H&E, hematoxylin and eosin. Also, small mineral deposits such as copper or calcium may dissolve and leach from tissues. Overexposure to xylene during processing can cause hardening of tissues. Connective and mesenchymal tissues with their stains, Bancroft’s Theory and Practice of Histological Techniques. A device that disinfects tissues to use in transplantation or allograft surgery. Fragments of tissue are embedded in melted agar, allowed to solidify and trimmed for routine processing. Conventional Processing - In Depth. A unique accession number or code should be assigned to every tissue sample as discussed in Chapter 5. Dehydration A. Rapid tissue processing protocol for dehydration and clearing for breast and kidney TOF data. They are not recommended for processing delicate tissues due to their hardening properties. The mold is placed on a small cooling area to allow the paraffin wax to solidify. • Embedding – orienting the tissue sample in a support medium and allowing it to solidify. Its main use is as a cohesive agent for small friable pieces of tissue after fixation, a process known as double embedding. Fixation of the tissue sample is essential to maintain cell and tissue morphology during … There are occasions when paraffin wax is an unsuitable medium for the type of tissue being processed including: • Processing reagents remove or destroy tissue components that are the object of investigation, e.g. Aseptic Processing: The processing of tissue using aseptic techniques when tissue, containers and/or devices are handled in a controlled environment in which the air supply, materials, equipment and personnel are regulated to prevent microbial contamination of tissue. To help in the visualization of small fragments of tissue during embedding, a few drops of 1% eosin can be added to the specimen container 30 minutes prior to processing. The tissue is impregnated with the medium, forming a matrix and preventing distortion of the tissue structure during microtomy. Artifacts interfere with histology by changing the tissues appearance and hiding structures. Dehydration should be accomplished slowly. “ Tissue processing ” describes the steps required to take animal or human tissue from fixation to the state where it is completely infiltrated with a suitable histological wax and can be embedded ready for section cutting on the microtome. Its properties are varied depending on the melting point used, ranging from 47 to 64°C. Embedding involves the enclosing of properly processed, correctly oriented specimens in a support medium that provides external support during microtomy. It is hydrophilic, miscible with water and other organic solvents, fast-acting and reliable. Acetone is a clear, colorless, flammable fluid that is miscible with water, ethanol and most organic solvents. For purposes of tissue processing it is used in the same manner as ethanol. In the fields of histology, pathology, and cell biology, fixation is the preservation of biological tissues from decay due to autolysis or putrefaction.It terminates any ongoing biochemical reactions and may also increase the treated tissues' mechanical strength or stability. For delicate tissue it is recommended that the processing starts in 30% ethanol. For this reason, specimens are processed through a graded series of reagents of increasing concentration. The medium should provide elasticity, resisting section distortion while facilitating sectioning. Identify the various factors affecting processing and compare how different tissue types require different processing cycles. When tissue is immersed in fluid, an interchange occurs between the fluid within the tissue and the surrounding fluid. • Multiple pieces of a tissue are oriented side by side with the epithelial surface facing in the same direction. Rationale: for paraffin embedding/sectioning C. Steps 1. wash out fixative 2. graded series of alcohol 70%, 95%, 100%, 100% 3. replace water by diffusion 4. not too long, not too short 9. Processing Steps Defined. Other than expressly provided for in this Agreement, Licensee shall not directly or indirectly reproduce, copy, alter, edit or otherwise modify or make or cause to be made any imitation of the Technology or the Tissue Processing. Relate how quality control measures ensure consistent tissue processing and identify tissue that is poorly processed and state a remedy for the problem. Tissue Processing. lymph nodes, The use of heat may adversely affect tissues or enzymes, The infiltrating medium is not sufficiently hard to support the tissue, Resin is used exclusively as the embedding medium for electron microscopy (see Chapter 22), ultra-thin sectioning for high resolution and also for undecalcified bone (see. Most of the solutions used in processing, dehydration and clearing, have similar viscosities, with the exception of cedar wood oil. Tissue processing. The size and type of specimen in the tissue cassette determines the time needed for complete fixation and processing. Tissue Processing means the separation of Adipose Stromal Vascular Fraction from fat tissue utilizing the Technology, which Tissue Processing is the patented process which the Medical Practice is licensing from the Technology Company as per this agreement. Diffusion results from the tendency of processing reagents to equalize concentrations both inside and outside blocks of tissue. Vacuum used on the automated processor should not exceed 50.79 kPa to prevent damage and deterioration to the tissue. (a) Average TOF trace for breast tissue (b) representative morphology for breast. Process definition, a systematic series of actions directed to some end: to devise a process for homogenizing milk. If possible, larger and smaller pieces of tissue should be separated and processed using different schedules. The techniques for processing the tissues, whether biopsies, larger specimens removed at surgery, or tissues from autopsy, are described below. Lower melting point paraffin wax is softer and provides less support for harder tissues. Embedding – orienting the tissue sample in a support medium and allowing it to solidify. Several factors influence the rate at which the interchange occurs: namely, agitation, heat, viscosity and vacuum. • Skin biopsies; shave punch or excisions, cross section of the epidermis, dermis and subcutaneous layers must be visible. If fixation is not complete prior to processing, stations should be designated on the processor for this purpose. Preserving cells and tissue components with minimal distortion is the most important aim of processing tissue samples. Heat must be used sparingly to reduce the possibility of shrinkage, hardening or embrittlement of the tissue sample. Clearing – removal of dehydrating solutions, making the tissue components receptive to the infiltrating medium. The quick cooling of the wax ensures a small crystalline structure, producing fewer artifacts when sectioning the tissue. Skin biopsies; shave punch or excisions, cross section of the epidermis, dermis and subcutaneous layers must be visible. Prolonged treatment with xylene during processing should be avoided, as tissue tends to become overhardened affects the following tissue the most: a. Regardless of whether an automated or manual labeling system is used, adequate policies and procedures must be in place to ensure positive identification of the tissue blocks and slides during processing, diagnosis, and filing. It is more flammable and volatile than xylene. Agitation increases the flow of fresh solutions around the tissue. Tissue fixation time is dependent on tissue size. Xylene substitutes are aliphatic hydrocarbons that exist in long- and short-chained forms. • Intestine, gallbladder, and other epithelial biopsies: cut in a plane at right angles to the surface, and oriented so the epithelial surface is cut last, minimizing compression and distortion of the epithelial layer. Factors influencing the rate of processing Times: All times in processing … The use of celloidin or LVN (low viscosity nitrocellulose) is discouraged because of the special requirements needed to house the processing reagents and the limited use these types of sections have in neuropathology. Tissues are exposed to a series of… Thicker tissue blocks can be processed, greater than 1 mm in thickness. Heating the paraffin wax to a high temperature alters the properties of the wax. They differ in the number of carbon atoms within the carbon chain. Embedding 8. This fluid has the same physical property as ethanol. Efficient agitation may reduce the overall processing time by up to 30%. When the dehydrating agent has been entirely replaced by most of these solvents the tissue has a translucent appearance: hence the term ‘clearing agent’. Many of these additives had a higher melting point than paraffin wax, consequently making the tissue more brittle. The amount of additive will impact the rate of infiltration. Phenol (4%) should be added to each of the 95% ethanol stations. Industrial methylated spirit (denatured alcohol), Processing reagents remove or destroy tissue components that are the object of investigation, e.g. Dehydration – removal of water and fixative from the tissue. Multiple pieces of a tissue are oriented side by side with the epithelial surface facing in the same direction. Infiltrating – permeating the tissue with a support medium. How to Fix and Prepare Tissue for Histology Submission Histology involves all processes from the collection of tissue from the animal to cover slipping the final slide product. This unique number should accompany the specimens throughout the entire laboratory process and may be electronically or manually generated. Tissue processing is concerned with the diffusion of various substances into and out of porous tissues. It is used in microwave processing schedules. Conversely, if the molecule size is larger, the rate of exchange is slower (high viscosity). If Licensee determines or attempts to determine the Technology for Tissue Processing. It also changes the tissues’ receptiveness to further processing. Isopropyl alcohol does not cause over-hardening or shrinkage of the tissue. Step Three-Tissue Processing. Tubular structures: cross section of the wall and lumen should be visible; arteries, veins, fallopian tube and vas deferens samples. Most laboratories use modular embedding centers, consisting of a paraffin dispenser, a cold plate, and a heated storage area for molds and tissue cassettes. Substances added to paraffin wax in the past include beeswax, rubber, ceresin, plastic polymers and diethylene glycol distearate. It has a wide range of melting points, which is important for use in the different climatic regions of the world. Tissue processing artifacts can include pigments formed by fixatives, shrinkage, washing out of cellular components, color changes in different tissues types and alterations of the structures in the tissue. 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