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In the chapter on cytology automation in the second edition of 
Comprehensive Cytopathology, published in 1997, Bartels, Bibbo, 
and Wied provide a prescient description of the challenges and 
strategies for the development of automated cervical cytology 
screening devices. In fact, writing a decade later, it is possible 
to follow their predictions directly to the instruments that are 
currently in clinical use. A thorough reading of that discussion 
will give the interested observer a background of the qualities 
and features that must be inherent in such devices in order to 
provide the accuracy necessary to the process, and also the pro-
gression of development necessary to meet the requirements 
of regulatory agencies. At present, this process has gone from 
a half century of developmental work to fruition in the form of 
primary screening instruments in clinical cytology laboratories. 
Along the way, enormous amounts of time and energy have been 
spent on development of specimen preparation methods and 
instrumentation, optical systems, computers, and algorithmic 
and other types of information-processing systems. This follows 
on the original work necessary to the process, that being the 
morphometric measurements of cells, both normal and abnor-
mal, that have provided the inputs necessary for these processes 
and devices. This discussion will highlight the historical features 
that have influenced the processes and the devices currently in 
Introduction Historical Attempts at Automation
As one of the last high-volume, manually performed procedures 
in the laboratory, cervical cytology has always been a target for 
automation. About a half-century of time and energy has been 
devoted to this task, beginning with developmental work nec-
essary to define what constituted morphologically, and more 
importantly morphometrically, the important, qualitative and 
quantitative features of cells—benign, reactive, and neoplastic—
obtained in a cervical cytology specimen.
In the days of Caspersson’s quantitative analysis of nucleic acid 
and proteins by microspectrophotometry, it was estimated that 
gathering information about one cell required a week’s work.
Detailed information on the relative or absolute number of 
measuring points per cell became available from the sophisti-
cated machines of Caspersson,1 Hyden and Larsson,2 Deeley,3 
Montgomery,4 Thornburg,5 and Tolles.6 However, despite this 
important and pioneering work, the potential for this detailed 
information remained largely unrecognized.
It was the widespread acceptance of the diagnostic capa-
bilities of cytology that eventually generated demands for the 
automation of this highly time-consuming and tedious man-
ual procedure. Both cytochemical and cytomorphologic cri-
teria were used in the cytoanalyzer project of the US National 
 Cancer Institute in an early attempt to develop an automated 
Automat
Contents
Introduction
Historical Attempts at Automation
The Rationale for Automation
Cytology Automation: Accuracy and Productivity
Currently Available Automation Platforms
Liquid-based preparation
automated Screening Devices
Laboratory Process Issues Associated with the Use of Automated 
Devices
reporting Issues
Issues with Specimens that Cannot Be Successfully processed
use, provide a practical presentation of their mode of operation, 
and detail their performance characteristics as presented in the 
medical literature. A closing discussion will speculate on what 
may be the next step in the evolution of this discipline.
C h a p t e r 
ion in Cervical Cytology
David C Wilbur and Marluce Bibbo
Stain Use in automated Systems
training required for Initiation of automated Methods
Laboratory Workflow Issues
Cost-effectiveness of Liquid-based Preparation and Automated 
Screening Devices
Concluding Remarks
34
1021
 process.7–10 It was discontinued because of its inability to be able 
to resolve the complexity of routinely prepared conventional 
cytology specimens due to cell overlap and obscuring factors. 
In addition, the robustness of the software was inadequate for 
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Special Techniques in CytologyPART THREE
the task. However, this project allowed investigators to appreci-
ate and develop approaches to the problems encountered in the 
early investigative process. Parts of the cytoanalyzer were used 
by Mendelsohn’s group to build CYDAC (Cytophotometric 
Data Conversion), a system designed toward computer-assisted 
description and identification of intracellular cell patterns.7,11 
In the 1960s, a number of cytochemical scanning methods and 
instruments of the integrating type were described.1,12–14 The 
most sophisticated optical and mechanical design entered into 
the Universal Recording Microspectrophotometer (UMSP-1) of 
Carl Zeiss,15 which incorporated an ingenious high-precision 
mechanical stage and achromat lenses corrected for the ultra-
violet and visible range. Limitations precluded clinical applica-
bility, however, including the slow scanning mechanism.
In 1968, Wied and collaborators introduced the taxonomic 
intracellular analytic system (TI-CAS),16 a device for cell identifi-
cation based on computer evaluation of the intracellular patterns 
of image scan data. This effort stimulated the interest of others 
in Europe and Japan, leading to later systems such as the VCSA,17 
Cerviscan,18 CYBEST,19 TUDAB,20 SAMBA,21 Diascanner,22 FAZY-
TAN,23 LEYTAS,24 Magiscan,25 McGill System,26 and TULIPS.27,28
Studies on the composition of cellular samples showed 
that the majority of abnormal cells in conventional Papanico-
laou smears were in clusters.29–33 This understanding led to the 
 conclusion that better cell presentation was needed to optimize 
the performance of scanning devices. Throughout the 1970s, the 
emphasis moved to development of techniques of cell sampling, 
preservation, disaggregation, and monolayering of cell prepara-
tions.34–37 Such specimens allowed for better visualization of cells 
for individual cell analysis, a process better suited to the compu-
ter hardware and software available. These processes have culmi-
nated in the present with successful introductions of automated 
cytology products into the marketplace. The 1980s saw the for-
mation of companies such as Neopath, Cytyc, Neuromedical Sys-
tems, and Roche Image Analysis Systems, which brought forward 
and tested the products that we now routinely utilize in daily 
practice. (Several organizations have sponsored and sustained 
those working in the field of automation for years. These include 
the Engineering Foundation of America, the Concerted Action 
on Automated and Analytical Cytometry of the European Eco-
nomic Community, and Professor George L. Wied of the Univer-
sity of Chicago, who organized academic meetings and specific 
 conferences on artificial intelligence and cytology automation.)
The Rationale for Automation
Cervical cytology has always been a high-volume test, one of the 
last such tests to be performed in a completely manual fashion. 
Because manual interpretation of cells for the identification of 
abnormalities has always followed a morphologic criteria-based 
approach, it was only reasonable to hypothesize that such a 
process would be amenable to an “image analysis”-based com-
puterization approach in which the computer software would be 
able to reliably “quantitate” widely recognized morphometric 
parameters such as nuclear size, nucleus to cytoplasmic ratios, 
chromatin texture, and distribution, and then sum (or otherwise 
analyze) such identified cellular changes into an interpretation, 
free of any human intervention. At first, such a hypothesis might 
seem to be very reasonable; however, this task was found to be 
far more complex due to a variety of factors. The conventional 
cervical cytology slide is very complicated.It may contain up to 
500 000 cells of various types that may be displayed in an innu-
merable array of patterns and variations, with complex over-
lapping, obscuration by blood and inflammation, and a wide 
variety of presentations in which benign or reactive cells may 
mimic malignancy and malignant cells can mimic the benign. 
As such, early attempts at automation, geared toward machine 
final interpretation, were generally unsuccessful. Philosophic 
changes in approach were necessary if automation was to be 
successfully introduced as a mainstream process. Therefore the 
concept of the device as an aid to human interpretation came to 
be regarded as a reasonable approach in distinction to methods 
seeking full device-only interpretation. This philosophic overlay 
is inherent in the two current approaches being utilized in auto-
mated screening: (1) the triage of slides by the device to those 
requiring manual review and those not requiring manual review 
(the Becton Dickinson/TriPath FocalPoint Primary Screening 
System, FPPS), and (2) the location-guided screening approach 
in which the device identifies cells or areas on the slide with 
high probability of abnormality for presentation to humans for 
final review and interpretation (Cytyc ThinPrep Imaging Sys-
tem, Neuromedical Systems PapNet, Becton Dickinson/TriPath 
FocalPoint GS).
Over this time, parallel efforts toward improving the cervi-
cal specimen were taking place. Clinical trials of liquid-based 
technology utilized new sampling devices such as endocervical 
brushes and “brooms” that provided a more cellular sample, 
particularly of the important transformation zone. Liquid-
based specimens are generally better distributed on the slide, 
with improved cell to cell separation or “segmentation.” Such 
specimens are also less prone to cellular obscuration by blood 
and inflammation. Hence liquid-based specimens create a more 
optimized “platform” for automated screening, and the most 
modern clinical devices take advantage of that fact.
Another important piece of the automation puzzle is the role 
of the regulatory agencies, most notably the US Food and Drug 
Administration (USFDA), which has to pass on the safety and 
effectiveness of such devices prior to commercial release. It was 
clear that the initial utility of automated screening devices des-
tined for commercial release would be tested and first released 
in a quality control (QC) use mode. Automated screeners would 
be utilized to “check” previously manually screened slides look-
ing for false-negative cases. During such activities, the overall 
performance of the devices could be tested. In addition, the for-
mulation of the types of trials necessary to adequately investigate 
and document these performance or operating characteristics 
without compromising patient care could also be developed. 
Professional societies published documents proposing per-
formance measures,38,39 and the USFDA and its advisory panels 
judged the data presented by manufacturers.
Following acceptable QC performance, the introduction and 
vetting of primary screening devices could be initiated.
Cytology Automation: Accuracy 
and Productivity
There are two ultimate goals of automation: (1) to improve the 
accuracy of the interpretation in terms of both false-negative 
and false-positive performance, and (2) to improve productivity 
of the workforce. A false-negative specimen is defined as one in 
which an interpretation is rendered of no disease being present 
when indeed such is not the case. False-negative specimens are 
Automation in Cervical Cytology
34
the most concerning, as they can lead to delayed detection and 
treatment with its inherent morbidity and mortality. However, 
false-positive results, defined as a positive result when no dis-
ease is present, are also important to avoid, as they can lead to 
overtreatment, which can be associated with morbidities such 
as hampered fertility and patient anxiety. In addition, false- 
positive results lead to inefficiencies in the system associated 
with greater than necessary time, workload, and expense. Cer-
vical cytology automation—the combination of liquid-based 
cytology and computerized automated screening—can theoreti-
cally enhance the accuracy of the Papanicolaou test in terms of 
both false-negative and false-positive parameters.
Liquid-based cytology enhances accuracy in the following 
ways.
• Complete (or nearly so) capture of all cellular material 
sampled from the patient. Compared with the making 
of a conventional slide, in which, on average, only about 
35% of sampled material is transferred to the glass slide 
for review, liquid-based technology captures most of the 
cells sampled.40 This diminishes the likelihood of false-
negative tests due to “transfer” error in which positive cells 
are left on the sampling device following the making of a 
conventional smear (Fig. 34.1).
 • Specimen randomization. Liquid-based slides contain 
a subsample of cells that have been randomized dur-
ing the preparation process. Randomization allows for 
a more representative cell pattern being present on the 
slide, containing all the types of cells sampled from the 
patient. This compares with the nonrandomized subsam-
ple routinely transferred in the making of a conventional 
smear. Randomization is important in reducing false-neg-
ative tests, particularly in the circumstances in which few 
abnormal cells are sampled or in circumstances in which 
abnormal cells may be geographically isolated to more 
poorly transferred areas of the sampling device (Fig. 34.1).
Fig. 34.1 the principles underlying improved performance by 
liquid-based specimen collection and preparation. Liquid-based methods 
capture all material collected from the patient. Conventional smears transfer 
only a portion of material collected (on average about 35%), the remainder 
being discarded with the sampling device. Conventional smear transfer is 
nonrandom and may be influenced by geographic location of the lesion and 
cells on the sampling device. Liquid-based specimens randomize cellular 
material, ensuring that each slide made is a representative sample.
 • Improved preservation of the cellular sample. Cells sam-
pled and then immediately immersed in liquid fixative 
transport media show, on average, better fixation, and 
thus better visual preservation, than will smeared 
cellular material on a conventional slide. Artifacts 
such as air-drying and other cellular degeneration chang-
es are virtually eliminated in liquid-collected specimens. 
Better preservation allows for more accurate assessments 
of nuclear size, nucleus to cytoplasmic ratios, and chro-
matin structure. This increases accuracy 
(both false negative and positive) via improved 
ability to correctly recognize and classify cellular 
changes.
 • Improved individual cell visualization. Because over-
all cellularity on the slide surface is controlled in the 
liquid-based processes, and because of the ability to 
equally distribute cells in a uniform fashion across the 
slide surface, liquid-based slides allow for improved vis-
ualization of cells due to lack of overlapping or piling 
up of cells. Again, this increases accuracy of cell classi-
fication (both false negative and positive) via improved 
ability to correctly recognize and classify morphologic 
changes.
 • Decrease in obscuring factors. The liquid-based proc-
esses generally improve cellular visualization via de-
creases in physiologic obscuring factors such as blood or 
inflammation. The cells in conventional smears may be 
obscured, leading to unsatisfactory or limited specimens 
when such factors are present. Liquid-based 
samples are largely immune (with caveats to be dis-
cussed below) to such encumbrances, leading to accu-
racy improvements directly tied to “visualaccessibility” 
of the cells.
 • Consistent location and limited size viewing area. 
Because liquid-based methods place cellular material in 
a limited area of the glass slide, always in the same place, 
screening (following training and initial experience) is 
more efficient and is associated with less viewer fatigue, 
leading to fewer “habituation,” or inattention, lapses. 
Such slides do not require “hunting down” 
cellular material at any location on the slide, including 
outside the cover slip area and adjacent to (or even under) 
the slide label.
Key features by which liquid-based preparation 
improves accuracy
• Complete cellular capture;
 • Specimen randomization;
 • Improved cellular preservation;
 • Improved individual cell visualization (improved segmen-
tation);
 • Decrease in obscuring factors (blood and inflammation); 
and
 • Consistent cell location and viewing size area.
All these features allow for standardization of the Papani-
colaou slide with uniformity of specimen and cell appearance 
from case to case. Overall, this uniformity leads to population-
wide specimen improvement. The process makes all prepara-
tions “optimal,” eliminating the “highs and lows” inherent in 
the highly variable conventional preparation method.
Computerized automated screening enhances accuracy in 
the following ways.
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1024
Special Techniques in CytologyPART THREE
 • The devices scan the entire cellular content of the 
slide without attention lapses or missed fields of 
view (FOVs).
 • The devices apply a standard set of analyses in a uniform 
fashion to all cells or FOVs on the slide. Theoretically, 
computerized cell or FOV measurements should lack the 
variability, or nonobjectivity, that is inherent in human 
analyses and which is subject to a wide variety of biases. 
Although not widely appreciated, human ability to 
“measure” an object, such as a nuclear size, is affected by 
the cell’s local environment, and biases such as precon-
ceptions about the case diagnosis (e.g. if an observer al-
ready “thinks” that a case represents a dysplasia, percep-
tion of size is likely to fall within the bounds of known 
“dysplasia criteria” regardless of the actual measured 
value41).
 • In the automated approach, in which slides are triaged 
into cases requiring review and those not requiring review, 
based on overall probability of abnormality on the slide, 
categorization as a low probability “no review” slide im-
proves specificity of abnormal detection. Put another way, 
if humans were allowed to screen this population of slides 
having very low probability of abnormality, a “finding” 
would more likely be a false, rather than a true, positive 
finding.42
 • In the location-guided screening approach, in which 
a limited number of “highest probability” areas of the 
slide are viewed by the human screener, the increased 
“signal to noise” ratio (meaning more abnormal cells 
are presented per normal cell) improves screener vigi-
lance and hence sensitivity to the abnormal cells present. 
This concept has been clearly shown in the literature 
about other types of screening procedures, such as visual 
screening for prohibited objects at airport checkpoints. 
If the prohibited objects are more prevalent in the total 
population of objects viewed, the sensitivity of detec-
tion increases.43 A decreased false-negative rate can be 
expected with the use of this focused screening approach. 
In addition, in a similar fashion to the previous con-
cept, when screeners do not view the “low probability” 
areas of the slide, the likelihood of false-positive results 
should be diminished.
Key features by which computerized automated screening 
improves accuracy are as follows.
• The device scans the entire slide without attention lapses 
or missed FOVs.
 • The device applies a standard set of analyses in a uniform 
fashion to all cells and FOVs.
 • In slide-ranking devices, slides triaged to no manual 
review increase specificity by decreasing false-positive 
tests.
 • In slide-ranking devices, increased screener vigilance in 
high-probability cases increases sensitivity by decreasing 
false-negative cases.
 • When devices are utilized in combination with liquid-
based preparation, improved cell visualization is syner-
gistic with the above four points, leading to yet further 
improvements in accuracy.
The combination of liquid-based cytology preparation tech-
niques with computerized automated screening has the potential 
to have a synergistic effect on accuracy. Better visualization of 
cells inherent in the liquid-based technique should allow for 
more accurate and efficient analyses by computer devices in a 
similar fashion to human observers. This hypothesis will be put 
to the test when data from the clinical trials and independent 
studies using these systems are presented below.
Productivity improvements with automated cytology methods 
and devices are as follow.
 • Liquid-based cytology specimens can be screened by 
humans and by automated devices at a faster speed than 
conventional cytology specimens. Studies have shown that 
productivity gains of about 20% for manual screening can 
be expected once personnel are trained and have gained 
experience.44
 • Devices that triage slides into categories requiring hu-
man review and those that do not, benefit from fewer 
slides entering the human review process. At present, this 
productivity gain can be as high as 25% based on USFDA 
regulatory operating parameters, but it is as much as 50% 
in use outside the United States.
 • Location-guided screening devices requiring only limited 
reviews of machine-selected fields substantially reduce 
the average time spent screening a slide. At present, in the 
currently approved Cytyc device, this productivity gain is 
reported to be as high as 100%.45
Suffice to say that automation provides theoretic and, in 
many cases, achieved improvements in accuracy, both sensitiv-
ity and specificity, as well as productivity gains. Such achieve-
ments are important in an era of declining resources, in both 
reimbursement and technologist labor, and the increased threat 
of costly litigation for inaccurate results.
Currently Available Automation Platforms
Liquid-based Preparation
Cytyc ThinPrep Pap Test
The ThinPrep Pap Test (Hologic, Marlborough, MA) was the 
first USFDA-approved method, receiving clearance in 1996. 
Collection of a ThinPrep specimen requires the use of either 
a broom-type device or the combination of an endocervical 
brush and spatula. Cells are washed off the sampling device(s) 
into a transport vial (Preservcyt, Hologic) containing a pro-
prietary preservative mixture that is methanol-based. The vial 
is received in the laboratory, where the cell suspension under-
goes homogenization, and therefore randomization, by a vor-
texing cell dispersion method. Cells are transferred by suction 
to a filter (Fig. 34.2). Cellularity of the specimen is controlled 
by monitoring of a pressure gradient across the filter. When 
this pressure gradient reaches a defined level, the suction stops 
and the cells are transferred to a glass slide by blotting and the 
application of gentle pressure across the filter in the opposite 
direction. The final result is a circle of deposited cells measur-
ing 20 mm in diameter (Fig. 34.3). There are two models of 
the ThinPrep processing device, both of which operate in a 
fundamentally similar fashion as described above; the T2000 
model (Fig. 34.4) is a single specimen load device, and the 
T3000 model (Fig. 34.5) is an automated multiload device 
designed for high-volume laboratories.
Automation in Cervical Cytology
34
Becton Dickinson/TriPath SurePath
The SurePath (Becton Dickinson/TriPath, Burlington, NC) method 
was approved by the USFDA in1999. Specimen collection 
requires a broom-type device or a combination of the endocer-
vical brush and spatula, in an identical fashion to the Thin-
Prep method. The collection device head is removed from the 
 handle and dropped into a preservative-containing vial, where 
it remains throughout the processing procedure. This is in con-
trast to the washing and discarding of the device in the ThinPrep 
method. Improved harvesting of all cells from the sampling 
device is the theoretic advantage of this procedure. The trans-
port vial contains an ethanol-based fixative. On receipt of the 
specimen in the laboratory, homogenization/randomization 
takes place via a syringing of the specimen. An aliquot of the 
homogenized sample is placed on a sucrose density column and 
is then subjected to a set of two centrifugation wash procedures 
shown to substantially reduce blood, proteinaceous debris, and 
inflammation without loss of diagnostic epithelial cellular-
ity (Fig. 34.6). These preprocessing steps use a semiautomated 
device (PrepMate) as shown in Fig. 34.7. The postprocessing cell 
suspension is loaded on to an automated pipetting/cell trans-
fer device (PrepStain), where cells are then placed into settling 
Fig. 34.2 the ThinPrep Pap Test preparation process. the thinprep 
method uses a plastic tube with a filter on its end as a “vortexing” device to 
homogenize the sample. Under negative pressure, cells are deposited on to 
the filter surface. Cellularity is controlled by pressure monitoring across the 
filter. When appropriate pressure is reached, the tube is withdrawn and cells 
are transferred under positive pressure by blotting on to the slide surface.
Fig. 34.3 The four types of gynecologic preparation. From left to right: 
the conventional smear, thinprep, Surepath, and Monoprep specimens.
chambers in batches of 48. Cells are allowed to descend on to 
the slide surface, where resultant cellularity is determined by an 
adhesive slide coating allowing only a single layer of cells to 
be deposited (Fig. 34.8). The finished slide contains a uniform 
13-mm circle of cellular material (Fig. 34.3). The slide can be 
stained or left unstained by the device.
Monogen MonoPrep
The Monogen MonoPrep (MonoGen, Arlington Heights, IL) 
slide-making system was approved by the USFDA in 2006. Cel-
lular material is collected in prebarcoded liquid fixative vials. 
The fixative is methanol-based. The vial cap has an integrated 
stirring device that is utilized for homogenization and randomi-
zation of the specimen. Following homogenization, the sample 
is drawn through a central hollow space and deposited on a fil-
ter. Cells are blotted to the slide surface, creating a 20-mm circle 
(Fig. 34.3). Preparation is entirely automated with a multiload 
device that will process up to 300 specimens per 8-h period in a 
“walk away” fashion (Fig. 34.9).
B
A
Fig. 34.4 (a) The ThinPrep T2000 device. this is the most commonly 
utilized thinprep device. It processes one preservative cell suspension vial 
at a time. (B) ThinPrep plastic filter/vortexing units and glass slides. 
Note the fiducial markings on the thinprep slide, which allow for accurate 
localization of fields of view when used with the thinprep Imaging System. 
1025
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Special Techniques in CytologyPART THREE
Operating Characteristics of Liquid-based Preparation
The data available to judge the performance of the liquid-based 
preparation methods come from clinical trials sponsored by the 
manufacturer and from independent reports by users. The former 
data receive rigorous regulatory review and determine the claims 
that a manufacturer can use in marketing the product. The latter 
reflect use in current practice, but such user reports have to be 
viewed with greater scrutiny due to wide variability of methods 
and rigor of study.
Data from initial clinical trials and follow-on studies, uti-
lized by the USFDA to determine the manufacturers’ product 
labeling, show remarkably similar results for the ThinPrep and 
SurePath methods. Each is considered a “replacement” for the 
 conventionally prepared specimen, and both show improved per-
formance over conventional cytology for the detection of squa-
mous dysplastic lesions. ThinPrep clinical trials showed an 18% 
increase,46 and the SurePath method showed a 7% increase,47 in 
detection of squamous intraepithelial lesions (SILs). Further stud-
ies showed increases in the detection of high-grade SILs in both 
systems, ThinPrep by 58%48 and SurePath by 64%.47 Both speci-
men types showed improvement in overall specimen adequacy, 
ThinPrep by 11% (albeit with a slight increase in unsatisfactory 
specimens, 1.9% versus 1.6%),46 and SurePath by 18% (with a 
reduction in unsatisfactory specimens, 0.6% versus 1%).47
In studies done by user laboratories, results are generally 
in support of the USFDA claims noted above but the vari-
ability is far greater. ThinPrep “direct to vial” studies show 
an average improved detection of SIL+ cases of 102% over 
conventional specimens (range 40–224%).49–55 An average 
improved detection of high-grade squamous intraepithelial 
lesion (HSIL)+ by 100% (range 59–112%) was noted.18,50,52 
Unsatisfactory rates also showed improvement, with an 
average of 24% reduction (range −81 to 215%).46,49,51,53–60 
SurePath direct to vial studies have also shown improvements, 
with SIL+ detection rates increasing by an average of 61% (range 
59–67%), with HSIL+ detection rates increasing by an average 
of 65% (range 46–78%),61–63 and unsatisfactory rates decreas-
ing by an average of 83% (range −100 to 9%) (Table 34.1).61–68 
(A direct to vial study is one in which the technique is used in its 
intended clinical mode as opposed to so-called “split sample” 
studies, in which conventional smears are made first and the 
residual cell material is used to make the liquid-based prepara-
tion. Split-sample studies directly compare performance on a 
single specimen but are biased against the sample made second 
(which in all liquid-based studies was that preparation). Direct 
to vial studies have the advantage of using all the cellular mate-
rial for the liquid-based preparation (intended use) but have the 
disadvantage of comparing performance with a historical popu-
lation of cases in which other variables—some known, some 
potentially not known—may come into play. Careful reading 
of historical population studies is required therefore in order to 
make useful judgments about performance of the test method.)
Table 34.1 summarizes studies showing the performance 
of ThinPrep and SurePath liquid-based preparation meth-
ods. Clinical trial data submitted to the USFDA and postap-
proval studies leading to USFDA labeling claims of improved 
HSIL-positive detection are illustrated in the top rows.47–50 
 Independent studies showing performance characteristics are 
shown in the bottom rows.51–71
Direct comparisons between the ThinPrep and SurePath tech-
nologies have not been reported to date; however, some data 
highlighting the differences between the techniques have 
emerged. Preliminary (non-peer reviewed) studies have sug-
gested that the higher unsatisfactory rates noted with ThinPrep 
as compared with SurePath may be due predominantly to the 
interference of blood in ThinPrep specimens.69,70 Blood in speci-
mens may displace epithelial cell material on ThinPrep filters, 
Fig. 34.5 The ThinPrep T3000 device. this is a multiload, “walk away” 
thinprep device designed for use in larger volume laboratories. It processes 
thinprep samples by an identical method as does the t2000. 
The vial, containing
10ml of cellular
material is mixed
A syringe device is inserted
through the membrane
in the vial’s cap
8ml of cellular material
is layered onto the density reagent
1st centrifugation
2 min
@ 200 xg
2nd centrifugation2 min
@ 8200 xg
Supermate
and
interface
Sedimentation
@ 1 x g
AutoCyte PREP
Sure
Path
Fig. 34.6 The SurePath preparation process. the Surepath 
process includes homogenization of the specimen via a syringing 
action, followed by passage through a sucrose density gradient 
column designed to substantially reduce blood and inflammatory 
cells. Final cell suspensions are allowed to gravity sediment on to 
the glass slide surface, where cellularity is controlled by the adhesive 
coating on the slide.
Membrane in vial cap
Enriched
diagnostic
material
Decant
Pellet
Automation in Cervical Cytology
34
leading to unsatisfactory samples and rarely to missed abnor-
malities.71,72 Caveats for reporting of bloody ThinPrep samples 
and the need for the use of pretreatment procedures, most com-
monly glacial acetic acid, to clear blood from these samples 
can improve the adequacy rates, and they have become routine 
procedures in many laboratories.73 SurePath, with its density 
gradient preprocessing and washing steps, eliminates this inter-
ference, accounting for the better adequacy rates noted when 
compared with unpreprocessed ThinPreps.
Clinical trials of the MonoPrep system included 10 739 
specimens with a split-sample design for comparison of con-
ventional smear with MonoPrep performance. Reference 
truth for each slide was determined by a masked independent 
pathologist review, and performance was determined in each 
reference diagnostic category. MonoPrep outperformed conven-
tional cytology in all categories. In the atypical squamous cells 
of undetermined significance (ASCUS)+ (all abnormal cases) 
group, MonoPrep identified 15% more cases. In the atypical 
squamous cells, cannot exclude HSIL/atypical glandular cells 
(AGC)+ category, MonoPrep identified 23% more cases. In the 
low-grade squamous intraepithelial lesion (LSIL)+ category, 
MonoPrep identified 26% more cases. All these differences were 
statistically significant. MonoPrep identified 4% more HSIL+, 
but this was not a statistically significant result. Unsatisfactory 
specimens were reduced by 58% with MonoPrep, with an over-
all unsatisfactory rate of 1.2% in the clinical trial (Table 34.2). 
Fig. 34.7 The PrepMate device. this device performs the syringing 
homogenization step and transfers the randomized sample to the 
density gradient column prior to centrifugation. It handles 12 specimens 
simultaneously in a “walk away” fashion.
Morphologic features of the MonoPrep slides are reported to be 
similar to other types of liquid-based specimens.74A,74B
Table 34.2 summarizes MonoPrep USFDA clinical trial results. 
These results led to MonoPrep USFDA approval as a replace-
ment for the conventional cervical cytology smear method.
B
A
Fig. 34.8 (a) The PrepStain device. this is an automated pipetting 
machine that transfers the final Surepath cell suspensions to the settling 
chambers where the final slides are prepared. In addition, the pipetting 
device can complete the staining process. (B) a close-up view of the settling 
chambers placed over the slides. Cell suspension and stain are added to the 
chambers by the prepStain device.
1027
10
Special Techniques in CytologyPART THREE
Based on the data presented, as well as our own experience 
utilizing liquid-based technology, it is clear that performance of 
the Papanicolaou test is substantially improved using these meth-
ods. The degree of improvement will vary from one laboratory 
to another, as illustrated by the high degree of variability in the 
studies presented. The achieved improvement is closely tied to the 
starting point for each facility. In those laboratories having opti-
mized conventional smear methods, improvements may be less 
than the norm, for laboratories receiving conventional slides from 
a widely varied sample-taker population, in which conventional 
specimen quality is diverse, the resultant technical standardiza-
tion of method and optimization of results will be maximized.
Automated Screening Devices
There are two fundamental approaches to automated screening. 
The first approach is for a device to categorize an entire slide 
as to the probability of abnormality being present anywhere on 
the slide. This process allows for the rank ordering of a group of 
slides, which allows machine decisions of “negative” based on 
A2 B
Fig. 34.9 (a1 & a2) MonoPrep preparation method. the Monoprep vial contains a hollow stirring rod that when rotated homogenizes the specimen. 
Following homogenization, the sample is drawn through the hollow center and deposited on a filter, which is then blotted on to the glass slide surface. (B) The 
MonoPrep processing device is an automated system, allowing multiple samples to be run in a “walk away” fashion.
 Initial clinical trials Postapproval studies
LSIL+ (%) Unsatisfactory rate (%) HSIL+ (%)
US Food and Drug Administration clinical trial dataa
thinprep +18 1.9 58
Surepath +7 0.6 64
Independent studiesa
thinprep +102 (+40 to +224) −24 (−81 to +215) +100 (+59 to +112)
Surepath +61 (+59 to +67) −83 (−100 to +9) +65 (+46 to +78)
hSIL, high-grade squamous intraepithelial lesion; LSIL, low-grade squamous intraepithelial lesion.
aIn comparison with conventional cytology.
Table 34.1 Liquid-based performance Data
2
A1
8
Automation in Cervical Cytology
34
a predetermined “slide score threshold” below which the prob-
ability of abnormality being present is negligible. On the oppo-
site high probability end of the scale, such an approach allows 
additional “vigilance” during the manual screening and/or QC 
processes, leading to improved sensitivity for abnormalities. This 
process accumulates and sums the total of data obtained from 
cellular analysis across the entire slide. This is the fundamental 
method utilized by the Becton Dickinson/TriPath FocalPoint Sys-
tem to be described below. In the second approach, the device 
identifies individual cells, or groups of cells on each slide, that 
have the highest probability of being abnormal, presenting these 
cells to human screeners for a final decision. This process is often 
referred to a “location-guided screening” and allows the device to 
sort the abnormal cell “needles” from the “haystack” of normal 
cells in a typical cervical cytology specimen. This is the approach 
utilized by the PapNet, ThinPrep Imaging, and FocalPoint GS 
(GS for “guided screening”) systems to be described below.
The evolution of the products currently available in the 
 marketplace necessarily went through an orderly progression 
of development and regulatory oversight. The radical change in 
the screening paradigm brought about by the introduction of 
computerized screening devices initially required acquisition 
of performance data in applications deemed to have the least 
impact on patient safety. In addition, regulatory agencies such 
as the USFDA needed to develop expertise in evaluating device 
performance in this early phase with low probability of unto-
ward effects on release into the clinical setting. As such, the first 
applications of the earliest devices were as QC rescreeners. It was 
recognized that basic functioning of the machines would be iden-
tical in this application as it would be in the more “risk-intense” 
primary screening mode. Therefore important performance data 
could be obtained and carefully analyzed prior to application 
as a primary screening device. Thus manufacturers engaged in 
the process from the beginning first developed QC applications, 
which were to be supplanted by primary screening devices.
The PapNet System
The PapNet system (Neuromedical Systems, Suffern, NY) con-
sists of an automated robotic microscope that acquires images 
from cervical cytology specimens for further analysis by its com-
puters (Fig. 34.10). The process begins with a low-magnificationslide scan that “maps” areas of cellular material for further high-
magnification image capture. During the second scan, objects 
are selected based on standard morphometric parameters such 
as size, shape, and optical density. High-risk objects (typically 
 
Percentage
Statistical 
significance
aSCUS+ +15 Significant
aSCh/aGC+ +23 Significant
LSIL+ +26 Significant
hSIL+ +4 Not significant
aGC, atypical glandular cells; aSCh, atypical squamous cells, cannot exclude 
high-grade squamous intraepithelial lesion; aSCUS, atypical squamous cells of 
undetermined significance; hSIL, high-grade squamous intraepithelial lesion; LSIL, 
low-grade squamous intraepithelial lesion.
aSensitivity compared with conventional cytology.
Table 34.2 Monoprep data: US Food and Drug administration clinical 
trial dataa
between 20 000 and 50 000 per slide) are sent to a “primary 
classifier” that further refines the abnormal selection process 
and sends the highest risk objects to a “neural network” com-
puting device that makes the final selection of objects to present 
to human screeners. This process is unique to the PapNet sys-
tem and allows “…the elements of the computer to enter into 
numerous flexible associations and therefore resembles the 
network of neurons in the human brain.”75 Through this proc-
ess, the software can “learn” to better discriminate between pre-
sented cellular features as to the type and degree of abnormality 
present. Selected final cells are presented to humans via a “tiled” 
video screen (64 images per case) on which final decisions are 
rendered.
The PapNet system was approved by the USFDA in 1995 
for QC rescreening of previously manually screened “nega-
tive” slides. A number of studies appeared shortly thereafter 
that indicated good performance of the system used in this 
modality. Koss et al. showed that using the device with known 
B
A
Fig. 34.10 (a) The PapNet system scanning device is currently utilized in 
a number of laboratories in europe and asia as a primary screening device. Fields 
of view containing potentially abnormal cells are displayed as a “tiled” array of 
images on a video screen on which they are reviewed. In this image, papNet 
screening stations are illustrated in use in Leiden, the Netherlands. (B) the 
PapNet system review station in use in Leiden. Images selected for review by 
the scanning device are tiled on to the monitor screen for review by cytologists. 
1029
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Special Techniques in CytologyPART THREE
 abnormal slides, reviewers recapitulated the abnormal result 
in 97% of cases.75 Important for the QC application, Kok 
and Boon showed that when known false-negative cases were 
reviewed using PapNet, all cases received at least a “suspicious” 
result.76 In a study of PapNet performance using varying num-
bers of “seeded” abnormal slides, Mitchell and Medley found 
that “seeding prevalence” played a role in device performance, 
low-prevalence seeding yielding lower sensitivities of detection 
and higher prevalence seeding yielded higher sensitivities.77 This 
phenomenon is directly related to vigilance of the observers, 
as was previously noted to be the case for the manual screen-
ing process. Other studies using PapNet in a QC rescreening 
mode have shown that more false-negative cases are identified 
using the system when compared with manual QC rescreening 
procedures alone.78–80
The PapNet system, by an identical technical process, could 
also be used as a primary screening device. In this mode, review 
of PapNet-obtained images as a means of triaging slides to a 
full manual review was found to be at least equivalent to man-
ual screening alone in two separate studies.81,82 In a study from 
Finland, PapNet was utilized in a large cervical cancer screen-
ing trial and no statistical difference compared with manual 
screening was identified.83 Prior to a USFDA approval for the 
primary screening application, Neuromedical Systems filed for 
bankruptcy and ceased its business operations in the late 1990s. 
Although PapNet is therefore not in use in the US market, devices 
are still present in laboratories in Europe and Asia, where they 
continue to perform primary screening of cervical cytology spec-
imens. At present, the intellectual property inherent to the Pap-
Net system resides at Becton Dickinson/TriPath, which acquired 
the rights to this technology following the bankruptcy.
Becton Dickinson/TriPath FocalPoint Device
The FocalPoint and FocalPoint GS devices (previously known 
as the Neopath AutoPap) (Becton Dickinson/TriPath, Burling-
ton, NC) are computerized scanning devices that can be config-
ured to utilize both of the slide analysis approaches described 
above: slide scoring with stratification triage and location-guided 
screening, respectively (Fig. 34.11). The FocalPoint scans slides 
at low and then high magnifications in a mapping and analysis 
progression similar to that described above for PapNet. Images 
obtained are analyzed by one of a series of parallel-processing 
computers housed within the device. Each slide receives a “device 
score” that is proportional to the probability that abnormal cells 
are located somewhere on the slide. Runs of slides (typically in 
groups of 100 or greater) are then stratified according to their 
“risk” for containing abnormality. In this fashion, the device 
orders a population from those at lowest risk of containing 
abnormality to those at highest risk. This feature is exploited in 
the slide-screening process, as will be described below. Inherent 
in the device is also the ability to identify locations on the slide 
where cells having the highest probability of being abnormal 
are located. This feature is exploited, in conjunction with slide 
rank ordering, in the location-guided screening application of 
the device (FocalPoint GS System).
Quality Control Application
As with PapNet, the first FocalPoint application tested and 
USFDA-approved in 1996 was as a QC rescreening device. In 
this mode of operation, previously manually screened slides 
that had been designated as “negative” are rescreened by the 
device. Because the highest ranking slides are at greatest risk 
for containing abnormal cells, it is in this population that 
false-negative slides are most commonly located. As illustrated 
in Fig. 34.12, the Clinical Laboratory Improvement Amend-
ments (CLIA) of 1988-mandated 10% random rescreening 
process has the potential to identify, at a maximum, only 10% 
of false-negative cases. This compares with the FocalPoint 
stratification process, which preferentially favors detection of 
such cases within the highest ranked group of slides. Using 
the USFDA-approved process of FocalPoint QC rescreening 
of the highest scoring 15% of slides, clinical trials showed that 
the FocalPoint QC system identified a four-fold increase in 
false-negative cases and a seven-fold increase in false-negative 
HSIL cases (Fig. 34.12).84,85 Following approval, other investi-
gators also confirmed the increased detection of false-negative 
cases using this method.86,87 However, as with PapNet’s QC 
application, the aim of FocalPoint QC was as a stepping stone 
to the more useful primary screening functionality. During the 
acquisition of data in the QC clinical trials and in-use applica-
tions, experience was gained on the operating characteristics 
of the device. The FocalPoint was shown to operate in a pre-
cise manner—indicating that slide scoring and ranking were 
reproducible from run to run.88 In addition, slides showing a 
wide variety of pathologic conditions were run on the device 
to ensure that the algorithms were robust for detection of the 
greatest range of abnormalities.84,85 This latter data-set was of 
significant importance in showing the efficiency needed for 
the FocalPoint primary screening application. Inthis data-set, 
approximately 90% of HSILs (n = 587) were scored in the top 
20% of ranked slides and 98% were present in the top 30%. 
For invasive carcinoma (n = 147), 98% were present in the top 
30% of ranked slides. Ranking data such as these for high-grade 
lesions has since been independently confirmed in a number 
of studies.89–91
Primary Screening Application
The FPPS incorporates the slide ranking of the QC system with 
the ability to triage low-scoring slides to a machine-designated 
“negative” status without further manual screening. As illus-
trated in Fig. 34.13, slides scanned by the device are ranked. 
Slides are then designated into two broad categories, the first 
of which includes those scoring in the higher rankings, which 
Fig. 34.11 The FocalPoint device is a robotic automated microscopy 
station with onboard computers. It can process about 300 conventional or 
Surepath slides in a 24-h period.
Automation in Cervical Cytology
34
are designated as “Review” and will be triaged to a full manual 
screening. Within the “Review” category, slides are further sub-
divided into “quintiles,” or groups, each constituting 20% of 
the “Review” total. Quintile 1 represents the highest scoring 
20%, quintile 2 the next highest scoring 20%, and so forth. The 
 second broad category includes those slides scoring in the low-
est portion of the rankings. These slides are compared with a 
fixed “primary” threshold that is unique to and determined in 
each laboratory based on preparation and staining characteris-
tics of that laboratory. This primary threshold is determined by 
a procedure referred to as a local process compatibility analy-
sis (LPCA), in which a series of at least 100 known “negative” 
slides are run and scored by the device in order to develop a 
“distribution curve” for normal slides. In this fashion, the pri-
mary threshold can then designate a predetermined percentage 
of normal slides in any subsequent clinical run that fall below 
this score as “No further review (NFR).” In the currently USFDA-
approved version of the FPPS device, the “primary” threshold 
is set at a point that allows a maximum of 25% of each slide 
run to be designated into this NFR category. The NFR slides can 
be reliably signed out as “Negative for intraepithelial lesion or 
malignancy (NILM)” with no manual screening or QC rescreen-
ing. QC rescreening is performed on only 15% of the highest 
scoring slides in the “Review” category that have been desig-
nated as NILM initially by manual review. As learned from the 
QC application of the FocalPoint device, the greatest concentra-
tion of false-negative cases will be present in this high-scoring 
population of slides. Interestingly, data show that performing 
QC rescreening on the NFR population will decrease the spe-
cificity of the overall process, as any putative “abnormal” case 
identified is likely to represent a false positive.42 One commonly 
held misconception regarding the FPPS process is that the rank-
ing process followed by triage into “Review” and NFR categories 
will “force” the device to label some abnormal slides as NFR, 
particularly if in any given run there is a high prevalence of 
abnormal cases. It is important to remember that the primary 
threshold is at a fixed score level, and the 25% NFR population is 
a maximum. If in a given run less than 25% of slides score below 
the primary threshold, the triaging process will place less than 
25% of slides into the NFR population. In the extreme circum-
stance in which all slides in a given run are abnormal, the FPPS 
would have an NFR population of 0%, as all slides should score 
at levels above the primary threshold.
To date, the USFDA clinical trial of the FPPS for convention-
ally prepared slides is the largest prospective cytology trial ever 
completed, with over 25 000 slides entering into the analysis. 
The trial directly compared manual screening with FocalPoint-
assisted screening, using the process as described above, in a 
two masked armed prospective fashion that was fully adjudi-
cated to allow for determination of “truth” when the two arms 
of the study were not in agreement. In that trial, the FocalPoint 
arm outperformed the manual screening arm for detection sen-
sitivity at every level of abnormality, as shown in Fig. 34.14. For 
the category of HSIL and above, the FPPS detected 97% of cases 
Fig. 34.12 Principles of the FocalPoint quality control (QC) case selection process. In the Clinical Laboratory Improvement amendments of 
1988-mandated 10% random rescreening process, the maximal number of false-negative cases that can possibly be identified is 10%. Using the 
Focalpoint-directed QC procedure, false-negative cases are stratified into high-scoring groups in which the 15% QC rescreening process has a better probability 
of detection. In a clinical trial setting, this process identified a seven-fold increase in false-negative high-grade squamous intraepithelial lesion cases than 
did the 10% random rescreening process.86,87
Manual practice*
Random QC selection
10% Random
rescreening
False negatives
Negatives
Ab
no
rm
al
 d
is
tri
bu
tio
n
in
 ra
nd
om
 p
op
ul
at
io
n
25% No
further review
≥75%
review
Focal point™ Assisted practice*
Enriched QC selection
Ab
no
rm
al
 d
is
tri
bu
tio
n 
in
 F
oc
al
Po
in
t 
cl
as
si
fie
d 
po
pu
la
tio
n
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Special Techniques in CytologyPART THREE
compared with manual screening HSIL-positive detection of 
93%, although this result was not statistically significant. For 
all abnormalities, the FPPS improved detection over manual 
screening by 7%, and this result was statistically significant.93,94 
As indicated above, the triage to an NFR category, in which no 
false-positive cases can be present, leads to improved specificity 
of the overall process—indicating that using the FPPS system 
moves the operator to a higher level receiver operator curve, 
indicating overall better accuracy. Studies following the USFDA 
approval have confirmed and extended the results obtained in 
the pivotal clinical trial. Studies have shown that few, if any, 
significantly abnormal slides are identified in the NFR popula-
tion.85,90,91,95,96 In one study, a single false-negative microinva-
sive carcinoma was identified in the NFR population, which 
was reclassified as “AGC” on review, from over 150 000 cases 
screened by the FocalPoint system. These authors, along with yet 
other studies, have concluded that the negative predictive value 
of the NFR population is extremely high and negates a need to 
perform QC rescreening in this group.97,98 Walts and Thomas 
showed that no cases that were high-risk human papillomavirus 
(HPV)-positive were triaged into the NFR population.99 In the 
US market, users are limited to a maximum of a 25% NFR pop-
ulation; however, users internationally have shown clinically 
acceptable results using the device with a primary threshold set 
to obtain as high as a 50% NFR population, further enhanc-
ing the productivity of the use of this device. (The pivotal clini-
cal trials of the FPPS had some interesting sidebars based on 
the overall data analysis. Interesting, the false-negative rate of 
manual screening for any abnormality [ASCUS and above] 
was 21%—very close to the 20% figure that has been “tossed 
Primary
threshold
QC
threshold
FocalPoint score0.0
Normal population
Abnormal population
1.0
Fig. 34.13 the principle of the FocalPoint slide scoring and ranking 
process. each slide processed by the Focalpoint receives a slide score ranging 
from 0 (very low probability of abnormality) to 1 (very high probability). 
abnormal slides “sort” toward the right side of the scale, whereas negative 
slides do the opposite. there is considerable overlapof these populations due 
to many benign “mimickers” of neoplasia that cannot be adequately resolved. 
however, setting a primary threshold below which cells are most likely to be 
negative improves productivity (these slides do not require manual screening) 
and accuracy (through reduction in false positives). Setting a quality control 
threshold allows directed rescreening of “high risk” cases deemed to be 
negative on initial manual screening. QC, quality control.
around” as a potential, but never well-documented, screening 
false-negative rate for cervical cytology. In addition, because 
the trial model utilized 10% random rescreening for QC in the 
manual screening arm, the detection rate for false-negatives in 
this process could be determined [as compared with identified 
abnormal cases in the FocalPoint arm]. When this calculation is 
performed, the false-negative rate for manual QC rescreening is 
75%! This is the first hard evidence substantiating claims that 
manual QC rescreening is a very inefficient process.92)
In 2001, additional USFDA studies using the FPPS with 
SurePath liquid-based slides were completed and successfully 
showed that performance compared with manual screening 
was increased using this type of preparation. With SurePath, 
the FPPS was found to improve HSIL+ detection at a level 
reaching statistical significance, and again the results showed 
overall performance improvement with better specificity of 
detection noted when compared with a manual screening pro-
cess.100 Studies following approval have shown that FocalPoint 
screening with SurePath samples is equivalent for the detection 
of HSIL+ but that detection of LSIL may be less than with man-
ual screening alone.101 In addition, FocalPoint studies using 
SurePath slides have shown that rank ordering of abnormality 
within the FocalPoint quintile system remains similar to that 
described for conventional slides, with HSIL+ cases virtually 
always showing very high scores (Table 34.3).
Table 34.3 shows FocalPoint ranking data for SurePath slides 
in each diagnostic category. Note the stratification of abnormal 
cases to the highest scoring quintiles. For high-grade squamous 
intraepithelial lesion+, cases were found predominantly in 
quintiles 1 and 2 and never lower than quintile 3. Occasional 
cases of low-grade squamous intraepithelial lesion and atypical 
squamous cells of undetermined significance were found in the 
“No further review” group and would be false negative; how-
ever, the rate in which they fall here is less than generally noted 
for manual screening false-negative cases.89,90
ASCUS+
CP Arm
AP Arm
LSIL+ HSIL+
0
25
50
75
100
%
 d
et
ec
tio
n
Fig. 34.14 FocalPoint/conventional smear clinical trial results. In 
this initial primary screening clinical trial, Focalpoint-assisted screening was 
shown to be more sensitive for the detection of abnormalities at every 
level. Fp, Focalpoint-assisted practice; aSCUS, atypical squamous cells of 
undetermined significance; Cp, routine clinical practice (manual screening 
only); hSIL, high-grade squamous intraepithelial lesion; LSIL, low-grade 
squamous intraepithelial lesion.
Automation in Cervical Cytology
34
l
Low cellularity cases automatically placed into Quintile 5. 
The FPPS has several other operational features worth not-
ing. Slides that cannot be successfully scanned are designated as 
“Process review.” This population of cases is generally less than 
3% with SurePath slides (D. Wilbur, unpublished data), and users 
are required to send such cases on for a full manual review. One 
other important feature is that the FPPS makes an assessment of 
squamous cellularity on all cases and is programmed to never 
place poorly cellular samples into the NFR population. This is 
important because invasive carcinoma samples, which may be 
poorly cellular with obscuring blood and diathesis material, 
may receive a low device score. As a safety measure, the FPPS 
always places such cases into the fifth quintile (the lowest cat-
egory requiring a manual review). Therefore these cases will 
always receive a manual screening despite a score that may have 
otherwise caused them to be placed below the primary thresh-
old. Data from a seeded study with large numbers of invasive 
cancers have shown the value of this system, as low-cellularity 
cancers have been reliably placed into this review category.89 
It therefore has been noted that the highest “risk” FocalPoint 
quintiles are the first (in which most HSILs are present) and 
the fifth (in which poorly cellular invasive carcinomas can be 
found) (Table 34.4).
Table 34.4 shows FocalPoint ranking data for SurePath slides 
and the importance of quintile 5 “scantly” cellular specimens. 
The FocalPoint system has a built in “safety net” algorithm 
designed to ensure that poorly cellular samples will always be 
reviewed by human screeners. This was designed as such because 
it is well known that poorly cellular samples may preferentially 
harbor malignancy because of dilutional effects caused by excess 
blood and necrotic material. As such, low-cellularity cases are 
always placed into quintile 5 to ensure a manual review. Note 
that in this study, which included 87 cancers, 18 were “low cel-
lularity” specimens placed into this group by the method noted 
above. This principle makes quintiles 1, 2, and 5 the “highest 
probability” quintiles—1 and 2 for high-grade squamous 
intraepithelial lesion-positive and 5 for cancer.
Location-guided Screening Application
As indicated above, the FocalPoint system has the ability to not 
only rank order slides but also to identify the locations on the 
slide where abnormal cells are most likely to be found. The slide 
score is a cumulative process in which the scores of individual 
FOVs are summed to achieve the total slide score. The highest 
scoring FOVs would therefore be most likely to contain abnor-
mal cells. The ability to present these FOVs to manual screen-
ers would be a further improvement in the overall process by: 
(1) increasing productivity and (2) increasing accuracy by apply-
ing the previously noted principle of “increased prevalence 
leads to increased detection sensitivity.” With the device hon-
ing down the population of potential “needles in the haystack,” 
the manual screener will have a higher likelihood of successfully 
identifying them. The process allows for FOV review to triage to 
a full manual review if abnormality or potential abnormality is 
identified, or to triage direct to sign out as NILM in the instance 
in which no potential abnormality is identified on FOV-only 
review. At present, the device (in its SurePath-mated configura-
tion) identifies 10 FOVs per case, which are rank ordered from 
highest to lowest probability of containing abnormality.
(after parker et al. 2004,89 with permission.)
 
First
 
Second
 
Third
hSIL+ 85 22 2
LSIL 47 21 11
aSCa/aGC 54 23 12
aGC, atypical glandular cells of undetermined significance; aSC, atypical squamous ce
LSIL, low-grade squamous intraepithelial lesion.
atotal aSC-positive over third rank: 84%.
(after Vassilakos et al. 2002,90 with permission.)
Table 34.3 Focalpoint/Surepath Study: 9665 Cases Studied 
Quintile HSIL+ (%) HSIL (%)
1 60 (58) 19 (66)
2 25 (24) 7 (24)
3 11 (11) 3 (10)
4 6 (6) 0 (0)
5 1 (1)–21a 0 (0)–3a
No further review 0 (0) 0 (0)
total 103–21 29–32
Quintiles 1 and 2 85 (83) 26 (90)
Quintiles 1–3 96 (93) 29 (100)
aIS, adenocarcinoma in situ; hSIL, high-grade squamous intraepithelial lesion.
a
Table 34.4 Focalpoint ranking of Surepath Slides 
 
Fourth
 
Fifth
No further 
review
0 0 0
10 5 6
5 4 2
ls of undetermined significance; hSIL, high-grade squamous intraepithelial lesion; 
AIS (%) Carcinoma (%)
0 (0) 41 (59)
1 (20)17 (25)
2 (40) 6 (9)
2 (40) 4 (6)
0 (0) 1 (1)–18a
0 (0) 0 (0)
5 69–87
1 (20) 58 (84)
3 (60) 64 (93)
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Special Techniques in CytologyPART THREE
Early after the FPPS was approved for use in the United 
States, this location-guided screening capability was exploited 
using a manual location transfer method by investigators in 
Taiwan and Hong Kong, with data showing overall abnormal 
detection performance improvement with substantial gains in 
screening productivity.102,103 The study by Chang et al. is also 
of interest because the final interpretation was made using the 
FOV review only, not triaging the slide to a full manual review 
when an abnormality was initially detected. This study showed 
the importance of the triaging process, because although an 
abnormal interpretation was capable of being made using FOV 
review alone, the final diagnosis rendered was in some cases not 
at the level of abnormality obtained when the entire slide was 
reviewed. This study illustrates the need for a full manual review 
procedure with this location-guided screening device in order to 
maximize the accuracy of the final interpretation.103
In a two-armed masked prospective study using this device 
with an integrated robotic microscopy station (Fig. 34.15) that 
allowed for automated localization and review of the device-
selected FOVs, data showed that 100% of adjudicated HSIL+ 
slides were appropriately triaged to manual review. After man-
ual review, 98% were correctly forwarded for pathologist review 
using this system, as compared with 91% with manual screen-
ing alone. In this study, again, a gain in specificity of abnormal 
case detection was also noted.104 Further studies have confirmed 
the equivalence of the FocalPoint location-guided screening sys-
tem to manual screening for the detection of HSIL+105,106 and 
SIL+.107
The device ensures that the location identified on the robotic 
microscope is as intended via the use of images captured at the 
selected FOV sites during the screening process. These images 
are displayed on the monitor for the reviewer at the work-
station to confirm that the proper location is being viewed 
in the microscope (Fig. 34.16).
At present, the FocalPoint GS location-guided screening 
 system is in clinical trials for use in the United States.
The Cytyc ThinPrep Imaging System
In 2003, an approval was granted by the USFDA for the Thin-
Prep Imaging System. This device is a location-guided screen-
ing instrument that is mated to ThinPrep liquid-based slides. 
The device is a personal computer-based system designed to be 
Fig. 34.15 FocalPoint review scope (Slide Wizard). In the Focalpoint GS 
model, locations of potentially abnormal cells are identified by the scanning 
device and transferred to this station for cytologist review.
located within the clinical cytology laboratory. The scanning 
computer (Fig. 34.17) is loaded with cassettes of ThinPrep slides 
and scanning proceeds in a walk away manner with a robotic 
microscope. The computer analysis is designed around a special 
Papanicolaou-like stain that is stoichiometric for the DNA con-
tent of cell nuclei. Thus the device primarily performs a DNA 
analysis on each cell, looking for cases with abnormal DNA con-
tent. As is well known from numerous prior studies dating back 
many years, the DNA content of dysplastic and neoplastic cells 
is not diploid and hence detection of nondiploid cells in the 
sample indicates a risk of abnormality. In addition, other mor-
phometric parameters such as cell size and shape are also report-
edly included in the cellular analysis. The device identifies 22 
FOVs per ThinPrep slide and transfers these locations to a com-
puterized microscopy review station (Fig. 34.18). The cytologist 
Fig. 34.16 FocalPoint review scope computer screen, showing 
navigation tools and reporting menus. the slide image showing locations 
is at the bottom of the field, and the black and white image saved by the 
Focalpoint ensures that the field of view presented by the review station is 
the actual area of high probability designated by the device.
Fig. 34.17 Cytyc ThinPrep Imaging System device. this is a personal 
computer–based multiload “walk away” device that consists of a robotic 
scanning microscopy station into which cassettes of slides are placed. 
Automation in Cervical Cytology
34
reviews the identified FOVs, which are relocated automatically 
by the use of a mouse-driven integrated robotic stage. FOVs are 
relocated geographically from one end of the cellular circle to 
the other (as opposed to the FocalPoint hierarchic ranking proc-
ess). If no abnormality is detected in the 22-FOV review, the slide 
may be signed out as NILM without further manual screening. If 
any potential abnormality is identified, the slide must go on for 
a complete manual screening, which is performed on the same 
microscope in an automated fashion. After a full manual screen-
ing, there is the option to automatically place ink dots on the 
slide for further non–device-aided slide review. An image of the 
microscopic review field with cell localization reticle is shown in 
Fig. 34.19. Fiducial markings (see Fig. 34.4B) present in the 
ThinPrep slide ensure proper localization of the device-selected 
FOV in the review microscope.
Clinical trials of the ThinPrep Imaging System have 
shown statistically equivalent sensitivity for the detection of 
HSIL and greater and LSIL and greater lesions and statistically 
Fig. 34.18 ThinPrep Imager System review scope station. In a fashion 
similar to the Focalpoint review station, the thinprep Imaging System 
review station receives coordinates from the imager and automatically 
directs the cytologist to high-probability fields of view (FOVs) for rapid 
examination. twenty-two FOVs are generally designated on each slide, and 
if no abnormality (or potential abnormality) is identified on that review, the 
slide can be designated as negative without further manual screening. If 
an potential abnormality is identified, complete slide review is required to 
ensure that the most accurate classification ensues. the microscope is fully 
automated and is controlled by a mouse-like “pod” device seen on the right 
side of the scope.
increased specificity for the detection of HSIL and greater 
lesions. In addition, statistically superior sensitivity for the 
detection of ASCUS and greater lesions and statistically equiv-
alent specificity for the detection of ASCUS and greater and 
LSIL and greater lesions were shown in the clinical trial data.45 
Productivity utilizing this process improved substantially, such 
that USFDA labeling of the device allowed cytology screeners 
to examine 200 FOV-only cases during an 8-h work period, as 
opposed to the current CLIA-mandated maximum of 100 slides 
per 8-h period. The process continues to implement the cur-
rent CLIA-mandated 10% random and targeted QC rescreening 
requirement, similar to fully manual screening.
Post approval, a number of clinical studies have docu-
mented field performance of the device. In two large stud-
ies comparing imager performance on approximately 
80 000–100 000 cases each with an historical control popula-
tion of similar numbers of manually screened cases, the Thin-
Prep Imaging System identified more LSILs (8 and 37%) and 
more HSILs (13 and 42%).109,110 One of the studies showed an 
estimated false-negative rate that was halved for the studied 
populations when using the imaging device.110 Another study 
showed similar increases in SIL cases identified but also docu-
mented a decrease in HPV positivity in cases of ASCUS identified 
using the imager, indicating that increases in detection of this 
equivocal category are most likely due to overcalls of reactive 
findings.111 One important utility gained by the automatedscreening process is improved productivity. A postimplementa-
tion timing study showed that overall screening time using the 
device was reduced by 42% over manual screening.112 Interest-
ingly, in this study a number of imager “false negative” cases 
were identified by manual screening. On review of each of these 
cases, all had abnormal cells within the device-identified FOVs, 
indicating that operator, and not device, error was responsi-
ble for the missed cases. The authors indicate the necessity for 
review of the entire FOV and not just the central areas in order 
Fig. 34.19 ThinPrep Imager System review scope microscopic field of 
view. the view in the microscope highlights the central area of interest as 
designated by the thinprep Imaging System scanner. the 
L-shaped reticle shows the location where the automated dotting function 
that is inherent in the thinprep Imaging System review scope will place a dot 
at the request of the cytologist on completion of the review process.
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1036
Special Techniques in CytologyPART THREE
to maintain maximal detection sensitivity in the imager-based 
process.112 The ability to make specific abnormal interpreta-
tions from the FOVs only (without triage to a full manual 
review when potential abnormality is detected) was addressed 
in another study. The authors found that the triage to full 
manual review is an essential part of the overall process, as 
FOV-only final interpretations were found to be statistically sig-
nificantly less sensitive and specific when compared with manual 
screening alone. In the same study, when the recommended 
procedure of triage to full manual review was utilized, the sen-
sitivity and specificity increased and was equivalent to manual 
screening alone.113 As was similarly described previously for the 
FocalPoint GS location-guided screening process, the ThinPrep 
Imaging System is not fully capable of making final interpreta-
tions in every case based on FOV review alone—the triage func-
tion is acceptable in order to make a determination of negative 
versus “possibly abnormal,” but a reliably accurate final inter-
pretation rests on full manual review.
Laboratory Process Issues Associated with 
the Use of Automated Devices
Implementation of automated systems in the cytology labo-
ratory requires cognizance of and adherence to an increased 
number of procedural and regulatory issues.
Fig. 34.20 Sample report of cytology specimen utilizing the ThinPrep 
Imaging System. In this Bethesda format patient report, note that the use of 
the thinprep Imaging System for initial slide screening is clearly 
indicated.
Reporting Issues
The Bethesda System indicates that the final report should 
indicate what type of specimen was processed: conventional 
slide, liquid-based preparation, or other.114 Some laboratories 
have chosen to indicate which type of liquid-based method 
was utilized, as this may have implications for further test-
ing procedures. In addition, if automated screening devices 
are utilized, the final report should indicate: (1) the type 
of instrumentation utilized, (2) whether the specimen was 
successfully processed, and (3) the result of the process, if 
applicable. For instance, use of the ThinPrep Imaging System 
requires some form of manual review in all cases, and there-
fore mention of the scanning device is all that is necessary. 
However, with the FPPS, a statement regarding the instrument 
result—NFR, “Review,” etc.—would be necessary. Examples 
of report verbiage for the use of these devices are shown in 
Figs 34.20 and 34.21.
Issues with Specimens that Cannot Be 
Successfully Processed
Not all specimens can be successfully examined and reported by 
automated instruments. In the early days of the FocalPoint QC 
and primary screening applications, many slides were rejected 
by the device because of technical issues related to slide quality 
or other characteristics. Rejections for “tilted cover slips,” une-
ven distribution of mounting media, slides with fingerprints, or 
extension of mounting media from beneath the cover slip were 
Fig. 34.21 Sample report of cytology specimen utilizing the FocalPoint 
System. In this Bethesda System format patient report, the use of the 
Focalpoint Screening System is indicated. In addition, the result of the 
screening process—review, no further review, or process review—is 
also indicated. this information indicates to the reader whether the slide has 
or has not received a manual review.
Automation in Cervical Cytology
34
all common, sometimes constituting as many as 15–20% of 
each run. In more recent FocalPoint versions, software upgrades 
have decreased these “Process review” cases to the range of 
2–3% of each run (D. Wilbur, unpublished data). For the Thin-
Prep Imaging System, “Process review” cases generally make up 
between 5 and 8% of all cases (D. Wilbur, unpublished data). 
For all such rejected specimens, full manual review of the slides 
is required.
Stain Use in Automated Systems
For liquid-based cytology, any standard Papanicolaou stain can 
be utilized. In the ThinPrep Papanicolaou test, slides are not 
stained by the preparation devices and are stained by whatever 
method the laboratory chooses. For the SurePath method, the 
slides can be stained by the PrepStain device using a propri-
etary stain mixture provided by Becton Dickinson/TriPath or 
one of the laboratory’s choosing. Automated screening with the 
ThinPrep imager, however, requires a special proprietary stain 
from Hologic that is specifically mated to imager performance. 
This stain is similar to the routine Papanicolaou method but 
has Feulgen-like properties allowing for DNA quantitation. In 
practical use, the ThinPrep imager stain shows darker nuclear 
staining than a standard Papanicolaou method, which requires 
a familiarization process.
Any routine Papanicolaou stain can be utilized with the 
FocalPoint System; however, the stain must fall within certain 
optical parameters. Compliance with these standards is contin-
uously monitored by the device during screening. Differences 
in stain between laboratories are accounted for by the LPCA 
process described above. Thus slides and the FocalPoint device 
are “mated” in a given laboratory, and hence slides not stained 
according to the laboratory protocol (e.g. consult slides stained 
elsewhere) should not be processed.
Training Required for Initiation of Automated 
Methods
The USFDA has, in their approvals for the liquid-based prepa-
ration methods, mandated that all users of the technology 
undergo company-sponsored operation and morphology 
training programs. These programs are generally given at cor-
porate training facilities and last between 1 and 3 days. Indi-
viduals trained in these programs are then “certified” as users 
and are able to train other individuals in their laboratories to 
operate the devices and manually interpret the slides. These 
programs generally consist of demonstrations and hands-on 
operation of the devices, reviewing of known and unknown 
slides, didactic sessions, and a final “proficiency examination.” 
Prior to initiation of liquid-based methods in a laboratory, it 
is recommended that users gain experience with the method 
via a series of split samples in which conventional smears are 
initially made with the residual cell material being used to 
make paired liquid-based slides. Comparison of morphology 
between the two methods during initiation will ensure correct 
device operation and will provide confidence to new users. 
Laboratories generally run between 100 and 500 split-sample 
studies prior to complete conversions. Fortunately, virtually all 
automated functions in the preparatory areas can be handled 
by trained preparatory technicians. Cytotechnologists are not 
required to perform any of the automated