Liquid-based Cytology (LBC)

method for Gynecologic cytology

Lenny Sari
RS Kanker Dharmais
Metode Liquid-based pada Gynecologic cytology
yaitu
Metode yang dilakukan dengan memasukkan sikat
endoserviks/ brush kedalam cairan pengawet yang
kemudian dilakukan prosesing melalui tahap
dispersi sel, pengumpulan sel dan transfer sel pada
gelas objek dan menghasilkan lapisan yang
monolayer dengan diameter 20mm.
The Cervical Smear
Impact of Pap Smear Screening

Number of Deaths Per Year

• A 50 year old test with
a remarkable history of
success most common
cancer
screening test
• Reduced death rate
significantly
1940 2000
Conventional Pap Smear
Sources of False Negative Results (FNR)1, 2, 3

• Screening error–1/3 of FNR’s

• Screening errors–abnormal cells are present on the
slide but missed by a cytologist
• Interpretive errors–cells not classified correctly
• Sampling/preparation errors–2/3 of FNR’s
• Cells not collected on the sampling device
• Cells collected are not transferred to the slide
• Poorly preserved cells

1 Gay JD, et al.: False-Negative Results in Cervical Cytologic Studies, Acta Cytologica 1985, Vol. 29(6):1043-6
2 Joseph MG, et al.: Cyto-Histological Correlates in a Colposcopic Clinic: A 1-Year Prospective Study,
Diagnostic Cytolopathology 1991, Vol. 7(5):477-81
3 Linder J, et al., ThinPrep Papanicolau Testing to Reduce False-Negative Cervical Cytology,
Arch Pathol Lab Med 1998, Vol. 122(2):139:44
Discover a More Accurate Test
Physician’s Office—Still a Simple Process
Step 1 Step 2 Step 3

Obtain Rinse Seal

In the Laboratory—A Significant Improvement

Step 1 Step 2 Step 3

Load Process Screen

 Sampling Is a Concern
Percentage of Obtained Sample Transferred onto the Slide

EPITHELIAL CELLS (000s)

Cells transferred to slide

1400
92%
1200
Cells rinsed from
collection device after
of cells
left on
collection
• More than 80% of the cell
smear prepared
1000 device
sample may be discarded
800

93%
with the collection device
82%
600 of cells
left on
of cells
left on
after the conventional Pap
400 collection

smear1.
collection
device device

200

0
Swab/Spatula Endocervical Broom-like
Brush/Spatula Device

1
Hutchinson ML, et al., Homogeneous sampling accounts for the increased diagnostic
accuracy using the ThinPrep® Processor, Am J Clin Patholo 1994; 215-219
The Problem
The Problem
The Conventional Cervical Smear

A cervical sample Non-randomized

containing precancerous portion of cells
cells (red)

Over 80% of
cells discarded

Sample may not reflect

patient’s actual condition

Smear spray-fixed
and sent to lab

Missing cells,
obscuring elements
The Solution
The Solution
Liquid base method Pap Test
Virtually 100% of
A cervical sample cells collected into
containing precancerous Liquid base vial
cells (red)

Cells
immediately
preserved and
sent to lab
Increased
opportunity to detect
early signs of
abnormality Filtration process disperses,
randomizes cells

More representative
and clear thin layer
of cells
More Homogeneous Sample
The Conventional Smear Liquid base method Pap Test

• Majority of cells not captured • Virtually all of the sample is

• Non-representative transfer of collected
cells • Randomized, representative
transfer of cells

1
Hutchinson ML, et al., Homogeneous sampling accounts for the increased
diagnostic accuracy using the ThinPrep® Processor, Am J Clin Patholo 1994; 101:215-219
 Pivotal Trial
Design
• 6,747 patients
• 6 sites – 3 Screening Centers and 3 Hospitals
• Screening population:
— Low rates of cervical abnormality (<5%)
— Used to demonstrate ThinPrep would be sensitive to
disease in a normal screening situation
• High-risk population:
— High rates of cervical abnormality (>10%)
— Used to show ThinPrep would detect disease effectively
when tested with a high number of patients with
abnormalities
• Split-sample, blinded, matched pair

Lee et al., Comparison of Conventional Papanicolaou Smears and a Fluid-Based, Thin-Layer System for Cervical
Cancer Screening, Obstetrics and Gynecology, 1997, Vol. 90(2):278-84
Pivotal Trial
Diagnostic Results

Screening Population High Risk Population

65% Increase 6% Increase
200
500
The ThinPrep® The ThinPrep®
191 Pap Test™ Pap Test™

Conventional 400 446 Conventional

Smear 422 Smear

300
100 116

200

100

0 0

OVERALL RESULTS OVERALL RESULTS

Lee et al., Comparison of Conventional Papanicolaou Smears and a Fluid-Based, Thin-Layer System for
Cervical Cancer Screening, Obstetrics and Gynecology, 1997, Vol. 90(2):278-84
Pivotal Trial
Specimen Adequacy Results

• Improves specimen 28%

54%
quality significantly over Reduction
conventional Pap smear
by reducing
13%
• Air drying artifact
• Mucus
• Blood
• Inflammation Conventional The ThinPrep®
Pap Test™
Smear

Lee et al., Comparison of Conventional Papanicolaou Smears and a Fluid-Based, Thin-Layer System for
Cervical Cancer Screening, Obstetrics and Gynecology, 1997, Vol. 90(2):278-84
Roberts , et al.
Laverty and Associates, Sydney, Australia

• 35,560 split-sample patients HGEA* Cases Detected

• 94% reduction of + 16%
unsatisfactory cases 273
• 12% increased detection of 236
significant abnormality
• 16% increased detection of
biopsy-proven HGEA
• No loss in specificity

Conventional The ThinPrep®

Smear Pap Test™

Medical Journal of Australia, 1997, Vol. 167(3):466-69

* HGEA=High Grade Epithelial Abnormalities
 Yeoh, et al.
Diagnostic Pathology Laboratory, Hong Kong
Increased HSIL
• 16,541 ThinPrep Pap Tests
Detection
results vs. 7,258 conventional
smears + 28%
• Improved sensitivity with a
higher positive predictive value
of 94.2%
• Higher yield of samples
containing endocervical cells
• Biopsy confirmation
Conventional The ThinPrep®
“Another advantage was the ability to Smear Pap Test™

perform HPV DNA assays on equivocal

cases.”
Yeoh GPS et al, Evaluation of the ThinPrep Papanicolaou test in clinical practice, HKMJ 1999; 5:233-9
 Weintraub, et al.
Laboratoire Weintraub, Geneva, Switzerland

• 39,864 ThinPrep results vs. Increased HSIL Detection

130,381 conventional smears

• Improved sensitivity with an
odds ratio of 1.86 for HSIL
and 3.41 for LSIL
• Decrease in the ASCUS:SIL + 186%
ratio
• Biopsy confirmation

“There was an excellent agreement between the cytologic

Conventional The ThinPrep®
Pap Test™
Smear
diagnosis of HSIL and the results of the subsequent
histological evaluation.”
Weintraub, J. Efficacy of a liquid-based thin layer method for cervical cancer screening in a population
with a low incidence of cervical cancer, Diagn Cytopathol 2000 Jan;22(1):52-59
 Diaz-Rosario, et al.
Quest Diagnostics, Inc. Cambridge, MA, USA
• 56,339 ThinPrep test results vs.
74,756 conventional slides Increased HSIL Detection

• Decreased ASCUS/SIL ratio by

39.11%
• Increased detection of HSIL by + 102.54%

102.54%
• Increased detection of LSIL by
71.65%
“ThinPrep produced increased detection of pre-
malignant precursors while improving the specimen Conventional The ThinPrep®
Smear Pap Test™
adequacy.”
Diaz-Rosario LA et al. Performance of a Fluid-Based, Thin-Layer Papanicolaou Smear Method in
Clinical Setting of an Independent Laboratory and an Outpatient Screening Population in New
England, Arch Patholo Lab Med – 1999, Vol. 123(9):817-21
Clinical Performance Summary
Benefits Over The Conventional Pap Smear

• The ThinPrep® Pap Test™ has improved specimen quality1

• The ThinPrep Pap Test has higher sensitivity and positive
predictive value2
• The ThinPrep Pap Test has a higher yield of samples containing
endocervical cells2
• The ThinPrep Pap Test produced increased detection of
pre-malignant precursors3

1 Lee KR et al. Comparison of Conventional Papanicolaou Smears and a Fluid-Based, Thin-Layer System
for Cervical Cancer Screening, Obstetrics and Gynecology, 1997 Vol. 90(2):278-84
2 Yeoh GPS et al. Evaluation of the ThinPrep Papanicolaou test in clinical practice, HKMJ 1999; 5:233-9
3 Diaz-Rosario LA et al. Performance of a Fluid-Based, Thin-Layer Papanicolaou Smear Method in Clinical
Setting of an Independent Laboratory and an Outpatient Screening Population in New England, Arch
Patholo Lab Med – 1999, Vol. 123(9):817-21
Summary
Liquid-based method
• A replacement for the conventional Pap smear
• Significantly more effective for the detection
of LSIL and more severe lesions
• Specimen quality is significantly improved

Additional Testing
• Residual Specimen Remains in the Vial
• Ability to perform Human Papilloma Virus (HPV) testing
from the vial, which may be useful to triage women in low-
risk or high-risk groups 1,2
• Currently evaluating adjunct testing for additional STD’s
e.g., chlamydia,3 gonorrhea, trichomonas and herpes.
Summary
Liquid-based method
• Is a more accurate test for the early detection of cervical
cancer precursors
• Results in a significant improvement in specimen
quality with a decreased number of unsatisfied cases
• Creates the opportunity for additional testing from the
patient’s sample
• Is the foundation for automation and a total system
approach

The best possible results from the best possible sample

□Automatic Capping System
□ Automatic Cell Transfer System
□ Automatic Filter Supply system
□ Membrane Filtration Method
(20mm Speculum Vision)
□ Convenient
(Operating Control by Touch Screen
LCD Panel)
□ Economical
□ Quickness
Cell prep the full automatic Liquid Based
Cytology solutions
Tahapan Process
More Accurate and Clean
Current Cancer Screening Methods

Methods macroscopic examination Pap Smear Liquid-Based Cytology

Equipment MRI, CT PET Smear CellPrep

specificity & above 3cm 96% High as 96%

Low as about 20~40%
screening ability below 1cm 24.8% (above 5/1000mm sizes)
5mm

Piled cell layers Even single layer

Cannot detect the early unnecessary bloods and debris Eliminate bloods and unnecessary
stage cancer remaining debris

Remarks Impossible to preserve cell Easy to preserve cells (3 years)

Impossible to do any
Additional tests available
additional tests

Possible to examine HPV or DNA

Morphology of the cells
 Easy to interpret
URINE
URINE((T.C.C
T.C.C))––Bladder
BladderCancer
Cancer SPUTUM
SPUTUM((S.C.C
S.C.C))––Lung
LungCancer
Cancer

HSIL
HSIL((CIN
CINIII
III))––Cervical
CervicalCancer
Cancer
LSIL
LSIL((HPV
HPV))––Cervical
CervicalCancer
Cancer
Liquid-based Characteristics

 Wet Fixation
 Cell Size
 Smear Pattern
 Specimen Background
 Similarities > Differences
Liquid-based Characteristics

Wet Fixation
– Enhanced Cytoplasmic Detail

– Enhanced Nuclear Detail

– Variability in Nuclear Staining

40x
40x 40x
Liquid-based Characteristics

Cell Size
– “Proportionately” Smaller

– Single Cells More Prominent

– Cells Round-Up in Solution

40x
40x
Liquid-based Characteristics

Smear Pattern
– Mechanical Artifact Eliminated

– Cellular Material Not Pulled Out

in Mucus

– Tissue Architecture Maintained

Liquid-based Characteristics

Specimen Background
– Cleaner Background
– Cellular Debris More Clumped
– TP “Clues” in the Background
– “RATTY” Background:
Infectious agent, Cytolysis, Blood (menses), DISEASE
Liquid-base Characteristics

Screening Technique
– Systematic

– Slow

– Slight Overlap
 Liquid based Characteristics
Cellular Composition for Adequacy

FN20 eyepiece/ FN20 eyepiece/ FN22 eyepiece/ FN22eyepiece/

10x obj. 40x obj. 10x obj. 40x obj.
THIN Area Fields @ Cells/ Fields @ Cells/ Fields @ Cells/ Fields @ Cells/
PREP FN20 Field for FN20 Field for FN22 Field FN22 Field for
DIAM MM 10x 5K 40X 5K 10X for 5K 40X 5K

20 314.2 100 Total 1600 Total 82.6 Total 132.2 Total

50.0 3.1 60.5 3.8
10X
FN 22
10x
FN 20
Endocervical Cells

 Honeycomb / Palisading
Arrangements Maintained
 Round-Up in Solution
 More Tightly Packed Groups
 Smaller Cell Groups/Single Cells
 Nuclei “Busier”
20x
40x
Squamous Metaplasia

 Sheets/Cobblestone Arrangements
 Dense, Homogeneous Cytoplasm
 Cells Often Single
 Cells Appear Smaller & Rounder
40x
Endometrial Cells

 Tight 3D Cell Clusters

 Loose Cell Clusters with Vacuolated
Cytoplasm
 Single Cells More Prominent
 Nuclei “Busier”
 Menstrual Blood Lysed
Atrophy

 Well Preserved Sheets of Parabasal

Cells
 Endocervical Cells May Be
Distinguished
 Bare Nuclei “Reduced”
 “Atrophic Vaginitis” Pattern More
Clumped
Trichomonas Vaginalis

 Frequently Smaller

 Internal Structure Readily Identifiable

 Classic “Trich” Pattern Maintained

Candida spp.

 Classic Cell Clumping

 Reactive Squamous Cells with
Engulfed WBC’s
 Distinguish Mucus Strands from
Pseudohyphae
Actinomyces

 Organized Clusters of Branching

Filamentous Bacteria

 Associated Blue Staining Bacteria

Herpes Simplex Virus

 Ground Glass Nuclei

 Multinucleation with Nuclear Molding

 Classic Eosinophilic Nuclear

Inclusions
 LSIL (Low Grade Squamous Intraepithelial
Lesion)
 Ukuran inti 3-4x inti sel intermediate
 Irreguler Nuclear membrane
 Increased Nuclear detail
 Sharp, irregular cytoplasmic cavitation (HPV)
 Binucleation, as seen here, may also be an indication of HPV.
 LSIL cells can be seen in groups and singly.
HSIL (High Grade Squamous Intra-
epithelial Lesion)
 Sheet&Syncytial groupings maintained
 Cytoplasmic border more distinc
 Nuclear membrane irregularities
 These squamous cells are small in size when compared to a
reference intermediate squamous cell.
 N/C ratio is very high. Nuclei are also hyperchromatic /coarse
chromatin and vary in size and shape.
 HSIL cells can appear both singly and in groups. This single small
cell in the background should serve as a clue to look for more
definitive HSIL material on the slide.
 Karsinoma sel skuamosa serviks
 The nuclei are predominantly centrally located but vary significantly in size
and shape.
 This grouping of atypical squamous cells exhibits a lack of polarity.
 The nuclei visible on the edge of the group have nuclear membrane
thickening and irregularities. Nucleoli prominen.
 The chromatin pattern is clumped and irregularly distributed.
 Prominent chromatin clearing
 Tad pole cells
 Keratinized cells
 Malignant squamous cells are caught up in a granular background
along with cellular debris, fibrin and lysed RBCs (Tumor diathesis)
 Adenocarcinoma cervix

 3D clusters maintained
 Groups of cells exhibiting acinar and papillary configurations.
 Crowded malignant nuclei vary in size and shape and many have
nucleoli.
 Tumor diathesis (-)
 Cluster of large epithelial cells with rounded, scalloped border to the
group, increased n/c ratio
 vacuolated cytoplasm
 Differential diagnosis includes endometrial versus endocervical
adenocarcinoma.