Comparison of the INNO-LiPA and PapType assays for detection of human papillomavirus in archival vulva dysplasia and/or neoplasia tissue biopsy specimens.
ABSTRACT INNO-LiPA and PapType human papillomavirus (HPV) genotyping assays were compared for detection of HPV genotypes on archival vulvar tissue. The INNO-LiPA assay detected 49 HPV-16 infections, compared with 47 detected by the PapType assay. The INNO-LiPA assay detected amplifiable DNA in 59 (91%) biopsy specimens, compared with 57 (88%) specimens for which amplifiable DNA was detected by the PapType assay. The two genotyping assays were highly comparable.
- [show abstract] [hide abstract]
ABSTRACT: Data on human papillomavirus (HPV) prevalence in vulvar and vaginal cancers are limited. These data are important to predict the potential future effect of prophylactic HPV vaccines. Our aim was to conduct a systematic review of HPV type distribution in vulvar and vaginal invasive carcinomas, vulvar intraepithelial neoplasia (VIN), and vaginal intraepithelial neoplasia. A MEDLINE search was conducted using the terms vulvar/vaginal cancer, intraepithelial neoplasia, and HPV/human papillomavirus through September 2007 with no specified start date or language restrictions. A total of 725 abstracts (564 vulvar, 161 vaginal) were reviewed, of which 67 studies (56 vulvar, 11 vaginal) met the inclusion criteria of using polymerase chain reaction (PCR) or hybrid capture assays for HPV DNA detection and having more than one case with HPV data available. This review identified 2,790 vulvar (1,379 invasive, 1,340 VIN2/3, 71 VIN1) and 315 vaginal cases (83 invasive, 166 vaginal intraepithelial neoplasia 2/3, 66 vaginal intraepithelial neoplasia 1). Most cases were from North America and Europe (87.2%), with few from Asia (5.5%) and South America (7.3%). Human papillomavirus prevalence in vulvar cancer, VIN2/3, and VIN1 was 40.1%, 80.4%, and 77.5%, respectively. HPV prevalence in vaginal cancer, vaginal intraepithelial neoplasia (VAIN)2/3, and VAIN1 was relatively higher at 65.5%, 92.6%, and 98.5%, respectively. HPV16 was the most common type in vulvar (29.3%) and vaginal (55.4%) cancers, VIN2/3 (71.2%) and VAIN2/3 (65.8%). Human papillomavirus prevalence was higher among vaginal than vulvar cases, and HPV16 accounted for most HPV-positive cases for both cancers. Although the potential effect of HPV vaccines on these gynecologic cancers may not be as high as for cervical cancer due to their more diverse causes, vaccinating young women against HPV16/18 may help to reduce the incidence of HPV-related cases.Obstetrics and Gynecology 05/2009; 113(4):917-24. · 4.80 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Molecular diagnostic applications that use microarrays to analyze large numbers of genes simultaneously require high-quality mRNA. As these genome-wide expression assays become more commonly used in medical practice, pathologists and oncologists will benefit from understanding the importance of obtaining high-quality RNA in order to generate reliable diagnostic and prognostic information, especially as these relate to cancer. To review the effects that different tissue preservation techniques have on RNA quality and to provide practical advice on changes in tissue acquisition and handling that may soon be needed for certain clinical situations. A review of recent literature on RNA quality, tissue fixation, cancer diagnosis, and gene expression analysis. Studies have consistently shown that frozen tissue yields more intact RNA than formalin-fixed, paraffin-embedded tissue. The chemical modification, cross-linking, and fragmentation caused by formalin fixation often render RNA unsuitable for microarray analysis. Thus, when expression analysis involving hundreds or more than 1000 gene markers is contemplated, pathologists should consider freezing a specimen within half an hour (preferably within minutes) of surgical resection and storing it at -80 degrees C or below. In coming years, pathologists will need to work closely with oncologists and other clinicians to determine when saving frozen tissue for microarray expression analysis is both practical and necessary. In select cases, the benefit of implementing a few extra tissue-handling steps may improve diagnostic and prognostic capability.Archives of pathology & laboratory medicine 01/2008; 131(12):1805-16. · 2.78 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The PGMY L1 consensus primer pair combined with the line blot assay allows the detection of 27 genital human papillomavirus (HPV) genotypes. We conducted an intralaboratory and interlaboratory agreement study to assess the accuracy and reproducibility of PCR for HPV DNA detection and typing using the PGMY primers and typing amplicons with the line blot (PGMY-LB) assay. A test panel of 109 samples consisting of 29 HPV-negative (10 buffer controls and 19 genital samples) and 80 HPV-positive samples (60 genital samples and 20 controls with small or large amounts of HPV DNA plasmids) were tested blindly in triplicate by three laboratories. Intralaboratory agreement ranged from 86 to 98% for HPV DNA detection. PGMY-LB assay results for samples with a low copy number of HPV DNA were less reproducible. The rate of intralaboratory agreement excluding negative results for HPV typing ranged from 78 to 96%. Interlaboratory reliability for HPV DNA positivity and HPV typing was very good, with levels of agreement of >95% and kappa values of >0.87. Again, low-copy-number samples were more prone to generating discrepant results. The accuracy varied from 91 to 100% for HPV DNA positivity and from 90 to 100% for HPV typing. HPV testing can thus be accomplished reliably with PCR by using a standardized written protocol and quality-controlled reagents. The use of validated HPV DNA detection and typing assays demonstrating excellent interlaboratory agreement will allow investigators to better compare results between epidemiological studies.Journal of Clinical Microbiology 03/2003; 41(3):1080-6. · 4.07 Impact Factor
JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 2011, p. 3980–3982
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 49, No. 11
Comparison of the INNO-LiPA and PapType Assays for Detection
of Human Papillomavirus in Archival Vulva Dysplasia and/or
Neoplasia Tissue Biopsy Specimens?
Sarah E. Tan,1,2,3* Suzanne M. Garland,1,2,3,4Alice R. Rumbold,5,6and Sepehr N. Tabrizi1,2,3,4
Department of Microbiology and Infectious Diseases, The Royal Women’s Hospital, Melbourne, Victoria, Australia1;
Department of Obstetrics and Gynaecology, The University of Melbourne, Melbourne, Victoria, Australia2;
WHO HPV LabNet Regional Reference Laboratory—Western Pacific Region, Melbourne, Victoria,
Australia3; Murdoch Children’s Research Institute, Melbourne, Victoria, Australia4;
Menzies School of Health Research, Darwin, Northern Territory, Australia5; and
Discipline of Obstetrics and Gynaecology, The University of
Adelaide, Adelaide, South Australia, Australia6
Received 15 March 2011/Returned for modification 16 June 2011/Accepted 6 September 2011
INNO-LiPA and PapType human papillomavirus (HPV) genotyping assays were compared for detection of
HPV genotypes on archival vulvar tissue. The INNO-LiPA assay detected 49 HPV-16 infections, compared with
47 detected by the PapType assay. The INNO-LiPA assay detected amplifiable DNA in 59 (91%) biopsy
specimens, compared with 57 (88%) specimens for which amplifiable DNA was detected by the PapType assay.
The two genotyping assays were highly comparable.
Human papillomavirus (HPV) is involved in the etiology of
a number of human mucosal cancers, particularly those of the
anogenital region (2, 15). With the recent introduction and
further development of HPV prophylactic vaccines (5, 8), ep-
idemiological studies are needed to determine the prevalence
of HPV genotypes present in HPV-related cancer and precan-
cerous lesions. This requires evaluation of HPV retrospectively
by using archival tissues. Challenges often arise when examin-
ing HPV genotypes in paraffin-embedded tissues. DNA can
degrade due to extensive or inappropriate storage conditions,
resulting in cross-linking, fragmentation, and chemical modi-
fication, which can affect the detection by PCR (1, 4, 7).
This study compared two commercially available HPV
genotyping detection kits, the INNO-LiPA HPV Genotyp-
ing Extra (INNO-LiPA) (Innogenetics, Ghent, Belgium)
(14) and PapType high-risk (HR) HPV detection and geno-
typing (PapType) (Genera Biosystems, Melbourne, Victo-
ria, Australia) kits, to amplify and detect HPV genotypes in
archival vulvar tissue.
The INNO-LiPA assay uses SPF10 primers to amplify a
region of the HPV L1 gene and detects 28 anogenital HPV
genotypes, including 12 HR HPVs, 1 probable HR HPV, 7
possible HR HPVs, and 8 low-risk types (9). In addition to
having an internal control, the INNO-LiPA assay also had two
HPV internal controls (both 65-bp products from the L1 re-
gion). This assay uses conventional PCR and reverse line blot
hybridization for the detection of controls and HPV genotypes.
The PapType HPV genotyping test directs the amplification
of a region of the HPV L1 gene and allows for the identifica-
tion of 12 HR genotypes, 1 probable HR HPV, 1 possible HR
HPV, and 2 low-risk genotypes (9). A conventional PCR prod-
uct is hybridized to the HPV type-specific probes bound to
labeled silica microspheres, and binding events are read by
Biopsy specimens stored as formalin-fixed paraffin-embed-
ded tissue were obtained from 65 histologically diagnosed
cases of high-grade vulvar intraepithelial neoplasia (VIN-2/3)
(n ? 59) and invasive vulvar cancer (n ? 6), with patients
between 18 and 50 years of age at diagnosis.
Seven-micrometer sections were processed for histological
analysis by using a sandwich sectioning method (5). Outer
sections were stained with hematoxylin and eosin to confirm
the initial histological diagnosis, while the three inner sections
remained unstained for DNA extraction and HPV detection
(5). To minimize cross-contamination, the microtome blade
was changed and the microtome surface was cleaned with
xylene after sectioning of each sample. Regular changing of
gloves was performed between each sample, and the sectioning
of HPV-negative tissue specimens (negative control) was car-
ried out in a random order to ensure no DNA carryover. One
section was deparaffinized and DNA extracted as previously
described (12). For the INNO-LiPA assay, typing strips were
visually interpreted according to the manufacturer’s instruc-
Comparison of INNO-LiPA and PapType assays for prepa-
ration, amplification, and detection of HPV genotypes in ar-
chival vulvar tissue is shown in Table 1. Comparison of the
amplification efficiencies of internal controls and the abilities
to detect single-HPV-genotype infections is shown in Table 2.
Agreement in detection of HPV genotypes was determined by
Cohen’s kappa statistics, which are a measure of agreement
which is in excess of that due to chance. Due to the cross-
reactivity of the probes in the INNO-LiPA test, coinfection
with other HPV genotypes cannot be ruled out, and hence,
coinfection has been noted.
* Corresponding author. Mailing address: Department of Microbi-
ology and Infectious Diseases, The Royal Women’s Hospital, Locked
Bag 300, Parkville, Victoria 3052, Australia. Phone: (61-3) 8345-3678.
Fax: (61-3) 8345-2225. E-mail: firstname.lastname@example.org.
?Published ahead of print on 21 September 2011.
demonstrated that the PapType assay takes half the amount of
hands-on time of the INNO-LiPA assay. The PapType assay
produces computer-generated results, whereas the INNO-LiPA
assay requires manual reading of line blot strips. Amplification of
the respective internal controls demonstrated that 22% (n ? 13)
were successfully amplified using the INNO-LiPA assay, com-
pared with 31% (n ? 20) amplified with the PapType assay (P ?
and detected in 56% (n ? 18) and 71% (n ? 46) for HPV control
1 and HPV control 2, respectively. There was almost perfect
agreement between assays in the detection of HPV-16 (? value ?
0.959) (P ? 0.84) and perfect agreement for the detection of
HPV-33 (? value ? 1.000), although the sample size was small for
the latter comparison (n ? 9). All biopsy specimens that were
internal control negative/HPV negative by the INNO-LiPA kit
(n ? 7) were also identified as internal control negative/HPV
negative by the PapType kit. Two cases were recorded as discor-
dant; both cases were HPV-16 positive using the INNO-LiPA
The INNO-LiPA assay detected 4 multiple-HPV-genotype infec-
tions (16 and 33, 16 and 33, 16 and 52, and 16 and 39), compared
with the PapType assay detecting 2 multiple-HPV-genotype in-
fections (16 and 33 and 16 and 39).
This study has shown that there is very good agreement
between the INNO-LiPA and PapType assays for the detection
of single infections, namely, HPV-16 and HPV-33, the only two
genotypes detected. This is in accordance with HPV-16 being
the most prevalent HPV genotype detected in high-grade VIN
and vulvar cancers, followed by HPV-33 (3, 10). HPV-18 was
not detected in any biopsy specimens in this study; this is in
accordance with other studies (11, 13), and this may be due to
a difference between HPV genotypes and tropisms for vulvar
epithelium. The PapType assay takes less hands-on time than
the INNO-LiPA assay, and classification of HPV genotype-
positive bands on the INNO-LiPA strip is subjective to inter-
pretation and may take additional time to complete, whereas
the PapType assay produces computer-generated results.
The discrepant results that were observed in this study may
be due to the amount of DNA added in each assay. The
INNO-LiPA assay requires 10 ?l, compared to 5 ?l for the
PapType assay, resulting in an initially higher concentration of
DNA in the PCR: HPV genotypes not detected may have been
present in low copy numbers (6). A number of specimens were
HPV genotype positive though negative for the respective in-
ternal control. This may be due to amplicon size of the internal
controls relative to HPV genotypes, as fragmentation and deg-
radation of DNA is often encountered with archival tissue, and
generation of larger amplicons is often difficult with archival
tissue (1, 12). In the small number of samples, the INNO-LiPA
assay detected 4 cases with multiple HPV types, whereas the
PapType assay detected 2 samples with multiple types; how-
ever, the two assays detected similar genotypes. Comparison of
the abilities of the two assays to detect multiple HPV infections
would need to be verified in a larger study.
In conclusion, the INNO-LiPA and PapType assays are com-
parable HPV genotyping tests when applied to vulvar archival
paraffin-embedded tissue for the detection of HPV genotypes.
The INNO-LiPA assay is able to detect a greater number of
low-risk HPV genotypes and employs a smaller amplicon; the
PapType assay has an automated result output and less
hands-on time. Both assays are efficient genotyping tests and
have contrasting benefits for future epidemiological studies in
relation to the detection of single and multiple HPV infections.
S.E.T. was supported by The Royal Women’s Hospital Post Grad-
uate Degree Scholarship from The Royal Women’s Hospital and the
Women’s Centre for Infectious Diseases, The Royal Women’s Hospi-
tal, Melbourne, Australia. A.R.R. is supported by the Jean B. Reid
Fellowship from the University of Adelaide Medical Endowment
We thank Tania Tabone for help in critical reading of this article.
Genera Biosystems provided PapType high-risk HPV detection and
S.M.G. and S.N.T. were previously members of the clinical devel-
opment advisory board at Genera Biosystems; however, this advisory
TABLE 2. Comparison of single infections detected using
INNO-LiPA and PapType HPV genotyping assays in
65 biopsy specimens from patients with high-grade
vulva dysplasia or vulva cancer
No. (%) of specimens with detection result
IC positive/HPV genotype negative
IC negative/HPV gentoype positive
HPV control 1d,e
HPV control 2d
IC and HPV negative
Total HPV genotypes detected58 56
aIC, internal control.
bAssessable indicates either positive for IC or an HPV genotype.
cEither MLC-1 positive by PapType or HLA-DPB1 positive by INNO-LiPA.
This parameter had a P value of 0.22.
dDetected by the INNO LiPA HPV genotyping test only.
eP ? 0.84.
fWe were unable to rule out coinfection with HPV-52 and -54.
TABLE 1. Comparison of INNO-LiPA and PapType kits for
preparation, amplification and detection of HPV genotypes
in 16 archival vulvar tissue biopsy specimens
ParameterINNO-LiPA kit PapType kit
Amount of DNA in
270-bp region of the
288-bp region of the alkali
myosin light chain
140-150 (depending on
Detection time (min)
Presentation of results
Reverse line blot assayFlow cytometry
Manual reading of line
blot strip (?1-2 min
VOL. 49, 2011NOTES 3981
board has ceased to exist. S.M.G. has received advisory board fees and
grant support from CSL Biotherapies and GlaxoSmithKline (GSK),
lecture fees from Merck, GSK, and Sanofi Pasteur, and funding
(through her employing institution) to conduct HPV vaccine studies
for Merck, Sharp and Dohme (MSD) and GSK and is a member of the
Merck Global Advisory Board and the Merck Scientific Advisory Com-
mittee for HPV. This relates to work outside this study. S.E.T. is the
recipient of a GSK Australian Postgraduate Support Grant for work
outside this study. CSL has provided funding to the authorship group
to support a workshop on genetic susceptibility to vulvar cancer.
1. Ben-Ezra, J., D. A. Johnson, J. Rossi, N. Cook, and A. Wu. 1991. Effect of
fixation on the amplification of nucleic acids from paraffin-embedded mate-
rial by the polymerase chain reaction. J. Histochem. Cytochem. 39:351–354.
2. Della Torre, G., et al. 1992. HPV DNA in intraepithelial neoplasia and
carcinoma of the vulva and penis. Diagn. Mol. Pathol. 1:25–30.
3. De Vuyst, H., G. M. Clifford, M. C. Nascimento, M. M. Madeleine, and S.
Franceschi. 2009. Prevalence and type distribution of human papillomavirus
in carcinoma and intraepithelial neoplasia of the vulva, vagina and anus: a
meta-analysis. Int. J. Cancer 124:1626–1636.
4. Ferrer, I., et al. 2007. Effects of formalin fixation, paraffin embedding, and
time of storage on DNA preservation in brain tissue: a BrainNet Europe
study. Brain Pathol. 17:297–303.
5. Garland, S. M., et al. 2007. Quadrivalent vaccine against human papilloma-
virus to prevent anogenital diseases. N. Engl. J. Med. 356:1928–1943.
6. Kornegay, J. R., et al. 2003. International proficiency study of a consensus L1
PCR assay for the detection and typing of human papillomavirus DNA:
evaluation of accuracy and intralaboratory and interlaboratory agreement.
J. Clin. Microbiol. 41:1080–1086.
7. Medeiros, F., C. T. Rigl, G. G. Anderson, S. H. Becker, and K. C. Halling.
2007. Tissue handling for genome-wide expression analysis: a review of the
issues, evidence, and opportunities. Arch. Pathol. Lab Med. 131:1805–1816.
8. Romanowski, B., et al. 2009. Sustained efficacy and immunogenicity of the
human papillomavirus (HPV)-16/18 AS04-adjuvanted vaccine: analysis of a
randomised placebo-controlled trial up to 6.4 years. Lancet 374:1975–1985.
9. Schiffman, M., G. Clifford, and F. M. Buonaguro. 2009. Classification of
weakly carcinogenic human papillomavirus types: addressing the limits of
epidemiology at the borderline. Infect. Agents Cancer 4:8.
10. Smith, J. S., D. M. Backes, B. E. Hoots, R. J. Kurman, and J. M. Pimenta.
2009. Human papillomavirus type-distribution in vulvar and vaginal cancers
and their associated precursors. Obstet. Gynecol. 113:917–924.
11. Srodon, M., M. H. Stoler, G. B. Baber, and R. J. Kurman. 2006. The
distribution of low and high-risk HPV types in vulvar and vaginal intraepi-
thelial neoplasia (VIN and VaIN). Am. J. Surg. Pathol. 30:1513–1518.
12. Tan, S. E., S. M. Garland, A. R. Rumbold, and S. N. Tabrizi. 2010. Human
papillomavirus genotyping using archival vulval dysplastic or neoplastic bi-
opsy tissues: comparison between the INNO-LiPA and linear array assays.
J. Clin. Microbiol. 48:1458–1460.
13. van Beurden, M., et al. 1995. Multifocal vulvar intraepithelial neoplasia
grade III and multicentric lower genital tract neoplasia is associated with
transcriptionally active human papillomavirus. Cancer 75:2879–2884.
14. van Hamont, D., M. A. van Ham, J. M. Bakkers, L. F. Massuger, and W. J.
Melchers. 2006. Evaluation of the SPF10-INNO LiPA human papillomavirus
(HPV) genotyping test and the roche linear array HPV genotyping test.
J. Clin. Microbiol. 44:3122–3129.
15. Walboomers, J. M., et al. 1999. Human papillomavirus is a necessary cause
of invasive cervical cancer worldwide. J. Pathol. 189:12–19.
3982 NOTESJ. CLIN. MICROBIOL.