Pap smear introduction Principle and method in brief Bethesda outlines Liquid based cytology

Types
Principles and methods Future advancements.

HISTORY

Dr.Georgios Papanikolaou (1920)

Idea of LBC -1970. 1987 NHS CSP (National Health Service cervical

Screening Programme) successful in decreasing the incidence and mortality(80% prevention)

PAP smear
The Papanicolau test (also called Pap smear,

Pap test, cervical smear, or smear test) is a screening test used in gynaecology to detect premalignant and malignant processes in the ectocervix.

Basic principle :
The technique of removing cells from the surface of the cervix scraping with a wooden spatula and smearing on the glass slide staining and examining them microscopically

Why a PAP?
The test aims to detect potentially pre-cancerous

changes (cervical intraepithelial neoplasia (CIN) or cervical dysplasia), which are usually caused by sexually transmitted HPV.
The test may also detect infections and

abnormalities in the endocervix.

Whom to screen?
Guidelines on whom to screen vary from country to

country. In general, screening starts at age 20 or 25 and continues until about age 50. No benefit in screening women aged 60 or over whose previous tests have been negative. Pap smear screening is still recommended for those who have been vaccinated against HPV, since the vaccines do not cover all of the HPV types that can cause cervical cancer . Guidelines on frequency of screening vary.Typically every three to five years for those who have not had previous abnormal smears, though some guidelines recommend testing as frequently as every year.

How to perform?
Pap test should not occur when a woman is menstruating but can be performed using a liquid-based test.
Insert a speculum into the patient's vagina, to allow access to the cervix. Samples are collected from the outer opening or os of the cervix using an ayres spatula (conventional PAP),an endocervical brush, or a plastic-fronded broom.(LBC)

Conventional pap smear slide preparation

Sensitivity-72% Specificity-94%

Reporting according to Bethesda classification

Specimen type : conventional

LBC
Adequacy :

Epithelial(8-12,000 cells well preserved and well visualised.For LBC -5000 squamous cells) Glandular components(10 well preserved cells in groups/single) General categorization: Negative for intraepithelial lesion or malignancy organisms, other non neoplastic findings. Epithelial cell abnormalities squamous cells, glandular cells.

Interpretation/result :
Squamous cell abnormalities (SIL)
Atypical squamous cells of undetermined significance (ASC-US)

Atypical squamous cells - cannot exclude HSIL (ASC-H)

Low-grade squamous intraepithelial lesion (LGSIL or LSIL) High-grade squamous intraepithelial lesion (HGSIL or HSIL) Squamous cell carcinoma.

Glandular epithelial cell abnormalities

Atypical Glandular Cells not otherwise specified (AGC or AGC-

NOS) of endometrial ,endocervical and glandular Atypical Glandular Cells favouring neoplastic endocervical and glandular Endocervical adenocarcinoma insitu Adenocarcinoma

False negatives main reasons

Using the incorrect sampler or the right sampler incorrectly Not sampling TZ Cellular transfer onto slide sampling error Quality of the smear (thickness, blood, exudate, poor fixation) Missing the abnormal cells. screening Interpreting the abnormal cells.

Reasons to improve the PAP smear

New collection devices (brooms and brushes rather

than spatulas/Q tips, etc) Liquid-based Pap Tests rather than smears Ancillary tests such as HPV detection Computerized screening devices.

Liquid based cytology

Since the mid-1990s, techniques based around placing the

sample into a vial containing a liquid medium that preserves the cells have been increasingly used. The media are primarily ethanol-based. Improvised method, can be used as a substitute. Works on different principle to ultimately give accurate results. Proper sample acquisition - a cell that is not in the sample cannot be evaluated.

SYSTEMS TO SCREEN
Two systems:

Sure path Thin prep These two have entirely different theory and methods produce similar results. sensitivity 61% to 66% specificity 82% to 91%

THIN PREP
ThinPrep Imaging System a dual-review computer system that screens for cancerous and precancerous cervical cells.
Dual Review means that your ThinPrep Pap Test gets screened twice first by the ThinPrep Imager, and then by a laboratory professional trained in looking for abnormal cervical cells.

Sample collection
Obtain cervical specimen prior to manual examination. Use an un-lubricated speculum (saline or warm water may be used). Vaginal discharge or secretion, when present in large amounts, should

be removed before obtaining the cervical sample so as not to disturb the epithelium Use of liquid-based specimen collection minimizes the interference from factors blood and mucous.
Different devices : Cervex-Brush The Pap Perfect plastic spatula Cytobrush Plus GT Rovers cervex-brush
Both systems require the sample taker to follow the same method of sample collection.

Cervical brush used for LBCs.

Thinprep and surepath

Apply gentle pressure until the surrounding bristles come into contact with the ectocervix and splay out across the cervix. Rotate the Cervex Broom(TM) 5 times in a CLOCKWISE direction to obtain the optimal amount of material. Note - Do not rotate the broom anti-clockwise as little or no sample will be collected rendering the sample inadequate. The bristles are 'D' shaped, and will only collect material if rotated clockwise.

ThinPrep(TM)
Once collected, immediately rinse the 'broom-head' in the preservative solution. This is best achieved by pushing the broom into the base of the vial 10 times, forcing the bristles apart. Finally, swirl the brush vigorously to release any trapped material and further break down the previoulsy released material

ThinPrep Pap Test slide preparation

THIN PREP
The vial is received and is processed in two ways:

semi-automated method (T2000 processor) fully automated method(T3000 processor) The vial is agitated and then the fluid is sucked up through a micropore filter. Neutrophils and RBCs are taken up by the filter but not the epithelial cells obstruct the pore. There is a pressure gradient developed and is sensed by the processor. This improves the cell yeild.

 The filter is then removed and dabbed on to a slide

which is electrically charged permits the transfer of the cells. The slides are then stained as for conventional methods. T3000 processor does not need the cells to be transferred manually done automatically.

Image analyser for thinprep

ThinPrep Pap Test liquid-based slide: Improvement over the conventional pap

The ThinPrep Imager scans every cell and cell cluster

on the slide, measuring DNA content. It then identifies the largest and darkest nuclei, highlighting cells of interest for pathologists' review. An Imaged ThinPrep Pap Test slide is placed in the Review Scope, which takes the pathologist to the appropriate coordinates of the areas of interest. If the pathologist determines the presence of an abnormality, then he or she will review the entire slide, marking any additional areas for further review.

Imaged slide from the ThinPrep Imaging System:

the ThinPrep Imager indicates area of interest for further human review

The area of interest is shown in the "crux" of the L-shape when identified by the ThinPrep Imager

Conventional Pap Smear

ThinPrep Pap Test

SURE PATH
Sample Collection:
Both systems require the sample taker

to follow the same method of sample collection. This test requires a SurePath special collection kit, which includes a SurePath preservative fluid collection vial and the sampling device(s).

Sure path

Cervex Brush (Broom)

SurePath liquid-based collection media

 The device should be twisted slowly. Transfer the entire sample by placing your thumb against the back of

the brush pad, and simply disconnect the entire brush from the stem into the SurePath preservative vial.
For spatula -To obtain an adequate sampling, scrape the ectocervix

using the Pap Perfect plastic spatula. Disconnect the spatula head into the SurePath Preservative vial. For cytobrush - Insert the Cytobrush Plus GT into the cervix until only the bottom most fibers are exposed. Slowly rotate or turn one half turn in one direction. DO NOT OVERROTATE. Disconnect the brush head and place in the SurePath preservative vial.

SurePath(TM) Once collected, transfer the material to the SurePath(TM) vial. To do this, place the thumb against the back of the 'brush-head' and push the entire 'broom-head' from the stem into the preservative solution.

 Recap the vial and tighten.

Record the patients first and last name, date of birth,

specimen source and date collected on the vial. Record the patients information and medical history . Place the vial and test request form in a specimen bag for transport to the laboratory.

SURE PATH
Works on the principle of density gradient. Density gradient centrifugation process. (DGC process)

Vials are received mixed to re-suspend the cells. A syringe is inserted in to the vial through the cap and the fluid is dispensed in to a centrifuge tube filled with a density reagent. First centrifugation 2min / 200g

Decant
Second centrifugation 10min / 800g Decant

 Transferred to a settling chamber mounted on the

microscope slide. Cells settle to form a thin layer and the excess fluid is discarded. Staining is an integral part of the process. Prep stain processor.

Pilot studies
In a UK setting largest in the number of samples studied.
It also studied the complete conversion to LBC for the

purpose of screening. Two centers used thinprep and the other sure path. Included detection of HPV. Study showed a decrease in inadequate samples from 9.1% to 1.6%. There was increase in the specificity of these tests.

Screening methods
Area to be screened is smaller and circular.
Screening must be 100% of the deposit. Must be done in 10x. For cellular details,40x may be used. Primary screening both of them have different look. Thin prep more widely spaced than sure path. After primary screening , all the negative and inadequate

samples are taken for a second review. Must be less than 90seconds.

Thin prep smear

Surepath smear

Morphology
Not much difference from the conventional smears. Due to rapid fixation, the cells are well preserved

evidenced by clarity of nuclear chromatin. Nuclear membranes are well visualized. Over all appearance of the cell - small due to rounding up effect of the liquid. Cells with low grade dyskaryosis are very easy to find. Severe dyskarosis often presents as single and dispersed cells (a feature less well recognized in the conventional smear)

 Hyperchromatic crowded cell groups (HCCG) difficult to assess in both LBC and conventional

smears. Nuclear clarity and small groups morphology in LBC. Glandular abnormalities misdiagnosed as severe squamous dyskaryosis. Thinprep small groups with radial arrangement of the cells with occasional feathering. Surepath single abnormal cells are more common. Criteria for the no. of abnormal cells in LBC have not been separately mentioned.

Appearances considered

inadequate for interpretation

Before the lab

-Cervix not fully visualized or sampled -No brush in vial(sure path) -Brush left in vial(thin pep) Only a cause of inadequacy in women who were treated or CIN-3 with endocervical margin involved. Extremely rare on LBC

No endocervical cells

Obscured by blood or polymorphs

Contamination

Use of inappropriate lubricant.

Advantages Vs Disadvantages of LBC

More representative sample with random distribution cells More costly than conventional pap smears

Multiple slides and/or ancillary tests possible Preparation is more labor intensive than conventional

Screening area reduced & cells in one 10x focal plane reduced screening time

Some differences in architecture and morphology

Cells better preserved and not obscured by blood, mucus, or inflammatory cells

Requires training for both the screeners and the smear takers

Infectious organisms retained and better preserved Smears ideal for Automated Cytology

A Standardised Smear is Obtained

Future advances
HPV testing on the same sample.
Automation subjective nature of morphological

interpretation replaced by numerical quantitative process which was not open to any ambiguity. Computer-assisted Pap test screening detects cervical cell abnormalities by having a computer analyze every square millimeter of a Pap test slide. There are currently two such devices that are FDAapproved and in use in theUnited States.

 FocalPoint device (Becton, Dickinson and Company,

Franklin Lakes, NJ). ThinPrep Imaging System (Cytic Corporation,Boxboro, MA) The imaging system scans a slide and selects 22 areas on the slide that are the most worrisome for a squamous intraepithelial lesion (SIL). And 20 worst cells and 2 worst cell groups are identified. Automated stage is used to take the screener to the identified feilds, allowing a pathologist to review those 22 areas and decide if abnormal cells are present or not.

 The FocalPoint system works in a different manner

and is called a primary screening instrument. but is not approved for ThinPrep slides.

It scans SurePath slides, as well as conventional slides,

At the end of the computer scanning a given set of

slides, it can declare up to 25% of the slides in the set to be normal and need no further review and can be auto-archived (or filed away). (considered the most abnormal) are ranked into quintiles from 1 to 5 (number 1 quintile being the most abnormal)

The remaining 75% of Pap test slides in a given set

 And receive a score related to the percent chance of having an

abnormality compared with the other Pap slides in that set.

This quintile and percentage information is given to the

pathologist who then completely screens the slide as usual.

Because of the computers prescreening and ranking capabilities,

review of slides by pathologists can pay additional attention to slides considered at higher risk for abnormalities.
This allows the reassurance of all Pap tests being reviewed at

least twice: by both a computer and one or two human beings.

Summary
LBC represents the first major technological change in

the cervical cytology. It is as effective as conventional cytology for the detection of high grade abnormalities. Offers improvements by decreasing rates of inadequacy and improving screener productivity. Two systems have been approved for use. It provides a platform for future advances in cervical screening.

References
Recent advances in histopathology Vol 22.

Cytologystuff.com