1.0    TITLE: Sample Staining
     
    2.0    POLICY:
    All gynecologic slides for cytologic examination should be stained by Papanicolaou.
    The Papanicolaou stain or onother appropriate permanent stain is used for non-gynecologic specimens.
     
    3.0    PROCEDURE PAPANICOLAOU STAINING 
    3.1    Materials
    Hematoxlin- Commercially prepared
    0.5 % Hcl (0.5 ml. Hcl and 95.5 ml. Distilled water.
    Absolute and 80% isopropanol (see preparation of graded alcohol)
    OG-6- Commercially prepared
     EA – 50- Commercially prepared
    Xylene
             
    3.2    Step by step Staining procedure: (Automated multistainer)
    1. 80% isopropanol 10 minutes (fixation)
    2. Rinse in tap water
    3. Harris or Gill Hematoxylin in 2-3 minutes (time vary with selection of hematoxylin solution)
    4. Rinse in tap water or Scott’s tap water
    5. 80% isopropanol 10 dips
    6. OG-6 stain for 1.5 minutes
    7. 80% isopropanol 10 dips
    8. EA-50, or modified EA-50 or EA-65 stain for 2.5 minutes
    9. 80% isopropanol 10 dips, 2 changes
    10. 95% isopropanol 1 minute
    11. Clear in 2 changes of xylene, 2 minutes each
    12. Mount with permanent mounting medium.
    Automated Multistainer Downtime: the same staining procedure is done manually.
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    4.0    QUALITY CONTROL OF STAINING
    To maintain the optimal quality of the cells staining for diagnostic interpretation.
    4.1    The stains are filtered each morning.
    4.2    Weekly stain changing and cleaning of set up is documented.
    4.3    One empty slide for each batch of stain is evaluated microscopically.
    4.4    Daily QC sheet is maintained for quality of staining on all specimens.
    4.5    All reagents are used within their indicated expiration date.
    4.6    Assigning the Expiry Date to any reagents that do not have a manufacturer-provided expiration date.  The assigned expiration
    date is 5 years from the opening date.
    4.7    Use of expired Reagents
    Expired reagents are used only under the following circumstances:
    1.                  The reagents are unique, rare or difficult to obtain; or
    2.                  Delivery of new shipments of reagents is delayed through causes not under control of the laboratory.
    3.                  The laboratory has a strict quality control and documents validation of the performance of all reagents on Daily QC sheet.
    4.                  Fill the form (Gen. 023) for each expired reagents used.
     
    5.0    ASSESSMENT OF STAINS
    Cytology stains undergoing a daily technical quality review are exempt from annual assessment. Where applicable, expiration date
    assigned by a manufacturer must be observed. However, most stains used in cytology laboratory are not subject to outdating, so that
    assignment of expiration date may have no meaning.
                     Procedure of stain assessment:
    1.   Cytology stains are assessed regularly for proper storage and acceptable quality.
    The acceptable performance of such stains is confirmed each time a new batch of stain is started.  (Technical assessment is done on
    actual case material and as part of the evaluation of cytopathology cases).
    A well stained Pap smear should demonstrate crisp blue/purple nuclei. Cytoplasmic staining should show a broad spectrum of color
    ranging from orange in highly keratinized cells through ranges of orange/pink in superficial cells and green/blue in intermediate and
    parabasal cells.
    2.   Whenever a new batch of stain is started a test slide is stained and presented to senior Cytotech to assess stain quality.
    3.   A record of daily QC sheet is maintained for regular check on quality of staining.
     
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    6.0    PROCEDURE: IMPORTANT FACTORS INFLUENCING STAINING RESULTS OF PAPANICOLAOU STAIN
    6.1    Maintenance of Solutions and Stains
    Solutions may be used over a longer period of time if the slide carrier is rested on several thickness of paper toweling for a few
    seconds after removing it from the solutions.  The life expectancy of stains may be increased by storing them in dark bottles when not
    in use and in keeping staining dishes covered.
    The frequency of replacement of solutions required to ensure crisp, well-stained slides, is dependent on the volume of slides
    processed daily.  Daily microscopic checks are recommended.  The following and nature of material processed.
    6.2    Hematoxylin
    Remains relatively constant in staining characteristics and seldom requires discarding if small amounts of fresh stain are added daily to
    replace stain loss due to evaporation.  However, the use of coating or Carbowax fixatives may result in contamination, making
    frequent changes necessary.
    6.3    OG-EA
    Lose strength more rapidly than Hematoxylin and should be replaced each week or as soon as the cells appear gray, dull or without
    crisp contrasting colors.
    6.4    Water Rinses: Should be changed after each use.  Alcohols used during the rehydrating and dehydrating process prior to the
    cytoplasmic stains should be replaced weekly or may be discarded each day to avoid the necessity of filtering these solutions. The
    alcohol rinses following the cytoplasmic stains are usually changed on a rotating basis after each use.
    6.5    Xylene
     Should be changed as soon as it appears tinted with any of the cytoplasmic stains. Water in the xylene will make the solution appear
    slightly milky. The clearing process may be disturbed, and tiny drops of water can be seen microscopically on a plane above the cell
    on a slide.
    6.6    Dipping Slides:
    Agitation of the slides by dipping is necessary to remove excess dye.  If slides are not rinsed properly, a dull rather than sharp, crisp
    picture results.  Slides should be dipped gently to avoid cell loss, and the slide carrier should not hit the bottom of the staining dish. 
    Each dip should last approximately one second.  Dipping too slowly will result in too much decolorization.  However, one or two
    dips more or less will not affect results.
    6.7    Intensity of Staining Reaction:
    The desired intensity of nuclear and cytoplasmic stains is one of personal preference and varies with different cell samples.  Individual
    experimentation is necessary.  The quality of the stained slides is also dependent on the solubility, percentage of dye concentration,
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    experimentation is necessary.  The quality of the stained slides is also dependent on the solubility, percentage of dye concentration,
    etc., of the dyes used in making EA, OG, and Hematoxylin, as discussed in the section of this chapter devoted to stain preparation. 
    Factors other than timing, however, may influence the nuclear and cytoplasmic staining intensity, quality of stain, aging, storage
    condition, temperature, etc…….
    6.8    Nuclear Stain Too Pale:
    Under staining of the nucleus may occur for one or more of the following reasons:
       1.  Contamination of Hematoxylin with Carbowax or coating fixatives.
       2.  Time in Hematoxylin is not increased for Carbowax-fixed specimens wherein.
       3.  Smears may have been permitted to air-dry before fixation.
       4.  The pH of the tap water not sufficiently alkaline to blue properly.
       5.  Single cells may appear understained if thick areas of the smears are correctly stained.
       6.  Stain may become diluted if water is not drained from racks prior to immersion in Hematoxylin.
       7.  If the timing of staining in Hematoxylin is based on material collected in intensity of staining.
       8.  Expiration date of commercially prepared stain may have been overlooked.
       9.  Stain may be too old and should be replaced.
      10.  Inadequate mixing of the contents of aerosol and spray fixatives can result in poorly distributed fixation.  Staining may be
           uneven and spray can result in poorly distributed fixation.  Staining may be uneven and muddy in appearance.  Shake all
           fixatives well prior to use.
      11.  Slides sprayed with aerosol fixatives at too close or too far a range result in pale, poorly stained slides. 
      12.  Waxes and oils from hairspray fixatives alter staining reactions if not adequately removed.  Some brands may require the
           soaking of slides overnight in 80% isopropanol alcohol, rather than merely rinsing them in alcohol prior to staining.
    6.9    Nuclear Stain Too Dark
    Over staining of the nucleus may occur for one or more of the following reasons:
    1.      Cells fixed for a few minutes in modified Carnoy’s shrink, causing some chromatin condensation.  Therefore, staining time in
    Hematoxylin must be decreased.
    2.      If time is based on staining slides prepared from fresh material, less time must be used for prefixed material.
    3.      If single cells are well stained, thick areas of the slide may appear overstained.
    4.      Nuclepore filters that are dissolved prior to staining sometimes require less staining time in Hematoxylin.
    5.      The smears may have been prepared directly from very bloody or high-protein fluids.  The sediments from these fluids should be
    washed with a balanced salt solution prior to slide preparation.
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