Liquid Based Cytology

7/14/2019 Liquid Based Cytology

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Pap smear –introduction Principle and method in brief

Bethesda – outlines

Liquid based cytology Types

Principles and methods

Future advancements.



HISTORY

Idea of LBC -1970.

1987 – NHS CSP (National Health Service cervical

Screening Programme) – successful in decreasing theincidence and mortality(80% prevention)

Dr.Georgios Papanikolaou

(1920)



PAP smear

The Papanicolau test (also called Pap smear,Pap test, cervical smear, or smear test) is ascreening test used in gynaecology to detectpremalignant 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) orcervical dysplasia), which are usually caused by

sexually transmitted HPV.

The test may also detect infections andabnormalities 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 continuesuntil about age 50.

No benefit in screening women aged 60 or over whoseprevious tests have been negative.

Pap smear screening is still recommended for those whohave been vaccinated against HPV, since the vaccines donot 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 previousabnormal 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 ayre’s spatula(conventional PAP),an endocervical brush, or aplastic-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 ingroups/single)

General categorization:

Negative for intraepithelial lesion or malignancy –organisms,other non neoplastic findings.

Epithelial cell abnormalities – squamous cells, glandularcells.



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 – endocervicaland 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 samplingerror

• 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 thatpreserves 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 accurateresults.

Proper sample acquisition - a cell that is not in the samplecannot 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 Testgets screened twice — first by the ThinPrep

Imager, and then by a laboratory professionaltrained 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 theepithelium

Use of liquid-based specimen collection minimizes the interference fromfactors blood and mucous.

Different devices :Cervex-Brush™The Pap Perfect® plastic spatulaCytobrush® Plus GT

Rover’s cervex-brush™ Both systems require the sample taker to follow the same method of sample

collection.



Cervical brush used forLBCs.



Thinprep and surepath Apply gentle pressure until the

surrounding bristles come into

contact with the ectocervix and splay

out across the cervix. Rotate theCervex 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 nosample 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 intothe 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 previoulsyreleased 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 amicropore filter.

Neutrophils and RBCs are taken up by the filter but not the epithelialcells – 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 conventionalmethods.

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 clusteron the slide, measuring DNA content. It thenidentifies the largest and darkest nuclei, highlighting

cells of interest for pathologists' review.

An Imaged ThinPrep Pap Test slide is placed in theReview Scope, which takes the pathologist to theappropriate coordinates of the areas of interest.

If the pathologist determines the presence of anabnormality, then he or she will review the entire slide,marking any additional areas for further review.



Imaged slide from the ThinPrep Imaging System:

The area of interest is shown in the

"crux"

of the L-shape when identified by the

ThinPrep Imager

the ThinPrep Imager indicates area of

interest for further human review



Conventional Pap Smear ThinPrep Pap Test



SURE PATH

Sample Collection:

Both systems require the sample takerto follow the same method of samplecollection.

This test requires a SurePath™ specialcollection kit, which includes aSurePath™ preservative fluid collection

vial and the sampling device(s).



Sure path

SurePath liquid-based

collection mediaCervex Brush (Broom)



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 steminto the SurePath™ preservative vial.

For spatula -To obtain an adequate sampling, scrape the ectocervixusing the Pap Perfect® plastic spatula. Disconnect the spatula head intothe SurePath™ Preservative vial.

For cytobrush - Insert the Cytobrush® Plus GT into the cervix untilonly the bottom most fibers are exposed.

Slowly rotate or turn one half turn in one direction. DO NOT OVER-ROTATE. Disconnect the brush head and place in the SurePath™preservative vial.



SurePath(TM) Once collected, transfer thematerial to the SurePath(TM)

vial. To do this, place thethumb against the back of the'brush-head' and push theentire 'broom-head' from thestem into the preservative

solution.



Recap the vial and tighten.

Record the patient’s first and last name, date of birth,specimen source and date collected on the vial. Recordthe patient’s information and medical history .

Place the vial and test request form in a specimen bagfor 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 thepurpose 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 dispersedcells (a feature less well recognized in the conventionalsmear)



Hyperchromatic crowded cell groups (HCCG) –

difficult to assess in both LBC and conventionalsmears.

Nuclear clarity and small groups morphology – inLBC.

Glandular abnormalities – misdiagnosed as severesquamous dyskaryosis.

Thinprep – small groups with radial arrangementof the cells with occasional feathering.

Surepath – single abnormal cells are morecommon.

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 orsampled-No brush in vial(sure path)-Brush left in vial(thin pep)

No endocervical cells Only a cause of inadequacy in women who were treated or CIN-3 withendocervical margin involved.

Obscured by blood or polymorphs Extremely rare on LBC

Contamination Use of inappropriate lubricant.



Advantages Vs Disadvantages of LBCMore representative sample with randomdistribution cells

More costly than conventional pap smears

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

Screening area reduced & cells in one 10xfocal plane reduced screening time

Some differences in architecture andmorphology

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

Requires training for both the screeners andthe 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 morphologicalinterpretation – replaced by numerical quantitativeprocess which was not open to any ambiguity.

Computer-assisted Pap test screening detects cervicalcell abnormalities by having a computer analyze every

square millimeter of a Pap test slide. There are currently two such devices that are FDA-

approved 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 areason the slide that are the most worrisome for asquamous intraepithelial lesion (SIL). And 20 worstcells and 2 worst cell groups are identified.

Automated stage is used to take the screener to the

identified feilds, allowing a pathologist to review those22 areas and decide if abnormal cells are present ornot.



The FocalPoint system works in a different mannerand is called a ‘‘primary screening’’ instrument.

It scans SurePath slides, as well as conventional slides,but is not approved for ThinPrep slides.

At the end of the computer scanning a given set of slides, it can declare up to 25% of the slides in the setto be normal and need no further review and can beauto-archived (or filed away).

The remaining 75% of Pap test slides in a given set(considered the ‘‘most abnormal’’) are ranked intoquintiles from 1 to 5 (number 1 quintile being the mostabnormal)



And receive a score related to the percent chance of having anabnormality compared with the other Pap slides in that set.

This quintile and percentage information is given to thepathologist who then completely screens the slide as usual.

Because of the computer’s prescreening and ranking capabilities,review of slides by pathologists can pay additional attention toslides considered at higher risk for abnormalities.

This allows the reassurance of all Pap tests being reviewed atleast 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 thedetection 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.



ReferencesRecent advances inhistopathology – Vol 22.

Cytologystuff.com

Liquid Based Cytology

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