The methods necessary to take an animal or human tissue from fixation to the point where it is entirely infiltrated with an appropriate histological wax and can be embedded ready for section cutting on the microtome are referred to as “tissue processing.” It can be done manually, but it is far more convenient and effective to use an automated tissue processing machine (a “tissue processor”) when dealing with several specimens.
These devices have been around since the 1940s and have gradually improved to be safer to operate, handle bigger specimen numbers, process faster, and generate higher-quality results. Tissue-transfer (or “dip and dunk”) machines transfer specimens from one container to another for processing, and fluid-transfer (or “enclosed”) machines, in which specimens are held in a single process chamber or retort and fluids are pumped in and out as needed, are the two main types of processors.
To improve processing and shorten processing times, the most advanced phantom rapid tissue processor uses higher temperatures, better fluid circulation, and vacuum/pressure cycles.
Steps in Tissue Processing
Fresh tissue samples will be obtained from a variety of sources. It’s worth noting that when they’re removed from a patient or an experimental animal, they’re easily injured. They must be treated with care and correctly repaired as soon as possible following dissection. Fixation should preferably take place at the time of removal, such as in the operating room, or as soon as possible after transport to the laboratory.
The specimen is fixed with a liquid fixative, such as formaldehyde solution (formalin). This will gradually enter the tissue, creating chemical and physical changes that will harden, preserve, and protect the tissue from further processing. There are just a few reagents that can be used for fixing since they must have specific qualities that make them suited for the job. Tissue components must retain some chemical reactivity to be stained with specialized staining processes later. The most common fixative for preserving tissues that will be processed to produce pamphlets is formalin, which is usually in the form of a phosphate-buffered solution. Ideally, specimens should be fixed for long enough for the fixative to penetrate every portion of the tissue, and then for another period to allow the fixation chemical reactions to reach equilibrium (fixation time). In most situations, this entails fixing the specimen for 6 to 24 hours. The first station on most laboratories’ processors will be a fixative step.
Following fixation, further dissection of the specimens may be necessary to determine acceptable examination areas. To keep processed specimens apart from other specimens, they will be stored inappropriately labeled cassettes (little perforated baskets). The kind and dimensions of the largest and smallest specimens, the processor utilized, the solvents chosen, the solvent temperatures, and other parameters will all influence the length of the processing schedule used to process the specimens.
The majority of the water in a specimen must be removed before the wax can be infiltrated since melted paraffin wax is hydrophobic (insoluble in water). Immersing specimens in a series of ethanol (alcohol) solutions of increasing concentration until pure, water-free alcohol is obtained is a typical method. Ethanol is miscible with water in all quantities, therefore the alcohol gradually replaces the water in the specimen. To minimize significant tissue distortion, a succession of escalating concentrations is used.
Although the tissue is now essentially water-free, we are still unable to permeate it with wax because wax and ethanol are highly incompatible. As a result, we’ll need a solvent that’s miscible with both ethanol and paraffin wax as an intermediary step. This solvent will replace the ethanol in the tissue, which will then be replaced by molten paraffin wax. The clearing is the term for this procedure, and the reagent used is called a “clearing agent. The word “clearing” was chosen because, due to their relatively high refractive index, many (but not all) clearing agents provide optical clarity or transparency to the tissue. The cleansing agent also removes a significant amount of fat from the tissue, which would otherwise act as a barrier to wax entry.
It takes numerous adjustments to completely replace ethanol with xylene, which is a typical clearing agent.
A suitable histological wax can now be injected into the tissue. The paraffin wax-based histology waxes are the most popular, although many alternative reagents have been investigated and employed for this purpose throughout the years. At 60°C, a common wax is liquid and can be infiltrated into the tissue, then cooled to 20°C, where it solidifies to a consistency that permits pieces to be cut regularly. These waxes are made out of pure paraffin wax and a variety of additives, such as styrene or polyethylene resins. These wax formulations have very specific physical properties that allow tissues infiltrated with the wax to be sectioned at a thickness of at least 2 m, to form ribbons as the sections are cut on the microtome, and to retain enough elasticity to flatten completely during flotation in a warm water bath.
After being substantially pierced with wax, the specimen must now be molded into a “block” that can be clamped into a microtome for section cutting. When a mold is filled with molten wax and the specimen is placed within, it is referred to as an “embedding center.” The specimen is precisely oriented in the mold because the “plane of section,” an important consideration in both diagnostic and research histology, is determined by its positioning on the top of the mold, a cassette is inserted, which is then filled with more wax and set on a cold plate to harden. After that, the block can be removed from the mold and inserted in the microtome, along with its cassette. It’s worth noting that, if tissue processing is done correctly, the wax blocks containing the tissue specimens are quite stable and can be used as archival material. click here more about.
The necessity of tissue processing is stressed by most laboratory supervisors to their personnel. It’s worth noting that following an incorrect processing schedule or making a fundamental error (such as forgetting to replace or sequence processing reagents) will result in tissue specimens that can’t be sectioned and thus won’t provide any valuable microscopic information. When dealing with diagnostic human tissue, where the entire specimen has been treated, this can be disastrous. Although mechanical or electrical problems can occur in tissue processors, the majority of processing errors that result in tissue compromise are caused by human mistakes. It’s critical to emphasize the importance of proper education and training for people performing tissue processing, as well as the importance of taking extra precautions when setting up a processor for every processing run. For ultra-absorbent Morgue stretcher Pad, get in touch today with Scigen