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/km² )• 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

• Ct’s 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:pEA-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 prior to fixation• Excessive time in hematoxylin colored cytoplasm• Inadequate rinsing of slidesInadequate rinsing of slides• PH of EA is not 4.5 -5• I d t st i i ti s• Inadequate staining times• Exhausted dyes

Troubleshooting

Cornflakes slide too dry before coverslipping (air)y pp g ( )

Adequate and satisfactory smears

• It is important to diagnose only adequate and ti f t satisfactory smears

• S l di f d h i d d • Several studies found that inadequate and unsatisfactory smears were more often from high risk patients and a significant number of these risk patients and a significant number of these smears were followed by a severe lesion

Specimen Adequacy:*Review of within normal limits cervical smears within five years before the diagnosis of cervical cancer

Review Lbcn (%)

Conventionaln (%)

Total n (%) ( ) ( ) ( )

Unsatisfactory 2 (18%) 21 (24%) 23 (24%)WNL 7 (64%) 31 (36%) 38 (39%)ASCUS/SIL 2 (18%) 16 (18%) 18 (18%)> HSIL 0 19 (22%) 19 (19%)Total 11 (100%) 87 (100%) 98 (100%)Total 11 (100%) 87 (100%) 98 (100%)

*De Bie RP et al. Patients with cervical cancer; why did screening not prevent these cases? Am J Obstet Gynecol 2011

Specimen Adequacy:

*Unsatisfactory smears previously diagnosed as WNL smears

Reasons for unsatisfactory Unsatisfactory (n)A (too much blood) 1B (too many leucocytes) 6C (few epithelial cells) 8D (b dl fi d) 1D (badly fixated) 1A+B 1A+C 2

13

A+C 2B+C 3A+B+C 1Total 23

*De Bie RP et al. Patients with cervcial cancer; why did screening not prevent these cases? Am J Obstet Gynecol 2011

Specimen Adequacy:Specimen Adequacy:Bethesda criteria for cellularity

• Satisfactory or unsatisfactory due to … (don’t use satisfactory but limited by is to confusing among limited by, is to confusing among clinicians)

• Conventional smears: estimated minimum of 8000-12000 well minimum of 8000 12000 well preserved and well-visualized squamous cells

• Liquid based preparations: estimated minimum of 5000 well

d d ll l d preserved and well-visualized squamous cells

Bethesda criteria for cellularityBethesda criteria for cellularityConventional smears

The amount of squamous cells should be estimated with the use of ”reference images”.C l i h ld h i i Cytologists should compare these images to specimens in question to determine if there are a sufficient number of fields with approximately equal or greater number of fields with approximately equal or greater cellularity than the reference image

Bethesda criteria for cellularity LBCBethesda criteria for cellularity LBCField Cell-count

• A minimum of 10 microscopic fields should be assessed along a diameter that includes the center of the preparation and an average number (4 9) of of the preparation and an average number (4-9) of well preserved squamous cells per field must be countedcounted

• Formula: number of cells required per field= 5000/(area of preparation/area of field)( p p )

Bethesda criteria for transformation zone component

For conventional smears and liquid based preparations :

• An adequate transformation zone component i s t l st 10 ll s d d i l requires at least 10 well-preserved endocervical or

squamous metaplastic cells, single or in clusters

• Give regular feed back to clinicians and smear Give regular feed-back to clinicians and smear takers on specimen quality

THE END