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Transcript of Gynecological Cytology: Technical aspects - Ausl Bo · PDF fileGynecological Cytology:...

  • Gynecological Cytology:Technical aspects

    Rietje Salet-van de PolDepartment of Pathologyp gyRadboud University Nijmegen Medical CentreNijmegenj gThe Netherlands

    Trieste June 6th 2011

  • The Netherlands

    16.400.000 inhabitants (395.6/km ) National population screening program for cervical

    i 1988cancer since 1988 Women between 30-60 years l 5 years interval 500.000 smears/year

    nijmegen

  • Radboud University Nijmegen Medical CentreRadboud University Nijmegen Medical Centre,Nijmegen, The Netherlands

  • Cervical smearsCervical smears

    Quality of the smear depends on cell sampling, fixation and staining

    Cell sampler is responsible for cell sampling and fi tifixation

    Gi l f d b k t li i i d Give regular feed-back to clinicians and smear takers on specimen quality

  • Cell sampling

  • Normal smearsUnsatisfactory

    Reason: Obscuring blood, inflammatory cells,

    overlapping cells Low cellularity Poor preservation Cytolysis

  • Unsatisfactory smears

    Obscuring bloodOverlapping cells

    Poor preservation Low cellularity

  • Conventional Liquid based

    Unevenly distributed Equally distributedy Cell overlapping Obscured by mucus,

    q y Thin layer Well preserved non-y ,

    blood or inflammatory cells

    pobscured well stained cells

    Insufficient cell fixation Unsatisfactory:CS 1,11% - LBC 0,33%

  • Conventional, Thinprep and Surepath

    The Netherlands:

    24 % conventional

    76 % liquid based:- 37 % Surepathp- 39 % Thinprep

  • Liquid based cytology (LBC)Liquid based cytology (LBC)

    C ll li i i Cell sampling in same way as in conventional method

    Cellular material is Cellular material is Cellular material is Cellular material is transferred into a vial with transferred into a vial with fixativefixativefixativefixative

    Cell suspension is Cell suspension is h i d d h i d d homogenized and put on a homogenized and put on a glass slideglass slide

  • Thinprep Hologic, Marlborough,USAUSFDA approved in 1996

    Transport vial contains a methanol-based fi ifixative

    Cell suspension undergoes homogenization b spinnin th i l (T3000)by spinning the vial (T3000)

    Cells are transferred to a filter by suction C ll l it i t ll d b Cellularity is controlled by pressure

    monitoring across the filter 20 mm circle of cellular material20 mm circle of cellular material

  • Thinprep Thinprep Hologic, Marlborough,USA

    Tp2000 Tp5000

    Tp3000

  • Surepath Becton Dickinson/Tripath,Burlington,USAUSFDA approved in 1999

    S i t t d it di t ti ith Semi automated gravity sedimentation process with two centrifugation steps

    Collection device head remains in vial l h l d f Vial contains ethanol-based fixative Homogenization via syringing of the specimen Passage trough a sucrose density gradient column to Passage trough a sucrose density gradient column to

    reduce blood and inflammatory cells Final cell suspension trough gravity sedimentation on

    l l d ( l d ll l l ll a glass slide (slide coating allows only a single cell layer)

    13 mm circle of cellular material13 mm circle of cellular material

  • Surepath pBecton Dickinson/Tripath,Burlington,USA

    PrepMate device fori i h i tisyringing homogenization

    step and tranferring the sample to density gradientsample to density gradientcolumn prior to centrifugation (2x)g ( )

    PrepStain device for finalslides and staining process

  • ResultsResultsResultsResults

    ConventionalConventional LBCLBC

  • Conventional vs Liquid based

    Uneven distribution of cellsCells obscured by numerous Cells obscured by numerous polymorphonuclear leukocytes

  • Liquid based cytology

    Higher costs of material Less unsatisfactory smears Reduced screening time due to better preservation

    and recognition of cells and reduction in screening area

    Cts favor to look at lbc (job satisfaction) Additional diagnostic techniques (HPV testing and

    immunocytochemistry)immunocytochemistry) Facilitates automated screening

  • Staining

    Prior to staining spray fixatives, which contain g p y ,alcohol and a waxy substance (carbowax), must be removed in ethanol 96%

    To visualize the cells and various cell components ( difi d) P i l i i d(modified) Papanicolaou stain is used

    Multichromatic staining and a very reliable technique

    The modification vary from staining times, use of tap or distilled water and progressive ( blue with tap or distilled water and progressive ( blue with water) or regressive blue (hydrochloric acid)

  • George Papanicolaou(1883 1962)(1883-1962)

    Kymi Greece

  • Papanicolaou stainPapanicolaou stain5 dyes:

    Hematoxylin Orange G Eosin Azure (EA)

    Eosin Y Light green SF yellowish Bismarck brown Y EA has 3 separate formulas:p

    EA-56, EA-50 and EA-36

  • Papanicolaou stain

    Hematoxylin for nuclear staining. Important to date hematoxylin, because the dye may oxidize overtime, especially in moist climates

    Orange G counter stain is an acidic dye and stains keratin and the Orange G counter stain is an acidic dye and stains keratin and the granules in superficial cells

    EA counter stain: Eosin Y stains superficial squamous cells,

    nucleoli and red blood cells Light Green SF yellowish stains cytoplasm Light Green SF yellowish stains cytoplasm

    of intermediate sq. cells, parabasal and columnar cells Bismarck brown Y stains nothing and is now often omitted Compositions of EA can differ between different vendors

  • Papanicolaou stain

    The dye should be filtered daily Stored in dark well-sealed bottles when not in use Pap stain is not fully standardized. The selection

    of staining times, type of method used (progressive or regressive), use of tap or distilled water and PH of water and temperature differ water and PH of water and temperature differ from lab to lab

    Whichever modification is used it is advisable to Whichever modification is used it is advisable to standardize the staining method as much as possible to achieve reproducible resultsp p

  • R l P l Results Papanicolaou stain

    Cell nuclei are crisp blue to black

    S fi i l ll Superficial cells are orange to pink

    Intermediate and parabasal Intermediate and parabasal cells are turquoise green to blueblue

    Metaplastic cells often stain green and pinkg p

    Cells with high content of keratin are yellow-orange

  • Cover slipping

    Under a well ventilated fume hood to avoid inhaling toxic vapors

    N 1 hi li ll ll No.1 thinness coverslip to cover all cells Mounting medium should harden rapidly, may not

    diss l th d nd must st l ft d indissolve the dye and must stay clear after drying

  • Lysing red blood cellsLysing red blood cells

    In fixative: all contain glacial-acetic acid (commercial CytoLyt or Cyto Rich Red)After fixation: by placing slides in lysing solution (methanol glacial acetic acid 10 %)(methanol-glacial-acetic acid 10 %)

    Red blood cells After lysis

  • Destaining and restaining

    Only good results when sample was properly fixed Coverslip and removal of mounting medium in

    l ( k l h h ld h lid xylene (may take several hours, the older the slide the more time it takes)

    T th d th slid is l d i dil t To remove the dye the slide is placed in a dilute hydrochloric acid in an aqueous or an alcohol-based solutionsolution

    Removal may take 5 20 minutes Prior to restaining the slide is rinsed in running tap Prior to restaining the slide is rinsed in running tap

    water for 5 10 minutes

  • Tips and troubleshooting

    Microscopically control step after every staining to anticipate on potential staining problems

    P i i i fi i b d Prior to staining spray fixative must be removed When slides are transferred from water to

    h t li l ss d l ts f t st dis hematoxylin glassy droplets of water must disappear otherwise the dye does nor penetrate the nucleus

    Hematoxylin changed after 2000 slides or 6 weeksHematoxylin changed after 2000 slides or 6 weeks PH of water following hematoxylin must be alkaline

    (PH 7 4)(PH 7.4) PH of EA should be between 4.5 and 5

  • Tips and troubleshooting

    Alcohols must be changed regularlyg g y Slides should not be left in alcohol solutions after OG

    and EA because stains are washed out in alcohol Xylene has to be replaced when it becomes milky or

    contains small bubbles (water) To avoid cross-contamination solutions must be

    filtered after every use Solutions should be kept covered when not in use and

    stored in a dark well-sealed bottle Wh th ll h h th i When the cells have a hazy appearance there is a

    water contamination in dehydrating solutions or the spray fixative is not removed from the cellsspray fixative is not removed from the cells

  • TroubleshootingTroubleshooting Nuclei too pale S l i d i d i t fi ti Sample air dried prior to fixation Carbowax from spray fixative was not properly

    removedremoved Time in hematoxylin not adequate PH of water followin hematoxylin was not PH of water following hematoxylin was not

    sufficiently alkaline Hematoxylin exhaustedHematoxylin exhausted Nuclei too darkNuclei too dark Overstaining in hematoxylin Excess dye not removedExcess dye not removed

  • TroubleshootingTroubleshooting

    Cytoplasmic color not satisfactoryCytoplasmic color not satisfactory Sample air dried prior to fixationSample air dried pr