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    OCULAR PATHOLOGY
    CERVICAL CYTOLOGY
    GENERAL PATHOLOGY
    TELEMEDICINE
    EDUCATION
    PATHOLOGY LABORATORY

    Cabinet d'anatomie pathologique
    28 ter, rue Guersant
    75017 Paris
    Phone : 01 45 72 30 00
    0033-145723000
    Fax : 01 45 72 43 43
    Mail : info@putterman.org

    GENERAL PATHOLOGY

    Tissue Processing


    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. The techniques
    for processing the tissues, whether biopsies, larger specimens removed
    at surgery, or tissues from autopsy, are described below. The persons
    who do the tissue processing and make the glass microscopic slides are
    histotechnologists.



    Specimen Accessioning


    Tissue specimens received in the surgical pathology laboratory have
    a request form that lists the patient information and history along
    with a description of the site of origin. The specimens are accessioned
    by giving them a number that will identify each specimen for each
    patient

    Gross Examination


    Tissues removed from the body for diagnosis arrive in the Pathology
    Department and are examined by a pathologist, pathology assistant, or
    pathology resident. Gross examination consists of describing the
    specimen and placing all or parts of it into a small plastic cassette
    which holds the tissue while it is being processed to a paraffin block.
    Initially, the cassettes are placed into a fixative.

    When a malignancy is suspected, then the specimen is often covered
    with ink in order to mark the margins of the specimen. Different
    colored inks can be used to identify different areas if needed. When
    sections are made and processed, the ink will mark the actual margin on
    the slide.

    Fixation - types of fixatives


    The purpose of fixation is to preserve tissues permanently in as
    life-like a state as possible. Fixation should be carried out as soon
    as possible after removal of the tissues (in the case of surgical
    pathology) or soon after death (with autopsy) to prevent autolysis.
    There is no perfect fixative, though formaldehyde comes the closest.
    Therefore, a variety of fixatives are available for use, depending on
    the type of tissue present and features to be demonstrated.


    There are five major groups of fixatives, classified according to mechanism
    of action:


    • Aldehydes
    • Mercurials
    • Alcohols
    • Oxidizing agents
    • Picrates

    Aldehydes include formaldehyde (formalin) and glutaraldehyde. Tissue
    is fixed by cross-linkages formed in the proteins, particularly between
    lysine residues. This cross-linkage does not harm the structure of
    proteins greatly, so that antigenicity is not lost. Therefore,
    formaldehyde is good for immunoperoxidase techniques. Formalin
    penetrates tissue well, but is relatively slow. The standard solution
    is 10% neutral buffered formalin. A buffer prevents acidity that would
    promote autolysis and cause precipitation of formol-heme pigment in the
    tissues.


    Glutaraldehyde causes deformation of alpha-helix structure in
    proteins so is not good for immunoperoxidase staining. However, it
    fixes very quickly so is good for electron microscopy. It penetrates
    very poorly, but gives best overall
    cytoplasmic and nuclear detail. The standard solution is a 2% buffered
    glutaraldehyde


    Mercurials fix tissue by an unknown mechanism. They contain mercuric
    chloride and include such well-known fixatives as B-5 and Zenker's.
    These fixatives penetrate relatively poorly and cause some tissue
    hardness, but are fast and give excellent nuclear detail. Their best
    application is for fixation of hematopoietic and reticuloendothelial
    tissues. Since they contain mercury, they must be disposed of carefully.


    Alcohols, including methyl alcohol (methanol) and ethyl alcohol
    (ethanol), are protein denaturants and are not used routinely for
    tissues because they cause too much brittleness and hardness. However,
    they are very good for cytologic smears because they act quickly and
    give good nuclear detail. Spray cans of alcohol fixatives are marketed
    to physicians doing PAP smears, but cheap hairsprays do just as well.


    Oxidizing agents include permanganate fixatives (potassium
    permanganate), dichromate fixatives (potassium dichromate), and osmium
    tetroxide. They cross-link proteins, but cause extensive denaturation.
    Some of them have specialized applications, but are used very
    infrequently.


    Picrates include fixatives with picric acid. Foremost among these is
    Bouin's solution. It has an unknown mechanism of action. It does almost
    as well as mercurials with nuclear detail but does not cause as much
    hardness. Picric acid is an explosion hazard in dry form. As a
    solution, it stains everything it touches yellow, including skin.


    Fixation - factors affecting fixation


    There are a number of factors that will affect the fixation process:


    • Buffering
    • Penetration
    • Volume
    • Temperature
    • Concentration
    • Time interval

    Fixation is best carried out close to neutral pH, in the range of
    6-8. Hypoxia of tissues lowers the pH, so there must be buffering
    capacity in the fixative to prevent excessive acidity. Acidity favors
    formation of
    formalin-heme pigment that appears as black, polarizable deposits in
    tissue. Common buffers include phosphate, bicarbonate, cacodylate, and
    veronal. Commercial formalin is buffered with phosphate at a pH of 7.


    Penetration of tissues depends upon the diffusability of each
    individual fixative, which is a constant. Formalin and alcohol
    penetrate the best, and glutaraldehyde the worst. Mercurials and others
    are somewhere in between. One
    way to get around this problem is sectioning the tissues thinly (2 to 3
    mm). Penetration into a thin section will occur more rapidly than for a
    thick section.


    The volume of fixative is important. There should be a 10:1 ratio of
    fixative to tissue. Obviously, we often get away with less than this,
    but may not get ideal fixation. One way to partially solve the problem
    is to change the
    fixative at intervals to avoid exhaustion of the fixative. Agitation of
    the specimen in the fixative will also enhance fixation.


    Increasing the temperature, as with all chemical reactions, will
    increase the speed of fixation, as long as you don't cook the tissue.
    Hot formalin will fix tissues faster, and this is often the first step
    on an automated tissue processor.


    Concentration of fixative should be adjusted down to the lowest
    level possible, because you will expend less money for the fixative.
    Formalin is best at 10%; glutaraldehyde is generally made up at 0.25%
    to 4%. Too high a
    concentration may adversely affect the tissues and produce artefact
    similar to excessive heat.


    Also very important is time interval from of removal of tissues to
    fixation. The faster you can get the tissue and fix it, the better.
    Artefact will be introduced by drying, so if tissue is left out, please
    keep it moist with saline. The longer you wait, the more cellular
    organelles will be lost and the more nuclear shrinkage and artefactual
    clumping will occur.


    Fixatives - general usage


    There are common usages for fixatives in the pathology laboratory
    based upon the nature of the fixatives, the type of tissue, and the
    histologic details to be demonstrated.


    Formalin is used for all routine surgical pathology and autopsy
    tissues when an H and E slide is to be produced. Formalin is the most
    forgiving of all fixatives when conditions are not ideal, and there is
    no tissue that it will harm significantly. Most clinicians and nurses
    can understand what formalin is and does and it smells bad enough that
    they are careful handling it.


    Zenker's fixatives are recommended for reticuloendothelial tissues
    including lymph nodes, spleen, thymus, and bone marrow. Zenker's fixes
    nuclei very well and gives good detail. However, the mercury deposits
    must be removed
    (dezenkerized) before staining or black deposits will result in the
    sections.


    Bouin's solution is sometimes recommended for fixation of testis, GI
    tract, and endocrine tissue. It does not do a bad job on hematopoietic
    tissues either, and doesn't require dezenkerizing before staining.


    Glutaraldehyde is recommended for fixation of tissues for electron
    microscopy. The glutaraldehyde must be cold and buffered and not more
    than 3 months old. The tissue must be as fresh as possible and
    preferably sectioned
    within the glutaraldehyde at a thickness no more than 1 mm to enhance
    fixation.


    Alcohols, specifically ethanol, are used primarily for cytologic
    smears. Ethanol (95%) is fast and cheap. Since smears are only a cell
    or so thick, there is no great problem from shrinkage, and since smears
    are not sectioned,
    there is no problem from induced brittleness.


    For fixing frozen sections, you can use just about anything--though methanol and ethanol are the best.


    Tissue Processing


    Once the tissue has been fixed, it must be processed into a form in
    which it can be made into thin microscopic sections. The usual way this
    is done is with paraffin. Tissues embedded in paraffin, which is
    similar in density to tissue, can be sectioned at anywhere from 3 to 10
    microns, usually 6-8 routinely. The technique of getting fixed tissue
    into paraffin is called tissue processing. The main steps in this
    process are dehydration and clearing.


    Wet fixed tissues (in aqueous solutions) cannot be directly
    infiltrated with paraffin. First, the water from the tissues must be
    removed by dehydration. This is usually done with a series of alcohols,
    say 70% to 95% to 100%. Sometimes the first step is a mixture of
    formalin and alcohol. Other dehydrants can be used, but have major
    disadvantages. Acetone is very fast, but a fire hazard, so is safe only
    for small, hand-processed sets of tissues. Dioxane can be used without
    clearing, but has toxic fumes.


    The next step is called "clearing" and consists of removal of the
    dehydrant with a substance that will be miscible with the embedding
    medium (paraffin). The commonest clearing agent is xylene. Toluene
    works well, and is more tolerant of small amounts of water left in the
    tissues, but is 3 times more expensive than xylene. Chloroform used to
    be used, but is a health hazard, and is slow. Methyl salicylate is
    rarely used because it is expensive, but it smells nice (it is oil of
    wintergreen).


    There are newer clearing agents available for use. Many of them are
    based on limolene, a volatile oil found in citrus peels. Another uses
    long chain aliphatic hydrocarbons (Clearite). Although they represent
    less of a health hazard, they are less forgiving with poorly fixed,
    dehydrated, or sectioned tissues.


    Finally, the tissue is infiltrated with the embedding agent, almost
    always paraffin. Paraffins can be purchased that differ in melting
    point, for various hardnesses, depending upon the way the
    histotechnologist likes them and upon the climate (warm vs. cold). A
    product called paraplast contains added plasticizers that make the
    paraffin blocks easier for some technicians to cut. A vacuum can be
    applied inside the tissue processor to assist penetration of the
    embedding agent.



    The above processes are almost always automated for the large
    volumes of routine tissues processed. Automation consists of an
    instrument that moves the tissues around through the various agents on
    a preset time scale. The "technicon" tissue processor is one of the
    commonest and most reliable (a mechanical processor with an electric
    motor that drives gears and cams), though no longer made. Newer
    processors have computers, not cam wheels, to control them and have
    sealed reagent wells to which a vacuum and/or heat can be applied.

    Tissues that come off the tissue processor are still in the
    cassettes and must be manually put into the blocks by a technician who
    must pick the tissues out of the cassette and pour molten paraffin over
    them. This "embedding" process is very important, because the tissues
    must be aligned, or oriented, properly in the block of paraffin.



    Alternatives to paraffin embedding include various plastics that
    allow thinner sections. Such plastics include methyl methacrylate,
    glycol methacrylate, araldite, and epon. Methyl methacrylate is very
    hard and
    therefore good for embedding undecalcified bone. Glycol methacrylate
    has the most widespread use since it is the easiest to work with.
    Araldite is about the same as methacrylate, but requires a more complex
    embedding process. Epon is routinely used for electron microscopy where
    very thin sections are required.


    Plastics require special reagents for deydration and clearing that
    are expensive. For this reason, and because few tissues are plastic
    embedded, the processing is usually done by hand. A special microtome
    is required for
    sectioning these blocks. Small blocks must be made, so the technique
    lends itself to small biopsies, such as bone marrow or liver.



    Sectioning


    Once the tissues have been embedded, they must be cut into sections
    that can be placed on a slide. This is done with a microtome. The
    microtome is nothing more than a knife with a mechanism for advancing a
    paraffin block standard distances across it. There are three important
    necessities for proper sectioning: (1) a very sharp knife, (2) a very
    sharp knife, and (3) a very sharp knife.


    Sectioning with microtome.
    MPEG movie [672k] demonstrating sectioning technique with microtome.

    Knives are either of the standard thick metal variety or thin
    disposable variety (like a disposable razor blade). The former type
    allows custom sharpening to one's own satisfaction, but is expensive
    (more than $100 per
    blade). The latter cost about $1 per blade and are nearly as good. The
    advantage of the disposable blade becomes apparent when sectioning a
    block in which is hidden a metal wire or suture.


    Plastic blocks (methacrylate, araldite, or epon) are sectioned with
    glass or diamond knives. A glass knife can section down to about 1
    micron. Thin sections for electron microscopy (1/4 micron) are best
    done with a diamond knife which is very expensive ($2500).


    Microtomes have a mechanism for advancing the block across the
    knife. Usually this distance can be set, for most paraffin embedded
    tissues at 6 to 8 microns. The more expensive the microtome ($15,000 to
    $20,000), the better and longer-lasting this mechanism will be.



    Sectioning tissues is a real art and takes much skill and practice.
    Histotechnologists are the artists of the laboratory. It is important
    to have a properly fixed and embedded block or much artefact can be
    introduced in the sectioning. Common artefacts include tearing,
    ripping, "venetian blinds", holes, folding, etc. Once sections are cut,
    they are floated on a warm water bath that helps remove wrinkles. Then
    they are picked up on a glass microscopic slide.


    Picking sections up from water bath.
    Unstained section on glass slide.

    The glass slides are then placed in a warm oven for about 15 minutes
    to help the section adhere to the slide. If this heat might harm such
    things as antigens for immunostaining, then this step can be bypassed
    and glue-coated slides used instead to pick up the sections.


    Tray of unstained slides in drying oven.


    Frozen Sections


    At times during performance of surgical procedures, it is necessary
    to get a rapid diagnosis of a pathologic process. The surgeon may want
    to know if the margins of his resection for a malignant neoplasm are
    clear before closing, or an unexpected disease process may be found and
    require diagnosis to decide what to do next, or it may be necessary to
    determine if the appropriate tissue has been obtained for further
    workup of a disease process. This is accomplished through use of a
    frozen section. The piece(s) of tissue to be studied are snap frozen in
    a cold liquid or cold environment (-20 to -70 Celsius). Freezing makes
    the tissue solid enough to section with a microtome.


    Frozen sections are performed with an instrument called a cryostat.
    The cryostat is just a refrigerated box containing a microtome. The
    temperature inside the cryostat is about -20 to -30 Celsius. The tissue
    sections are cut and picked up on a glass slide. The sections are then
    ready for staining.




    Staining


    The embedding process must be reversed in order to get the paraffin
    wax out of the tissue and allow water soluble dyes to penetrate the
    sections. Therefore, before any staining can be done, the slides are
    "deparaffinized" by running them through xylenes (or substitutes) to
    alcohols to water. There are no stains that can be done on tissues
    containing paraffin.


    The staining process makes use of a variety of dyes that have been
    chosen for their ability to stain various cellular components of
    tissue. The routine stain is that of hematoxylin and eosion (H and E).
    Other stains are referred to as "special stains" because they are
    employed in specific situations according to the diagnostic need.



    Frozen sections are stained by hand, because this is faster for one
    or a few individual sections. The stain is a "progressive" stain in
    which the section is left in contact with the stain until the desired
    tint is achieved.


    H and E staining


    Hematoxylin is the oxidized product of the logwood tree known as
    hematein. Since this tree is very rare nowadays, most hematein is of
    the synthetic variety. In order to use it as a stain it must be
    "ripened" or oxidized. This can be done naturally by putting the
    hematein solution on the shelf and waiting several months, or by buying
    commercially ripened hematoxylin or by putting ripening agents in the
    hematein solution.


    Hematoxylin will not directly stain tissues, but needs a "mordant"
    or link to the tissues. This is provided by a metal cation such as
    iron, aluminum, or tungsten. The variety of hematoxylins available for
    use is based partially on choice of metal ion used. They vary in
    intensity or hue. Hematoxylin, being a basic dye, has an affinity for
    the nucleic acids of the cell nucleus.


    Hematoxylin stains are either "regressive" or "progressive". With a
    regressive stain, the slides are left in the solution for a set period
    of time and then taken back through a solution such as acid-alcohol
    that removes
    part of the stain. This method works best for large batches of slides
    to be stained and is more predictable on a day to day basis. With a
    progressive stain the slide is dipped in the hematoxylin until the
    desired intensity of staining is achieved, such as with a frozen
    section. This is simple for a single slide, but lends itself poorly to
    batch processing.


    Eosin is an acidic dye with an affinity for cytoplasmic components
    of the cell. There are a variety of eosins that can be synthesized for
    use, varying in their hue, but they all work about the same. Eosin is
    much more forgiving than hematoxylin and is less of a problem in the
    lab. About the only problem you
    will see is overstaining, especially with decalcified tissues.



    Coverslipping


    The stained section on the slide must be covered with a thin piece
    plastic or glass to protect the tissue from being scratched, to provide
    better optical quality for viewing under the microscope, and to
    preserve the tissue section for years to come. The stained slide must
    go through the reverse process that it went through from paraffin
    section to water. The stained slide is taken through a series of
    alcohol solutions to remove the water, then through clearing agents to
    a point at which a permanent resinous substance beneath the glass
    coverslip, or a plastic film, can be placed over the section.




    Decalcification


    Some tissues contain calcium deposits which are extremely firm and
    which will not section properly with paraffin embedding owing to the
    difference in densities between calcium and parffin. Bone specimens are
    the most likely type
    here, but other tissues may contain calcified areas as well. This
    calcium must be removed prior to embedding to allow sectioning. A
    variety of agents or techniques have been used to decalcify tissue and
    none of them work perfectly. Mineral acids, organic acids, EDTA, and
    electrolysis have all been used.


    Strong mineral acids such as nitric and hydrochloric acids are used
    with dense cortical bone because they will remove large quantities of
    calcium at a rapid rate. Unfortunately, these strong acids also damage
    cellular morphology, so are not recommended for delicate tissues such
    as bone marrow.


    Organic acids such as acetic and formic acid are better suited to
    bone marrow, since they are not as harsh. However, they act more slowly
    on dense cortical bone. Formic acid in a 10% concentration is the best
    all-around decalcifier. Some commercial solutions are available that
    combine formic acid with formalin to fix and decalcify tissues at the
    same time.


    EDTA can remove calcium and is not harsh (it is not an acid) but it
    penetrates tissue poorly and works slowly and is expensive in large
    amounts.


    Electrolysis has been tried in experimental situations where calcium
    had to be removed with the least tissue damage. It is slow and not
    suited for routine daily use.



    Artefacts in Histologic Sections


    A number of artefacts that appear in stained slides may result from
    improper fixation, from the type of fixative, from poor dehydration and
    paraffin infiltration, improper reagents, and poor microtome sectioning.


    The presence of a fine black precipitate on the slides, often with
    no relationship to the tissue (i.e., the precipitate appears adjacent
    to tissues or within interstices or vessels) suggests formalin-heme
    pigment has formed. This can be confirmed by polarized light
    microscopy, because this pigment will polarize a bright white (and the
    slide will look like many stars in the sky). Formalin-heme pigment is
    most often seen in very cellular or bloody tissues, or in autopsy
    tissues, because this pigment forms when the formalin buffer is
    exhausted and the tissue becomes acidic, promoting the formation of a
    complex of heme (from red blood cells) and formalin. Tissues such as
    spleen and lymph node are particularly prone to this artefact. Making
    thin sections and using enough neutral-buffered formalin (10 to 1 ratio
    of fixative to tissue) will help. If the fixative solution in which the
    tissues are sitting is grossly murky brown to red, then place the
    tissues in new fixative.


    The presence of large irregular clumps of black precipitate on
    slides of tissues fixed in a mercurial fixative such as B-5 suggests
    that the tissues were not "dezenkerized" prior to staining. These black
    precipitates will also appear white with polarized light microscopy.


    Tissues that are insufficiently dehydrated prior to clearing and
    infiltration with paraffin wax will be hard to section on the
    microtome, with tearing artefacts and holes in the sections. Tissue
    processor cycles should allow sufficient time for dehydration, and
    final ethanol dehydrant solution should be at 100% concentration. In
    humid climates, this is difficult to achieve. Covering or sealing the
    solutions from ambient air will help. Air conditioning (with
    refrigerants, not with evaporative coolers) will also reduce humidity
    in the laboratory. Toluene as a clearing agent is more forgiving of
    poorly dehydrated tissues, but it is more expensive and presents more
    of a health hazard than other non-xylene clearing agents


    Though alcohols such as ethanol make excellent fixatives for
    cytologic smears, they tend to make tissue sections brittle, resulting
    in microtome sectioning artefacts with chattering and a "venetian
    blind" appearance.


    Bubbles under the coverslip may form when the mounting media is too
    thin, and as it dries air is sucked in under the coverslip.
    Contamination of clearing agents or coverslipping media may also
    produce a bubbled appearance under the microscope.




    Problems in Tissue Processing


    "Floaters" are small pieces of tissue that appear on a slide that do
    not belong there--they have floated in during processing. Floaters may
    arise from sloppy procedure on the cutting bench-- dirty towels,
    instruments, or gloves can have tissue that is carried over to the next
    case. Therefore, it is essential that you do only one specimen at a
    time and clean thoroughly before opening the container of the next case.


    The best way to guard against unrecognized floaters is to always
    separate like specimens in the numbering sequence. For example, if you
    have three cases with prostate chips, separate them in accessioning
    with totally different specimens such as uterus or stomach. That way,
    if numbers are transposed or labels written wrong or tissue carried
    over, then you will have an obvious mismatch. Carrying over one
    prostate to another, or transposing the numbers of identical tissues
    may never be recognized.


    If reusable cassettes are employed, you must be aware that tissue
    may potentially be carried over and appear as "floaters" even several
    days later, when the cassette is re-used. The problem arises when,
    during embedding, not all the tissue is removed from the cassette.
    Then, in the cleaning process, not all of the wax is removed. Then, the
    next person using the cassette does not pay attention to the fact that
    there is tissue already in the cassette and puts his specimen in it.
    The floater that appears on the slide will look well-preserved--it
    should, because it was processed to paraffin.


    Always be sure that you properly identify the tissue! This means
    that you make sure that the patient label on the specimen container
    matches that of the request slip. An accession number is given to the
    specimen. This number must
    appear with the tissue at all times. You must never submit a cassette
    of tissue without a label. You must never submit a cassette of tissue
    with the wrong label. Mislabelling or unlabelling of tissues is
    courting disaster.


    Safety in the Lab


    The lab should be well-ventilated. There are regulations governing
    formalin and hydrocarbonds such as xylene and toluene. There are limits
    set by the Occupational Safety and Health Administration (OSHA) that
    should not be exceeded. These limits have recently been revised to
    reduced levels.


    Every chemical compound used in the laboratory should have a
    materials safety data sheet on file that specifies the nature,
    toxicity, and safety precautions to be taken when handling the compound.


    The laboratory must have a method for disposal of hazardous wastes.
    Health care facilities processing tissues often contract this to a
    waste management company. Tissues that are collected should be stored
    in formalin and may be disposed by incineration or by putting them
    through a "tissue grinder" attached to a large sink (similar to a large
    garbage disposal unit).


    Every instrument used in the laboratory should meet electrical
    safety specifications (be U.L. approved) and have written instructions
    regarding its use.


    Flammable materials may only be stored in approved rooms and only in storage cabinets that are designed for this purpose.


    Fire safety procedures are to be posted. Safety equipment including
    fire extinguishers, fire blankets, and fire alarms should be within
    easy access. A shower and eyewash should be readily available.


    Laboratory accidents must be documented and investigated with incident reports and industrial accident reports.


    Specific hazards that you should know about include:


    • Bouin's solution is made with picric acid. This acid is only sold in the aqueous state. When it dries out, it becomes explosive.


    • Many reagent kits have sodium azide as a preservative. You
      are supposed to flush solutions containing sodium azide down the drain
      with lots of water, or there is a tendency for the azide to form metal
      azides in the plumbing. These are also explosive.


    • Benzidine, benzene, anthracene, and napthol containing compounds are carcinogens and should not be used.


    • Mercury-containing solutions (Zenker's or B-5) should
      always be discarded into proper containers. Mercury, if poured down a
      drain, will form amalgams with the metal that build up and cannot be
      removed.


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