Liquid based cytology ( l b c)

LIQUID BASED CYTOLOGY
• Presented by Dr. Vikram Saraswat
• Moderator- Dr. Abha Mathur

Introduction
Cervical cytology was introduced by George
Papanicolaou into clinical practice in 1940. In 1945,
the Papanicolaou smear received the endorsement of
the American cancer society as an effective method
for the prevention of cervical cancer . Center of cytology
in Vancouver, British Columbia published data which
confirmed that cytologic screening leads to a reduction
in the rate of invasive cancer of the uterine cervix.
It was later established that the sensitivity of the
conventional Pap smears for the detection of cervical
cancer precursors was less than 50%. Several
limitations of conventional smear were identified. Thus
Liquid based, thin layer technology was developed
to address the limitation of Pap smear.

Limitations of conventional pap smear
• Inadequate samples constitute about 8% of the specimens received.
• False-negative results as high as 20-30% have been reported, which
occurred due to clumping of cells when the cells are not uniformly spread
on the glass slide.
• Sometimes, other contents of the cervical specimen such as blood, mucus,
bacteria and yeasts contaminate the sample and prevent the detection of
abnormal cells.
• If exposed to air for too long before being fixed on the slide, cervical cells
can become distorted.
• Human error is probably the primary threat to accurate interpretation. An
average Pap smear slide contains 50,000-300,000 cells that must be
examined and if the sample contains only a few abnormal cells within a
crowded background of healthy cells, the abnormal cells may be missed.
• Due to limited capacity, long back-logs for reporting cervical cytology were
often seen. An automated system was needed to improve screening
productivity and reduce manual workload.

LIQUID BASED CYTOLOGY ( L B C)
• It is a method for preparing cytological specimens—in
particular from the cervix—for microscopic evaluation in
which the patient specimen is suspended in a liquid, which
is used to produce a thin layer of cells.
Traditional Pap smears are conducted by physicians who
use a spatula to collect cervical cells for analysis. A small
sample of cells is lightly scraped from the cervix, placed on
a microscope slide, and sent to a laboratory for staining and
analysis by a cytologist . Unlike a traditional Pap smear,
where the cells are placed directly on a microscope slide, in
liquid based cytology the head of the spatula is detached
and placed into a vial containing a preservative fluid. Most
liquid preservatives for liquid based cytology are ethanol-
based

• Liquid based cytology has become increasingly more
common in hospitals and clinics since the 1990s. The
centrifuging process allows for clearer, more uniform
samples that are easier for cytologists to analyze. This
has also helped to reduce the number of unreadable or
inconclusive tests, requiring fewer women to return to
clinics for repeat testing. Liquid samples are also more
suitable for human papillomavirus(HPV) testing.
• Some studies also indicate that liquid based cytology
increases the sensitivity and specificity of cytological
reports, making the Pap smears more effective in
detecting cancers and other diseases

• Currently, two technical methods, which use
automats, were validated by Food and Drug
Administration (FDA,america) and are used
frequently. One is proceeding by filtration and
collection of vacuum-packed cells on a
membrane and transferring to the glass slide
(ThinPrep®, Cytyc®). The other is proceeding by
centrifugation and sedimentation
through a gradient of density (Surepath®,
Tripath Imaging®,AutoCyte PREP)
®Stands for registered trademark names

• Methods Not approved by FDA
Cytoscreen
Turbitec
Cellslide®
Papspin®
• Centrifugation and sedimentation are
performed by manual techniques in these
methods, no automate is used.

Advantages of LBC
• Simplifies the collection process for the smear taker
• Reduces inadequate rate
• Improves cellular preservation
• All the collected cells are transferred to the vial
• Quicker for the laboratory to screen and report as the
area to be screened is smaller and circular.
• Multiple slides can be produced allowing further
testing or for teaching and Quality Assurance purposes
• Residual material in the vial could be made available
for associated tests e.g. HPV assay
• Facilitates Computer Assisted Screening which may
also be available in the future
• Enhances screening processes by improving and
standardising the service to the patient

Disadvantages of LBC
• -More expensive than conventional cytology
• -Cytological interpretation differs slightly from
conventional slides with resultant training
issues for laboratory staff
- Smear taking procedures require a standard
approach
• -Transport of the vials requires a more
specialised approach with specialised packing

The limitation of conventional pap
smear

With the conventional Pap smear method,
cells can be obscured by blood, mucus, and
inflammation

The LBC method preserves the cells
and
minimizes cell overlap, blood, mucus,
and inflammation.

History of Thin Prep method
• Cytyc Corporation is a company engaged in the design,
development, manufacture, and marketing of clinical
products that focus on women’s health since 1987. In
order to overcome limitations on conventional pap,
the company introduced the ThinPrep Pap Test. In
May 1996, the Food and Drug Administration (FDA) in
the USA approved the ThinPrep 2000( T 2000 ) as a
replacement for the conventional Pap smear method
in USA for use in screening for the presence of
atypical cells, cervical cancer or its precursor lesions
(Low Grade Squamous Intraepithelial Lesions, High
Grade Squamous Intraepithelial Lesions), as well as all
other cytologic categories

method
• The heart of the ThinPrep® System is the
ThinPrep 2000 Processor, a semi automated
slide preparation unit that produces
remarkably uniform thin-layer slides, virtually
free of obscuring artifacts such as blood,
mucous and inflammation.
Step 1
A gynecologic sample is
collected using a broom-type
or cytobrush/spatula cervical
sampling device.

Step 2
Instead of smearing the cells
on a slide, the sampling device
is rinsed into a ThinPrep vial
containing PreservCyt®
transport medium(buffered
methanol soln). The device is
then discarded
Step 3
The sample vial is capped,
labelled, and sent to the
laboratory for slide
preparation.

Step 4
At the laboratory, the vial is placed into the ThinPrep
2000 Processor. First, a gentle dispersion step breaks
up blood, mucous, non-diagnostic debris, and then
thoroughly mixes the sample. A negative pressure pulse
is generated which draws fluid though a TransCyt®
Filter( a micropore filter) that collects a thin, even
layer of diagnostic cellular material. The ThinPrep 2000
Processor constantly monitors the rate of flow through
the TransCyt Filter during the collection process to
prevent the cellular presentation from being too scant
or too dense. The filter is then removed and dabbed
onto an electrically charged slide causing cells to
transfer onto the glass slide. This is then stained in a
separate process using same staining machine as is
used for conventional samples.

The gentle mixing of the sample before preparing the slides ensures a
thorough sampling of the cells present. As part of the validation of the
ThinPrep Pap Test, ten or more slides were made from single vials.
These studies showed that the first slide prepared from the vial is
representative of the remainder of the material.
With a conventional Pap smear there is tremendous
variability in the number of cells transferred to the slide,
from as few as 4,000 to up to 300,000.
With the ThinPrep Pap Test, approximately 70,000 diagnostic
cells are collected that provide a more representative sampling
of the specimen taken from cervix. These are consistently
displayed to the cytologist in a high quality preparation
Currently a fully automated T3000 system is being used UK
and USA, it readily converts vials into slides ready for staining
with no intervention.

Basic principle of thin prep processor
(T3000 fully automated)
• The ThinPrep Processor makes use of mechanical, pneumatic,
and fluidic principles for cell dispersion, collection, and
transfer. A rotary drive mechanism gently disperses samples.
A pneumatic/fluidic system, controlled by a microprocessor,
monitors cell collection. Electrochemical principles, the
pneumatic and fluidic systems, the natural binding qualities of
cells,and the qualities of the ThinPrep Pap Test Filter are
responsible for cell transfer.
• The ThinPrep Processor slide preparation process can be
divided into the following phases
Sample preparation/Instrument loading
• Start of cycle
• Fluid level detection
• Dispersion
• Filter wetting

• Cell collection
• Waste clearing
• Bubble point
• Cell transfer
• Slide ejection
• Completion of cycle
Sample Preparation/Instrument Loading
In preparation for sample processing, the operator loads four essential items
into the ThinPrep Processor: a Sample vial, a ThinPrep Pap Test Filter attached
to the filter cap, a ThinPrep Slide and a fixative vial containing a standard
laboratory fixative.
Start of Cycle
When the operator initiates a sequence, the
ThinPrep Processor verifies installation
Fluid Level Detection
The cap seal lowers to seal the filter assembly
and the sample vial is raised towards the
filter membrane. The sample vial stops when
the filter membrane makes contact with the
surface of fluid.

Dispersion
The cap seal lifts and the dispersion system rotates the ThinPrep Pap Test Filter
assembly within the cell suspension, creating shear forces in the fluid that are
strong enough to separate randomly joined material and disperse mucus.
Filter Wetting
The head seal lowers to seal the filter assembly. Negative pressure is briefly applied,
drawing a small amount of fluid through the ThinPrep Pap Test Filter to wet it. Following
wetting, the system gently blows out the liquid in the ThinPrep Pap Test Filter. This clears
any cellular material from the filter surface.
Cell Collection
The filter membrane is biologically neutral and is mounted at one end of the ThinPrep
Pap Test Filter cylinder. The membrane is a flat, smooth, porous surface that collects the
cellular material on one plane.
The pneumatic system applies negative pressure to the filter in a series of pulses. These
negative pressure pulses (sips) draw PreservCyt Solution through the filter membrane
and collect suspended cellular material onto the outer membrane surface.
The collection process ceases when a target filter coverage, predetermined by the
processor sequence, is attained. Cell collection is controlled by an embedded
microprocessor that monitors the pressure in the ThinPrep Pap Test Filter cylinder. After
collection, the cells sit on a single plane over the pores, ready for transfer to the slide.

Waste Clearing
When collection ends, the ThinPrep Pap Test Filter is withdrawn from the sample vial
and the filtrate is aspirated into the waste bottle as the filter is inverted. The collected
cells remain on the ThinPrep Pap Test Filter due to the negative holding pressure.
Bubble Point
Bubble point removes excess fluid from the filter membrane prior to transferring cells
onto the slide to enhance cell adhesion to the slide.
Bubble point is performed after all of the fluid is evacuated. This is evident by the
bubbling activity on the inside of the filter membrane. Cells do not air-dry during
bubble point.
Cell Transfer
When bubble point is complete, the slide handler moves the slide into contact with
the inverted ThinPrep Pap Test Filter.
The natural adhesion properties of cells and the electrochemical charge of the glass
slide are responsible for the transfer of cells from the filter membrane to the slide.
The cells have a higher affinity for the glass slide than for the membrane; slight
positive air pressure behind the filter membrane enhances cell transfer.

Slide Ejection
Once cell transfer is complete, the slide is removed from contact with the
filter and automatically ejected into the fixative vial.
Cycle Completion
All the motorized mechanisms return to their initial positions and the
display returns to the Main Menu. If the system detects an error during
the process, a message will be displayed and an audible alarm will sound

The prepared ThinPrep smear is shown in the diagram compared
with the conventional Pap smear. The sample prepared by the
ThinPrep is evenly distributed on the circle of the slide

Microscopically, the uneven distribution of
cellular material associated with the
Conventional Pap Smear pattern is evident.

This slide is the from the same patient as the
previous slide. Tissue architecture is
maintained. ThinPrep® rearranges the
relationship of cell groups on the glass slide. A
group/sheet of endocervical cells present
represents this.

THIN PREP IMAGING SYSTEM
(recent advancement)

The ThinPrep Imaging System is the only system that provides Dual Review
screening. With this capability, every slide is analyzed by the ThinPrep Imager and
screened by an experienced cytotechnologist, thereby combining human expertise
with advanced technology to achieve more accurate clinical diagnoses.
the ThinPrep Imager scans every cell and cell cluster on the slide, measuring DNA
content. The largest and darkest nuclei are identified so that cells can be more
accurately assessed for abnormalities by highly skilled cytotechnologists. Cells of
interest are highlighted for cytotechnologists' review, helping them to better focus
their interpretive skills where it counts most
The area of interest is shown
in the "crux" of the L-shape
when identified by the
ThinPrep Imager

A comparison of disease detection with the ThinPrep Imaging System vs.
manually reviewed ThinPrep Pap Test slides reported a 37% increase in LSIL
detection and a 42% increase in HSIL detection with the ThinPrep Imaging
System
The ThinPrep Imaging System showed a 67% decrease in unsatisfactory
and a 15% decrease in ASC-US rates compared to manually reviewed
ThinPrep Pap Test slides with 50% reduction in false negative fraction

SurePath™ Liquid-Based Pap
Test (AutoCyte PREP™
System) (cytorich/prep stain
slide processor)

Pap Smear Specimen Collection Using SurePath
Pap Test Method
• Option 1: SurePath Sample Collection with Broom-
Type Device with Detachable Head
• 1. Insert the cervix-brush into the endocervical canal. Apply gentle pressure until
the bristles
• form against the cervix. Maintaining gentle pressure, hold the stem between the
thumb and
• forefinger. Rotate the brush five times in a clockwise direction.
• 2. Place your thumb against the back of the removable collection device tip and
disconnect
• the entire tip from the stem and place in the SurePath preservative vial.
• 3. The collection device tip should be transferred in the vial. One to three different
sampling
• tips can be left in the SurePath vial. Place the cap on the vial and tighten.
• 4. Record two patient identifiers, physician name and collection date on the vial.
Also record
• the patient information and medical history on the requisition form.
• 5. Complete a laboratory requisition form with complete patient information and
medical
• history.
• 6. Place the vial and requisition in a specimen bag for transport to the laboratory.

Option 2: SurePath Sample Collection with Combination
Brush/Plastic Spatula Device
with Detachable Heads
• 1. Insert the contoured end of the plastic spatula and rotate 360 degrees around
entire exocervix.
• 2. Snap the device handle and drop the detachable head of the device into the
SurePath vial.
• 3. Insert Cytobrush into the endocervix until only the bottom-most bristles are
exposed at the os. Slowly rotate one-fourth to one-half turn in one direction. To
reduce unnecessary bleeding, do not over-rotate brush.
• 4. Snap the device handle and drop the detachable head of the device into the
SurePath vial. Place the cap on the vial and tighten.
• 5. Record two patient identifiers, physician name and collection date on the vial. Also
record the patient information and medical history on the requisition form.
• 6. Complete a laboratory requisition form with complete patient information and
medical
• history.
• 7. Place the vial and requisition in a specimen bag for transport to the laboratory

Collect
using either a broom-like device
or combination brush/plastic
spatula.
Drop the detachable
head
device(s) into the
SurePath™
test vial.
Place the cap on the vial
and
tighten. Send the BD
SurePath™
test vial to the lab for
processing

Method and principle
• The Autocyte Prep system/ Sure path method
converts liquid suspension of cervical cell sample into
a discretely stained homogenous thin layer of cells
while maintaining diagnostic cell clusters. The
process includes
• 1) cell preservation (preservative fluid is dil. Soln. of
denatured ethanol)
• 2) randomisation
• 3)enrichment of diagnostic material
• 4) automated pipetting
• 5) sedimentation
• 6) staining

• After collection and preservation, the sample is mixed by
vortexing ( to re-suspend cells) and dispersed onto a density
reagent (contains sodium azide). An enrichment step
consisting of centrifugal sedimentation through density
reagent partially removes non diagnostic debris and excess
inflammatory cells from sample. After centrifugation, the tube
containing the enriched cellular component is placed onto the
instrument where the pelleted cells are robotically
suspended, mixed and transferred to settling chamber. Here a
specialised slide coat is applied to enhance cell adhesion. Cells
are sedimented by gravity and then stained using modified
papanicolaou staining process. The slide is cleared in xylene
and coverslipped.

trapped
debris
SurePath™
test
enriched
cell pellet

• The final product is a well preserved
population of stained cells present within
13mm diameter circle on slide. Air drying
artefact, obscuring , Overlapping cellular
material and debris are largely eliminated. The
number of white clood cells is reduced
allowing easier visualisation of epithelial cells,
diagnostically relevant cells and infectious
organisms.

AutoCyte PREP System can also be
used for preparing non-
gynaecological specimens including
bronchial and fine-needle
aspiration specimens.
Stained cytology smears of
bronchial specimen (prepared by
AutoCyte PREP System)

FOCAL POINT SLIDE PROFILER
FOCAL POINT GS IMAGE CAPTURER
(recent advancement)

• Focal point slide profiler , an automated screening
system, scans slides prepared by SUREPATH method and
can also be used on conventional smears. It identifies cells
which are potentially abnormal and then ranks the whole
sample on its likelihood of being abnormal. This is the only
fully automated computerised slide reading application
that has been given FDA approval and is in routine use in
USA.
• The other development is the Focal point GS system that
captures images,records the location of potentially
abnormal cells and then via an automated microscope
stage,guides the screener to 10 selected locations. This
method has not yet received approval as it has always been
recommended that a screener must screen the whole slide.

THIN PREP v/s SUREPATH
sample collection
THIN PREP
(broom rinsed in the vial)
SUREPATH
(broom head snapped off
And retained in the vial)

THIN PREP v/s SUREPATH
reception and numbering
• One of the advantages of the THIN PREP system is
that it automatically prints a bar code onto the
slide, thus making it a very secure process.
• With SUREPATH, it must be ensured that samples
are labelled correctly throughout the process.
Staff receiving specimens must ensure the correct
vial and form are matched ( a process analogous
to histology specimen reception)

Preparation technique
thinprep v/s surepath
THINPREP
• PreserveCyt fluid
• Methanol
• No vortexing
• No gradient centrifugation
• Filter
• No sedimentation
SUREPATH
• CytoRich fluid
• Ethanol
• Vortexing
• Gradient centrifugation
• No filter
• Sedimentation

Staining
• With Thin prep, slide preparation is automated
and yields an unstained slide which can be
inserted into a range of automated staining
machines.
• With Surepath, staining is an integral part of the
process.
• The Prep stain processor ( trade name), employed in
surepath technique, has higher capacity than Thin prep
T3000 ( it produces 48 slides per hour), but it is not fully
automated and reuires more skilled manual operation.

13mm
20mm
SUREPATH
THIN PREP
Result -

screening
• At primary screening, the two methods do have different
appearances. The cells in THINPREP are more widely
spaced and there is less depth to the field.
• Glandulare abnormalities show smilar nuclear features but
architecture and distribution exhibit significant differences.
In THINPREP, the characteristic appearance are of group of
cells showing radial alignment of cells with occasional
feathering. Palisaded strips with nuclei at multiple levels
are seen and occasionally micro acinar structures such as
rosettes are visible. Single cells are rarely seen. On
SUREPATH, though groups of cells are seen,single abnormal
cells are also common. Cells show abnormal nuclei
distending the cytoplasm. These cells may be arranged in
strips which often show a shared or community border.

Cell block preparation from SurePath
specimen containing glandular epithelium

Other LBC methods
• Though not as popular and effective as the
previously mentioned methods, the
CYTOSCREEN and CELL SLIDE PROCESSOR are
other two notable LBC techniques.

CYTOSCREEN method
• In this method after sample collection,the brush
is sunk in a preservation liquid vial.
• The following stages can be mentioned:
• – the vial with the removed device is placed in a
shaker to take the cells out of the removing device
and get an homogenous cell suspension.
• – automate reading of the cell sample density by
using a nephelometer
• – cells deposition in after centrifugation, resulting
a round shaped smear with a diameter of 17 mm.

Cytoprep brush and vial
with conservation liquid
shaker
Nephelometer

CELL SLIDE PROCESSOR
CellSlide Processor is an automatic computer-controlled
instrument, designed to prepare standardized, thin-layer
cytological preparations using a filtration process. The
processor filters cells from a liquid fixative suspension
and transfers them onto a rectangular area of the
microscope slide. The instrument operates using
membrane filters in a closed circuit system that
offers operational security with all types of specimens. All
phases of processing, including filtration, residual fluid
recovery, membrane transfer and pressure are
automatically performed. The instrument is equipped with
two filtering towers, which can be operated either
independently or in parallel.

From a diagnostic standpoint, it is preferable to use a fresh specimen,
since cytological samples may lose their integrity if they are processed
when a long period of time has elapsed between sample collection and
slide processing. However, the CellSlide Pap test solution, a buffered,
alcohol-based preservative solution, has been designed to maintain cell
integrity from the time of sample collection until the slide is processed
(maximum 4 wk) at temperatures between 4 and 30 degree C.
Also, a second fixative solution, CellOfix is automatically added to the
sample before filtration. This fixative solution contains a polymeric agent
with a high viscosity power, dissolved in an alcoholic solution containing
colouring agents. The purpose of this agent is to embed the cells,to
increase fixative power during filtration and to facilitate the long-term
storage of the specimen. The purpose of the colouring agents is to
differentiate the processed samples from the non-processed preservative
solution. CellOfix is delivered in a concentrated form, which is then diluted
in ethyl alcohol or isopropyl alcohol.

LBC v/s conventional pap
major differences

Conventional Pap Smear Liquid Based Cytology
Majority of cells not captured.
Non-representative transfer of cells.
Clumping and overlapping cells.
Obscuring material.
Virtually all of the sample is collected.
Randomized, representative transfer of
cells.
Even distribution of cells.
Minimizing Obscuring material.
The difference is obvious

LBC v/s CONVENTIONAL PAP
screening and morphology
Using LBC, modifications must be made to the conventional
screening method. The area to be screened is smaller and
circular, thus screening must cover 100% of the deposit and
should be intensive. There are no breaks in the sample, as
there are in conventional smears, meaning that the screener
must conentrate throughout the process.
The prompt fixation of LBC samples leads to good preservation
and this is particularly seen in the clarity of presentation of
chromatin. Nuclear membranes are also well visualised. In
general, cells appear slightly smaller due to rounding up effect
of fixation in a liquid. The clarity of nuclear features is helpful
in diagnosing dyskaryosis but training needs to address the
tendency to overcall dyskaryosis as subtle variations in
chromatin and membrane pattern, invisible on conventional
smears, can now be seen.

• Cells showing Low grade dyskaryosis, esp. those with koilocytosis,
are usually easy to find and look identical to similar cells in
conventional smear. High grade dyskaryosis (moderate and severe)
often appears similar to that seen in conventional smear. In LBC ,
sometimes severe dyskaryosis may present as dispersed single cells,
a recognised but less frequent feature on conventional smear.
• Hyperchromatic crowded cell groups are a problem both in
conventional and LBC cytology, but in latter the size of groups is
reduced and in most cases even if grop is too dense to assess
nuclear features,the cells on the edge are fully assessable.
• The improved nuclear clarity also helps in differentiation of benign
and abnormal conditions.

Future of LBC
• LBC represents the first major technological
change in cervical cytology.
• Various studies have shown it to be more
effective than conventional smear in detecting
abnormalities, decreasing inadequacy rates and
improving screener productivity.
• It provides platform for future advances in
cervical screening which includes HPV testing and
introduction of automated cervical screening
system.

Liquid based cytology ( l b c)

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