Journal of Clinical Virology 75 (2016) 33–36

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Journal of Clinical Virology

journal homepage: www.elsevier.com/locate/jcv

Laboratory audit as part of the quality assessment of a primary

HPV-screening program
Maria Hortlund a , Karin Sundström a , Helena Lamin b , Anders Hjerpe a , Joakim Dillner a,∗
a
Department of Laboratory Medicine, Karolinska Institutet, Sweden
b
Department of Laboratory Medicine, Karolinska University Hospital, Sweden

a r t i c l e i n f o a b s t r a c t

Article history: Background: As primary HPV screening programs are rolled out, methods are needed for routine quality
Received 30 October 2015 assurance of HPV laboratory analyzes.
Received in revised form 2 December 2015 Objective: To explore the use of similar design for audit as currently used in cytology-based screening, to
Accepted 22 December 2015
estimate the clinical sensitivity to identify women at risk for CIN 3 or worse (CIN3+).
Study design: Population-based cohort study conducted within the cervical screening program in Stock-
Keywords:
holm, Sweden, in 2011–2012. All women with histopathologically confirmed CIN3+ in the following two
Primary screening
years were identified by registry analysis. Primary HPV and cytology screening results were collected.
Audit
HPV
For women who had not been HPV tested, biobanked cytology samples were HPV-tested. If the original
Sensitivity HPV result had been negative, the sample and subsequent biopsies were analyzed with broad HPV typing
(general primer PCR and Luminex).
Results: 154 women had a biobanked prediagnostic cytology sample taken up to 2 years before a
histopathologically confirmed CIN3+. The high-risk HPV-positivity was 97% (148/154 women), whereas
143/154 (94%) women had had a cytological abnormality. Among the six HPV-negative samples, one
sample was HPV 33 positive in repeat testing whereas the other five cases were HPV-negative also on
repeat testing, but HPV-positive in the subsequent tumor tissue.
Conclusions: A sensitivity of the HPV test that is higher than the sensitivity of cytology suggests adequate
quality of the testing. Regular audits of clinical sensitivity, similar to those of cytology-based screening,
should be used also in HPV-based screening programs, in order to continuously monitor the performance
of the analyzes.
© 2015 Elsevier B.V. All rights reserved.

1. Background
Cervical screening programs reduce the incidence of cervical
cancer and are globally recommended as a public cancer control
policy [1,2]. Originally, cervical screening programs were based
on cytological examinations. Human papillomavirus (HPV) is the
major cause of cervical cancer [3] and detection of such infection
has now formed the basis for a novel screening technology. HPV-
based screening is more sensitive than cytology in detecting CIN
grade 2 or worse (CIN2+) and provides a greater and more long-
lasting protection against invasive disease [4–7].
There are detailed international guidelines for quality assurance
of cytology-based screening [1,2] and there are also international
∗ Corresponding author at: Department of Laboratory Medicine, Karolinska Insti-
tutet, SE-141 86, Stockholm, Sweden.
E-mail address: joakim.dillner@ki.se (J. Dillner).
laboratory manuals for HPV testing [8]. However, clinical guidelines
for quality assurance of HPV testing when used for primary cervical
screening are much less developed than for cervical cytology. This
could conceivably result in the gains of the ongoing switch to HPV-
based screening programs are diminished. There is thus an urgent
need for research toward development of such quality assurance
systems.
Routine estimation of the clinical sensitivity of cytology for
detecting cases of CIN3 or invasive cancer (CIN3+) by analyzing
smears taken before diagnosis of CIN3+ is routinely used by many
laboratories that perform cytology-based screening, for example in
the Swedish organized cervical screening program [9]. We inves-
tigated the feasibility of using exactly the same laboratory audit
procedures, as currently in use for cytology-based screening, also
in the real-life HPV-based screening used in the population-based
cervical screening program of the greater Stockholm County in
Sweden.

http://dx.doi.org/10.1016/j.jcv.2015.12.007
1386-6532/© 2015 Elsevier B.V. All rights reserved.

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34 M. Hortlund et al. / Journal of Clinical Virology 75 (2016) 33–36
2. Objective
As primary HPV screening programs are rolled out, methods are
needed for routine quality assurance of the HPV laboratory ana-
lyzes. We studied the feasibility of using the same study design as is
currently used in cytology-based screening to estimate the clinical
sensitivity to identify women at risk for CIN3+.
3. Study design
The Swedish cervical screening program invites all women res-
ident in Sweden for screening every third year (23–49 years of age)
or fifth year (50–60 years of age) [10]. The 2008 EU guidelines
on cervical screening recommended that HPV-based screening
should be confined to controlled implementation trials [11] and
in the greater Stockholm County, an implementation trial that
invites 50% of the population each to either cytology-based or HPV-
based screening is ongoing [12]. The organized cervical screening
program has centralized all cytology and all HPV testing to the
Karolinska University Hospital’s Laboratory in Huddinge. The Cen-
ter for Cervical Cancer Prevention of the Karolinska University
Hospital (i) performs all the HPV testing for the screening pro-
gram of the greater Stockholm County, (ii) is responsible for the
Swedish National Cervical Screening Registry and (iii) performs
systematic biobanking of the liquid cervical samples taken [13,14].
All cervical screening samples are taken using liquid-based cytol-
ogy (LBC/ThinPrep® ), which suspends the cervical cells in 20 ml of
the methanol-based medium PreservCyt (ThinPrep® Hologic, Marl-
borough, MA). Whereas the screening biobank contains all cervical
samples, including those collected in clinical contexts, the current
study is restricted to samples collected from women participating
in the organized screening program.
The Karolinska University Hospital’s Laboratory registry was
searched to identify all women who had a cervical biopsy diagnosed
with CIN3+ taken 1-JAN-2013 to 31-JAN-2014. The idea is that LBC
sample preceding this diagnosis should be abnormal by a screen-
ing test, forming a basis for estimation of diagnostic sensitivity. This
search identified 381 women.
Out of those, 193 women had had a prediagnostic LBC sample
taken up to two years before the histopathological CIN3+ diagnosis.
Among 154 out of 193 women the LBC sample had been biobanked
and these samples were included in this study (Fig. 1). The sam-
ples had been biobanked as previously described [14] and stored
in 96-well microplates (0.75 ml Tracker 2D in Loborack-96w low
cover, MPW52337BC3, Nordic Biolabs AB) at −25 ◦ C. As the liquid
preservative is methanol-based, the cells are not frozen at this tem-
perature. The sample preservation procedure is annually quality
assured at inspections by SWEDAC (the Swedish Board for Accred-
itation and Conformity Assessment) [14].
Of the 154 LBC samples included in this study, 91 had not been
HPV-tested and were retrieved from the biobank for HPV test-
ing. 63 had been HPV-analyzed in routine screening by the cobas
4800 platform (Roche Diagnostics), the HPV assay purchased by the
Stockholm County routine screening program. The test is a qualita-
tive detection of DNA by amplification of a 200 bp fragment of the
polymorphic L1 region of the HPV genome with multiplex real-time
PCR for simultaneous detection of HPV 16, 18 and a pool of 12 other
high-risk HPV-containing genotypes 31, 33, 35, 39, 45, 51, 52, 56,
58, 59, 66 and 68. If the LBC sample from the women with CIN3+
was HPV-negative, the sample was further analyzed by a modi-
fied GP5+/6+-general primer PCR method with HPV typing using
Luminex [15]. The sensitivity of HPV analyzes performed with cobas
4800 was then compared to the sensitivity of cytology, as defined
by the original reading/diagnosis of the smear. Prediagnostic LBC
samples originally read as cytologically negative were retrieved and
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Table 1
Sensitivity of the HPV-analyzes in LBC by cobas 4800 compared to cytology to iden-
tify women with subsequent CIN3+ in biopsy.
Method True positive False negativea
Cobas 4800 b
148/154 (97%) 6/154 (3%)
Cytology diagnosis 144/154 (94%) 11/154 (6%)
a
5 LBC samples negative in cobas 4800 testing were further analyzed with
GP5+/6+ -PCR by Luminex, where 4 samples were HPV negative. 1 sample was
positive for HPV33, 1/154 (1%).
b
HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68.
Table 2
HPV types detected by Cobas and Luminex in the LBC and the tissue samples.
Method N LBC samples (HPV type) N tissue (HPV type)
a 1 2 (31)
Luminex
(33) 1 (52)
1 (53)
1 (67)
a
HPV types 6, 11, 16, 18, 26, 30, 31, 33, 35, 39, 40, 42, 43, 45, 51–54, 56, 58, 59,
61, 66–69, 70, 73, 74, 81–83, 86, 87, 89, and 90.
re-read. For the statistical analysis we used the chi-square test with
the Yates correction for continuity.
The study end-point was histopathologically diagnosed CIN3+.
The audit was restricted to women who had had a cervical LBC
sample within two years prior to the CIN3+ lesion. CIN3+ lesions
diagnosed as a result of the LBC screen were included. Women were
excluded if no LBC sample had been taken within two years before
CIN3+ or if the LBC sample was not available in the biobank. For
the statistical analysis we used the chi-square test with the Yates
correction for continuity.
The investigation forms part of the Stockholm County organised
screening program’s routine quality follow-up. Ethical permission
was obtained from the ethical regional board of Stockholm.
4. Results
The cobas 4800HPV analyzes (either the original testing or sub-
sequent testing of a biobanked sample) of the 154 LBC samples
taken before CIN3+ identified 149HPV positive cases, correspond-
ing to a sensitivity of 149/154 (97%) (Table 1). Cytology performed
on the same samples identified abnormal cells in 144/154 cases
(94%). Comparing the outcomes of virology and cytology in a two-
by-two contingency test (including Yate’s correction for continuity)
demonstrates a statistical difference between the two measures
(2 df=1 = 5.01 > 4.03 = 2 df=1; ˛ = 0.05). The corresponding figure for
the 84 samples taken simultaneously with the diagnostic biopsy,
i.e., independent of the decision to make a biopsy and with-
out knowledge of the histological diagnosis, was 74 cytologically
abnormal samples (88%). Further HPV analysis of the five HPV-
negative samples by general primer PCR and Luminex found only
one HPV-positive LBC sample (containing HPV 33) (Table 1). The
formalin-fixed paraffin-embedded biopsies of the five remaining
cases with HPV-negative prediagnostic LBC samples were retrieved
and analyzed by the modified general primer GP5+/6+-PCR method
with typing using Luminex. We found HPV types 31 (in two cases),
52, 53 and 67 in these samples (Table 2). HPV types 53 and 67
are not tested for with the cobas 4800 platform, so prediagnos-
tic HPV-negativity was not unexpected in these samples. All six
HPV-negative samples were positive by cytology (five cases were
diagnosed as CIN3/carcinoma in situ and one case as CIN2). Among
the 144 samples diagnosed as cytologically abnormal, 53 samples
were diagnosed as low-grade lesions and 91 as high-grade lesions.
The 10 samples that had been negative by original cytology reading
 M. Hortlund et al. / Journal of Clinical Virology 75 (2016) 33–36 35

Histopathological diagnosis of CIN3+ at the

Karolinska Hospital during 2013-2014 (n=381)
Excluded: No LBC sample
in the previous 2 years
(n=188)
Had an LBC sample taken up to two years prior
to diagnosis (n=193)
Excluded: LBC sample
not biobanked (n=39)

Had a biobanked LBC sample (n=154)

HPV analyzed in routine Biobanked sample HPV-

screening (n=63) analyzed in study (n=91)

Fig. 1. Flow-chart of sample collection.

were re-examined and found to be positive in five cases: one case
of HSIL, one case of LSIL and three cases of ASCUS.
5. Discussion
Primary HPV-based screening should have higher clinical sen-
sitivity than cytology, which has been reported in many studies
[4,16–18]. A suggested target guideline for HPV testing [19] is that
the sensitivity for detecting CIN2+ among women over 30 years
should be over 88%. Our current study found 97% sensitivity for HPV
testing in CIN3+, while the corresponding sensitivity for cytology
was 94%. Although this latter figure is high, the viral test showed
a still higher value, far above that recommended in the guide-
lines and better than cytology when analyzed on exactly the same
samples. The cytological diagnoses had been established indepen-
dently, without knowledge of findings in histology or from the virus
analyzes.
We chose not to include estimation of specificity in the routine
audit. First, HPV-prevalences are highly age-dependent, resulting
in that large numbers of normal samples would need to be included
and results adjusted to the world standard population. HPV preva-
lences in the general population are also highly variable between
countries. Second, as the program uses triaging of HPV-positive
women with cytology, the consequences of a false positive test
are not severe (mostly limited to an unnecessary cytology triage
test being performed). Third, most women who are continuously
HPV-positive over several years will ultimately develop CIN2+ [7],
resulting in that specificity estimates are strongly dependent on the
length of follow-up time.
In this study, we have given an example of how an audit of the
clinical sensitivity of the screening analysis in a real-life screening
program can be carried out in a simple, structured and quality-
assured manner. The same regular audit of the clinical sensitivity is
already routinely used in the cytology-based organized screening
program by many laboratories. We propose that such audits should
also be a regular part of HPV-based screening programs to warrant
a preserved high sensitivity for the HPV testing. Our audit used the
laboratory database on histologies and other cervical tests to iden-
tify the cases and the samples taken before histopathology-verified
CIN3+ to be included in the audit. Similar laboratory database are
likely to be available in most pathology laboratories, suggesting
that the proposed audit scheme could be generally useful. Well-
structured audits can systematically identify errors and analyse
artifacts [20], as well as assess the quality of the HPV-analysis per-
formed and monitor possible shift of HPV types and possible new
variants [21,22]. The proposed audit of clinical performance will be
an important part in an overarching quality framework that will
be required for laboratories performing the testing in HPV-based
screening programs. Other important components of the quality
framework will include a quality assurance system (with reporting
of quality deviations, inspections and accreditations by authorized
accreditation bodies), validation and verification of assays as well
as robust training and associated competency assessments. Such
audits will be important tools in the post HPV-vaccination era, to
monitor which types of HPV that continue to cause disease in the
population. We suggest that laboratories performing organised pri-
mary HPV screening should perform an audit of this type every
year; that the results of the audit should be reported to the national
screening registry of the country, and be made openly available as
parts of the annual reporting of quality indicators of the program.
Funding
This study was performed using the resources of the organized
screening program. Supplementary funding was obtained from the
Swedish Foundation for Strategic Research.
Competing interest
None declared.
Ethical approval
Research Ethics Committee approval was obtained from the
Ethics Review Board in Stockholm, Sweden.
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