conventional and lbc similarities and differences in...

Conventional Smears and LBC

Similarities And Differences

Dr. Mina DesaiFRCPath; CBE

Head of the Service/ Manchester Cytology CentreDirector/Northwest Cytology Training Centre

Past President/ British Society For Clinical CytologyU.K.

Manchester

Manchester Cytology Center• One of the largest Lab In UK

With Affiliated Training School• Fully converted to LBC in 2005• Started with 70% TP, 30% SP• Hub for processing of both systems

of LBC• Laboratory workload 300,000/annum

for processing • Screening workload 225,000/annum. • Changed to 70% SP, 30% TP• Converted to SP only in October

2010• Workload reduced to 135,000/year• Majority of Laboratory staff trained

for both systems of LBC• Introduced Imager and Focalpoint

for automated screening for National Trial

Manchester Cytology LabExperience with Automation

Cytyc®Imager BD FocalPoint GS™ Imaging System

National HTA funded MAVARIC Trial

Manchester Cytology Training CentreTeaching activities

NW Regional Training School

Conventional and LBCCOMPARATIVE FEATURES

Conventional Smear• Heterogeneous • Graphic cell localization • 300-500 k cells/slide• Variable fixation• Thick uneven groups

need frequent focusing• Dirty background• Variable preservation

LBC Slides• Homogeneous • Random cell presentation• 50-70 k cells/slide• Uniform fixation• Uniform thin layer

Not single cell monolayers• Clean background• Well preserved cells

Liquid Based Cytology Morphological Appearances Comparison of

Conventional and LBC

• Well preserved cells in a liquid medium – more rounded single cells.

• Cell aggregates where present are smaller and evenly spread• Blood and polymorphs in the background not obscuring the cells• Improved staining – more nuclear detail visible• Homogeneous cell distribution –• loss of cell to cell associations (e.g. streaks of histiocytes)• Same criteria used for grading dyskaryosis/SIL

Surepath Conventional Thinprep

General appearance of ThinPrepslides

• 1.9cm diameter circle• 70k vs 250k cells per sample• Well-demarcated edge – no ‘drift’• Cells evenly distributed • Holes in between cells• Good Fixation and nuclear detail• Cleaner background• Traditional smear pattern lost• Rounded up, small, single cells

and small clusters

General appearance of SurePathslides

• 1.3cm diameter circle of cells• Densely cellular• More 3D effect seen• Need to focus more often than with

Thinprep slides• Not as many “holes” as with Thinprep• Need to use high power more often• Other appearances are similar to

Thinprep

Advantages Small No. of cells 70,000 or less (conventional

250,000 cells)Disadvantages Traditional smear pattern absent Mechanical artefact is eliminated Cells have no company Cellular material not pulled out in mucus Cells round up Well preserved single cells, therefore need to go on

high power too often Preparation artefact

LBC Advantages and Disadvantages – screening

Advantages Small area to screen Good fixation Good nuclear detailDisadvantages Nuclear detail too good More borderline/ASC

reports during learning curve

LBC Advantages and Disadvantages

System Specific Advantages and Disadvantages

SurepathGood Points

• Small area to screen

• ‘In-your-face’ chromatin

• Low grade dysk /LSIL easy

Not-so-good

• Cell crowding with 3D effect

• difficult areas are Small cell and HCCG

• Atypical glandular can be in single cell

• Monotone/two-tone staining with loss of orange colour

System Specific Advantages and Disadvantages

ThinprepGood Points

• Monolayer effect

• Flexibility with staining

• Gaps in-between cells give rest to the eyes

Not-so-good

• Larger area to screen

• Difficult areas are Metaplastic vs.HSIL, pale cell and bland cell

• Atypical glandular can be without feathering

Conventional & Liquid Based CytologyCommon Errors

• Screening Errors

• Interpretation Errors+

• Preparation Errors

Sparse dyskaryosis/SILA feature of conventional and

Both systems of LBC

Litigation cell

LBC Screening Error

LBCSCREENING METHOD

COMPARISON

CONVENTIONAL ---- 50mm vertically or horizontallySUREPATH----- 13mm diameter circle screen vertically and

horizontally during learning period THINPREP----- 19mm diameter circle screen one way and

around the periphery during learning periodWith all 3 systems need to screen Systemic, Slowly and with

Significant overlap. Focus and use high power more often ( Remember 3S )

Significant overlap = (1/3 to >4/5)

Surepath Primary Screening technique

Screen at half speed of conventional screening i.e. x 10

Screen smears in one direction looking for single abnormal cells and screen second time across at 90º studying sheets/groups i.e. x 10

Screen initially under x 20 until you are familiar with the smear pattern of that particular slide

Use one of the following screening methods initiallyduring learning period :

Surepath Screening technique

Key to good screening technique is maximum overlapping of fields at x10 and slow speed. Screen Systemic, Slowly and with Significant overlap. Focus and use high power more often ( Remember 3S )

Significant overlap = (1/3 to >4/5)

ThinPrepPrimary Screening Technique

during learning curve• Systematic

• Slow at x10

• 1/3 to 4/5

Overlap

• Frequent use of

High power

Up/Down and periphery OR

Left/Right and periphery

For Routine use No need to screen periphery separately

Interpretation errorNo difference between conventional and

LBCPattern recognition and experience

Liquid Based CytologyInterpretation errors

• Small cell• Pale cell• Bland cell• Syncitial group• Hyperchromatic crowded groups• Microbiopsies• Small Keratinized cells• Scanty Dyskaryosis ( HSIL)• Dysk in metaplastic cells• Koilocyte or NOT • Multinucleate cellsSame pitfalls as in conventional smears. Need to change the

baseline criteria of abnormality due to ‘ in your face chromatin’ in LBC

LBC Normal CellsClassic Criteria is retained.

Squamous cells• More folded cells – “v” shaped cells• Nuclear chromatin granular

Often nuclear folding or grooving with notches in the nuclear membrane Reason for increase ASC/Borderline reports during learning curve

Metaplastic cellsNuclei appear larger, more granular and hyperchromaticNormal mitoses not uncommonReason for increase ASC/BNC reports during learning curve

Endocervical cellsArtefact associated with cervex broom

Individual cells, smaller groups or in sheets – may be honeycomb Starburst or cartwheel arrangements common Cilia often seen

Endometrial cellTight cell ball (top hat) or

Well preserved loose cell groupings with depth of focus and disordered arrangement (3D clusters)

Major pitfall of HCCG with false +ve diagnosis of HSIL/ Severe dyskaryosis

LBC Normal CellsClassic Criteria is retained.

Squamous cells

Metaplastic cells

•More folded cells – “v” shaped cells•Nuclear chromatin granular •Often nuclear folding or grooving with notches in the nuclear membrane•Reason for increase ASC/Borderline reports during learning curve

•Nuclei appear larger, more granular and hyperchromatic• Normal mitoses not uncommon• Reason for increase ASC/BNC reports during learning curve

LBC Normal CellsClassic criteria are retained

Endocervical cells•Artefact associated with cervex broom•Individual cells, smaller groups or in sheets – may be honeycomb•Starburst or cartwheel arrangements common •Cilia often seen

Endometrial cells•Tight cell ball (top hat) or•Well preserved loose cell groupings with depth of focus and disordered arrangement (3D clusters)•Major pitfall of HCCG with false +ve diagnosis of HSIL/ Severe dyskaryosis•More LUS cells and combination of EC andEM in same cluster

LBC OrganismsThinprep Surepath

No change

LBCLow Grade Dyskaryosis/LSIL

• Abnormal cells appear either singly or in sheets

• Easy to find if Hyperchromatic

• Pale cell dysk (SIL) easy to miss

• Low grade dysk (LSIL) in rounded cells in LBC-misinterpreted as HSIL/Mod. Dyskaryosis resulting in Low PPV

LBCHigh Grade Dyskaryosis/HSIL

• More Isolated cells rather than streaks

• More three dimensional groups• May have smaller nuclei but with

higher N/C ratio than found in conventional

• Small isolated cells stand out due to clean background.

• Nuclear membrane and chromatin distribution easily identified.

• Surepath more dysk (SIL) in HCCG

• Thinprep more Dysk (SIL) in metaplastic

LBCSquamous carcinoma

• Tumour diathesis still present

• Fibre Cells still present

• Prominent Nucleoli easily seen

HCCGMajor Pitfall in Surepath LBC

ALL of the following cells are normal!

HCCGMajor Pitfall in Surepath LBC

ALL of the following cells are abnormal !

Metaplastic CellsMajor Pitfall in Thinprep LBC

Differential diagnosis is often between normal reactive or moderate (HSIL) dyskaryosis


Spot the Difference


• Dyskaryosis/SIL • Metaplastic cells

LBCGlandular lesions/CGIN/AIS

• More single cells and small strips with community borders

• Psuedostratification and Hyperchromasia

• Changes more subtle in individual cells due to better fixation

• Prominent Macro nucleoli, Gland openings and rosettes

• Feathering present but not as pronounced

• Vaculation of the cytoplasm• Scalloped borders

LBC Normal Cells

Additional Pitfalls Due Tosampling technique

• Broom artefact – distorted tangles of disrupted cells and streaked DNA

• Endocervical sampling in the atrophic cervix– Unexpected– May be wrongly interpreted – often as high-grade squamous

dyskaryosis (HSIL)

• Lower uterine segment sampling in the post-surgical cervix– Interpreted as residual/recurrent high-grade disease

LBCUnanswered Questions

The adequacy dilemma: How many cells are enough?

Cellularity0 5K 10K 15K 20K

Abnormaldetectionrate %

1.0% Threshold

NB: Hypothetical graph

The adequacy dilemma: where should we set the threshold?

Cellularity0 5K 10K 15K 20K

1.0% Threshold

NB: Hypothetical graph

Higher threshold=higher detection ratesbuthigher inadequate rates and higher cost

Lower threshold=lower inadequate rates,lower costbutlower detection ratesAbnormal

detectionrate %

Bethesda

The Bethesda System GuidelinesMinimum of 10 microscopic fields at 40x should be

counted along a diameter that includes the centre of the preparation

The average cell number per 40x to achieve 5000 minimum is shown in the table below

FN20 eyepiece/ 10X obj.




PREP DIAM(mm)

AREAFields@ FN20 10X

Cells/ fields for 5000 Total

Fields@ FN20 40X

Cells/ fields for 5000 Total

Fields@ FN22 10X

Cells/ field for 5000 Total

Fields@FN2240X

Cells/ field for 5000 Total

13 132.7 42.3 118.3 676 7.4 34.9 143.2 559 9

SurePath adequacy: macro and micro

27 cells per x40 field = 15K

Total material must be more than 50%

2/3 of this material should be well preserved and visible squamous cells

Thinprep

Definition of adequacy Bethesda minimum 5,000 cells UK uses different definitions for each

system and different centres are using different cellular cut off

5,000, 10,000, 15,000 in different centres HTA funded trial in UK will report next

year Dr. Desai and Dr. Turnbull are lead

investigators

Traditional and LBCConclusions

• Major difference between conventional and LBC• Cytotechnologists do NOT want to go back to conventionals • There are major differences between Surepath and Thinprep

in sample preparation ,cell presentation and screening methods.

• Finding abnormal small cells in Surepath requires different screening techniques for different slide presentations

• Generally, SP requires slower search strategy and greater overlap than ThinPrep and conventional smears

• Only minor differences are present in Interpretation of cells between two methods

• Be extra vigilant with HCGs in Surepath and Metaplastic cells in Thinprep

• Modify your existing interpretative skills otherwise more ASC and more HG reports with reduced PPV

• Be careful with Preperation artifact• LBC has reduced inadequate smears but adequacy is not

defined• LBC has opened the door for HPV and molecular testing

AcknowledgementMy special thanks to Dr. Persad & Dr. Bijal shah

Jean Mather &

Andrew EveredFor

Producing images and drawingsand

Writing some of the texts

I have also used images from Cytyc, Surepath and NHS CSP Atlas

The End

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