1. Obtaining a fresh specimen
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Fresh tissue specimens will come from various sources. It should be noted that they can very easily be damaged during removal from the patient or experimental animal. It is important that they are handled carefully and appropriately fixed as soon as possible after dissection. Ideally, fixation should take place at the site of removal, perhaps in the operating theater, or, if this is not possible, immediately following transport to the laboratory.
2. Fixation
The specimen is placed in a liquid fixing agent (fixative) such as formaldehyde solution (formalin). This will slowly penetrate the tissue causing chemical and physical changes that will harden and preserve the tissue and protect it against subsequent processing steps.2 There are a limited number of reagents that can be used for fixation as they must possess particular properties that make them suitable for this purpose. For example, tissue components must retain some chemical reactivity so that specific staining techniques can be applied subsequently.3 Formalin, usually as a phosphate-buffered solution, is the most popular fixative for preserving tissues that will be processed to prepare paraffin sections. Ideally, specimens should remain in fixative for long enough for the fixative to penetrate into every part of the tissue and then for an additional period to allow the chemical reactions of fixation to reach equilibrium (fixation time). Generally, this will mean that the specimen should fix for between 6 and 24 hours. Most laboratories will use a fixative step as the first station on their processor.
Following fixation, the specimens may require further dissection to select appropriate areas for examination. Specimens that are to be processed will be placed in suitably labeled cassettes (small perforated baskets) to segregate them from other specimens. The duration of the processing schedule used to process the specimens will depend on the type and dimensions of the largest and smallest specimens, the particular processor employed, the solvents chosen, the solvent temperatures, and other factors. The following example is based on a six-hour schedule suitable for use on a Leica Peloris™ rapid tissue processor.
3. Dehydration
Because melted paraffin wax is hydrophobic (immiscible with water), most of the water in a specimen must be removed before it can be infiltrated with wax. This process is commonly carried out by immersing specimens in a series of ethanol (alcohol) solutions of increasing concentration until pure, water-free alcohol is reached. Ethanol is miscible with water in all proportions so that the water in the specimen is progressively replaced by the alcohol. A series of increasing concentrations is used to avoid excessive distortion of the tissue.
A typical dehydration sequence for specimens not more than 4mm thick would be:
At this point, all but a tiny residue of tightly bound (molecular) water should have been removed from the specimen.
4. Clearing
Unfortunately, although the tissue is now essentially water-free, we still cannot infiltrate it with wax because wax and ethanol are largely immiscible. We, therefore, have to use an intermediate solvent that is fully miscible with both ethanol and paraffin wax. This solvent will displace the ethanol in the tissue, then this, in turn, will be displaced by molten paraffin wax. This stage in the process is called “clearing” and the reagent used is called a “clearing agent”. The term “clearing” was chosen because many (but not all) clearing agents impart an optical clarity or transparency to the tissue due to their relatively high refractive index. Another important role of the clearing agent is to remove a substantial amount of fat from the tissue, which otherwise presents a barrier to wax infiltration.
A popular clearing agent is xylene, and multiple changes are required to completely displace ethanol.
A typical clearing sequence for specimens not more than 4mm thick would be:
5. Wax infiltration
The tissue can now be infiltrated with a suitable histological wax. Although many different reagents have been evaluated and used for this purpose over many years, the paraffin wax-based histological waxes are the most popular. A typical wax is liquid at 60°C and can be infiltrated into tissue at this temperature then allowed to cool to 20°C, where it solidifies to a consistency that allows sections to be consistently cut. These waxes are mixtures of purified paraffin wax and various additives that may include resins such as styrene or polyethylene. It should be appreciated that these wax formulations have very particular physical properties which allow tissues infiltrated with the wax to be sectioned at a thickness down to at least 2 µm, to form ribbons as the sections are cut on the microtome, and to retain sufficient elasticity to flatten fully during flotation on a warm water bath.
A typical infiltration sequence for specimens not more than 4mm thick would be:
6. Embedding or blocking out
Tissue Processing Systems Market: Introduction
According to the report, the global tissue processing systems market was valued at US$ 318.5 Mn in and is projected to expand at a CAGR of ~5% from to . Tissue processing involves steps needed to take animal or human tissue from fixation to a state that an appropriate histological wax is thoroughly penetrated and can be embedded on the microtome ready for section cutting. Tissue processing is a method to remove water from cells and replace it with a medium that solidifies to cut thin sections on a microtome. Once tissue is properly fixed, it goes through a process, which involves steps such as dehydration, clearing, and infiltration. Tissue processing can be accomplished manually (hand processing); however, it is more practical and much more effective to use an automatic tissue processing unit when several specimens need to be dealt with. These instruments have been available since the s and have slowly improved to be safer in operation, manage greater quantities of specimens, process faster, and achieve higher quality performance.
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North America dominated the global tissue processing systems market in . The trend is anticipated to continue during the forecast period. Strategies adopted by key players such as product approval & launch and mergers & acquisitions, well-established healthcare infrastructure, and early adoption of advanced technologies drive the tissue processing systems market in the region. Asia Pacific accounted for a significant share of the global tissue processing systems market in . The market in the region is likely to expand at a moderate CAGR during the forecast period. Government initiatives in developing countries toward modernization of healthcare infrastructure and rise in awareness about advanced tissue processing systems are the key factors projected to propel the tissue processing systems market in Asia Pacific.
Adoption of Advanced Technologies to Drive Tissue Processing Systems Market
Tissue processing techniques have been greatly revolutionized over the past few years. There are a number of advantages to tissue processor automation such as effective fluid circulation, time saving, and temperature adjustment. The other benefits of automated tissue processors include dramatically increased tissue processing with the aid of automated processors, automatic cleaning cycles, and reagent management system. Moreover, it helps minimize human error. This improves the acceptance of technologically advanced tissue processors, which, in turn, increases the demand for tissue processing systems.
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Small Volume Tissue Processors to Dominate Tissue Processing Systems Market
In terms of product type, the global market for tissue processing systems has been categorized into small volume tissue processors, medium volume tissue processors, and high volume tissue processors. The small volume tissue processors segment dominated the global market accounting for a major share in . The trend is projected to continue during the forecast period. It is anticipated to be highly attractive segment from to . Due to the small amount of reagent required for processing, small volume tissue processors offer excellent balance between durability and reagent saving. In the tissue processing sector, planned introduction and acceptance of small volume tissue processors to meet unmet medical needs is expected to propel the segment during the forecast period.
Bench-top Tissue Processing Units to Drive Tissue Processing Systems Market
Based on modality, the global tissue processing systems market has been bifurcated into bench-top tissue processing unit and portable tissue processing unit. The bench-top tissue processing unit segment dominated the market, accounting for a major share of the market in . The trend is projected to continue during the forecast period. It is anticipated to be a highly attractive segment from to . The bench-top tissue processing unit provides large collections with high-speed processing, and has an established track record of high efficiency, intuitive feature that decreases training time, and greater productivity. These factors are expected to drive the segment.
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Vacuum Tissue Processors to Witness High Demand
In terms of technology, the global market for tissue processing systems has been classified into microwave tissue processors and vacuum tissue processors. The vacuum tissue processors segment dominated the market, accounting for a key share of the market in . The trend is projected to continue during the forecast period. It is anticipated to be a highly attractive segment from to .
Research Laboratories to be Key End Users
Based on end user, the global tissue processing systems market has been divided into hospitals, pharmaceutical companies, research laboratories, and others. The research laboratories segment is driven by increase in the number of research activities, which help innovate new drug products for life-threatening diseases.
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North America to Dominate Tissue Processing Systems Market
In terms of region, the global tissue processing systems market has been segmented into North America, Europe, Asia Pacific, and Rest of the World. North America dominated the global tissue processing systems market in , followed by Europe. North America accounted for a major share of the global tissue processing systems market in . Presence of key players, adoption of advanced tissue processing systems, and focus on research & development are the key factors anticipated to drive the tissue processing systems market in the region. The tissue processing systems market in Asia Pacific is likely to expand at a moderate CAGR during the forecast period. Strategies adopted by key players to address the unmet needs of cancer treatment in developing regions are likely to boost the market in the region.
Competition Landscape
The global tissue processing systems market is fragmented in terms of number of players. Key players in the global tissue processing systems market include Thermo Fisher Scientific, JOKOH, Danaher (Leica Biosystems Nussloch GmbH), MEDITE Medical GmbH, MILESTONE Srl, Sakura Finetek USA, Inc., SLEE medical GmbH, Bio-Optica Milano Spa, Histo-Line Laboratories, and Intelsint Srl.
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