Outcome 3 Guide PDF

Outcome 3 Describe the Preparation of Clinical Specimens for Histological Examination 1 Table of Contents Section 1 - Specimen Acceptance, Gross Description, and Gross Dissection..................................5 1.1 Definitions............................................................................................................................. 6 1.2 Why Specimens are Sent to the Laboratory......................................................................... 6 1.3 Sources of Tissue Specimens................................................................................................ 7 1.4 Pathway of a Surgical Specimen........................................................................................... 7 1.5 Specimen Preparation........................................................................................................... 8 1.6 Tissue Examination Methods................................................................................................ 8 1.7 Treatment of Tissue of Brightfield Microscopy..................................................................... 9 1.8 The Role of the Histotechnologist....................................................................................... 11 1.9 Specimen Accessioning....................................................................................................... 12 1.10 Grossing Introduction......................................................................................................... 15 1.11 Gross Description................................................................................................................ 15 1.12 Gross Dissection.................................................................................................................. 16 1.13 Gross Dissection Equipment............................................................................................... 17 1.14 Safety Precautions.............................................................................................................. 21 1.15 Tissue Cassettes.................................................................................................................. 22 1.16 Clean up of Gross Dissection Area...................................................................................... 24 1.17 Specimen Transportation.................................................................................................... 25 Section 2: Fixation....................................................................................................................26 2.1 Definition and Purpose of Fixation..................................................................................... 28 2.2 Autolysis and Putrefaction.................................................................................................. 28 2.3 Criteria of a Good Fixative................................................................................................... 29 2.4 Causes of Improper Fixation............................................................................................... 29 2.5 Physical Fixation Methods.................................................................................................. 29 2.6 Chemical Methods of Fixation............................................................................................ 30 2.7 Factors Involved in Fixation................................................................................................ 30 2.8 Chemical Action on Proteins............................................................................................... 32 2.9 Fixing Agents....................................................................................................................... 34 2.10 Properties Unique to Formaldehyde.................................................................................. 35 2.11 Compound Fixatives............................................................................................................ 36 2.12 Neutral Buffered Formalin (NBF)........................................................................................ 36 2 2.13 Zinc Formalin....................................................................................................................... 37 2.14 Lillie’s B-5 Fixative............................................................................................................... 37 2.15 Bouin’s Fluid........................................................................................................................ 38 2.17 Secondary Fixation (Post Fixation)...................................................................................... 38 2.18 Fixation Artifacts................................................................................................................. 39 2.19 Fixation Artifact Pigments................................................................................................... 39 2.20 Fixation for Electron Microscopy........................................................................................ 40 2.21 Fixation for Immunohistochemical Techniques.................................................................. 41 2.22 Fixation of Cytological Specimens....................................................................................... 41 Section 3: Principles of Tissue Processing..................................................................................44 3.1 Introduction to Processing.................................................................................................. 45 3.2 Dehydrants.......................................................................................................................... 45 3.3 Criteria of Good Clearing Agents........................................................................................ 45 3.4 Clearing Agents (Clearants)................................................................................................. 46 3.5 Xylene Substitutes and Other Clearing Agents................................................................... 46 3.6 Universal Solvents............................................................................................................... 46 3.7 Safe Handling and Recycling of Clearing Agents................................................................. 47 3.8 Factors Affecting Dehydration and Clearing....................................................................... 47 3.9 Common Problems in Dehydration and Clearing............................................................... 47 3.10 Advantages and Disadvantages of Paraffin Infiltration...................................................... 48 3.11 Composition, Characteristics, and Properties of Paraffin Wax........................................... 49 3.12 Commercial Paraffin Waxes................................................................................................ 49 3.13 Factors Affecting Infiltration............................................................................................... 49 3.14 Vacuum Infiltration............................................................................................................. 50 3.15 Developments in Automatic Tissue Processing.................................................................. 50 3.16 Tissue-Tek V.I.P. Vacuum Infiltration Processor................................................................. 51 3.17 Recycling Reagents.............................................................................................................. 56 Section 4: Decalcification..........................................................................................................58 4.1 Purpose of Decalcification.................................................................................................. 59 4.2 Criteria of a Good Decalcifying Solution............................................................................. 59 4.3 Procedure for Handling Tissue Requiring Decalcification................................................... 60 4.4 Types of Bone Specimens................................................................................................... 61 3 4.5 Types and Characteristics of Decalcifying Agents............................................................... 61 4.6 Commercially Available Decalcifying Fluids........................................................................ 63 4.7 Methods to Determine the Endpoint of Decalcification..................................................... 63 4.8 SURFACE DECALCIFICATION................................................................................................ 64 References...................................................................................................................................... 65 4 Section 1 - Specimen Acceptance, Gross Description, and Gross Dissection Upon completion of this section you will be able to discuss the role of the histotechnologist in specimen entry, gross description, and gross dissection. Learning Objectives To successfully complete this section, you should be able to: 1.1 Define histology, pathology, histopathology, histopathological technique, histochemistry, and cytology. 1.2 Describe why tissue specimens are sent to the laboratory. 1.3 Discuss the source of tissues submitted. 1.4 Outline the pathway of a surgical specimen. 1.5 List and briefly define the methods of specimen preparation which may be utilized in the laboratory. 1.6 List and briefly discuss methods of tissue examination. 1.7 List the major steps in treatment of tissue for study using the brightfield microscope. 1.8 Describe the role of the histotechnologist. 1.9 List the basic steps that result in the accurate accessioning of a tissue specimen. 1.10 Describe gross description and gross dissection. 1.11 Discuss the gross description of tissue specimens as it pertains to the clinical laboratory. 1.12 Describe the general procedure for gross dissection as it pertains to a MLT. 1.13 List and briefly describe the equipment used for gross dissection. 1.14 Describe the safety precautions that should be followed at gross dissection. 1.15 Describe the use and accurate labelling of tissue cassettes. 1.16 Describe the clean-up procedures used in the gross dissection area. 1.17 List and briefly describe the factors involved in specimen transportation. 5 1.1 Definitions Histology The study of the microscopic anatomy of cells and tissues. Pathology A medical specialty concerned with the diagnosis of disease. Histopathology The study of disease from the perspective of structural, particularly histological abnormalities of cells and tissues. Histological Technique/Histotechnology The branch of medical laboratory technology involved in the preparation of normal and diseased tissue for microscopic examination by pathologists. Histochemistry The identification and distribution of the chemical constituents of tissues by means of stains, indicators, and microscopy. Diagnostic Cytology The study of cells to identify disease. It may be used to diagnose various types of cancer and certain infections. 1.2 Why Specimens are Sent to the Laboratory Provides information about the patient: o Is the specimen normal? o Is the lesion present benign or malignant? o What is the prognosis for the patient? o Is anything unexpected detected in the specimen? o Is there correlation with other findings? Required by accrediting bodies: According to the College of American Pathologists (CAP)2 “each institution, in conjunction with the pathologist and appropriate medical staff departments, should develop a written policy that addresses which specimens do not need to be submitted to the pathology department and which specimens may be exempt from a requirement for microscopic examination. The policy should clearly state that all specimens not specifically exempted must be submitted to the pathology department for examination. It should also state that a microscopic examination will be performed whenever there is a request by the attending physician, or when the pathologist determines a microscopic examination is indicated by the gross findings or clinical history.” 6 1.3 Sources of Tissue Specimens Autopsy Curettage Surgical Tissue Amputation Whole organ Products of conception Partial organ Biopsy Excised Tumor Exfoliated Cells Excised Cyst 1.4 Pathway of a Surgical Specimen The pathway of a surgical specimen may be divided into the following categories: 1. Specimen arrival at lab a. Identification b. Accessioning 2. Technical preparatory steps a. Gross examination and description b. Specimen preparation 3. Initial pathologist viewing a. Visual (gross specimen) b. Microscopic (routinely stained sections) 4. Additional requirements a. Recuts b. Special stains c. Consultation 5. Reporting steps a. Verbal b. Word processing 6. Filing a. Wet specimens b. Blocks c. Stained slides d. Requisition 7 1.5 Specimen Preparation Sectional Methods Cut sections into thin, translucent slices of tissue. The microanatomy of the tissue is retained and the relationship of cells to each other is demonstrated. Touch Preparation The cut side of a tissue specimen is touched to a slide and an imprint is made of the architectural arrangement. This procedure may be used to detect malignancy, e.g. in brain, spleen, nodes. It is also used when microorganisms are suspected, e.g. Tuberculosis. Fresh Cells Cells suspended in their own fluid may be examined directly using the microscope, e.g. lymph, urine, CSF, semen. Dissociation Material may be "teased apart" to carefully break up the tissue and allow individual cells to be examined. This is not routinely done though may have application in research. Fluid Spread on a Slide Cells may be "fixed" to a slide to preserve their appearance. They are usually stained and may be mounted before they are examined. e.g. Cytology, Hematology 1.6 Tissue Examination Methods Gross Examination Initial macroscopic examination of the specimen by a pathologist/technologist. Brightfield Microscope Routinely used to examine tissue sections. Polarizing Microscope Useful in identification of birefringent materials found in tissue. Fluorescent Microscope Useful in immunohistochemistry where antigens in tissue are demonstrated. e.g. kidney biopsies in glomerulonephritis. Can be used to identify Mycobacterium tuberculosis. Specimen components to be demonstrated are combined with a fluorochrome which glows in UV light. Electron Microscope Magnification approximately 200,000 times. Use short wavelength beam of electrons producing high resolution with high magnification. Transmission EM may be used for liver, kidney biopsies, etc. 8 1.7 Treatment of Tissue of Brightfield Microscopy Tissue must be treated in order that it can be viewed with the brightfield microscope. The steps below outline the processes a specimen will follow in an anatomical pathology laboratory. FIXATION: Tissue is immersed in a FIXATIVE immediately after collection to preserve the tissue. ACCESSIONING: Specimens are received in the lab and are given a unique lab identification number for tracking the specimen while in the laboratory. GROSS DISSECTION: Tissue is examined and clearly described for the health record then the tissue is GROSSED and placed into cassettes. PROCESSING: Tissue is chemically treated to prepare the tissue for paraffin wax embedding. EMBEDDING: Tissue is inlaid in paraffin wax to provide support during sectioning. SECTIONING: Tissue is sliced which produces uniform very thin slices and places on a glass slide. STAINING: Tissue is treated with dyes to enhance various tissue components. MOUNTING: Tissue is preserved onto the slide using a resin and coverslip resulting in a permanent preparation. Overview of Technical Steps - Laboratory Demonstration Following accessioning of the tissue, the specimen is carried through a series of technical steps to produce a slide ready for microscopic study by the pathologist. Step 1 - Gross Dissection Blocks are selected and placed in a numbered tissue cassette and immersed in fixative. “Gross dissection” © NAIT 9 STEP 2 - Processing Tissue cassettes are placed in a basket on a processor to exchange fluids in and around the tissue from fixative to molten wax. “Tissue processor” © NAIT STEP 3 - Embedding Specimens are embedded into a block of paraffin wax using a small mold. The numbered part of the tissue cassette becomes an integral part of the embedded block. “Embedding centre” © NAIT “Embedded block” © NAIT STEP 4 - Microtomy Blocks are sliced on a microtome to obtain 4 - 6µm thick sections which are mounted onto glass slides. “Microtomy station” © NAIT 10 STEP 5 - Routine Staining All sections are stained by the H & E method for initial viewing by the pathologist. This method stains cell nuclei with hematoxylin and cell cytoplasm with eosin. H & E of Kidney © NAIT 1.8 The Role of the Histotechnologist The histotechnologist prepares tissue selected by the prosector (pathologist, pathology assistant, or technologist) in order that a diagnosis can be made. The end product of tissue preparation is a tissue section mounted on a glass slide. A histological section may be described as: only part of an individual living animal a two-dimensional slice out of four-dimensional system minus what has been lost in preparation plus, what has been added in its preparation The preparation of a tissue section involves the production of an agreed set of artifacts, meaning the processes used in the preparation of the tissues will have known effects on the tissues that are expected. The skill of the technologist is to be consistent in producing these artifacts. Pathologists are trained to recognize the most minute changes and this skill must not be adversely affected by the section quality. Compliance with the Quality Control and Quality Assurance standards is essential. 11 1.9 Specimen Accessioning Computerization Most hospital laboratories have a computer Laboratory Information System (LIS) which is used in the documentation of each case. This computerization has replaced the traditional laboratory ledger which was used to record incoming specimens. Today, basic computer keyboard skills are mandatory for the technologist working at the gross dissection bench. Specimen and Requisition Requirements Any specimen arriving in the laboratory, whether it be from the hospital or a clinic in a different location, must be fully and accurately labelled. Specimens must contain the following information entering the laboratory as a minimum requirement for Alberta Health Services (2018): Major Requirements: Patients full first and last name At least one assigned unique identifiers – Unique lifetime Identifier (ULI), Personal Health Number (PHN), Government Issued Identification number or Medical Record Number Exact body site –including quadrant, laterality, lobes, etc. Relevant clinical history Date and time the tissue was removed – also called devitalization Fixation time – the exact time the tissue was placed into fixative Minor Requirements: Date of Birth (DOB) Gender Collection date and time, if applicable Patient location Patient phone number Tests/Procedures ordered Full first and last name of the requestor Report location for the requestor Full first and last name, location of any “copy to” recipient Labeling of the specimen container and completion of the requisition is performed at the site of procurement. The major requirements must be present on the sample container and the requisition. The information must match exactly on the sample and requisition. Devitalization and tissue fixation time, and collection date and time must appear on either the sample label or requisition. It is the responsibility of the laboratory worker accessioning specimens to check that the information on the specimen label corresponds with that on the requisition. Pathology samples are not rejected if minimum acceptance criteria are not met. However, the laboratory will not process the sample until a Laboratory Sample Request Deficiency Resolution 12 Request or Waiver has been completed by the person who procured the sample, and returned to the laboratory. Technologist Entry on LIS Following receipt of the specimen and requisition, the technologist retrieves the patient file on the computer. This is usually done using the patient's hospital identification number or personal health number. At this point, the specimen is assigned a laboratory number. The numerical system begins at number one, January 1st each year and so the number must include a year designation. The accession number is recorded on the specimen label, the requisition, and in the patient's case information on the LIS system. The initial entry on the LIS system includes: patient's name patient's hospital identification number surgeon specimen type(s) - note there may be more than one specimen per case modifier level: for statistical purposes, the technologist entry also includes the Anatomical Pathology specimen modifier level for each specimen. This level pertains to the complexity of the case. Each laboratory has a common procedures list with the designated levels. In order to assign the correct level, the worker checks the requisition for specimen type/tentative diagnosis and then locates this specimen type on the alphabetical listing. Note: levels can vary for the same organ - example: o uterus (with or without tubes and ovaries) for prolapse - level IV o uterus (with or without tubes and ovaries) other than prolapse/neoplastic -level V o uterus (with or without tubes and ovaries) neoplastic - level VI Accession numbers Once all the patient and sample information is entered into the LIS, the LIS will generate a unique laboratory identification number called an ACCESSION number. In the histopathology department the accession number usually has a letter assignment. S = surgical specimen A = autopsy specimen The letter designation is followed by the year in which the specimen was collected. S20- A20- Following the letter designation and year is a case number. This combination creates the accession number which is unique and is never repeated. S20-1223 A20-3456 13 There are times when multiple specimens may be submitted on the same patient. The accession number would be unique for the patient, but then additional digits or letters are added on to the accession number for multiple specimens. Example: Smith, Joe – 2 biopsies were submitted to the lab, lung and liver Lung Specimen: S19 -1234 – 1 Liver Specimen: S19 -1234 – 2 If it was required that the specimen could not fit in to one cassette and more than one cassette was required, an additional distinguishing digit is required. This will be further discussed in gross dissection. Example: Smith, Joe – 2 biopsies were submitted to the lab, lung and liver Lung Specimen: S19 -1234 – 1 3 cassettes required S19 -1234 -1A S19 -1234- 1B S19 -1234 -1C Liver Specimen: S19 -1234 – 2 During and After Gross Dissection The technologist entry is updated on the LIS systems to include: number of blocks selected per specimen special instructions for embedding, cutting, and staining pathologist assigned to view the slides Generation of a Worksheet After the completion of gross dissection, the laboratory worker requests the production of the computer-generated worksheet. It is this worksheet that is used to guide the work to be done on a given specimen. The pathologist's initials are on the worksheet to aid sorting the completed slides. 14 1.10 Grossing Introduction The pathologist gains much knowledge about each case by thorough examination of the gross specimen and careful block selection. It is the role of the technologist to prepare and maintain the work area with emphasis on efficiency and safety. The technologist may be required to describe specimens that are processed “in toto”, and to dissect simple cases. The technologist may perform grossing on simple cases and assist the pathologist on more complicated cases. The pathologist describes and selects blocks from complex organs. The assistant ensures accurate documentation of the specimen by competent use of the Laboratory Information System. Recording block selection, identification of each block selected, and preparation of worksheets are important tasks as any errors, especially in specimen identification can have serious consequences. 1.11 Gross Description There are 3 key responsibilities of the medical laboratory technologist in the histology gross room: 1. Prevent autolysis/putrefaction 2. Describe and dissect uncomplicated specimens 3. Assist the pathologist or pathology assistant on complicated cases. All histopathology specimens received in the laboratory are described and this description is recorded for the patient’s file. Features of the sample are dictated by means of a dictaphone. Specimens are handled outside of their containers in a fume hood or well ventilated area. The dictaphone is set up in the specimen handling area. The dictaphone consists of a microphone, a recording device, and a foot pedal. The laboratory professional will use the foot pedal to start and stop recording. SD cards are used to record the information about the specimens. Dictations capture valuable information about the sample. Dictations must always include the following information: Confirmation of the accession number Fixation time (each specimen must have a from the sample container and the minimum 8 hours in fixative prior to requisition processing) Patient first and last name Details of the gross examination or Clinical history description (can include weight) Collection date and time Submission details including number of cassettes submitted and details for each Number of Containers cassette Any additional information or features of the tissue discovered during gross dissection 15 Gross Description Specimen descriptions should be brief but accurate. Ensure the description includes number tissue of fragments, colour, textures, and dimensions or volumes. Only one specimen is handled at one time. Specimens are removed from their originating containers. The fluid from containers holding small specimens should be filtered through a fine mesh strainer, sponge, or biopsy bag to ensure the entire specimen is captured. Specimens that do not require any dissection are known in Edmonton as “pick and transfer” specimens and are submitted “in toto” meaning altogether. Medical laboratory technologists are trained to perform grossing on simple cases and must recognize the need to consult with a pathologist or pathology assistant as required. Grossing tips: Always rinse containers that contained small fragments with water and pour the rinse water through a filter Only gross one specimen at a time, finishing one before starting the next Tubular structures are always measured in length and diameter 1.12 Gross Dissection Orientation of the specimen is the most important aspect of gross dissection. Pathologists, pathology assistants, and histotechnologists must always attempt to determine the specimen orientation prior to making any cuts. Orientation of the specimen and the description of the orientation is key in relating the following: The location of a pathological finding in relation to the rest of the specimen Surgical margins are identified by marking reagents and evaluated if neoplastic processes are suspected Layers of tissues within the specimen that must be demonstrated in relation to one another Specimens arriving in the lab will come in all shapes and sizes. Small specimens like biopsies could be submitted “in toto” in one cassette while larger specimens will need to be dissected. Some small specimens may need to be dissected into smaller pieces to enhance the fixation and processing of the sample. Representative sections of larger specimens are dissected, removed, described, and submitted for processing. The representative section of the specimen is used to represent other grossly similar areas of the sample. Many organs have standard sections which are always collected during grossing, for example: Appendix: the proximal end and distal tip are always submitted along with any irregular areas and a cross-section or two. Placenta: Sections of the membranes, a cross-section of the umbilical cord, and at least 2 full thickness sections (including fetal and maternal surfaces) should also be included. 16 Grossing templates are used to ensure consistency in gross description and dissection between staff performing grossing procedures. Staff are trained to use the same terminology and format for description. The level of complexity of a specimen determines who will be performing the grossing. Larger more complicated specimens must be grossed by a pathologist or pathology assistant. The higher level of complexity, the more education and training the worker must have. Larger specimens must be opened (bread-loafing, opening specimens with lumens) to ensure that fixative can reach all layers, preventing autolysis and/or putrefaction. Suspicious areas of the specimen are noted (any tumors/masses, ulcerations or pigmented areas) and avoid cutting these if possible. Submit any lesions intact if possible for the pathologist or pathology assistant. Once a section of a specimen has been selected for processing, the sample(s) are placed into cassette(s). The specimen is placed into the cassette with the surface of interest facing down in the cassette. This ensures the specimen area of interest will be handled first in further steps. Labelling of the cassettes must be double checked against the requisition. Each cassette is logged (either on paper or in the LIS), and information provided will be used in later histological processes. Further dictation on the submitted cassettes is performed to give the pathologist additional information when reviewing the final slides. All cassettes are placed into fixative and must sit in the fixative for a specified amount of time prior to processing. NOTE: the most common cause of poor processing is overfilling the cassette. Keep your sections thin and do not overfill the cassette. 1.13 Gross Dissection Equipment Bench Area Protection from splashes is important and some bench areas have a movable plexiglass screen which can be positioned to protect the pathologist. Board, Tray, and Sink The material that is used for the gross dissection board must be impervious and readily cleaned. Dissecting boards of high-density polypropylene are commonly used. These are available in a variety of sizes and may have a trough extending around the perimeter to contain fluids. The board is usually housed in a stainless-steel tray which drains to a sink. It is important to have a sink as part of the gross dissection area. Specimens may be rinsed here, and fluids may be drained from a specimen using a sieve. Many prosectors use paper towel between the board and the specimen, changing it between specimens. This practice helps absorb some of the liquid and aids in preventing carry over between cases. This means that an adequate supply of towels should be easily accessible. There should also be a large waste container available for discarding the towels. As this waste contains tissue fragments, the waste container should be lined with a biological waste bag and disposed of appropriately. 17 Cutting Tools The stainless scalpel handle and disposable blade is widely used. The grooved tip of the handle takes slotted disposable blades. The length and shape of the blade varies, though blade size 22 is common. Some laboratories also stock longer blades that allows a larger specimen to be sliced in single strokes. “Scalpel Handle and Blade” ©NAIT Small plastic scalpel removal devices are available for safe removal of scalpel blades. Slicing of large specimens is done using dissecting knives, which are similar in size to a kitchen carving knife. These require sharpening to maintain the knife edge, therefore it is not uncommon to find a sharpening steel as part of the gross dissection equipment. Some laboratories prefer to use a handle which accepts a disposable blade approximately 15 cm in length and 3 cm in width. Bone specimens may require the use of a saw to remove blocks for examination. Amputation specimens are often dissected in the autopsy suite as there is the appropriate equipment there. Usually larger blocks are taken, fully fixed and decalcified, and then blocks of suitable size for processing are dissected. Scissors A wide variety of dissecting scissors are available though most pathologists are satisfied with surgical grade scissors with pointed tips, operating scissors with one sharp and one blunt point, and dissecting scissors with one blade longer than the other. “Scissors” ©NAIT 18 Forceps The type of forceps used depends on the pathologist’s preference. Some tissue forceps have a sharply cut tooth and notch at the tip which helps grasp the tissue firmly. Standard dissecting forceps may have fine tips or medium rounded tips and are usually 130 mm long. Some set ups may include extra-long forceps specially designed for removing specimens from deep containers. Another useful addition is a pair of hemostatic forceps with fully serrated tips and box-lock construction. “Hemostatic Forceps ©NAIT” Use clean forceps for each specimen’s container when possible. At minimum ,the forceps should be rinsed or washed in water between cases. Ensure no tissue fragments are caught in the grooves of the forceps after tissue handling. Measurement Tools Measurement of the specimen is an important part of the gross description. Stainless steel rulers are frequently used though some cutting boards have dimensional scales along the edge for quick sizing of specimens. As well as linear measurement, weight can be significant and so a scale is also part of a gross dissection set up. Marking Reagents There are several reasons why a specimen may be marked. The first reason is to identify the resection margins which are the cut edges of the specimen. This is done by marking the very edge of the specimen using a permanent ink which will remain after the specimen is processed. Some laboratories use tattoo ink. Marking resection lines is done to aid the pathologist in reconstructing the whole specimen in their mind from the blocks which were selected. When blocks are selected, they are taken from the excision margins as well as the tumor area of the specimen. A second reason for marking tissue is as a guide to embedding. It is sometimes extremely important to embed tissue so that a specific surface is sectioned. The tissue is marked on the opposite side from the area of interest. This process helps guide further technical steps. Some pathologists may choose to make a small “v” shaped groove as an alternative to marking. The permanent ink is most often applied using a swab. The basic procedure is to dry the specimen, apply the ink, pat off excess ink, place the specimen into Bouin’s fluid or a 4% acetic acid solution to help set the ink and help it bind to the tissue. Dry the specimen again before cutting. If the ink is still wet, it will be drawn into the specimen obscuring the margins. 19 Some very tiny biopsy specimens may be coloured with a dye such as eosin to facilitate embedding following tissue processing. Alternate colouring agents are silver nitrate and Mrs. Stuart’s Bluing Agent. Artifacts in Histology rroij.com NOTE: The marking dye is placed on the tissue at the time of grossing. It is not the natural color of the tissue margins. The marking agent can minimally penetrate into the tissue. Wax Trays Bowel specimens are often opened and pinned out flat to ensure proper fixation of the lining and to aid in block selection. The most common way to do this is to use a wax tray. Wax trays are made by filling a suitable polyethylene container with about an inch of molten wax which is allowed to solidify. It is practical for a lab to have several sizes on hand. When used, the specimen is pinned out on the wax board and covered with fixative. Fixative Any specimens which arrive at the laboratory without adequate fixative should be topped up. This is best done by having a container of routine fixative on the gross bench. The container should have a spigot with a tube for easy dispensing. Cassettes Most laboratories use small, perforated containers called cassettes for processing the routine specimens. These are identified using a mechanical pencil, a special pen which is not removed by processing reagents, or a special marking device. Sometimes the perforations may allow fragments of tissue to escape. In these cases, the specimens may be wrapped in suitable papers. Another alternative is to place the specimen in a fine mesh bag. Computer Terminal The computer terminal is an extremely important part of the gross dissection bench. As it is usually operated with gloved hands which have been in contact with tissue and fixative, the keyboard is protected with a cover. This would be considered an area which could possibly be contaminated, and the cover should be such that it can be wiped with a suitable disinfectant. 20 1.14 Safety Precautions Personal Protection All unfixed or partially fixed tissue specimens must be considered potentially infectious. Standard precautions should be followed which includes the use of protective clothing. Lab coats or gowns should be worn plus a protective apron. Disposable plastic aprons are often used though some laboratories find a heavy plastic apron which can be wiped with disinfectant more economical and does not produce waste which requires special disposal. Suitable, well- fitting latex gloves must be used as well as eye protection. Although recommended in Standard Precautions, it is not common to see masks being worn at gross dissection. A plexiglass screen can be positioned to protect the worker from splashes and aerosols. Ventilation It is important to minimize the exposure to formalin fumes and so a safe environment for gross dissection should have adequate ventilation in good working order. Handling Specimens When removing and returning specimens to their containers, care must be taken to avoid aerosol production and splashing. Handling Cutting Blades Disposal of scalpel blades should be carried out with care into an appropriate sharps container which is designated biohazardous waste. New industry standards also include wearing cut resistant gloves under the disposable nitrile or vinyl gloves while using scalpels or other cutting blades. Special Handling Standard precautions procedures do not include flagging specimens which are known to be from patients with hepatitis, tuberculosis, or AIDS. Should you be aware of any of these, you may decide to ensure that fixation is complete by extending fixation time. Some workers fix a liver biopsy from a hepatitis patient for 24 hours rather than 4 hours. Tuberculosis is also resistant and benefits from extended fixation. Although highly publicized, routinely fixed specimens from a patient with AIDS do not present any special problems. Creutzfeldt-Jacob Disease There is a rare contagious neurodegenerative disorder called Creutzfeldt-Jacob disease (CJD) which all pathology laboratory workers must be aware of and be prepared to handle. The infectious agent, now known as a prion, appears to be a protein. This prion may remain in the host for a period (months to years) until clinical symptoms appear. There is no cure for CJD and is typically fatal within a year after the onset of symptoms. This prion has great resistance to inactivation by 10% formalin or 70% alcohol fixation and infectivity may even be preserved in the paraffin wax embedded block. Should tissue arrive at the laboratory from a suspected/confirmed CJD case, the tissue should be fixed for a minimum of one week in formalin followed by treatment in concentrated formic acid and then tissue processing. Disinfection using sodium hydroxide is recommended for equipment which cannot be steam sterilized in the autoclave. 21 1.15 Tissue Cassettes Description of a Cassette Tissue cassettes are perforated containers which are 4 x 3 x 0.5 cm in size. Some manufacturers produce a cassette with an attached lid which is snapped into place, whereas other manufacturers produce the plastic cassette base and a reusable metal lid. The lids have a lip or tag which aids in opening easily. Most large laboratories may use several different coloured cassettes to identify specimens which may need special treatment e.g., specimens fixed in a mercury containing fixative, or to aid in sorting surgical cases from autopsy cases. The biopsy cassette has much smaller perforations than the routine cassette to ensure that the small tissue specimen is not lost. Biopsy specimens may also be processed on a nylon mesh, or placed between special sponges which fit the cassette. If a biopsy cassette is not available or the specimen is very friable, the specimen may be wrapped to prevent any tissue from being lost. The routine cassettes are 0.5 cm deep which will comfortably accept a specimen 0.3 cm deep. Occasionally, a deeper cassette may be required. This 1 cm deep cassette would only be used with a thin walled specimen to ensure that it would process in the same time as specimens cut 0.3 cm thick. An example of a specimen which would benefit by being processed in a deep cassette is a cross section of an eye. It is extremely difficult to cut a 0.3 cm slice of an eye; a 0.6 cm slice works better and would process in the same time as a 0.3 slice of a solid tissue. Wrapping of Tissue Should a specimen be very small or friable, it should be wrapped in paper, placed between porous sponges, or placed in a fine nylon mesh bag. Commercial companies produce papers suitably packaged for easy dispensing. Histowrap is one example. When wrapping a specimen, it is important to do it in such a way that the tissue will not escape but also consider the ease in which the package can be unwrapped at the embedding station when it is impregnated with paraffin wax. The “butcher’s” wrap is a common protocol used. ©NAIT Labelling Labelling should be done with a pencil or a special pen (water and solvent resistant.) This is very important. Should any ordinary type of pen be used, water soluble or permanent, the ink would be lost during the processing cycle. To ensure that a sharp, fine point is always available, a 22 mechanical pencil is the pencil of choice by many workers. Remember, this pencil would be used in an area which is potentially infectious and so should remain at the gross cutting bench. Most institutions follow the protocol where the number is recorded on the cassette the way it is indicated below. The size of the labelling area is 28 mm x 8 mm. This is the protocol used at NAIT. ©NAIT Labelling a Cassette Other institutions follow the protocol where the number is recorded on the cassette the way it is indicated below. This enables the label to be easily read when the blocks are filed. S19-2467-1 Labelling of routine surgical specimens includes: S19 – 2467 -1 S 19 - 2467 - 1 surgical year case # block # Some laboratories use a letter designation for the block. S19 - 2467 - A In a case where there is more than one specimen, e.g., breast tissue and axillary tissue and more than one block is taken on each, then this would require the need of and additional digit. S19 – 2467 – 1 – 1 or S19 – 2467 – 1 - A S 19 - 2467 - 1- 1 surgical year case # specimen # block # 23 Note that the label space only measures 28 x 8 mm and so it is essential to write legibly and small. If an error is made, a complete erasure is essential. Occasionally, a laboratory may develop a protocol where additional information may be recorded on the long side of the cassette, e.g., embedding and cutting instructions , number of pieces, specimen type, TURP, Tnote (technical notes – more detailed notes are found in LIS). Some laboratories use an automated labelling system that uses a barcode to permanently mark cassettes (VANTAGE system). ©Scientific Laboratory Supplies Ltd 1.16 Clean up of Gross Dissection Area Instruments Disposable blades are placed in an appropriate sharps container which is designated as biohazardous waste and will be disposed of by incineration when filled. Each laboratory adopts a protocol for cleaning instruments which meets or surpasses safety standards. Several different protocols are listed below: Protocol A - Soak the instruments in a commercial laboratory detergent containing a phenolic disinfectant such as Sanikleen. A stainless steel lidded tray is a suitable container. Following soaking in the detergent/disinfectant, the container and instruments are steam sterilized in an autoclave prior to washing. Protocol B - Soak the instruments in Sanikleen followed by washing in hot, soapy water. Protocol C - Soak in a glutaraldehyde disinfectant such as Cidex for the recommended time, followed by thorough washing in hot, soapy water. Protocol D – Spray work area and all instruments (e.g. board, scalpel handle, ruler etc.) with a commercial disinfectant such as Viralex or Accel ® TB. There is no reason to dry the area because it will leave a film over the area which acts as a protective barrier to ensure all organisms remain dead. Bench The bench area including the dissecting board used to be disinfected using a hypochlorite solution, but it is now known that gaseous chlorine reacts with paraformaldehyde, a polymer of formaldehyde, to form phosgene or carbon monoxide, therefore most histology laboratories are bleach free zones. Hypochlorite should never be used on the metal drain tray as it corrodes metal. 24 Current practice is to clean the bench with a commercial product, such as Conflikt®, Accel ® TB, and Percept™, or 70% alcohol. Accel®TB and Percept™ contain hydrogen peroxide. Conflikt® is an ammonium-based disinfectant. Waste Disposal The gross dissection area generates a considerable amount of contaminated waste such as paper towel, gloves, and disposable aprons. These should be collected in a large container lined with a biohazardous waste bag. At the end of the working day, the bag should be sealed and removed to the site where it is disposed of by incineration. Specimen Filing Completed specimens are checked to see that they are covered with fixative and are then filed numerically on shelves. The length of time varies with each institution though 1 to 3 months is the norm in a routine hospital laboratory. Some legal cases are identified and kept indefinitely. Specimen Disposal Specimens no longer required are discarded as follows: the 10% formalin is drained from the specimen and disposed of separately. The specimens are then appropriately bagged and disposed of by incineration. The 10% formalin should be neutralized prior to disposal. 1.17 Specimen Transportation If specimens are to be transported from one laboratory to another, several factors must be considered, which include: Container Size Specimen container must be large enough to accommodate the size of the specimen and contain a reasonable volume of fixative. The container should have a tightly fitted lid to prevent leakage. Fixative Appropriate fixative must be selected. For routine examination and for most histochemical examination, as well as medico-legal purpose, 10% neutral buffered formalin is recommended because a tolerant fixation fluid should be used. Labelling Specimen container must be properly labelled, and information on it should correspond to the information on the accompanying requisition. Packaging Packing material around the specimen container must be adequate to prevent breaking or cracking in the event of dropping package in transit. Shipping Labels Special shipping labels should be used such as: fragile, biological material, do not allow to freeze, handle with care. 25 Section 2: Fixation Upon completion of this section, you will be able to apply the theory of fixation and decalcification to the selection of the appropriate chemical agent(s) for any given clinical specimen and demonstration technique. Learning Objectives To successfully complete this section you should be able to: 2.1 Define fixation and state its purpose. 2.2 Define autolysis and putrefaction and describe the microscopic appearance of an autolyzed cell. 2.3 List the criteria of a good fixative. 2.4 List causes of improper fixation. 2.5 Describe methods of fixation by physical methods. 2.6 Describe chemical fixation including vapor fixation, direct immersion, perfusion, and effects on tissue. 2.7 Describe the factors involved in fixation. 2.8 Classify chemicals used for fixation according to their action on proteins. 2.9 For each of the following simple fixatives, state the molecular formula, concentration, effects on proteins, fats and carbohydrates, tolerance on tissue, subsequent staining reactions, disposal, and hazards: formaldehyde, mercuric chloride, alcohol, picric acid and acetic acid. 2.10 Describe properties unique to formaldehyde. 2.11 Define the terms compound fixative and microanatomical fixative. 2.12 Briefly describe Neutral buffered formalin. 2.13 Briefly describe Zinc formalin. 2.14 Briefly describe Lillie's B-5 fixative. 2.15 Briefly describe Bouin's Fluid. 2.16 Describe washing procedures following fixation. 26 2.17 Briefly describe secondary fixation. 2.18 Describe artifacts produced by fixation. 2.19 Describe fixation artifact pigments. 2.20 Briefly describe fixation for electron microscopy. 2.21 Discuss fixation for immunohistochemical techniques. 27 2.1 Definition and Purpose of Fixation Definition of Fixation Fixation is the foundation for subsequent stages in preparation of tissue for diagnosis. It preserves cells and tissue constituents in as life-like a condition as possible. This treatment of tissue must be done to allow the preparation of thin, stained sections. Changes that occur are physical and chemical and can usually be demonstrated within minutes. Purposes of Fixation prevent desiccation prevent osmotic swelling and shrinkage prevent putrefaction prevent autolysis tissue becomes firmer and easier to cut tissue elements are stabilized tissue constituents are more easily stained refractive index of cellular components is changed 2.2 Autolysis and Putrefaction Autolysis: A self-digestion caused after cell death by the action of intracellular enzymes causing the breakdown of protein and eventual cell liquefaction. Factors Affecting Autolysis: Temperature o Cold (4°C) slows action o Heat (37°C) hastens action o Heat (57°C) inhibits action Types of Cells - Highly specialized cells of complex organs (e.g. brain, kidney) which have a high rate of metabolic activity are more rapidly and seriously affected. Elastic and collagen fibres remain unaffected. Speed of decomposition is proportional to the natural metabolic activity of tissue. Microscopic Appearance: Cell nucleus o condensed (pyknosis) o fragmented or lysed (karyorrhexis) o eventual disappearance (karyolysis) Cytoplasm - swollen and granular Overall cellular effect - a granular, homogeneous mass with altered staining reactions 28 Putrefaction/Postmortem Decomposition Bacterial decomposition, often with gas production leading to eventual liquefaction. Occurs with the presence and multiplication of bacteria in diseased tissues at time of death, e.g., in septicemia, or by bacteria normally present in the body site during life. bacteria contain enzymes which affect tissue acid production occurs which also contributes to putrefaction the odour of putrefaction is caused by putrescin and a cadaverine which are amine byproducts of the decomposition process. 2.3 Criteria of a Good Fixative Must not shrink, swell, dissolve or distort tissue. Must kill bacteria and molds. Must render autolytic enzymes inactive. Make tissue resistant to subsequent treatments. Make many tissue components readily colourable by suitable dyes. NOTE: Some tissue constituents cannot be fixed with certain fixation fluids. They may be retained in tissue because of their large molecular size or because they are insoluble, e.g., glycogen, neutral lipid 2.4 Causes of Improper Fixation Delay in fixation Inadequate amount of fixative fluid Poor penetration Use of fixing fluid, which is not suitable for a particular tissue type Prolonged fixation time in intolerant fixative Use of improper storage fluid 2.5 Physical Fixation Methods Heat - Seldom used for tissue because it causes distortion. Dry heat is used in bacterial smears. One advantage is that nothing dissolves out of the cell. Microwave Fixation - Microwaves cause water molecules to oscillate very quickly producing heat. The advantage of using microwaves to create heat is more even penetration, less time required, also some enzymes are preserved. 29 2.6 Chemical Methods of Fixation Vapor Fixation Volatile fixatives are used; fumes react with the tissue on the slide. Tissue must be fresh and must not be in contact with the liquid fixative as it is too concentrated. Cryostat sections can be fixed in formaldehyde this way. Liquid Fixation Chemical solids in solution, are used. The diluent is usually water. Direct Immersion Penetrates tissue by diffusion. The rate of penetration varies with the type of fluid and tissue. Facilitated by gentle heat (37°C) and limited by the size of the tissue. Perfusion Injection of liquid fixative into vessels or airways. It is the method of choice for fixing the whole brain. Embalming and preparing whole organs for museum purposes is referred to as "in toto" fixation. Effects of Chemicals on Tissue Tissue shrinks or swells Consistency of tissue - may harden, soften, or make brittle Affects subsequent staining reactions Dissolves out some intracellular materials Chemical alterations (may mask antigenic sites) Produces artifacts 2.7 Factors Involved in Fixation Buffer and Hydrogen Ion Concentration The pH values vary with different fixing agents. The hydrogen ion concentration is usually adjusted with suitable buffer to pH range 6-8. Outside this range, detrimental changes may occur to the ultrastructure. The most common buffer employed is phosphate buffer, others include barbital and acetate. For some specialized purposes a specific pH may be used. Temperature Fixation routinely occurs at room temperature. An increase in heat increases the rate of penetration but it also increases the rate of autolysis. Gentle heat may be used as fixation is completed on the tissue processor. Heat may also be used for STAT procedures. For electron microscopy and some histochemistry studies, cold temperatures of 0-4°C are used to retard autolysis and slow down diffusion of cellular components. 30 Speed of Penetration Rate is relatively slow, but varies with different fluids, size of tissue, and density of tissue. Examples: Formaldehyde 3mm/hour Mercuric chloride 2mm/hour Picric acid 1mm/hour Note that some fixing agents impede their own penetration. Vacuum The use of vacuum increases the rate of fixation. Vacuum is used on the tissue processor to ensure complete fixation of tissue blocks. Osmolality of Fixative An isotonic fluid has the same osmotic pressure as the solution inside the cell membrane (e.g. 0.85% NaCl) and so shrinkage or swelling is avoided. Hypotonic solutions are less concentrated than solution inside the cell membrane and give rise to swelling of cells. Hypertonic solutions are more concentrated than the solution inside the cell and result in shrinkage. Example: Red Blood Cell a) Isotonic b) Hypotonic c) Hypertonic Size of Tissue Block Selected This should not exceed 5 mm in thickness. The thinner the specimen the less time required for fixation. Density of Tissue A denser tissue requires a longer time for fixation to occur. Volume of Fixative in Relation to Tissue It is recommended that the fixative volume be at least 20-50 times that of tissue. This is rarely possible with large surgical specimens, 5 times being more realistic. 31 Time of Tissue Exposure to Fixative Prolonged tissue exposure to most fixatives is detrimental. Formaldehyde, the routine fixative, is tolerant, most fixatives are intolerant. Gross specimens must be stored in a tolerant fixative. Agitation This speeds up the fixation process. 2.8 Chemical Action on Proteins The most important reaction of a fixative is to stabilize proteins, which play a role in maintaining the proper relationships between cells and extracellular substances. This permanent change allows subsequent tissue treatment to occur without allowing excessive damage to take place. Proteins can be stabilized by using physical and chemical methods. This process is called denaturation. Chemical fixation is the primary method of stabilizing proteins in histology. The chemical agents used in fixation can be classified as additive or non-additive and coagulating or non-coagulating. Additive Molecules of fixative combine chemically with the something in the tissue (proteins, lipids or nucleic acids) forming “bridges” Usually very difficult to remove from the tissue Most routine fixatives are of this type. o e.g. picric acid + protein ---------> protein/picrate complexes Binding of a fixative molecule to the tissue can change the electrical charge at the site Formaldehyde is a negatively charged chemical therefore it will cross-link with the positively charged amino acids in the tissue forming methylene bridges. Formalin is not a particularly good nuclear fixative because there are a lot of positively charged amino acids in the cytoplasm and very few in the nucleus Non-Additive Two categories: 1. Dehydrants – alcohols (methanol, ethanol and isopropanol), acetone Do not chemically attach to the tissue Act by removing the water from between the protein layers in the tissue Can result in shrinkage and hardening of the tissue if exposure is prolonged. 32 2. Acids – acetic acid Have the opposite effect of the dehydrants Do not chemically bind to tissue Breaks naturally occurring crosslinks between amino acids which allows water to enter and causes a change in protein structure Coagulating Fixatives Transform the watery protoplasm into a microscopic sponge-work creating a mesh network of proteins allowing other solutions to easily penetrate the mesh. Organelles, e.g., mitochondria, secretory granules, are destroyed or distorted. Examples: metallic salts (mercuric oxide, zinc sulfate), alcohols, acetone, picric acid Non-Coagulating Fixatives Cause the proteins to form a gel thus making it more difficult for solutions to penetrate. Organelles may be well preserved. Example : aldehydes, (formaldehyde/formalin, glutaraldehyde, paraformaldehyde), potassium dichromate, osmium tetroxide/osmic acid The concept of coagulating and non-coagulating fixation is believed to be more of a physical observation of what is occurring with the tissue proteins, rather than what is happening chemically. 33 2.9 Fixing Agents Non-Coagulating Fixatives Formula Formaldehyde CH2O General Description colourless gas dissolved in water Usual Concentration 4% aqueous (10% formalin) Reactions with Proteins non-coagulating additive; methylene bridge formation Reactions with Carbohydrates no fixation (traps glycogen in protein meshwork) Reactions with Lipids no fixation Tolerance tolerant Effect on Staining Results: Anionic dyes: does not enhance acidophilic staining Cationic dyes: good Hazards toxic, allergic dermatitis, irritation to mucus membranes Use as Simple Fixative useful Additional Comments wear gloves and safety glasses; proper ventilation is essential; optimum fixation around 7 days Coagulating Fixatives Formula Ethanol C2H5OH Picric Acid C6H2(NO2)3OH General Description colourless liquid yellow crystals Usual Concentration 70 - 100% saturated aqueous 1-2% Reactions with Proteins strong coagulant; non additive coagulant additive Reactions with Carbohydrates precipitates glycogen no fixation; but binds glycogen to protein therefore classified as best Reactions with Lipids may dissolve some lipids no reaction Tolerance intolerant, causes shrinkage intolerant, causes shrinkage Effect on Staining Results: Results: Anionic dyes: little change good Cationic dyes: satisfactory Hazards flammable Crystals are explosive when dry. Store under a layer of water. Use as Simple Fixative used extensively for cytology; not used as a simple fixative Additional Comments used mainly as a fixative for blood films used as a fixative, a dye and a differentiating and smears agent; forms picrate’s with protein, some being soluble in water. 34 Coagulating Fixatives Formula Mercuric Chloride HgCl2 Acetic Acid CH3COOH General Description white crystals soluble to approximately pungent, colourless liquid 7% Usual Concentration saturated aqueous solution 5% aqueous Reactions with Proteins powerful coagulant; additive no effect on cytoplasmic proteins; non- additive Reactions with Carbohydrates no reaction no effect Reactions with Lipids do not make insoluble no effect Tolerance intolerant, makes brittle not applicable Effect on Staining Results: Results: Anionic dyes: good poor Cationic dyes: satisfactory good Hazards toxic irritant Use as Simple Fixative not suitable not used as a simple fixative Additional Comments reacts with metal; produces artifact excellent fixation of nuclei; termed glacial as pigment; zone/layer fixation may occur it is solid at temperatures below 17oC; swells tissue counteracting shrinkage 2.10 Properties Unique to Formaldehyde Commercial formaldehyde (strong formalin) is a saturated solution of formaldehyde gas with the formula CH2O. In water it is approximately 37 – 40 % gas by weight. This is designated as 100% or concentrated formaldehyde. To prepare 100 mL of 10% formalin you would need: 10 mL formaldehyde 90 mL laboratory grade water The exact concentration of formaldehyde in 10% formalin is 4% (40% x 1/10 = 4%) Paraformaldehyde Commercial formaldehyde may become turbid when stored in the cold or at room temperature because formaldehyde polymerizes to form paraformaldehyde. This white precipitate is not harmful to tissue, but the concentration of the working solution decreases slightly. The manufacturer prevents this by adding methanol. In the laboratory, paraformaldehyde may be 35 removed by applying gentle heat (60oC) at a slightly alkaline pH (7.4). Filtration can also be used, but concentration is reduced. Acid Formaldehyde Hematin (AFH) If formalin is oxidized, formic acid is produced. This occurs when the pH of the solution is below 5.6. When using acidic formalin, the formic acid reacts with hemoglobin in tissue, producing a brown-black compound known as acid formaldehyde hematin. This is known as a fixation artifact. It occurs readily around and over cells, and may be found inside the cell. To prevent formation of AHF, formalin is buffered using a phosphate buffer. This is referred to as 10% neutral buffered formalin (NBF). 2.11 2.11 Compound Fixatives Compound fixatives are composed of the combination of fixing agents and other chemicals. Shortcomings of one fixing agent is compensated for by the addition of one or more other fixing agents. Fixatives to be discussed in this section are: Neutral Buffered Formalin, Zinc Formalin, Lillie’s B-5, and Bouin's Fluid. Fixatives suitable for routine use are described as microanatomical. These fixatives preserve the microanatomy of tissue which involves maintaining cell relationships to each other and the noncellular tissue components. In addition to fixing agents, compound fixatives may contain other types of chemicals. In total, a compound fixative can consist of a combination of the following. non-coagulating fixing agent salt or buffer coagulating fixing agent diluent agent to counteract shrinkage 2.12 Neutral Buffered Formalin (NBF) The 10% solution is buffered to pH 7.0 - 7.2 at room temperature. A small piece of tissue in a cassette will fix in 4-8 hours, though crosslinking may continue for weeks. On the tissue processor, agitation, gentle heat, and the vacuum/pressure cycle reduce this time. Ingredient Role formalin non-coagulating fixative laboratory grade water solvent/diluent acid sodium phosphate monohydrate (NaH2PO4 H2O) buffer anhydrous disodium phosphate (Na2 HPO4) buffer 36 Formalin is a widely used routine fixative which is easy to prepare. It imparts a firm consistency to tissue without excessive hardening and is therefore tolerant. It is possible to restore the natural color of tissue after fixation by placing in 80% alcohol for a limited time. Use of formalin allows various other methods of tissue preparation besides paraffin processing, e.g. frozen sections. Formalin preserves some enzymes. Glycogen demonstration is also possible. Formalin fixation is suitable for many standard staining techniques. 2.13 Zinc Formalin Ingredient Role 10% formalin non-coagulating fixative 1% zinc sulphate coagulating fixative This fixative combines a coagulating and non-coagulating fixative. There is improved general morphology and superior nuclear detail. Zinc sulphate as a substitute for mercury salts avoids mercury disposal problems. Zinc formalin can also be used be used for post fixation. Zinc Formalin is commercially available. As this fixative is not buffered, acid formaldehyde hematin pigment can be produced. 2.14 Lillie’s B-5 Fixative Ingredient Role mercuric chloride coagulating fixative distilled water solvent/diluent sodium acetate salt concentrated non-coagulating fixative formaldehyde Lillie’s B-5 is primarily used for the fixation of small biopsy specimens. It preserves a wide variety of tissue antigens and is therefore suitable for immunological staining techniques. Mercury containing fixatives are not suitable for frozen sections and the mercuric chloride also severely corrodes metals. Mercuric salts also require special disposal. Tissue cytoplasm and nuclei are well fixed and acid dyes and metachromatic staining are enhanced. Some silver impregnation techniques are inferior. B-5 is an excellent secondary fixative following buffered formalin fixation; however mercury precipitate always forms and must be removed prior to staining. This is an intolerant fixative, sometimes resulting in layer fixation in specimens that are not thin. To avoid the use of mercury containing fixatives, many laboratories have opted to use B Plus fixative because it does not contain mercury. It uses buffered formalin and 0.5% zinc chloride. B Plus is an ideal fixative for hematologic and lymphoid tissues (i.e., lymph nodes and bone marrow biopsies). 37 2.15 Bouin’s Fluid Ingredient Role picric acid, saturated aqueous coagulating fixative solution concentrated formaldehyde non-coagulating fixative agent to counteract shrinkage; glacial acetic acid nuclear fixative This stable microanatomical fixative penetrates rapidly and evenly. Bouin’s fixation results in brilliant staining with the Trichrome staining methods. Glycogen is also well preserved by Bouin’s fluid. The fixation results in a yellow colour which is advantageous with very small specimens, making them easier to see. This fixative is intolerant, and tissues become brittle if left more than 12 hours. 2.16 Washing out a Fixative In some cases, it is necessary to wash a fixative out of the tissue before proceeding with processing. Some fixatives are washed out with water, others with alcohol. In the routine processing schedule, tissues are transferred directly to a low-grade alcohol following neutral buffered formalin. Low grade alcohol is required to prevent undesirable precipitate formation which can occur between the buffer salts and absolute alcohol. Following fixation containing picric acid, it is recommended that tissues are transferred to 70% alcohol. Water may cause the loss of some protein/picrate complexes which may be water soluble. 2.17 Secondary Fixation (Post Fixation) Treatment of tissue in a second fixing fluid, after initial treatment in a fixative is often called post fixation. These tissues are fixed with two different fixatives in succession. Examples include 10% neutral buffered formalin followed by a mercury containing fixative or Bouin’s fluid. This process increases the number of reactive positive tissue groups, therefore increasing tissue affinity for acid dyes. Secondary fixation also improves protein fixation. Remember that formalin fixation is progressive and that proteins are not totally crosslinked when placed in the secondary fixative. 38 2.18 Fixation Artifacts An artifact is a structure not naturally present in cells or tissue but is formed by reagents during treatment or by changes taking place in the cell as it dies. Fixation Artifacts as a result of as a result of the use of delayed fixation particular chemicals autolysis putrefaction fixation protein pigments denaturation Nucleus: normal flora condensation infected tissue membrane not crisp fragmentation karyolysis cellular damage Cytoplasm: desquamation swollen gas formation granular eventual liquefaction loss of membranes desquamation loss of substances 2.19 Fixation Artifact Pigments Formalin Pigment Formalin pigment is often found in tissues containing large amounts of blood, particularly when acidic formalin is used. Acidic formalin contains some formic acid, which interacts with the heme molecule of the protein hemoglobin. Formalin pigment is more correctly termed acid formaldehyde hematin. The acronym AFH is commonly used. In the H & E section, formalin pigment appears as dark brown to black opaque precipitate, and is birefringent when viewed with polarized light. The use of buffered formalin usually avoids the production of AFH, though it is relatively common in postmortem tissue specimens. AFH should be removed as part of the staining sequence. The use of alcoholic picric acid is the method of choice for the removal of AFH. Times vary depending on the amount of artifact present. The advantage of using picric acid over other methods that use strong alkalis is that the section does not tend to wash off the slide. 39 Mercury Pigment These deposits appear as brown to black opaque precipitate. This precipitate always occurs when a mercury containing fixative is used. The precipitate is distributed uniformly throughout the tissue. The chemical nature is not known with certainty, but is generally assumed to be mercurous chloride (Hg2Cl2). Mercury pigment can be removed during processing by adding 0.5% iodine to the 70% alcohol however this procedure is not common. Mercury pigment is usually removed by treating sections at the beginning of the staining method with an alcoholic iodine/sodium thiosulphate sequence. mercury pigment + alcoholic iodine ------> mercuric iodide which is soluble in alcohol iodine discoloration + sodium thiosulphate ------> sodium tetrathionate which is colourless 2.20 Fixation for Electron Microscopy When fixing tissue for electron microscopy studies, it is essential that cell organelles are well preserved. The most common procedure is initial fixation in glutaraldehyde followed by secondary fixation in osmium tetroxide. Osmium tetroxide blackens lipids greatly increasing the contrast in the “black and white” images seen. Only very small pieces of tissue can be used as the penetration rate of the fixative is limited. Also, the tissue must be fixed before organelles degenerate. Tissue is usually finely diced to 1 mm cubes with a very sharp razor blade. Fixing Agents Used in Electron Microscopy Glutaraldehyde Osmium Tetroxide Formula (CH2)3 CHO·CHO OsO4 General Description gas which dissolves in water pale yellow crystals to a maximum of 25% Reactions with Proteins non-coagulating non-coagulating additive additive Reactions with Carbohydrates traps glycogen in protein no reaction meshwork Reactions with Lipids similar to formaldehyde oxidizes some lipids resulting in a blackening Effects on organelles well preserved well preserved Rate of Penetration slow therefore very tiny slow specimens are used Change in Volume of Tissue none none Tolerance tolerant intolerant Hazards toxic harmful vapours, toxic 40 2.21 Fixation for Immunohistochemical Techniques Fixation for tissues undergoing immunohistochemical staining must preserve tissue antigens. A suitable fixative must give good morphological preservation and must not interfere with subsequent antigen/antibody reactions used to demonstrate the antigen. There is no ideal fixative for immunohistochemistry. Some reactions work better on frozen sections and other results are better with formalin, B-5, or alcohol fixation. Fixation times must be carefully controlled as over fixation may cause masking of antigenic sites or loss of immunoreactivity. Neutral buffered formalin is an acceptable fixative for many antigenic sites which can be demonstrated in paraffin sections. It has been hypothesized that formalin and other aldehydes cross link proteins around antigenic sites resulting in a masking effect which interferes with the binding of antibodies. This effect is progressive over time and while tissue fixed 12-24 hours may be satisfactory; those left longer than 24 hours may be irreversibly damaged for immunohistochemical studies. Tissues left less than the optimal time may show the “halo” effect where the outer edges will be fixed in formalin, but the inner cores will be alcohol fixed on the tissue processor. This will result in different staining in different areas of the tissue. Following formalin fixation, it is common to use a digestion procedure using a proteolytic enzyme prior to the antigen/antibody reaction to unmask more antigenic sites. Suitable enzymes are trypsin, pepsin, and pronase. This is also achieved by heating in a buffer solution in the microwave. Zinc formalin or B-5 fixation (B plus™) is recommended for lymph node biopsies. Bouin’s fluid has limited use. Many antigens are destroyed by routine fixation and paraffin processing. Heat destroys many antigenic sites. To demonstrate these antigens, cryostat sections of fresh tissue are prepared. This is particularly important for lymphoma studies as many lymphoma markers do not survive after treatment with most fixatives. It is common practice to fix the section in acetone prior to staining. 2.22 Fixation of Cytological Specimens Cytological specimens are broadly divided into two main groups: gynecological specimens (Pap smear) non-gynecological specimens Traditionally, a Pap smear was prepared in the clinician’s office as a direct smear and immediately fixed. The standard fixative of a direct smear is 95% ethanol. Fixation produces artifactual changes related to cell shrinkage. If a substitute fixative is selected, it must provide an equivalent amount of shrinkage. Examples of substitute fixatives include 100% methanol, 95% denatured alcohol, or 80% isopropanol. It is important to remember that as well as fixing the cellular components, the cells are adhered or “fixed” to the slide. 41 For convenience, it was common practice to use commercial aerosol sprays as a substitute for 95% alcohol. Cytospray is a commercial product that contains 95% alcohol and 2% carbowax. The carbowax provides a water-soluble protective coating for the cells on the slide. Direct smears, such as the conventional cervical smear (Pap smear), must be fixed immediately, within 1-2 seconds of procurement to avoid air-drying artifact. Today, gyne specimens are processed as liquid based specimens. Liquid based cytology (LBC) is the preferred method of collection for cervical (gyne) samples. The sample is collected using a spatula, broom or brush and then the head of the device is broken off into a vial which contains a proprietary liquid medium that preserves the cells. The sample is then sent to the laboratory where it undergoes a series of treatments to remove obscuring material, such as mucous or pus. After the technical preparation, a thin monolayered sample is created on a specified area of the glass slide that is now ready for staining. Currently there are two types of liquid-based cytology: Sure-Path (TriPath Imaging) and Thin-Prep (Hologic Corp.) Non-gynecological specimens are specimens from any other body site. Non-gyne specimens can include direct smears, fluids, and fine needle aspirates. At the site or procurement, the clinician has the option of preparing a direct smear or placing the cells in a liquid based cytology vial. Fluid specimens may include sputum, pleural fluid, ascites fluid, bronchial washings, bronchoalveolar lavage (BAL), CSF, needle rinse fluids, and biopsy supernatants. Fresh unfixed specimens are preferred and should be transferred directly to the laboratory. If a delay in processing is anticipated, a collection fixative with a lower concentration of ethanol (40 or 50% ethanol) is recommended. Commercial fluid or Saccomano’s fluid are commonly used. All fluids are processed to isolate the cells from the fluid. Processing of fluids vary according to the volume and cellularity of the specimen. Large volume specimens are centrifuged and then the supernatant is discarded leaving the sediment (cell button) and a very small amount of fluid. If a sediment is obvious, it can be spread on a slide and fixed with 95% ethanol. More frequently, 7-10 drops of fluid would be centrifuged in a cytospin which concentrates cells in a small area on the slide. This cytospin preparation must be fixed immediately in 95% ethanol. The cytospin is also used for specimens with low volume and low cellularity, such as CSF. Sputum specimens generate direct smears in which particles are selected using the pick and smear technique and spread on fully frosted slides. The prepared slides are fixed with 95% ethanol. The remainder of the specimen is processed as a cell block. Cell blocks can be prepared if a specimen contains visible flecks of tissue, large amount of sediment, mucoid, or clotted specimens from an exudate. Visible flecks or clots can be wrapped in gauze or lens paper and the placed in a labelled tissue cassette. The cassette is then placed in formalin and follows the protocol for preparation of histology specimens. Fluid specimens without visible particles or flecks are prepared using the trypticase soy agar (TSA) or plasma/thrombin clot method. For the plasma /thrombin clot method, the fluid specimens are first centrifuged in a large centrifuge tube to concentrate the cells. Most of the supernatant is removed without disrupting the cell button and then a couple of drops of plasma 42 is added and mixed with the sediment followed by the addition of fibrinogen. The addition of equal number of drops of fibrinogen will result in the formation of a clot that can be wrapped and placed inside a tissue cassette where fixation continues in formalin as part of the processing cycle to form a paraffin embedded block for sectioning. Fine needle aspiration biopsy can generate a variety of different specimens. Direct smears may be made at the time of collection. These smears may be fixed with 95% alcohol to be stained by the Papanicolaou technique or air dried to be stained by a hematological technique (Romanowsky Wright’s). The needle is then rinsed with a collection fluid or physiologic saline. The rinse fluid is processed as a fluid. If tissue fragments are present, they should be converted to a cell block for processing. Some facilities may require the needle to be rinsed directly into a liquid base vial and sent to the laboratory for processing. 43 Section 3: Principles of Tissue Processing Upon completion of this section, you will be able to discuss the processing of clinical specimens using paraffin wax as the infiltration medium. Learning Objectives To successfully complete this section you should be able to: 3.1 List and briefly describe the major steps in paraffin processing. 3.2 Describe and compare the dehydrants: ethanol and isopropanol. 3.3 List the criteria of a good clearing agent. 3.4 Describe and compare the clearing agent: xylene, toluene and benzene. 3.5 Briefly describe xylene substitute and list other clearing agents. 3.6 Briefly describe universal solvents. 3.7 Describe the safe handling and recycling of clearing agents. 3.8 List the factors affecting dehydration and clearing. 3.9 Describe common problems in dehydration and clearing. 3.10 List the advantages and disadvantages of paraffin infiltration. 3.11 Briefly describe the composition, characteristics and properties of paraffin wax. 3.12 List some additives of commercial paraffin waxes. 3.13 List three factors affecting infiltration. 3.14 Briefly describe vacuum infiltration. 3.15 Describe the developments in automated tissue processing. 3.16 Briefly describe the Tissue-Tek V.I.P. tissue processor. 3.17 Briefly describe recycling reagents. 44 3.1 Introduction to Processing Fresh, excised tissue must undergo a series of treatments before slices thin enough to be studied microscopically can be obtained. This treatment is referred to as processing. Tissue preparation always begins with fixation followed by paraffin processing, which consists of three steps: 1) Dehydration is the process in which water is removed from tissue. Since most fixatives are aqueous solutions and because the paraffin wax method is most commonly employed routinely, the water must be removed because water and paraffin are not miscible. 2) Clearing is the process where alcohol is removed and the solvent of the wax is introduced. Since alcohol and paraffin are not miscible, the alcohol must be removed by a solution which is miscible with paraffin. Since most solutions which are miscible with paraffin wax make tissue more transparent, the process has come to be known as "clearing", but the main purpose is to remove the alcohol and introduce the wax solvent. 3) Infiltration is the process by which molten paraffin wax infiltrates and permeates the cells and tissue spaces. When cooled and solidified, paraffin wax supports the tissue, enabling 5 micrometer (μm) thick sections to be cut. 3.2 Dehydrants Ethanol C2H5OH BP 78.3°C, flammable most commonly used, and considered the best graded alcohols are used to prevent shrinkage (70%, 80%, 95%, 100%); it is also important to prevent salts from fixation precipitating out in tissue an intolerant reagent usage strictly controlled by the government Excise Act relatively inexpensive for laboratories exempt from tax denatured ethanol is used by some laboratories. This has been made unfit for human consumption by the addition of methanol which is poisonous Isopropanol C3H7OH BP 82.3ºC, flammable excellent substitute for ethanol slightly more tolerant than ethanol causes less shrinkage and hardening than ethanol may be less expensive than ethanol 3.3 Criteria of Good Clearing Agents totally miscible with dehydrating agents and paraffin wax displaces alcohol relatively quickly from tissue does not over harden tissue easily eliminated from wax 45 3.4 Clearing Agents (Clearants) Comparative Chart of Clearing Agents Xylene Toluene Benzene (not recommended for use) Formula C8H10 C7H8 C6H6 Refractive Index (RI) 1.5; makes tissue 1.5; makes tissue 1.5; makes tissue transparent transparent transparent Boiling Point 138°C 110.6°C 80°C Flammability flammable flammable flammable Threshold Limit Value 100 ppm 100 ppm 10 ppm (TLV) Speed Fast, 3mm/hour fast fast Tolerance Intolerant; distorts and More tolerant than More tolerant than shrinks tissue if excess xylene xylene and toluene time used Ease of elimination

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