equal merit because, on the one hand, the inclusion of blood and inflammatory cells provides important information and, on the other, because these inclusions can make microscopic examination more difficult.