Liquid-based Cytology (LBC)method for Gynecologic cytologyLenny Sari
RS Kanker Dharmais
Metode Liquid-based pada Gynecologic cytology yaituMetode 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
• A 50 year old test with a remarkable history of success most common cancerscreening test
• Reduced death rate significantly
Impact of Pap Smear Screening
Number of Deaths Per Year
1940 2000
Conventional Pap Smear
• 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):1043-62 Joseph MG, et al.: Cyto-Histological Correlates in a Colposcopic Clinic: A 1-Year Prospective Study, Diagnostic Cytolopathology 1991, Vol. 7(5):477-813 Linder J, et al., ThinPrep Papanicolau Testing to Reduce False-Negative Cervical Cytology, Arch Pathol Lab Med 1998, Vol. 122(2):139:44
Sources of False Negative Results (FNR)1, 2, 3
Discover a More Accurate Test
Physician’s Office—Still a Simple Process
In the Laboratory—A Significant Improvement
Obtain Rinse Seal
Load Process Screen
Step 1 Step 2 Step 3
Step 1 Step 2 Step 3
Sampling Is a ConcernPercentage of Obtained Sample Transferred onto the Slide
1 Hutchinson ML, et al., Homogeneous sampling accounts for the increased diagnostic accuracy using the ThinPrep® Processor, Am J Clin Patholo 1994; 215-219
• More than 80% of the cell sample may be discarded with the collection device after the conventional Pap smear1.
0
200
400
600
800
1000
1200
1400
EPITHELIAL CELLS (000s)
Cells transferred to slide
Cells rinsed from collection device after smear prepared
92%of cells left on
collection device
82%of cells left on
collection device
93%of cells left on
collection device
Swab/Spatula EndocervicalBrush/Spatula
Broom-like Device
The Problem
The ProblemThe Conventional Cervical Smear
A cervical sample containing precancerous
cells (red)
Non-randomized portion of cells
Over 80% of cells discarded
Smear spray-fixed and sent to lab
Missing cells,obscuring elements
Sample may not reflect patient’s actual condition
The Solution
The SolutionLiquid base method Pap Test
A cervical sample containing precancerous
cells (red)
Virtually 100% of cells collected into
Liquid base vial
Cells immediately
preserved and sent to lab
Filtration process disperses, randomizes cells
More representative and clear thin layer
of cells
Increasedopportunity to detect
early signs of abnormality
More Homogeneous Sample
• Majority of cells not captured
• Non-representative transfer of cells
• Virtually all of the sample is collected
• Randomized, representative transfer of cells
Liquid base method Pap Test
The Conventional Smear
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
• 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
Design
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
Pivotal TrialDiagnostic Results
Screening Population65% Increase
High Risk Population6% Increase
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
0
100
200
Conventional Smear
The ThinPrep® Pap Test™
0
100
200
300
400
500
Conventional Smear
The ThinPrep® Pap Test™
116
191
422446
OVERALL RESULTS OVERALL RESULTS
• Improves specimen quality significantly over conventional Pap smear by reducing• Air drying artifact • Mucus • Blood • Inflammation
Specimen Adequacy 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
28%
13%
54% Reduction
ConventionalSmear
The ThinPrep®Pap Test™
Pivotal Trial
Roberts , et al.
• 35,560 split-sample patients
• 94% reduction of unsatisfactory cases
• 12% increased detection of significant abnormality
• 16% increased detection of biopsy-proven HGEA
• No loss in specificity
Laverty and Associates, Sydney, Australia
Medical Journal of Australia, 1997, Vol. 167(3):466-69* HGEA=High Grade Epithelial Abnormalities
HGEA* Cases Detected
236
273
+ 16%
ConventionalSmear
The ThinPrep®Pap Test™
Yeoh, et al.
• 16,541 ThinPrep Pap Tests results vs. 7,258 conventional smears
• Improved sensitivity with a higher positive predictive value of 94.2%
• Higher yield of samples containing endocervical cells
• Biopsy confirmation
Diagnostic Pathology Laboratory, Hong Kong
Yeoh GPS et al, Evaluation of the ThinPrep Papanicolaou test in clinical practice, HKMJ 1999; 5:233-9
“Another advantage was the ability to perform HPV DNA assays on equivocal cases.”
Increased HSIL Detection
ConventionalSmear
The ThinPrep®Pap Test™
+ 28%
Weintraub, et al.Laboratoire Weintraub, Geneva, Switzerland
• 39,864 ThinPrep results vs. 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 ratio
• Biopsy confirmation
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
“There was an excellent agreement between the cytologic diagnosis of HSIL and the results of the subsequent histological evaluation.”
Increased HSIL Detection
ConventionalSmear
The ThinPrep®Pap Test™
+ 186%
Diaz-Rosario, et al.Quest Diagnostics, Inc. Cambridge, MA, USA
• 56,339 ThinPrep test results vs. 74,756 conventional slides
• Decreased ASCUS/SIL ratio by 39.11%
• Increased detection of HSIL by 102.54%
• Increased detection of LSIL by 71.65%
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
“ThinPrep produced increased detection of pre-malignant precursors while improving the specimen adequacy.”
Increased HSIL Detection
ConventionalSmear
The ThinPrep®Pap Test™
+ 102.54%
Clinical Performance Summary
• 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
Benefits Over The Conventional Pap Smear
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):278-842 Yeoh GPS et al. Evaluation of the ThinPrep Papanicolaou test in clinical practice, HKMJ 1999; 5:233-93 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
• 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
Summary
• 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
Liquid-based method
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 & screening ability
above 3cm 96%below 1cm 24.8%
5mmLow as about 20~40%
High as 96%(above 5/1000mm sizes)
Remarks
Piled cell layers Even single layer
Cannot detect the early stage cancer
unnecessary bloods and debris remaining
Eliminate bloods and unnecessary debris
Impossible to preserve cell Easy to preserve cells (3 years)
Impossible to do any additional tests
Additional tests available
Possible to examine HPV or DNA
Morphology of the cells
Easy to interpret
URINE ( T.C.C ) – Bladder CancerURINE ( T.C.C ) – Bladder Cancer
LSIL ( HPV ) – Cervical CancerLSIL ( HPV ) – Cervical Cancer
SPUTUM ( S.C.C ) – Lung Cancer SPUTUM ( S.C.C ) – Lung Cancer
HSIL ( CIN III ) – Cervical CancerHSIL ( CIN III ) – Cervical Cancer
Liquid-based Characteristics
Wet FixationCell SizeSmear PatternSpecimen BackgroundSimilarities > Differences
Liquid-based Characteristics
Wet Fixation– Enhanced Cytoplasmic Detail
– Enhanced Nuclear Detail
– Variability in Nuclear Staining
40x
40x40x
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/ 10x obj.
FN20 eyepiece/40x obj.
FN22 eyepiece/ 10x obj.
FN22eyepiece/ 40x obj.
THIN PREP
DIAM MM
Area Fields @ FN20 10x
Cells/Field for 5K
Fields @ FN20 40X
Cells/Field for 5K
Fields @ FN22 10X
Cells/Field for 5K
Fields @ FN22 40X
Cells/Field for 5K
20 314.2 100 Total50.0
1600 Total3.1
82.6 Total60.5
132.2 Total3.8
10XFN 22
10xFN 20
Endocervical Cells
Honeycomb / Palisading Arrangements Maintained
Round-Up in SolutionMore Tightly Packed GroupsSmaller Cell Groups/Single CellsNuclei “Busier”
20x
40x
Squamous Metaplasia
Sheets/Cobblestone ArrangementsDense, Homogeneous CytoplasmCells Often SingleCells Appear Smaller & Rounder
40x
Endometrial Cells
Tight 3D Cell ClustersLoose Cell Clusters with Vacuolated
CytoplasmSingle Cells More ProminentNuclei “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 ClumpingReactive Squamous Cells with
Engulfed WBC’sDistinguish 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.