Journal of Clinical Virology 45, S1 (2009) S13 S17

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Journal of Clinical Virology

j o u r n a l h o m e p a g e : www.elsevier.com/locate/jcv

Principles and analytical performance of Abbott RealTime High Risk

HPV test
Shihai Huanga, *, Ning Tanga , Wai-Bing Maka , Brian Ericksona , John Salituroa , Yuhong Lia , Evelyn Krumpeb ,
George Schneidera , Hong Yua , John Robinsona , Klara Abravayaa
a
Abbott Molecular Inc., Des Plaines, Illinois, USA
b
Abbott GmbH & Co. KG, 65205 Wiesbaden-Delkenheim, Germany

ARTICLE INFO ABSTRACT

Keywords: Background: Abbott RealTime High Risk (HR) HPV is a new automated, qualitative real-time
m2000 PCR test for detection of DNA from 14 high-risk human papillomavirus (HPV) types (16, 18,
Abbott RealTime High Risk HPV 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68) in cervical specimens. The test can also
HPV 16/18 typing differentiate between HPV 16, HPV 18 and non-HPV 16/18 types in a single reaction.
real-time PCR Objectives: This article describes the principles of assay design and the analytical
cervical cancer screening performance of Abbott RealTime HR HPV.
Study Design: The analytical performance characteristics of Abbott RealTime HR HPV were
evaluated in terms of its sensitivity for each of the 14 high-risk types included in the test,
specificity (cross-reactivity), potential for interference by substances that may be present in
cervical specimens, and reproducibility.
Results: Abbott RealTime HR HPV provided sensitive detection of the 14 high-risk HPV types
included in the test. It was also highly specific to the HPV types targeted by the test and
did not show cross-reactivity with 15 low-risk HPV types tested, or non-specific reactivity
with other common microorganisms that may be present in the female anogenital tract. Test
results were not impacted by potential interfering substances evaluated in the study. The
test generated highly reproducible results in an in-house study and in studies carried out at
13 external evaluation sites.
Conclusions: Abbott RealTime HR HPV demonstrated a robust analytical performance with
reproducible and reliable results.
© 2009 Elsevier B.V. All rights reserved.

1. Abbreviations progression relative to other high-risk types.5 Therefore, type-

Ct: threshold cycle number
HPV: human papillomavirus
HR: high risk
IC: internal control
2. Introduction
Human papillomavirus (HPV) is the etiological agent for
cervical cancer.1,2 Persistent cervical infection of high-risk HPV
types has been well established as the main underlying cause
of cervical cancer and its immediate precursors.3 HPV 16 and
18 together account for approximately 70% of cervical cancers
worldwide.4 There is considerable evidence that HPV 16 and
18 are associated with a much higher risk of cervical disease
* Corresponding author. Abbott Molecular Inc.,
1300 E Touhy Ave., 1D, Des Plaines, IL 60018-3315, USA.
Tel.: +1 224 361 7362; fax: +1 224 361 7507.
E-mail address: shihai.huang@abbott.com (S. Huang).
specific identification of HPV 16 and 18 may be valuable in
routine clinical practice. The utility of HPV testing has been
demonstrated in various areas dealing with cervical cancer pre-
vention strategies, including primary cervical cancer screening,
triaging of patients with equivocal cytological abnormality, and
post-treatment follow-up.6 In addition, in the era of HPV vac-
cination, HPV testing will assume a greater role in screening
and surveillance and will be essential for monitoring vaccine
efficacy.7,8 In this regard, further incorporation of new molec-
ular technologies into HPV testing may lead to improvements
in testing modalities and patient care and management.
Abbott RealTime High Risk (HR) HPV is a recently developed
test for the detection of 14 high-risk HPV types, 16, 18, 31, 33,
35, 39, 45, 51, 52, 56, 58, 59, 66, and 68, in cervical specimens.
It is an automated, qualitative in vitro test, and utilizes real-
time PCR technology. By using a multi-color detection system,
this test can also differentiate between signals from HPV 16,
HPV 18 and the non-HPV 16/18 high-risk types in the same re-

1590-8658/ $ see front matter © 2009 Elsevier B.V. All rights reserved.
S14 S. Huang et al. / Journal of Clinical Virology 45 (2009) S13 S17

action, thus providing value-added information in HPV testing. Table 2, when DNA containing sequences from different HPV
The test also features an internal control (IC) for specimen ade- types was tested at a concentration of 107 copies per PCR
quacy by detecting an endogenous human b-globin sequence. In reaction, DNA from each type was only detected with the
addition, external positive and negative controls are included corresponding type specific probe.
in each run to assess run validity. Test results are automatically
reported by assay software that evaluates key characteristics 3.2. Assay procedure
of real-time PCR data against quality assurance parameters to Abbott RealTime HR HPV test procedure consists of sample
assess result validity.9 Abbott RealTime HR HPV can be per- preparation, reaction assembly, real-time PCR, and result
formed on three sample preparation platforms that satisfy lab- reporting. During sample preparation, 0.4 mL of sample is
oratory needs for low, medium and high levels of automation. processed using the Abbott mSample Preparation SystemDNA
The principles and the analytical performance charac- where sample is lysed with chaotropic reagents, and DNA is
teristics of Abbott RealTime HR HPV assay are described captured with magnetic microparticle technology. Unbound
in this article. Two initial studies evaluating the clinical sample components and inhibitors are washed away and the
performance characteristics of the test are also published in bound purified DNA is eluted and is ready for amplification.
this supplement.10,11 Abbott RealTime HR HPV is currently validated for use with
cervical specimens collected in PreservCyt solution (Hologic
3. Overview of the assay Inc., Marlborough, MA, USA). Specimens can be transported
3.1. Primers and probes at room temperature or refrigerated, and may be stored up to
4 months at room temperature or up to 6 months refrigerated
The HPV target sequence for Abbott RealTime HR HPV is located
or at 10ºC or colder following collection. At the completion
in the conserved L1 region of the genome. A modified GP5+/6+
of sample preparation, an amplification master mix is created
primer mix consisting of three forward primers and two reverse
with AmpliTaq Gold enzyme (Roche Molecular Systems, Inc.
primers is designed to hybridize to an approximately 150 base
Branchburg, NJ, USA), magnesium chloride, and oligonu-
HPV consensus region. The IC target sequence is amplified using
cleotide reagent containing primers, probes and dNTPs. The
a primer set targeting a region of 136 bases in the human
PCR reaction is then assembled in a 96-well optical reaction
b-globin gene. The HPV and IC probes are single-stranded DNA
plate by combining aliquots of the master mix and extracted
oligonucleotides modified with a fluorescent moiety covalently
DNA eluate. Three sample preparation methods can be used
linked to one end of the probe and a quenching moiety to
with Abbott RealTime HR HPV: the fully automated m2000sp for
the other end. Through the distinct labels, signals for HPV 16,
medium-to-high test volume that processes up to 96 samples
HPV 18, non-HPV 16/18 types, and IC can be simultaneously
in a run, the automated m24sp for low-to-medium test volume
detected and distinguished in a single reaction (Table 1).
that processes up to 24 samples in a run, and a manual sample
Fourteen type specific HPV probes were designed where each
preparation method. All three sample preparation methods
probe only detects the corresponding HPV type. As shown in
use the same reagents and the same procedure. In addition
to automated sample preparation, the m2000sp also provides
Table 1 automated sample eluate transfer and PCR plate assembly.
Probe labeling
Thermocycling and fluorescence detection of the amplified
Fluorescent label Type specific probe products are carried out in the m2000rt real-time PCR
VIC 16 instrument. Assay results are automatically reported by the
NED 18 m2000rt at the end of real-time PCR. For each sample, all
FAM 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68 three HPV signals, corresponding to HPV 16, HPV 18, and non-
Quasar Internal Control (human b-globin) HPV 16/18 types, and the IC signal are evaluated. Samples
with any of the three HPV signals detected will have an
interpretation of “HR HPV Detected”. In addition, each
Table 2 detected signal is reported individually. Samples with all three
Hybridization specificity of high-risk HPV probes a
HPV signals not detected will have an interpretation of “Not
HPV HPV probe type Detected”. Error codes or flags are reported or displayed for
target 16 18 31 33 35 39 45 51 52 56 58 59 66 68 results exceeding validity specifications. Assay results can be
type
delivered via a laboratory information system.
16 + With the fully automated m2000sp/rt, the turnaround time
18 + (from sample preparation to reporting results) is approximately
31 + 6 hours for 96 samples. The same system is also used for
33 +
multiple Abbott RealTime assays including HIV-1, HCV, HBV,
35 +
CT/NG and CT.
39 +
45 +
3.3. Controls
51 +
52 + A negative control and a positive control are required for each
56 + run to verify that the sample processing, amplification, and
58 + detection steps are performed correctly. The negative control
59 + is formulated with DNA containing the b-globin sequence. The
66 + positive control is formulated with DNA containing the HPV 16,
68 +
HPV 18, HPV 58, and IC sequences. Both controls contain poly
a
“+” denotes Detected; “ ” denotes Not Detected. dA:dT as carrier DNA.
 S. Huang et al. / Journal of Clinical Virology 45 (2009) S13 S17 S15

Table 3
Cross-reactivity panel
Organisms Concentration Organisms Concentration

Bacteroides fragilis 107 genomic copies HPV 6 107 genomic copies

Candida albicansa 107 CFUb HPV 11 107 genomic copies
Chlamydia trachomatisa 107 EBsc HPV 13 107 genomic copies
Corynebacterium genitalium 107 genomic copies HPV 26 107 genomic copies
Enterobacter cloacae 107 genomic copies HPV 30 107 genomic copies
Enterococcus faecalis 107 genomic copies HPV 32 107 genomic copies
Escherichia coli 107 genomic copies HPV 40 107 genomic copies
7
Gardnerella vaginalis 10 genomic copies HPV 42 107 genomic copies
7
Haemophilis ducreyi 10 genomic copies HPV 43 107 genomic copies
7
Lactobacilllus acidophilus 10 genomic copies HPV 44 107 genomic copies
7
Mycoplasma genitalium 10 genomic copies HPV 53 107 genomic copies
7
Mycoplasma hominis 10 genomic copies HPV 54 107 genomic copies
7
Neisseria gonorrhoeae 10 genomic copies HPV 55 107 genomic copies
7
Neisseria meningitides 10 genomic copies HPV 57 107 genomic copies
7
Proteus mirabilis 10 genomic copies HPV 61 107 genomic copies
7
Staphylococcus aureus 10 genomic copies HSV-I 107 genomic copies
7
Staphylococcus epidermidis 10 genomic copies HSV-II 107 genomic copies
7
Streptococcus pneumoniae 10 genomic copies HBV 107 genomic copies
6 d
Trichomonas vaginalis 10 genomic copies HCV 106 viral RNA copies
7
Ureaplasma urealyticum 10 genomic copies HIV-1 106 viral RNA copies
7
Human cellular DNA 10 genomic copies
a b c d
Cultured microorganisms. Colony forming units. Elementary bodies. Clinical specimen.

4. Analytical performance
4.1. Analytical sensitivity
Analytical sensitivity was determined by testing HPV DNA from
each of the 14 high-risk types included in the test. DNA was
quantified with optical density measurement and was further
diluted in PreservCyt solution in the presence of human cellular
DNA. For each type, a minimum of 4 levels, with 9 replicates
at each level were tested. Testing was performed with three
lots of amplification reagents on three m2000 Systems. Probit
analysis determined that with a probability of greater than
95%, HPV 16, 18, 35, 39, 45, 51, 59, 66, and 68 can be detected
at 500 copies per assay, HPV 31, 33, 52, and 56 at 2,000 copies
per assay and HPV 58 at 5,000 copies per assay.
4.2. Analytical specificity (cross-reactivity)
A panel of forty-one bacteria, viruses and fungi were evaluated
for potential cross-reactivity (Table 3), including 15 low-risk
HPV types and other organisms that can be found in the female
anogenital tract. Human cellular DNA was also evaluated.
Each potential cross-reactant was spiked into assay negative
controls at concentrations (per 0.4 mL sample input) shown in
Table 3. Purified nucleic acids were used except where noted.
Cross-reactivity was not observed with any of the organisms
tested.
4.3. Potential interfering substances
The potential for interference in the test results was assessed
with substances that may be present in cervical specimens.
High-risk HPV negative and HPV positive samples were tested
in the presence or absence of each of the substances listed
in Table 4. Blood and mucus were spiked into PreservCyt
solution at a concentration of 5% and all other substances at
a concentration of 0.5%. Interference or inhibition was not
observed with any of the substances tested.

Table 4
Potential interfering substances tested

Blood Monistat-1 Day or Night Treatment

Mucus Norforms Deodorant Suppositories
Clotrimazole Vaginal Cream (2%) Terazol-3 Vaginal Cream
Delfen Vaginal Contraceptive Foam Vagi-gard Povidone Iodine Medicated Douche
Gynecort 1% Hydrocortisone Anti-itch Creme Vagisil Anti-Itch Creme
KY Jelly Vagisil Intimate Lubricant
Lubrin Yeast-gard Homeopathic Vaginal Suppositories
MetroGel-Vaginal Zovirax Cream (Acyclovir) 5%
Miconazole Nitrate Suppository
S16 S. Huang et al. / Journal of Clinical Virology 45 (2009) S13 S17

30 30
HPV 16 HPV 18
25 25
HPV 16 Cycle Number

HPV 18 Cycle Number

20 20

15 15

10 10

5 5
(a) (b)
0 0
1 2 3 4 5 6 7 8 9 10 11 12 13 1 2 3 4 5 6 7 8 9 10 11 12 13
Evaluation site Evaluation site

30 30
HPV 58 β-Globin
25 25

β-Globin Cycle Number

HPV 58 Cycle Number

20 20

15 15

10 10

5 5
(c) (d)
0 0
1 2 3 4 5 6 7 8 9 10 11 12 13 1 2 3 4 5 6 7 8 9 10 11 12 13
Evaluation site Evaluation site

Fig. 1. Cycle numbers (Cts) of positive control signals across 13 external evaluation sites: (a) HPV 16; (b) HPV 18; (c) HPV 58; and (d) b-globin.

4.4. Reproducibility concurrently in a single assay. The choice of the conserved L1

Reproducibility of the test was evaluated by testing a panel
of 20 well-characterized cervical specimen pools in PreservCyt
(10 HR HPV positive and 10 HR HPV negative). The twenty panel
members were tested by two operators. Each operator, using a
unique combination of reagent lot and instrument pair, tested
two replicates of each panel member per day for four days for a
total of eight replicates. The overall agreement for 319 results
compared with expected results was 100%. The agreement
for 159 comparisons between the two operators using two
different reagent lots and two instruments was 100%. For
positive samples, results for each HPV signal were accurately
reported for all replicates.
Reproducibility was also assessed with data collected from a
multi-site evaluation study involving 13 sites in 11 countries.
Each site conducted multiple runs (up to 15 runs), each run
containing one or multiple replicates of negative control and
positive controls. Results from a total of 151 replicates of
positive or negative controls were included in the analysis. One
lot of control reagents and one lot of amplification reagents
were used at all sites. As shown in Fig. 1, threshold cycle
numbers (Cts) for HPV 16, 18, 58, and b-globin signals in the
positive control were tightly grouped within each site and
across different sites. The intra- and inter-site SDs in Ct were
calculated to be 0.35 and 0.46 for HPV 16, 0.30 and 0.43
for HPV 18, 0.55 and 0.59 for HPV 58, and 0.32 and 0.37 for
b-globin signals. Tight distribution of b-globin signals was also
obtained with the negative control (data not shown), with
intra- and inter-site SDs at 0.26 and 0.43.
5. Conclusions and Discussion
Abbott RealTime HR HPV test has been designed to detect
DNA from 14 high-risk HPV types and to type HPV 16 and 18
region and the use of a modified GP5+/6+ consensus primer mix
ensured broad coverage of relevant HPV genomes. Each probe-
binding region chosen was highly specific to the corresponding
type.
A high-risk HPV test should ideally detect infections that are
associated with high-grade cervical disease while minimizing
positive results due to transient HPV infections. This can
be achieved by optimally balancing clinical sensitivity and
specificity in detecting disease.12 Relative to other technologies
involving one-point determination at the end of a signal or
target amplification reaction, the sensitivity and specificity of
real-time PCR can be optimized much more easily by adjusting
the assay cut-off in Ct. During the development of Abbott
RealTime HR HPV, the impact of different Ct cut-off values
on clinical sensitivity and specificity for detecting high-grade
cervical intraepithelial neoplasia was evaluated. The assay
cut-off of Abbott RealTime HR HPV was established based on
optimal clinical performance for detection of disease, and
not necessarily a higher analytical sensitivity in detecting
high-risk HPV.
Abbott RealTime HR HPV provides robust analytical perfor-
mance and generates reproducible and reliable results. This
study showed an analytical sensitivity of 500 5,000 copies
per assay depending on the type. The test was shown to
be highly specific to the 14 high-risk HPV types included
in the test. No cross-reactivity with 15 low-risk types and
other potential cross-reactants was observed. Test results
were not impacted by various substances tested that may
exist in cervical specimens. Furthermore, highly reproducible
results were demonstrated with clinical specimen pools and
assay controls. Several aspects of the assay design ensure
necessary quality assurance of the test results. The internal
control is designed to control for sample adequacy, DNA
 S. Huang et al. / Journal of Clinical Virology 45 (2009) S13 S17
extraction, and PCR amplification while positive and negative
controls are designed to control for run validity. Sophisticated
data reduction algorithms analyze outputs and assess result
validity and test results are automatically reported without
subjective interpretation. Abbott RealTime HR HPV offers
flexibility in the level of automation and throughput. The fully
automated m2000 System provides 6-hour turnaround time
for 96 samples and minimizes operator errors associated with
manual procedures.
Abbott RealTime HR HPV has the feature of detecting high-
risk HPV and typing HPV 16 and 18 in single or mixed infections
simultaneously. HPV 16/18 typing has been proposed as one
way to increase specificity of HPV testing and to aid in risk
stratification of high-risk HPV positive populations.6,13,14 The
utility of HPV 16/18 typing in vaccine monitoring has also been
recommended.7,8,13,14 Combining the ability of high-risk HPV
detection and HPV 16/18 typing in a single reaction, Abbott
RealTime HR HPV may prove to be a useful tool in evaluating
and optimizing clinical algorithms and may further improve
cost-effectiveness of cervical cancer screening and patient
management.
Acknowledgements: We would like to thank Lei Zhang, Tracy
Miller, and Yuanhe Li for statistical analysis. We would
also like to thank Sam Ratnam for critical review of the
manuscript.
Competing interests: The authors are employees of Abbott
Molecular Inc.
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