LIQUID BASED CYTOLOGY

&
AUTOMATION
LIQUID BASED CYTOLOGY
LIQUID-BASED CYTOLOGY(LBC)
• For last few decades, cervical cancer screening is based on conventional preparation (CP) of cervical smear.
• Conventional cervical smear is a quick, relatively cheap technique and so far it has helped to reduce the
incidence of cervical cancer significantly in many developed countries.
• However, the false negative rate of conventional cervical smear is as high as 29% and sensitivity of this test
varies from 47% to 62%.
• Samples taken by CP may often be inadequate, non-representative and may be obscured by blood, debris or
may show air drying artifact.
• LBC technique is an important step in the preparation of cervical smear. It has overcome many limitations of
conventional cervical cytology. Presently, LBC is widely accepted in many laboratories despite the high cost of
this technique.
• The types of LBC include Sure Path (SP), and Thin Prep (TP), and Millipore Filter and Liqui-PREP. Currently, only
SP and TP have FDA approval since they are the more commonly used LBC.
• The TP and SP preparations were approved for cervicovaginal (Pap test) cytology in 1996 and 1999,
respectively, and both have since also been used for non-gynecological cytology including fine needle
aspiration (FNA). Both TP and SP are also FDA-approved for high-risk human papillomavirus (HPV) testing of
Pap tests.
• In conventional smears, the collected patient sample is smeared on glass slides, and the collection device,
containing some of the diagnostic cell sample, is discarded. Moreover, the collected sample is non-uniformly
distributed on the glass slide and may span the entire surface area of the slide.
• In LBP, unlike the conventional smears, the acquired patient sample, from various anatomic sites, is rinsed in
specially designated collection preservative media ensuring collection of potentially 100 % of the cell sample.
COLLECTION PROCEDURE OF LBC
• The cervical sample is obtained using a combination of plastic spatula and cervical broom type device in the same manner
of conventional screening.
• Alternatively, sample is collected by cervix brush supplied by the company.
• The sample is then rinsed into a vial containing preservative solution. The collection media for TP is CytoLyt and that for SP
is CytoRich Red.
• The head of the device is kept in the vial containing preservative solution and the handle is removed. The sample is sent to
the cytology laboratory for further processing. The cells are made discrete and a monolayer preparation is prepared.
 ThinPrep
PRINCIPLE
• ThinPrep preparation is a filter-based cell concentration technique.

INSTRUMENTS
• Two types are available, TP 2000 and a newer version TP 5000 for use only with TP. Both are FDA-approved for
processing Pap test and non-gyn specimens.

• The TP 2000 processor is a semiautomated device which processes one specimen at a time. TP 5000 is a fully
automated benchtop instrument that processes 25 specimens at a time. The latter device requires less hands-on
technician time in preparation. Multiple preparations can be made from a single specimen vial.

SPECIMEN PRESERVATION
The sample is preserved in CytoLyt® solution (methanol-based fixative which is both hemolytic and mucolytic). Here, the
cells are collected by a plastic spatula or cytobrush and then transferred to the transport media provided by the company.

SLIDES
The microscopic slides are provided by the company and are marked with a 20-mm diameter circle.
STEPS IN PREPARATION
• Specimen is first centrifuged at 1500 rpm, and the cell pellet is then resuspended in 30 mL of CytoLyt and again
centrifuged.
• Two to three drops of the cell pellet are transferred to PreservCyt (a methanol-based preservative solution). The vial and a
labeled slide are placed into the TP processor.
• Preparatory steps are similar for both TP 2000 and TP 5000 and include specimen dispersion, collection, transfer, and
staining.
➢ 1. Dispersion: A disposable cylinder with a polycarbonate filter attached to one end is introduced into the vial. The pore
size of the filter is 5.5 μm for non-gyn specimens and 8 μm for Pap test specimens. The instrument is rotated creating a
current that disaggregates blood, mucus, and other debris and breaks up large cell clusters, mixing and homogenizing the
cell suspension.
➢ 2. Collection: A gentle vacuum is applied to the cylinder which aspirates the cell suspension through the filter. Most of the
broken red blood cells (RBCs) and debris are allowed to pass through while the diagnostic cells attach to the external
surface of the filter. The instrument monitors cell density across the filter, and the flow rate decreases when cells are
evenly distributed on the filter with minimal overlap.
➢ 3. Transfer: The cylinder moves out of the specimen, is inverted 180°, and then gently pressed against a positively charged
slide and with slight positive pressure, transferring the cells (~70,000) to the glass slide. The result is a 20-mm circular
smear with even distribution of cells and minimal overlap. The slide is immediately dropped into 95 % ethanol fixative.
Preparation time ranges between 30 and 90 s depending on cell concentration.
➢ 4. Staining: Papanicolaou (Pap) staining is either performed manually or on an automatic stainer. The staining process
takes 30 min. A Papanicolaou stain of fixed samples offers the best option for judging the fine details of cell structure.
➢ A cylinder with a polycarbonate filter attached to one end is introduced into the specimen vial and gently rotated
creating a current that disaggregates mucus, blood, and other debris, breaks up large cell clusters, and mixes and
homogenizes the cell suspension.

➢ A gentle vacuum is then applied to the cylinder; most of the broken erythrocytes and debris pass through the filter
pores, while the cells of interest adhere to the filter.

➢ The instrument monitors cell density across the filter and the flow rate decreases when cells are evenly distributed on
the filter with minimal overlap.

➢ The cylinder then moves out of the specimen and is lightly pressed against a positively charged slide. A slight positive air
pressure is applied to transfer the cells to the slide.

➢ The slide is immediately dropped into 95 % ethanol fixative. The slide is removed from the processor and may be stained
either manually or by an automatic stainer
 SurePath
PRINCIPLE
SP is a density gradient-based cell enrichment process. It is a semiautomated technique. The specimen is processed on
the PrepStain processor. Multiple preparations can be made from a single specimen vial.

SPECIMEN PRESERVATION
Specimens are preserved in CytoRich® Red Preservative Fluid (an ethanol-based medium which also lyses blood).

SLIDES
Pre-coated slides are provided by the company and are marked with a 13-mm diameter circle. The slides can also be
freshly prepared in the laboratory for use within 48 h. The slides are coated with a modified poly-L-lysine and air-dried.
These positively charged slides allow diagnostic cells to settle out of solution and adhere to the surface.
➢ The cells are collected with the help of plastic device and transferred to a transport fluid.

➢ A syringe is used to disaggregate larger cell fragments.

➢ In the laboratory, the vial is vortexed and strained to break up mucus and large cell groups and the cell suspension is
treated through a density gradient centrifugation process(the specimen is dispersed onto a density gradient reagent,
a polysaccharide solution that acts to trap small particulates and debris) to remove the blood and other insignificant
material.

➢ The cell pellet is resuspended and is then allowed to sediment onto a glass slide
ADVANTAGES OF LIQUID-BASED PREPARATIONS
• There are several advantages to LBP compared to conventional smears.
➢ Both LBP techniques reduce debris and cell clumps and also homogenize the specimen,
➢ Increase specimen adequacy—Uniform collection technique with theoretical collection of 100 % of the
sample.
➢ Result in uniform cell distribution in a smaller screening area (the screening area of a conventional smear
is 2.5 × 1.5 cm, the size of the glass slide),
➢ Show cleaner background, reduce slide screening time.
➢ Remove air-drying artifact due to immediate liquid fixation. Other artifacts of smearing such as crush
artifact, cell overlap, and thick areas are usually lacking. Easier to interpret due to less or no obscuring
elements and less cellular overlap.
➢ Enhance cellular and nuclear details for improved diagnostic accuracy, and increase productivity. Cytologic
features of malignancy are retained.
➢ Moreover, residual material from LBP can be successfully used to process cell blocks, which provides
additional diagnostic information, including architectural pattern. Both LBP and cell block material can also
be utilized for ancillary studies such as immunocytochemistry, special stains, and molecular HPV tests.
➢ If LBP are used for ancillary studies, additional slides can be prepared from the specimen vial.
➢ Reproducibility with less variability in specimen preservation, staining, and quality.
➢ Increased cell yield, even with low cellularity samples.
➢ In Pap test cytology, processed as TP or SP, automated computerized screening devices with programmed
algorithms locate and mark worrisome cells making screening more accurate and increasing productivity.
DISADVANTAGES OF LBP
These are predominantly due to preparation techniques.
• Liquid-based cytology instrument is very costly. The overall processing cost of LBC is also much more than
conventional preparation.
• Pathologists need to become familiar with LBP morphology due to alterations in background, architectural,
and cytological features to avoid misdiagnosis.
• As mentioned before, the laboratory technical staff also needs adequate training for interpreting LBC smear.
• Fragmentation of papillae and cell groups and slightly more dyscohesion of cells may be more pronounced in
TP. Background material may be lost, reduced, or altered and may be more pronounced in TP. In TP, excess
blood may remain.
• In SP, multiple processing steps and settling of cells under the influence of gravity result in cellular elongation,
cells in different planes of focus, and increased three-dimensional appearance.
• Cell and nuclear size may become smaller in both TP and SP.
AUTOMATION
• Computer science has progressed remarkably in the last few decades and like every other field, it has also a significant
impact on cervical cytology.
• Automated devices have now been applied in the field of cervical cytology with the intention to have a more efficient
system in smear preparation, recognition and interpretation of the slide.
• The reduction of false negative cytology is the major aim of automation. There may be two types of error in a case of false
negative cytology:
A. Sampling error: This means that the lesion is present in the body but is not properly sampled. Sampling error is one of the
major sources of error. Remedies: It is not entirely possible to avoid the sampling error, but with the help of liquid-based
cytology (LBC) we can get the maximum number of cells on the smear.
B. Screening errors: Screening error may be due to failure to find out the representative cells on the smear or it may be due
to interpretation error.
➢ 1. RECOGNITION ERRORS: It is just like to find out the “needle in the haystack”. The representative cells may be too
few to find out even after a laborious search or they may be obscured within the blood, mucus or inflammatory cells
and necrotic debris. Cyto-screener may overlook these cells due to fatigue or due to the above mentioned technical
difficulties. Remedies:
➢ (1) Liquid-based cytology (LBC) may provide a clean background of the smear which is free of mucus or blood.
Moreover the proportionate amounts of cells are concentrated in a small part of the slide as a monolayer form.
This may help to find out the abnormal cell more easily.
➢ (2) The automated adjunctive technique helps the manual screening by locating the path of the screening on the
computer screen. Automated screening may also help to find out the abnormal cells.

➢ 2. INTERPRETATION ERRORS: Another possible source of errors is non-recognition of abnormal cells. This may be
because of professional incompetence or inadequate training. Remedies:
➢ Automated screening devices may initially help to find out the abnormal cells. However, trained cytopathologist is
needed for further confirmation of the abnormal cells detected by automated machines.
ADJUNCTIVE DEVICES TO MANUAL SCREENING
PATHFINDER
• It is an additional device for manual screening. The system is attached to the stage of the microscope and traces the screening
pathway of the smear and records the time taken by the cytotechnologist to screen each smear. The pathfinder is now obsolete
and out of the market.
TRAC CELL 2000 SYSTEM
• Trac cell 2000 system (Accumed) is a fully automated prescreening system that supports the screener. This system guides the
screener about the areas of the slides to be screened. It locates the areas that need to be reviewed and also maps the pathway of
the slide during screening. It also adjusts the speed, calculates the optimal focal plane and also the speed of the screening. The
company claims that Trac cell 2000 system increases the productivity of the screener and also contributes strict vigilance over the
cytoscreener.

AUTOMATED SCREENING
• Automated screening procedure should be designed in such a way that no dysplastic or malignant cells should be passed as
normal or negative and it should not label the normal cell as malignant cells.
• In addition, the automated screening technique should be economically cost-effective and it should shorten the period of
screening. Therefore, the two main goals of automated screening are to increase the accuracy and productivity of the cervical
screening.

AUTOMATED SCREENING DEVICES

• Till date, there is no fully automated system available in the market. Presently, only two semi-automated systems are available BD
FocalPoint GS Imaging System (previously known as Tri-Path AutoPap system) and HOLOGIC ThinPrep Imaging System (formerly
known as Cytyc ThinPrep Imaging System).
• However first two FDA approved technologies, were i.e. PAPNET and AutoPap 300 QC system.
REDUNDANT
SYSTEMS
PAPNET
• PAPNET system was designed and introduced by neuromedical system . PAPNET was approved by FDA in 1995. Later on, in
the year 1999, the company was declared as bankrupt. Presently, PAPNET is out of the market.
• The PAPNET system applied artificial neural network for the detection of abnormal cells. The PAPNET system consisted of
two parts: (1) scanning station and (2) review station.

SCANNING STATION:
➢ The cervical smears were sent to the central scanning station for screening. The scanning system is composed of an
automated microscope system that is electronically programmed to scan the smear and capture cell images for
computer processing.
➢ The smears were scanned by the microscope with attached electronic camera in different magnifications.
➢ The duties of the scanning station were to identify and record the potential abnormal cells with the help of artificial
neural network.
➢ The images of the abnormal areas were stored digitally and sent to the review station for final interpretation.

PAPNET review station:

• The stored images along with the slides were sent to the referral laboratory for further decision. The images of the slides
were examined on the computer screen and simultaneously the slides were reviewed under the microscope for final
confirmation.
AUTOPAP 300 QC SYSTEM
• The AutoPap system was first introduced by Neopath in the year 1995 and was approved by FDA. Presently, this system is no
longer available. This was an independent device, which automatically scans slides using image analysis algorithms.
• The AutoPap system generated an evaluation score ranging in value from 0 to 1.0 for each slide depending on the
probability of abnormality of the cells in the smear. The higher the evaluation score, the more likely that abnormal cell is
present.

Algorithmic Architecture of AutoPap System

• The images are analyzed by five algorithms:
(1) Stripe detection algorithm to detect dirt or glass scratches,
(2) focus check algorithm to assume that the object is optimally focused,
(3) single cell algorithm which classifies the cell and assesses the quality of the stain,
(4) group algorithm for classification of the group, and
(5) thick group algorithm for classification of thick groups of cells.
• All the information is collected and finally the score is made to assess the likelihood of abnormality of each object.
 CURRENT
AUTOMATED
SYSTEMS
BD FOCALPOINT GS IMAGING SYSTEM (FPGS):
• BD FPGS is FDA approved automated device. This system includes BD Focal PointTM Slide Profiler and the BD Focal Point
GS review station.
• BD Focal PointTM Slide Profiler is a computer based automated screening devices that scans the slide in low and high
magnification. More than 100 object analysis features are included in the screening algorithm of the system.
• The images are interpreted by an image analyzer software program and each slide is given a “device score” according to
the probability of abnormality. Subsequently, the slide processing data is transferred to the review station.
• The system always categorizes the slide into no further review and review group.

BD FOCALPOINT GS REVIEW STATION:

• Here, the slides “for review” group are reviewed by the cytotechnologists.
• The slides for specimen adequacy are also reviewed.
• If no abnormality is detected by manual screening then the slides are labeled as normal and can be kept in archives.
• If any abnormality is detected then the slides are reviewed further by cytopathologists for final interpretation.
HOLOGIC THINPREP IMAGING SYSTEM:
This system includes ThinPrep imaging system and ThinPrep review system.

THINPREP IMAGE PROCESSOR:

• Here the system acquires and process data from the ThinPrep test slides.
• This computer based system scans slide to find out the nuclei that are the largest and darkest as these nuclei are
considered as abnormal.
• The imaging system always excludes the overlapping cells or the objects other than nuclei.
• The system screens the slide and records the locations of 22 microscopic fields that contain the potential abnormal cells.

REVIEW SCOPE:
• Review scope is an automated microscope with a motorized stage. It guides the cytotechnologist to review the 22
microscopic field of interest.
• If there are no abnormal cells in the 22 field of views then the slides are passed as normal.
• However, if there is any abnormal cell present then the whole slide is screened thoroughly.
AUTOMATIC
VS
MANUAL
COMPARISON OF MANUAL AND AUTOMATED DEVICES
• Several studies claimed that automated screening done by either of these two techniques is more sensitive than routine
manual screening.
• However, these studies were not randomized and done in a small number of patients. Moreover, the studies are often
sponsored by the company itself.
• In a large randomized control study, the automation-assisted reading of cervical cytology was compared with manual report
of cervical cytology smear considering histology as an end point.
• Both BD Focal Point GS imaging system and Thin Prep imaging system were applied in two groups of patients and in both
groups manual screening were also done.
• It was noted that automation-assisted reading was 8% less sensitive compared to manual report. Cervical smear
categorized as “no further review” by automated screening was very reliable.
• Kitchener HC et al.17 also noted that automated screening and manual screening were almost similar in cost-effectiveness
issue.
• However, there was 60–80% increase in productivity for automated screening.
• It was concluded that there is no justification to introduce an automated screening technique in relation to sensitivity and
cost effectiveness of cervical screening.
PROBLEMS WITH
IMPLEMENTING
AUTOMATION
TECHNICAL
• DATA: Present generation computer technology can handle large amount of data. However, still it is difficult for a
computer to handle the immense amount of data gathered during the screening of the individual slides.
• AUTOFOCUS: Continuous focusing of the cells, particularly overlapping cells, is a challenge.

DIAGNOSTIC
• It is still very difficult for an automated system to make individual decisions in single cases.

FINANCIAL
• Automated systems are commercially available but they are very costly for routine laboratories for daily use. This is
particularly true for developing countries.

MEDICOLEGAL
• It is still not settled about the medicolegal issues related to the machine given report in primary screening.