HISTOPATHOLOGY TECHNIQUES Dr. K. Premkumar Associate Professor Dept of Biomedical Science Bharathidasan University Course :Human Pathology. This document is not intended to be, and should not be construed as medical advice. ... tissue-processing-on-immunocytochemistry/ Effects of Fixation and Tissue Proce ssing on Immunocytochemistry, Peter Jackson. Differential shrinkage of the various elements in these blocks during fixation and processing contributes to the problems that might be experienced when they are being sectioned. It is worthwhile to stress that use of an inappropriate processing schedule or the making of a fundamental mistake (perhaps in replenishing or sequencing of processing reagents) can result in the production of tissue specimens that cannot be sectioned and therefore will not provide any useful microscopic information. Problems in tissue processing Introduction There are 3 main techniques which are used in preparing microscopical sections from tissues: The paraffin technique … A series of increasing concentrations is used to avoid excessive distortion of the tissue. For light microscopy, three techniques can be used: the paraffin technique, frozen sections, and semithin sections. Send us a submission and we'll be in touch! 2. Ideally fixation should take place at the site of removal, perhaps in the operating theatre, or, if this is not possible, immediately following transport to the laboratory. Tissue Processing HISTOLOGY AND CYTOLOGY MODULE Histology and Cytology Notes 7 TISSUE PROCESSING 7.1 INTRODUCTION The technique of getting fixed tissues into paraffin is called tissue processing. Most laboratory supervisors would emphasise to their staff the importance of tissue processing. Molds are over-filled, requiring scraping of the back and edges of the cassette prior to microtomy. HISTOPATHOLOGICAL TECHNIQUES • Histopathology is the branch of pathology which concerns with the demonstration of minute structural alterations in tissues as a result of disease. The following example is based on a six hour schedule suitable for use on a Leica Peloris™ rapid tissue processor. The specimen is very carefully orientated in the mould because its placement will determine the “plane of section”, an important consideration in both diagnostic and research histology. This step is carried out using an “embedding centre” where a mould is filled with molten wax and the specimen placed into it. This provides a safer laboratory environment without compromising processing quality. Specimens are carefully orientated. www.imb‐mainz.de Microscopy Core Facility Different kinds and combinations of fixatives W.J. Want to see all 101 Steps to Better Histology? This can result in loss of tissue as re-embedding is required. Tissues can remain in cedarwood oil indefinitely without harm. “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. Fresh tissue specimens will come from various sources. Processing of tissue is an important step because poorly processed tissue badly affects the section cutting and staining. When this is completed the block with its attached cassette can be removed from the mould and is ready for microtomy. The synergy between increasingly sophisticated specialty services and evolving techniques in digital imaging yields opportunities for emerging companion diagnostics. Unfortunately, although the tissue is now essentially water-free, we still cannot infiltrate it with wax because wax and ethanol are largely immiscible. A. Some tissue can be fractured by this process. In Bancroft J and Stevens A eds. If you have viewed this educational webinar, training or tutorial on Knowledge Pathway and would like to apply for continuing education credits with your certifying organization, please download the form to assist you in adding self-reported educational credits to your transcript. The basic aim of processing is to remove water from the tissue section and to impregnate the tissue with another medium that can give support to the tissue. Specimens are handled gently during embedding. Paraffin sections It is important that they are handled carefully and appropriately fixed as soon as possible after dissection. Even at this stage of processing specimens can be damaged by excessive local heat. Histopathological examination of tissues starts with surgery, biopsy, or autopsy. The same mold size is used for every specimen. HISTOPATHOLOGY-It refers to the microscopic examination of tissue to study the manifestations of the disease. Our scientists are well-versed in this method, based on binding of nucleic acid and other acidic components of the tissue to the basic hematoxylin stain. Histological techniques are the techniques which have been developed for the processing of the specimens, mainly tissues, for the proper diagnosis of the diseases associated. It can also enhance tissue staining. The small pieces of the tissues or sometimes whole organs are submitted to the histopathology laboratory for the diagnosis of any abnormalities if present. There is no spare tissue. For optimal processing and good morphology tissue should be well fixed before processing. This reference document is presented as a service to health care professionals by Leica Biosystems and has been compiled from available literature. This can be disastrous if you are dealing with diagnostic human tissue where the whole of the specimen has been processed (“all in”). Histopathology It is the branch of science which deals with the gross and microscopic study of tissue affected by disease Tissue for study can be obtained from •Biopsies •Autopsies. Often the tissue touches the edge of the mold. High quality wax is used for infiltration and especially for embedding (blocking out) to ensure high quality blocks that are easy to cut. The tissue is removed from the body or plant, and then, often following expert dissection in the fresh state, placed in a fixative which stabilizes the tissues to prevent decay. Specimens are handled forcefully during embedding to make them lie flat in the mold. These sections are called. We are looking for more great writers to feature here. Cheap, poor quality wax from little-known sources is used for infiltration and embedding. Every microscopic examination is preceded by the processing and preservation of cells and tissues (embedding and cutting procedures). The technique of getting fixed tissue into paraffin for histological study is called tissue processing. Histopathological techniques -sectioning, STAINING, EMBEDDING, fixaton, microtomy, 1. Most modern fluid-transfer processors employ raised temperatures, effective fluid circulation and incorporate vacuum/pressure cycles to enhance processing and reduce processing times. • Most of histopathological techniques simulating to those of applied for study the normal histological … Unsubscribe at any time. Winsor L. Tissue processing. This solvent will displace the ethanol in the tissue, then this in turn will be displaced by molten paraffin wax. Eosin will stain in three shades of pink, provide contrast to the nuclear stain and show many cytoplasmic and tissue elements 83. A typical clearing sequence for specimens not more than 4mm thick would be: The tissue can now be infiltrated with a suitable histological wax. This stain is routinely used in diagnostic labs to evaluate liver diseases, such as cirrhosis. These waxes are mixtures of purified paraffin wax and various additives that may include resins such as styrene or polyethylene. In theory and in practice the paraffin blocks that will be easiest to section contain relatively homogenous tissue of uniform soft consistency (such as kidney), which, when infiltrated with wax, have a consistency similar to that of solidified wax alone (not containing tissue). Low viscosity refined oil should be used for clearing. There is no diagnosis. Over-filled blocks may sit unevenly in the microtome chuck causing instability that may lead to the tissue becoming damaged during microtomy. Microscopic analysis of cells and tissues requires the preparation of very thin, high quality sections (slices) mounted on glass slides and appropriately stained to demonstrate normal and abnormal structures. 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. In the histopathology laboratory, the term “routine staining” refers to the hematoxylin and eosin stain (H&E) that is used “routinely” with all tissue specimens to reveal the underlying tissue structures and conditions. Poor quality wax produces blocks that are difficult to cut. Most laboratories will use a fixative step as the first station on their processor. Hopwood D. Fixation and fixatives. Histopathology is the branch of pathology which concerns with the demonstration of minute structural alterations in tissues as a result of disease Sources for tissue study in Histology Cadavers Autopsy -Post-mortem examination 2. 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. Staff performing embedding have ready access to each specimen description and are appropriately trained. NUCLEAR AND CYTOPLASMIC: Hematoxylin and Eosin: Giemsa: Toluidine Blue: CARBOHYDRATES : Alican Blue (acid mucosubstances and mucins) Alican Blue PAS (acid mucosubstances and neutral polysaccharides) Congo Red (amyloid) Mucicarmine: PAS (glycogen, basement membrane) CONNECTIVE TISSUE: Masson’s Trichrome (muscle, collagen) Reticulin … Ethanol is miscible with water in all proportions so that the water in the specimen is progressively replaced by the alcohol. Histology,[help 1] also known as microscopic anatomy or microanatomy, is the branch of biology which studies the microscopic anatomy of biological tissues. This is a classic standard tissue section staining method widely used for the inspection of tissue components for pathological analysis that’s applicable in all organs and disease models. He is a former Senior Lecturer in histopathology in the Department of Laboratory Medicine, RMIT University in Melbourne, Australia. It has been estimated that tissues shrink as much as 20% or more by the time they are infiltrated with wax4. For any use, the product information guides, inserts and operation manuals of the various drugs and devices should be consulted. Where possible, xylene-free protocols are used (such as those available when using Leica Biosystems’ PELORIS). 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. An appropriate schedule is chosen for the tissue type and size. Routinely, tissues are fixed with neutral formalin 10%, embedded in paraffin, and then manually sectioned with a microtome to obtain 4-5 μm-thick paraffin sections. An inappropriate schedule is chosen. This describes the steps required to take animal and human tissues from fixation to the state where it is completely infiltrated with a suitable wax i.e. The term “special stains” has long been used to refer to a large number of alternative staining techniques that are used when the H&E does not provide all the information the … Although mechanical or electrical faults occasionally occur in tissue processors, processing mishaps where tissues are actually compromised, mainly occur because of human error. Where specimens are incompletely fixed additional formalin fixation is provided in the processing schedule. A typical infiltration sequence for specimens not more than 4mm thick would be: Now that the specimen is thoroughly infiltrated with wax it must be formed into a “block” which can be clamped into a microtome for section cutting. Cerba Research has invested in innovation through a fully equipped histopathology laboratory. Tissues of a dense or fibrous nature, or a specimen where both hard and soft tissue are present in discrete layers can pose more of a challenge because parts of them are not so well supported by the solidified wax. Molds are filled to an optimum level and do not overflow. For this method to be successful higher wax temperatures are required so that isopropanol can be eliminated from specimens during infiltration. 1B: H&E Staining-A comparison of Progressive and Regressive Techniques There are advantages and disadvantages in both techniques. 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. This stage in the process is called “clearing” and the reagent used is called a “clearing agent”. 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. From patient to pathologist, preparing tissue specimens for histological examination requires care, skill and sound procedures. Histology is the microscopic counterpart to gross anatomy, which looks at larger structures visible without a microscope. 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