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.