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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
Metaplastic CellsMajor Pitfall in Thinprep LBC
Spot the Difference
Metaplastic CellsMajor Pitfall in Thinprep LBC
• 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.
FN20 eyepiece/ 40X obj.
FN22 eyepiece/ 10X obj.
FN22 eyepiece/ 40X 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