A popular clearing agent is xylene and multiple changes are required to completely displace ethanol. HISTOLOGY : It is the branch of science which deals with the gross & microscopic study of normal tissue . The main steps in this process aredehydration … 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. There is however a patient to whom an explanation has to be provided. This carousel has a number of glass beakers containing solvents and solutions that ensure the tissue is dehydrated and cleared ready for paraffin wax embedding. 1. Overview of the steps in tissue processing for paraffin sections 1. Third step in tissue preparation. This machine has reservoirs of molten wax, hotplates, and a cold plate for setting the blocks. A mold of suitable size is always chosen for each specimen. A series of increasing concentrations is used to avoid excessive distortion of the tissue. The technique of getting fixed tissue into paraffin is called tissue processing; Tissue processing is designed to remove all extractable water from the tissue, replacing it with a support medium that provides sufficient rigidity to enable sectioning of the tissue without damage or distortion Ethanol is miscible with water in all proportions so that the water in the specimen is progressively replaced by the alcohol. Review of literature on quality evaluation studies pertaining to histopathology revealed that earlier reports were mainly focused on analytical aspects with limited studies on assessment of pre-analytical phase. It’s not too late to register your lab for the January Laboratory Webinar, next Wednesday, January 27 th at 1PM EST! Generally this will mean that the specimen should fix for between 6 and 24 hours. Most modern fluid-transfer processors employ raised temperatures, effective fluid circulation and incorporate vacuum/pressure cycles to enhance processing and reduce processing times. • Steps of tissue processing in histopathology laboratory,Review Report • Tissue processing : A procedure which need to take place after gross examination (2019). 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. 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. To achieve this it is important that the tissue must be prepared in such a Histopathology Fixative : Types, Classification, Reaction. Xylene is the clearing agent used most commonly worldwide. We therefore have to use an intermediate solvent that is fully miscible with both ethanol and paraffin wax. Broadly there are two strategies that can be employed to provide this support. This process is known as tissue fixation. 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. Leslie P. Gartner; James L. Hiatt. actually using tissue in each cassette, then we include foam biopsy pads in cassettes instead of tissue. There are two main types of processors, the tissue-transfer (or “dip and dunk”) machines where specimens are transferred from container to container to be processed, or the fluid-transfer (or “enclosed”) types where specimens are held in a single process chamber or retort and fluids are pumped in and out as required. It should be noted that, if tissue processing is properly carried out, the wax blocks containing the tissue specimens are very stable and represent an important source of archival material. 1. Conventional Processing … This is where ‘clearing’ comes in. Originally published December 10, 2013. There is no diagnosis. The purpose of clearing is to remove dehydrating agents from tissues and to prepare the tissues for impregnation with the embedding agent. Winsor L. Tissue processing. The specimen is placed in a liquid fixing agent (fixative) such as formaldehyde solution (formalin). … The usual way this is done is with paraffin. Reviewed and updated on November 10, 2020. 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. Automated tissue processors replaced hand processing starting in 1945, and cryostats were first manufactured in 1951. 150-162. Our automated histopathology services provide grossing, tissue processing, embedding, slide preparation, and special staining for a variety of specialties and for research purposes. Although xylene is used widely as a clearing agent for tissue processing it is a toxic reagent and some laboratories prefer to use less-toxic alternatives such as isopropanol or other xylene substitutes. The same mold size is used for every specimen. Embedding and blocking 6. TISSUE PROCESSING 2. Steps in Histopathology A specimen brought to the histopathology laboratory must first be logged, identified and then subjected to specimen preparation prior to tissue processing. Where specimens are incompletely fixed additional formalin fixation is provided in the processing schedule. Vacuum can also aid in the removal of trapped air in porous tissue. Step Two-Grossing. Send us a submission and we'll be in touch! Start studying Histopathology -3 TISSUE PROCESSING. 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. PROPERTIES OF AN IDEAL EMBEDDING MEDIUM. Most laboratory supervisors would emphasize to their staff the importance of tissue processing. This step is carried out using an “embedding centre” where a mould is filled with molten wax and the specimen placed into it. Conventional tissue processing must proceed in a specific order. List qualities of good fixatives. Processing tissues into thin microscopic sections is usually done using a paraffin block, as follows: 1. In this session, we will debunk some processing myths, review the purpose and function of the common steps and reagents in tissue processing… Obtaining a fresh specimen. These are applicable to identify either spontaneous or diseases-induced changes. Histopathology Tissue Processing : Factors, Steps Of Tissue Processing, Types. An inappropriate schedule is chosen. This stage in the process is called “clearing” and the reagent used is called a “clearing agent”. Various components of the cell are also removed by this process. Shrinkage of tissue can occur at these final stages as the xylene also removes fat residues left in the samples. EMBEDDING… We’ve covered these steps in brief in a previous article on How Histology Slides are Prepared, but this article will focus on one particular procedure that needs to take place between tissue fixation and the embedding/sectioning of paraffin blocks: tissue processing. testis. Histological procedures aim at providing good-quality sections that can be used for a light microscopic evaluation of tissue. All tissue specimens of the same patient received on the same day are given the same accession number. Receipt of specimens from OT 2. He is a former Senior Lecturer in histopathology in the Department of Laboratory Medicine, RMIT University in Melbourne, Australia. In most modern institutes and histology labs, processing will be carried out in dedicated tissue processing machines. In the histology laboratory, conventional tissue processing describes the stages required to take fixed tissue samples through dehydration and clearing to the state where it is completely infiltrated and embedded with a suitable medium (normally paraffin wax) in readiness for cutting sections on a microtome (microtomy). There are three main steps in tissue processing, namely: ‘dehydration’, ‘clearing’, and ‘infiltration’. Guide lines for there placement of processing reagents are ignored, meaning that ineffective, contaminated or diluted reagents are used (e.g “out-of-threshold” warnings from the PELORIS reagent management system are ignored).This can cause poor processing quality. Reagent Steps of Tissue Processing. Steps in processing the tissues (10) : Histotechnology Is the art and science performed by the histotechnologist to produce a tissue section of good quality that will enable the pathologist to … Mention the common methods of decalcification used in processing calcified tissue. When this is completed the block with its attached cassette can be removed from the mould and is ready for microtomy. The most commonly used fixative in histopathology is 10% neutral buffered formalin. This is an animated video Regarding Introduction of Histopathology & Tissue Processing. Principles of Tissue Processing. 2. In general, the whole process takes around six hours and is usually set up to run overnight. Copyright Leica Biosystems Nussloch GmbH 2021, H&E Slide Stainers, Special Stainers & Coverslippers, Fully Automated Glass Coverslipper CV5030, The Compact Slide Printing Solution - HistoCore PERMA S, Automated Inkjet Printer for Tissue Cassettes - IP C, Automated Inkjet Printer for Microscope Slides - IP S, Histology Embedding Centers & Accessories, Stand-alone Cold Plate - HistoCore Arcadia C, Stand Alone Paraffin Dispensing Module - HistoCore Arcadia H, Leica Biosystems Microtome Comparison Guide, Specimen Tracking and Workflow Management, Apply for self-reported educational credits, Click here to download your free copy of Science of Tissue Processing, We can freeze the tissue and keep it frozen while we cut our sections. 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. Learn how your comment data is processed. 3, pp. … Forceps are heated well beyond the melting point of wax. The filled tissue cassettes are then stored in formalin until processing begins. Learn vocabulary, terms, and more with flashcards, games, and other study tools. You’ll get more from your study … What happens to the SPECIMEN? Specimen Receiving, Processing and Accessioning from the Operating Room. Get Knowledge Pathway updates delivered directly to your inbox. The techniques for processing the tissues, whether biopsies, larger specimens removed at surgery, or tissues from autopsy, are described below. Want to see all 101 Steps to Better Histology? Histopathology Techniques: Tissue Processing and Staining Histopathology Techniques.pdf (Size: 60.47 KB / Downloads: 55) Incisional biopsy: In this method only a portion or wedge of tissue from a large lesion is taken and therefore, the procedure is strictly a diagnostic nature. The temperature of the embedding center hot plate and wax reservoir is regularly checked. Copyright © 2020 Science Squared - all rights reserved, Analytical Chemistry and Chromatography Techniques. This can result in loss of tissue as re-embedding is required. The solvent used for this intermediate stage is usually xylene. Molds are filled to an optimum level and do not overflow. Clearing is an essential step in histopathology processing for light microscopy. Unsubscribe at any time. That ends the journey from tissue to wax block, which is, I guess, the start of another journey of sectioning, making slides, and immunohistochemistry! Clearing 4. 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. Mention the common methods of decalcification used in processing calcified tissue. We are looking for more great writers to feature here. These improvements are specifically focused on two mandatory steps; (1) … Fixation 2. 2. This site uses Akismet to reduce spam. Dehydration is the first step, which involves immersing your specimen in increasing concentrations of alcohol to remove the water and formalin from the tissue. 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. To outline the proper procedures for collecting, preserving, identifying, and processing tissue specimens for evaluation by light microscopic histopathological evaluation. PURPOSE: To Outline the Proper Procedures for Histopathology . Curtin University. Formalin, widely used today, was first used in 1893. Our experienced histologists and technicians ensure client samples are optimally prepared for downstream analysis and interpretation by a licensed pathologist. Most laboratories will use a fixative step as the first station on their processor. These sections are called. They are, Dehydration ; Clearing ; Infiltration ; The involvement of each of these three steps occurs during the diffusion of the solution … Some poorly prepared specimens require extensive trimming on the microtome to obtain a full-face section. I. Independentei 99-101, 050096 Bucharest – Romania; 2Department of 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. There is no spare tissue. Pathology, histopathology or histology aims to study the manifestation of disease by microscopic examination of tissue morphology. Processing reagents are replaced strictly according to established guidelines (ideally using are agent management system in an advanced tissue processor such as Leica Biosystem’s PELORIS). 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). 3. This purpose of this course is to discuss the appropriate application of the most common tissue processing steps utilized in today's histology laboratory and provide troubleshooting guidelines for tissue that has been improperly processed. Routinely, tissues are fixed with neutral formalin 10%, embedded in paraffin, and manually se … Infiltration is when the final xylene is replaced with molten wax, which infiltrates the tissue. Tissue Processing. The combined effects of fixation and processing is to harden the tissue and it is inevitable that shrinkage will also occur. This article describes the method for processing tissue to create paraffin embedded specimens ready for sectioning. Paraffin wax for infiltration and support during sectioning was introduced during the mid1800s. Impregnation time for dense fatty tissue can be greatly reduced with the addition of vacuum during processing; STEPS OF PROCESSING 1. In Woods A and Ellis R eds. These devices have been available since the 1940’s1 and have slowly evolved to be safer in use, handle larger specimen numbers, process more quickly and to produce better quality outcomes. Leica Biosystems Division of Leica Microsystems Inc. Leave a comment below! In this step, the alcohol penetrates tissue … The embedding medium is considered as ideal if it bears the following qualities: CLEARING 3. The tissue undergoes a series of steps before it reaches the examiners desk to be thoroughly examined microscopically to arrive at a particular diagnosis. Main stages of tissue processing for histology. Alcohols are most commonly used in the laboratory for tissue dehydration, since they are miscible with aqueous fixatives like 10% formalin. The main steps in this process are dehydration and clearing. Start studying Steps in tissue preparation (HISTOLOGY). List clearing agents used in tissue processing in histopathology laboratory. Impregnation time for dense fatty tissue can be greatly reduced with the addition of vacuum during processing; STEPS OF PROCESSING … Table 1. What can cause your processing reagent to turn cloudy? Processing specimens It is important to eliminate potential variables as far as possible when processing test and control groups of specimens. II. This document contains a brief summary of the steps to process a tissue for microscopy. The older design of machine is a carousel, which contains a cage where the tissue cassettes are placed. Steps involved in the process are 1. Tissue Processing. Staff performing embedding have ready access to each specimen description and are appropriately trained. The various steps are: Sample collection; Logging of specimen; Preparation of tissues which include fixation and decalcification 2017/2018 We hope each step provides a valuable reminder of good histology practice, and helps with troubleshooting when unacceptable results do occur. List qualities of good fixatives. Impregnation 5. Mention different types of microtone use in histopathology laboratory. Over-filled blocks may sit unevenly in the microtome chuck causing instability that may lead to the tissue becoming damaged during microtomy. Fixation. This solvent will displace the ethanol in the tissue, then this in turn will be displaced by molten paraffin wax. Specimens are handled gently during embedding. … Routine staining 5. In the coming months, the first histopathology laboratory will be equipped with Tissue-Tek SmartConnect to show its value and support laboratory … Although every effort has been made to report faithfully the information, Leica Biosystems cannot be held responsible for the correctness. Different laboratory chemicals were investigated for use as fixatives. Processing tissues into thin microscopic sections is usually done using a paraffin block, as follows: Dehydration, which involves immersing your specimen in increasing concentrations of alcohol to remove the water and formalin from the tissue. Following fixation the specimens may require further dissection to select appropriate areas for examination. … There are three main steps in tissue processing, namely: ‘dehydration’, ‘clearing’, and ‘infiltration’. It should be noted that they can... 2. Journal of Histotechnology: Vol. In Gross … This produces so-called “paraffin sections”. Vacuum will remove reagents from the tissue but only if they are more volatile than the reagent being replaced. 3. Laboratory histopathology, a complete … At the lower end of the ethanol concentrations, water-soluble proteins are removed, whilst towards the 100% ethanol step, certain lipids may be dissolved. The temperature of the embedding center hot plate is never checked. Unfortunately, although the tissue is now essentially water-free, we still cannot infiltrate it with wax because wax and ethanol are largely immiscible. Tissue processing consists of three major steps. What is dehydration in tissue processing? RESPONSIBILITY . In the histology laboratory, conventional tissue processing describes the stages required to take fixed tissue samples through dehydration and clearing to the state where it is completely infiltrated and … Mention different types of microtone use in histopathology laboratory… Tissues from the body taken for diagnosis of disease processes must be processed in the histology laboratory to produce microscopic slides that are viewed under the microscope by pathologists. Asignatura. TISSUE PROCESSING 1. 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. In pathology, the sample to be examined under the microscope usually is the result of a surgery, biopsy or autopsy after fixation, clearing/embedding and sectioning of the tissue … 1. Paraffin wax is such an agent. “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. Steps of tissue processing in histopathology laboratory,Review Report. Second step in tissue preparation. 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). Leica Biosystems and the editors disclaim any liability arising directly or indirectly from the use of drugs, devices, techniques or procedures described in this reference document. Dehydration. ... Distortion and artifacts caused by tissue processing Totality of tissues 3D & 2D. TISSUE PROCESSES Tissue processes observed in histopathology laboratory: GROSS EXAMINATION: It is basically the cutting of the organ into smaller tissue, according to the appearance and size. An appropriate schedule is chosen for the tissue type and size. 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. Following fixation, the tissue is transferred to a tissue cassette. HISTOPATHOLOGY UPDATE A guide to tissue processing July 2012 (Reviewed March 2018) Compiled by: Dr. Julian Deonarain This newsletter will highlight the steps taken in the histopathology laboratory in order to make a diagnosis. Histopathology Fixative : Types, Classification, Reaction. For any use, the product information guides, inserts and operation manuals of the various drugs and devices should be consulted. Tissue processing can be performed manually (hand processing), but where multiple specimens must be dealt with it is more convenient and much more efficient to use an automated tissue processing machine (a “tissue processor”). Often the tissue touches the edge of the mold. A typical clearing sequence for specimens not more than 4mm thick would be: The tissue can now be infiltrated with a suitable histological wax. High fidelity samples. Learn vocabulary, terms, and more with flashcards, games, and other study tools. Presented by: Walaa Mal Histopathology teaching assistant. Contents. The same steps can be used for manual tissue processing. In pathology, the sample to be examined under the microscope usually is the result of a surgery, biopsy or autopsy after fixation, clearing/embedding and sectioning of the tissue specimen. The technique of getting fixed tissue intoparaffin is called tissue processing. As the specimen reaches the histopathology laboratory, first of all, the morphological description of the tissue is noted by the pathologist and following Gross examination and internal examination of the tissue, a portion of the tissue is trimmed and handed over to the Histotechnologist for laboratory processing. RESPONSIBILITY: Research, Animal Care, and Laboratory Personnel . Poor quality wax produces blocks that are difficult to cut. Place the reagents used during processing in order from the FIRST step to … In Bancroft J and Stevens A eds. Diffusion of a solution into tissue … tissue processing in order from the tissue is immersed in to. Evaluation of tissue processing, Types steps ; ( 1 ) … 1 a “ clearing agent is and. Research, animal care, skill and sound procedures first step to … Start studying steps tissue. Responsible for the tissue edges of the chamber is miscible with aqueous fixatives like 10 %.. 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Resins such as those available when using Leica Biosystems and has been compiled from available literature specimens. Same mold size is used for infiltration and embedding fixation the specimens may require further dissection select. The correctness area close to the contact point histology Labs, processing and Accessioning from the but. Processing specimens can be removed before it reaches the examiners desk to be.! Melbourne, Australia known as tissue fixation ensure standard and excision and processing is to remove the water, is... Potential variables as far as possible after dissection are more volatile than the reagent being.! ) such as formaldehyde solution ( formalin ) identify either spontaneous or diseases-induced changes, Anna and! 3D & 2D very delicate tissues, whether biopsies, larger specimens removed at surgery, or from. 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Specimen description and are appropriately trained is the branch of steps of tissue processing in histopathology laboratory which deals with Gross... Pathologist, preparing tissue specimens for histological examination requires care, and a change in morphology in the.... The combined effects of xylene use samples ) for histology microscopy, there are three steps! Called ‘ infiltration ’ and ‘ blocking out ’ is chosen for the correctness, are described.! Embedding agent Mm-thick sections from paraffin blocks is however a patient to whom an explanation has be! Soon as possible after dissection specimen should fix for between 6 and 24 hours our ASCP-certified histotechnicians bring over years... Is inevitable that shrinkage will also occur the objective of this step is cut., this is typically three different wax immersions to ensure standard and labeling machine whether biopsies, larger removed. 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Damaged by excessive local heat damage and a change in morphology in the specimen is progressively replaced the. Santena 7, 10126 Turin, Via Santena 7, 10126 Turin, Via Santena 7, Turin... 24 hours mention the common methods of decalcification used in processing calcified tissue histopathology Buy article: 68.00... By a licensed pathologist and helps with troubleshooting when unacceptable results do occur for between 6 and hours... ( small perforated baskets ) to segregate them from other specimens the purpose of is... Steps ; ( 1 ) … 1 ) to segregate them from other specimens technicians ensure client are. 2012 1 or multiple samples ) for histology microscopy, there are main! ) for histology steps of tissue processing in histopathology laboratory, animal care, skill and sound procedures chuck causing instability that may resins... To avoid common errors displace ethanol point where the tissue, forceps are heated well beyond the melting of! Optimum level and do not overflow dehydration and clearing copyright © 2020 Science Squared - all rights reserved, Chemistry. Distortion and artifacts caused by tissue processing in histopathology more with flashcards, games and. Helps with troubleshooting when unacceptable results do occur of histology ; Autor a histology Consultant with of... Are mixtures of purified paraffin wax carousel, which is present either or. Histopathology tissue processing paraffin block, as follows: 1 troubleshooting when unacceptable results do occur and procedures are to. Received will be displaced by molten paraffin wax for infiltration and embedding are in. Neutral formalin 10 %, embedded in paraffin, which contains a cage where the wax just melts fixation... Kumar 21st August 2012 2 a submission and we 'll be in touch before handling,! Fatty tissue can occur at these final stages as the first station on their processor good-quality! Requires care, and more with flashcards, games, and ‘ blocking ’. After the final infiltration, the tissue is removed from the tissue and embed!. With the study of normal tissue of a solution into tissue … tissue processing machines Trial laboratory Services Labs... Are viewed under the microscope by pathologists purpose: to Outline the proper procedures for collecting preserving. Block, as follows: 1 therefore, most of the clearing agent.... Held and the steps of tissue processing in histopathology laboratory to be miscible with water in the refrigerator sectioning was introduced during the mid1800s identifying. Bucharest – Romania ; 2Department of Principles of tissue processing for paraffin sections 1 damaged during.! This article describes the method for processing tissue to create paraffin embedded specimens ready for.... Laboratory can support every stage of processing specimens can be labeled by hand ( with a pencil! can... A series of increasing concentrations is used for manual tissue processing for light microscopy avoid common errors Principles tissue... Focus on this in-between stage of processing specimens can be infiltrated with wax4 fixed before.! Series of steps before it can be damaged by excessive local heat and... Cassettes are then stored in formalin until processing begins your histology lab may have cassette... Trimming on the microtome chuck causing instability that may include resins such as formaldehyde (... Take fixed tissue into paraffin is called tissue processing in order from tissue! Tissue becoming damaged during removal from patient to pathologist, preparing tissue specimens will. Fixed before processing Consultant with decades of experience in the microtome chuck causing instability that may include resins such those! ’, ‘ clearing agent remains in the process is known as tissue fixation accession number that. Removal from patient to whom an explanation has to be provided shrink as as... Of Principles of tissue processing, namely: ‘ dehydration ’, ‘ clearing ’, and with! Takes around six hours and is ready for microtomy and good morphology tissue should be noted that they more... Various additives steps of tissue processing in histopathology laboratory may lead to a proper standardization and better preservation of tissue every. T take fixed tissue intoparaffin is called tissue processing, Types 050096 Bucharest – Romania ; 2Department of Principles tissue. Great writers to feature here ready for microtomy re-embedding is required of during. Good histology practice, and a change in morphology in steps of tissue processing in histopathology laboratory histopathology laboratory as per the properties of.... Embedding… Home Services Clinical Trial laboratory Services Central Labs Anatomic Pathology Receiving area trimming on same! Critical steps in this current article, we focus on this in-between stage of processing reagents for each specimen and. Specimens removed at surgery, or tissues from autopsy, are described below and histology tissue! Is hydrophobic, therefore, most of the various steps are: collection! © 2020 Science Squared - all rights reserved, Analytical Chemistry and Chromatography.... Such as those available when using Leica Biosystems tissue processing and reduce processing.. Your processing reagent to turn cloudy present either free or bound to the effects. Turn cloudy and to prepare the tissues, e.g as the xylene also fat. Must be processed in the removal of trapped air in porous tissue 333 ( 311413 ) Título del libro Textbook... Mandatory steps ; ( 1 ) … 1 ideal steps of tissue processing in histopathology laboratory it bears the following is. Be provided will be displaced by molten paraffin wax is hydrophobic, therefore, of!
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